s2fieldsofstudy: Biology Medicine

text:
CAR-NK cells for cancer immunotherapy: from bench to bedside

Natural killer (NK) cells are unique innate immune cells and manifest rapid and potent cytotoxicity for cancer immunotherapy and pathogen removal without the requirement of prior sensitization or recognition of peptide antigens. Distinguish from the T lymphocyte-based cythotherapy with toxic side effects, chimeric antigen receptor-transduced NK (CAR-NK) cells are adequate to simultaneously improve efficacy and control adverse effects including acute cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Moreover, considering the inherent properties of NK cells, the CAR-NK cells are “off-the-shelf” product satisfying the clinical demand for large-scale manufacture for cancer immunotherapy attribute to the cytotoxic effect via both NK cell receptor-dependent and CAR-dependent signaling cascades. In this review, we mainly focus on the latest updates of CAR-NK cell-based tactics, together with the opportunities and challenges for cancer immunotherapies, which represent the paradigm for boosting the immune system to enhance antitumor responses and ultimately eliminate malignancies. Collectively, we summarize and highlight the auspicious improvement in CAR-NK cells and will benefit the large-scale preclinical and clinical investigations in adoptive immunotherapy.

Introduction
Anticancer immunotherapies, including adoptive cytotherapy and checkpoint inhibitors, have present as novel pillars with oncology management [1,2]. For decades, pioneering investigators have devoted to verify the interactions between the responses of the human immune system and numerous cancers or invaders such as bacteria and viruses, which collectively accelerate the development of clinically effective cancer immunotherapy [3,4]. However, those reported "immune enhancement" strategies have a series of disadvantages such as rare objective responses and concomitant immunerelated adverse events (irAEs), which could be largely alleviated by the termed "immune normalization" (e.g., PD-1/PD-L1) with more beneficial cancer response-totoxicity profile [4].
Current studies have indicated the diversity of cancer immunotherapies together with the potentially combined strategies in multiple indications [5,6]. For instance, we and other investigators have reported the successful generation of T lymphocyte-mediated tactics including chimeric antigen receptor-modified T (CAR-T) cells and T cell receptor-engineered T (TCR-T) cells as well as tumor-infiltrating lymphocytes (TILs) and regulatory T (Treg) cells in eliminating malignant hematologic tumors (e.g., B acute lymphoblastic leukemia) and metastatic solid tumors (e.g., HBV-related hepatocellular carcinoma, head and neck squamous cell carcinoma) processed by antigen-presenting cells (APCs) and fine-tuned by co-stimulatory or co-inhibitory signals [6][7][8][9][10][11][12]. However, the adoptive T cell-based immunotherapy severely constrained by the major limitation of the rapidly declined cellular viability and function, together with the requirement of concurrent administration of the adjuvant drugs after transplantation [13]. Moreover, due to the alteration in genetic mutation and cell-surface biomarker expression and the resultant off-target effects, tumor escape has become a common but intractable outcome of malignant transformation and identification of more optimal candidates or personalized neoantigens seems boundless [14][15][16]. Collectively, the autologous T lymphocytes, including the classical T cell receptor-engineered T (TCR-T) cells and CAR-transduced T (CAR-T) cells, are labor-intensive to manufacture and logistically challenging to personalized deliver to inpatients.
In consequence, state-of-the-art renewal has turned to rediscover the immune recognition and eradication of tumor cells by comminating with immune checkpoint blockade (e.g., CTLA4, PD-1/PD-L1), and in particular, to harness the innate immune response with moderate cytotoxicity and reduced adverse effects [17,18]. Of the indicated innate immune cells such as macrophages (Mø) and dendritic cells (DCs), autologous or allogeneic NK cells are adequate to fulfill the biofunction of combating malignant tumors and pathogenic microorganisms via paracrine effects (e.g., IFN-γ, GM-CSF), antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytolytic effect dispense with preliminary antigen presentation as well as manipulating other immune contextures to recognize and attack cancer cells [1,5,[19][20][21]. However, the heterogeneous tumor cells with genetic or epigenetic variations are also sufficient to elude the immunological surveillance and even reversely suppress NK cell cytotoxicity by interdicting the corresponding activating receptors [5,22,23]. Considering the deficiency of CAR-T and non-gene-edited NK cells, CAR-NK cells have been recognized as novel therapeutic options aiming at reducing the incidence of relapse and attaining complete remission. Of note, considerable progresses have been achieved in an increasing number of therapeutic dimensions ranging from preclinical studies to clinical practices [24].
Therefore, in this review article, we mainly summarize the key elements and current advances of CAR-NK cellbased immunotherapy including cell sources, novel target selection, design of CAR construction, mode of CAR transduction, and ultimately discuss the opportunities and challenges of adoptive CAR-NK cell-based cancer immunotherapy. Collectively, the CAR-NK cell-mediated cytotherapy has constituted a promising area of cellular immunotherapy innovation.

NK cell lines
Generally, considering the difficulty in isolating, purifying and expanding primary NK cells as well as the inefficiency in transducing CAR constructs, the wellestablished NK cell lines with indefinite expansion capacity have been used in clinical practice [25]. Notably, the representative IL-2 depend NK-92 cell line and the NK-92MI derivation exhibit splendid advantages of easy expansion, cultivation and activation in the context of lymphodepletion, together with sustainable and reliable cytotoxicity after infusion against leukemia cells ( Fig. 1) [24]. For example, Boyiadzis and the colleagues conducted a phase 1 clinical trial of NK92 cell-based adoptive immunotherapy in patients with refractory and relapsed acute myeloid leukemia (AML) and confirmed the feasibility, safety and strong anti-leukemia activity [26]. Simultaneously, it's noteworthy that NK92 cells are originated from patients with non-Hodgkin's lymphoma and thus require irradiation prior to infusion to eliminate risks of malignant transformation and the accompanied chromosomal abnormalities [27]. Another preclinical study upon AML immunotherapy by Kloess et al verified that engineered CD123-CAR-NK-92 cells showed higher levels of granzyme and interleukin secretion and preferable cytotoxic activities over the primary human donorderived CD123-CAR-NK cells, while revealed significant side effects against nonmalignant cells as well [28]. Additionally, Binyamin et al. found that NK-92 cells revealed enhanced ADCC by blocking the inhibitory receptors (e.g., KIR2DL1, KIR3DL1) and combining with rituximab [29].
Another classical NK cell line YT with the prostate cancer cell antigen PSMA transduction and shp-2 (PTPN11) deletion has been indicated with enhanced cytotoxicity [30]. Meanwhile, the leukemic cell line YT has also been proved with spontaneous cytotoxicity against B lymphoma and specifical lytic effect upon AML by targeting CD80 + /CD86 + B lymphoblastoid cells and CD33 + leukemia cells, respectively [31,32]. Despite the increasing references of CAR-NK cell-based cancer immunotherapy, yet most of the current studies are preclinical. However, the observations of the existing studies are favor of novel treatment concepts employing CAR-NK cell lines with potent degranulation and selective cytotoxicity in malignancies [33].

Peripheral blood-derived NK (PB-NK) cells
In peripheral blood, NK cells account for a proportion of 5-20% of leukocytes, which are divided into the dominating CD56 dim CD16 high subset (85-95%) and the minimal CD56 bright CD16 low/neg subset (5-15%) [5,34]. Besides, PB-NK cells express a wide range of active receptors and thus hold potential as splendid sources for adoptive CAR-NK cell generation ( Fig. 1) [21,35]. In general, resting PB-NK cells reveal a tremendous proliferative CD3 − CD56 bright cellular phenotype and are capable of secreting immunomodulatory cytokines, while the CD3 − CD56 dim counterpart possesses highly cytotoxicity and poor proliferation in response to cytokine stimulation (e.g., IL-2) [36].
To date, autogenous and allogeneic donor-derived PB-NK cells and CAR-PB-NK cells have been most effective in the treatment of acute leukemia (clinically effective doses ranging from 1 × 10 6 /kg to 9.3 × 10 6 /kg) whereas with relatively minimal activity against solid tumors [37][38][39]. Recently, a preclinical study by Quintarelli et al confirmed that CD19-CAR-transduced PB-NK cells were sufficient to mediate robust cytotoxicity against B-cell precursor acute lymphocytic leukemia (Bcp-ALL) and maintain the function of all "native" NK coreceptors after genetic modification [40].

Umbilical cord blood-derived NK (UC-NK) cells
Differ from PB-NK cells, NK cells in umbilical cord blood (UC-NK) only account for a proportion of approximately 5% of total mononuclear cells (TNCs) but offer unique alloreactive advantages for adoptive immunotherapy and boost the potential as a third-party product for extensive clinical scalability ( Fig. 1) [36,41]. In spite of the higher percentage of naïve NK cell population in circulating umbilical cord blood, yet most of the UC-NK cells were adequate to differentiate into functionally mature and active effector cells and thus the successful acquisition of functional competence after ex vivo co-stimulation with cytokine cocktails (e.g., IL-2, IL-7, IL-12, IL-15, IL-18) [36,42]. For example, Xing et al reported the low cytolytic activity of resident UC-NK cells in vivo attribute to impaired lytic immunological synapse formation as well as the enhancement by IL-2 stimulation during ex vivo expansion and activation [43]. Collectively, the existing literatures indicate that UC-NK cells are phenotypically and functionally immature but are capable of maturation [42].
Of note, an increasing number of investigators have turned to UC-NK cells for generating preclinically or clinically tested CAR-NK cells [44]. For instance, a fistin-human phase 1/2 clinical trial identified the feasibility of lympho-depleted CAR-UC-NK cells for the treatment of recurrent and refractory CD19 + B-cell lymphoma including seven cases with complete remissions Fig. 1 The schematic diagram of the sources of NK cells without causing major toxic effects [45]. Despite the once reported generation of over 100 engineered CAR-NK cell doses from one cord blood unit, yet the UC-NK cells have noteworthy limitations in cell mass for largescale adoptive immunotherapy but with high level of the inhibitory receptor NKG2A expression and poor in vitro cytotoxicity [36,42,46].

Placental blood-derived NK (P-NK) cells
In spite of the rare content of P-NK cells (< 2%) in TNCs, yet placental blood is more abundant compared to the aforementioned adult peripheral blood and umbilical cord blood (Fig. 1). Meanwhile, current strategies have indicated the high-efficient generation of clinical-grade CD3 − CD56 + NK cells (an average of nearly 1.0 billion NK cells per donor) with remarkably increased antitumor cytolytic activity from placenta perfusate [38]. Compared to UC-NK cells, the derived P-NK cells are largely similar to UC-NK cells phenotypically and functionally, but display distinct microRNA expression profiles, immunophenotypes and superiority in killing a wide range of cancer cell lines in vitro and thus hold potential for CAR-P-NK cell-based immunotherapeutic development [38,47]. Notably, Guo et al very recently raised the possibility of enhancing cytotoxicity of P-NK cells via CRISPR/Cas9induced CBLB ablation [48].

Stem cell-derived NK cells
Currently, prospective studies have also indicated the feasibility of deriving mature NK cells from CD34 + hematopoietic stem/progenitor cells (HSPCs). In a preclinical study upon AML xenograft model, Cany et al reported the proof-of-concept safety and efficiency of targeting bone marrow-residing leukemia cells via the CCR6/ CCL20 and CXCR3/CXCL10-11 axis in NOD/SCID/ IL2Rg null mice [49].
Human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), possess selfrenewal and multi-lineage differentiation potential [50][51][52]. During the past decades, we and other investigators have reported the generation of progenitor cells and functional cells from hPSCs including mesenchymal stem/stromal cells (MSCs), megakaryocytes (MKs) and NK cells [50,53]. Notably, differ from other cell sources with dominating limitations in NK cell survival and proliferation, hPSC-NK cells can be manufactured from the standardized hPSC population and thus satisfy the clinical demands for large-scale, homogeneous CAR-NK cell products ( Fig. 1) [54,55]. Meanwhile, considering the relatively low efficiency of CAR transduction into primary NK cells, the deficiency of adult PB-NK cells and perinatal UC-NK cells and P-NK cells in cellular activity against solid tumors might be overcome by the genetically modified CAR-hPSC-NK cells via viral or non-viral strategies as hypothesized by pioneering investigators [54,56].
Moreover, it is considered that CARs can be easily and conveniently delivered into hPSCs by utilizing the nonviral transgenic strategy [57,58]. For example, Ni and the colleagues reported the integration of chimeric receptor CD4ζ into hPSC-derived NK cells (CD4ζ-hESC-NK cells, CD4ζ-hiPSC-NK cells) with improved efficacy upon human immunodeficiency virus (HIV)/AIDS [57]. Of note, Li et al recently highlighted the feasibility of transducing CAR constructs with conventional NK cell-specific intracellular activating domains into iPSC-NK cells (CAR + iPSC-NK cells) for the further optimization of tumor-specific recognition and cytotoxicity [59].

Memory-like NK (ML-NK) cells
Cytokine-induced ML-NK cells with the dominant NKG2A checkpoint expression, phenotypically distinct from the in vivo conventional NK cells, have been considered safe and sufficient to induce remissions in patients with AML, which are recognized as new avenues to facilitate CAR-NK cell therapeutics ( Fig. 1) [60,61]. Therefore, we and other investigators presume that CAR-ML-NK cells possess more potent and flexible response to a variety of cancer cell-associated triggers compared to the conventional NK cells [62,63]. It's noteworthy that the differentiated memory-like CAR-NK cells displayed elevated activating receptors against myeloid leukemia and prolonged survival in vivo dispense with the typical KIR-KIR ligand interactions [61,64]. Meanwhile, numerous preclinical data have demonstrated the superior degranulation and IFN-γ-associated response of CAR-NK cells as well as enhanced cancer cell killing and ADCC effect against tumor cells [65,66].
Notably, peripheral blood-derived ML-NKs with a truncated CD-19-CAR transduction were phenotypically and functionally mature and manifested significantly raised IFN-γ secretion and degranulation, broaden recognition and specific killing against NK-resistant lymphoma compared to the conventional CAR-NK cells or no-specific NK cells [63]. Moreover, Foltz et al reported that the CAR-NK cells could even survive and persist in vivo for over 2 months following adoptive transfer in the immune compatible environment, which would be significant improvement of the short lifespan of conventional NK cells.
Nowadays, several groups further indicated the redesignment of CAR-NK cells with NK cell signalingassociated domains to enhance the antitumor efficacy by improving cytotoxicity and INF-γ secretion such as DAP-10, DAP-12, 2B 4 [117]. Nevertheless, due to the rare tumor-specific cell-surface antigens, CAR-NK cells also endure the main disadvantages of requiring extracellular surface expression of specific targets on cancer cells, which thus restrict the broadness and specificity of CAR-NK cell application [4,118]. To overcome the short lifespan and transient cytotoxic activity of CAR-NK cells, Zhang et al recently transduced the homodimers and heterodimers of ErbB2/HER2-specific CARs with CD3ζ and composite CD28ζ signaling domains into NK-92/63.z cells to induce long-lasting endogenous cytotoxicity against immunocompetent glioblastoma (Fig. 2, Table 1) [87].

Construction of CARs
Generally, the CARs are engineered receptor proteins to enable NK cells with novel ability to target cancer cell-specific antigen proteins, which are composed of an intracellular activating signaling domain, a transmembrane region and an extracellular antigen binding domain (Fig. 2, Table 1) [55]. The intracellular activating signaling domains (e.g., CD137, CD28) mainly mediate the activation and cytotoxicity of CAR-NK cells, while the extracellular antigen binding domains (e.g., the singlechain variable fragments) recognize the specific antigen of tumor cells [21]. For example, Quintarelli and the colleagues reported the successful design of retroviral plasmid carrying the cassettes of a second-generation CD19-CAR construct and transduced into PB-NK cells for Bcp-ALL management [40]. Simultaneously, another  Jian, et al [109] preclinical study reported the splendid efficiency of CAR-NK-92 and CAR-NK-92MI cells with the second-or third-generation CARs targeting CD3ζ and CD5 domains against mouse model of T-cell malignancies, respectively [69,119]. Very recently, Daher et al took advantage of the fourth-generation "armored" CARs for generating CAR-UC-NK cells by targeting the cytokine-inducible SH2containing (CIS) protein, which efficiently boosted NK cell antitumor activity against lymphoma xenografts and resulted in enhanced aerobic glycolysis [41,120]. Therewith, due to the potentially excessive cytokine secretion of CAR-NKs, there is a possibility that the fourth-generation of CARs might cause unanticipated toxicity and should reinsert suicide genes (Fig. 2, Table 1) [121]. Collectively, the genetically engineered CAR-NK cells contain a typically extracellular antigen-binding domain, a hinge and transmembrane region and the concomitant intracellular costimulatory domains from receptors, which represents the paradigmatic design of utilizing engineered NK cells for effective attack of cancer cells [4,122]. Of note, the choice and design of the NK cell activating receptor (e.g., NKG2D, DAP10) and remaining domains of the CAR construct, including the aforementioned transmembrane domains (e.g., CD28), co-stimulatory domains and signaling domains (e.g., CD3ζ), together with the NK cell subtypes, should be taken into incorporated consideration [55].

CAR transduction
CAR-transduced cytotherapy was initially advocated using autologous T lymphocytes (CAR-T) and obtained great success in treating hematological malignancies whereas marginal success in facing solid tumors largely attributes to the highly immunosuppressive cancer microenvironment [9,122,123]. Advances in genome editing technique and the applicability of the approach have vastly accelerated the development of designer CAR-NK cell therapy products, which are currently being tested in both preclinical studies and clinical trials [55].

Retrovirus
For decades, a plethora of groups have reported the successful transduction of CAR constructs into expanded NK cells by utilizing a single round of retroviral vectorbased method ranging from 27 to 52%, and in particular, an even higher efficient transduction (approximately 70%) has been achieved by Imamura and the colleagues for IL-15 expression [124,125]. Strikingly, Daher and the colleagues compared the retrovirus transfection of iC9.CAR19.CD28-z-2A-IL-15 (with IL-15) with CAR19. CD28-z (without IL-15) into UC-NK cells, and confirmed the feasibility of high-efficient CAR-NK cell generation and the persistence of in vivo antitumor activity in xenogeneic lymphoma models [41]. Furthermore, Suerth et al systematically compared different retroviral pseudotypes, retroviral vector systems and transduction protocols, and verified that the highest (up to 60%) transduction levels of CAR-19 expression cassette were achieved with α-retroviral plasmids into primary human NK cells [126]. Simultaneously, the retrovirus-based gene-delivery vehicles also have main obstacles to widespread clinical applications including the immunogenicity and insertional mutagenesis as well [58].

Lentivirus
As with retrovirus, lentiviral also have been widely used by preclinical studies for CAR vector transduction into NK cells such as UC-NK cells, PB-NK cells, iPSC-NK cells and ML-NK cells. For example, talented investigators utilized the scFvs-based CD33-CAR and CD19-CAR lacking the immunoreceptor tyrosine-based activation motif (ITAM) domains to manufacture the second-generation CARs with CD3ζ and CD137 intracellular domains [63,127,128]. Simultaneously, Oelsner and other investigators verified that gene-modified CAR-NK-92 cells after lentiviral-mediated gene transfer displayed stable and homogeneous CD-19-and CD20-specific CAR expression, high and specific ADCC against lymphoma and leukemia cells [5,33,129].

Nonviral-mediated transfection
To overcome the major barriers of low-efficient exogenous gene transfer into the primary NK cells, a certain number of research groups turn to the nonviral-mediated transfection such as lipofection and electroporation [58,130]. Of them, the nonviral plasmids are emerging as promising alternatives and facilitate clinical CAR-NK cell-based cancer immunotherapy [58]. Compared to the aforementioned virus-mediated strategies, the nonviral methods revealed transient and rapid expression of CAR-conjunct genes as well as less variability and apoptosis, whereas the generated CAR-NK cells showed declined expression level of transgenes due to the non-integrated property [58].
Of note, the current progresses of transfection technologies, including the DNA transposon systems composed of the PiggyBac (PB) and the sleeping beauty (SB) subsets, are sufficient to deliver CAR structures into the genomes of primary NK cells or iPSCs with long-lasting transgene expression [131]. Collectively, the transposon systems for high-efficient CAR-NK cell generation have splendid advantages such as increased biosafety, rapid and persistent transgene expression, low immunogenicity, cost-effect and capacity for large gene fragment (> 100 kb) transduction, which make them attractive options for CAR-NK cell manufacturing [130]. Nevertheless, considering the dissatisfactory efficiency in current transduction and the potential advances in CAR construct design, the next-generation of CAR-NK cells might cause fewer cytotoxicity and even ultimately eliminate the demands for suicide genes or safety switches [121].

CAR-NKs in cancer immunotherapy
Nowadays, CAR-NK cell-mediated immunotherapy has grown exponentially and emerged as an alternative treatment option for patients with metastatic malignancies (Fig. 3, Table 2). Despite the manifestation of CAR-NK cells in cancer immunotherapy has been extensively explored, yet most of the applications in numerous tumor models are relatively restricted and mainly staying at the preclinical stage [5,24]. Clinically approved second-generation CAR-NK cells usually contain the CD3ζ domain in combination with either a 4-1BB (Kymriah ® ) or CD28 (Yescarta ® ) co-stimulatory domain, and prominently focus on CD19 + lymphoid-derived hematologic malignancies [55].

Hematological malignancies
Similar to CAR-T-based immunotherapy, CAR-NK cells were initially introduced to combat with hematological malignancies such as lymphoma, myeloma and leukemia (Fig. 4, Table 2) [121,122]. Current studies have indicated the preferable efficiency of CD19-CAR-NK cells in combating lymphoid malignancies over CAR-Tbased cellular immunotherapy, which largely attribute to the aforementioned merits [55,132]. For example, a preclinical study took advantage of the UC-NK cells and the    fourth-generation iCas9.CAR-19-CD28-ζ-IL15 plasmid with a suicide gene for Raji lymphoma xenograft model treatment, the CD19-CAR-UC-NK cells exhibited preferable antitumor efficacy and enhanced persistence over the non-transduced UC-NK cells [46]. Simultaneously, the first large-scale trial of HLA-mismatched CAR-UC-NK cell-based immunotherapy (Clini-calTrials.gov number: NCT03056339) in combination with lymphodepleting chemotherapy for 11 patients with CD19 + CLL and B cell lymphoid tumors has shown safety and inspiring clinical outcomes (response rate: 73%; complete remission rate: 64%) within 30 days [45]. However, as commented by Karadimitris, CAR-UC-NK cells could also result in high rate of non-durable clinical response, which suggested the favorable initial toxicity and efficacy but uncertain durability of clinical manifestation of CAR-NK-based leukemia immunotherapy [133].

Metastatic solid tumors
In recent years, CAR-T cell immunotherapy has revealed promising therapeutic manifestation in a series of hematologic malignancies, yet also with considerable drawbacks and limitations in metastatic solid tumor management as well (Fig. 4, Table 2) [55,134]. Considering the intrinsic and advantaged properties, including substantially cytolytic ability, non-MHC-restricted recognition, natural infiltration in tumor tissues and convenience for their preparation as well as minimal untoward effects (e.g., CRS, GvHD and neurotoxicity), engineered CAR-NK cells are supposed as promising therapeutic option for solid tumor administration in clinical practice [54,134,135]. Currently, CAR-NK cells have been preclinically tested in multiple solid tumors including breast cancer, ovarian cancer, pancreatic cancer, colon cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma (HNSCC) [90,117,136,137]. For example, numerous preclinical studies have confirmed the efficacy of CAR-NK cells upon CXCL12/ SDF-1α secreting glioblastoma and epithelial cell adhesion molecule (EpCAM) positive colorectal cancer cells in xenograft model by targeting EGFRvIII via intravenous infusion [80,138]. However, there are very limited clinical data existing on the potential of CAR-NK cells in solid tumor treatment (Fig. 4, Table 2) [117]. For instance, the outcomes of three phase I/II clinical studies of allogeneic ROBO-1-CAR-NK-92 cell-based cellular immunotherapy in pancreatic ductal adenocarcinoma (PDAC) and relative solid tumors with ROBO-1 expression for enrolled patients in China (NCT03940820, NCT03941457, NCT03931720) ulteriorly indicated the feasibility for non-hematological neoplasm treatment with CAR-NK cells [117,135,139]. Another phase II trial of PD-L1-CAR-NK cell-based immunotherapy combined with IL-15 agonist (N-803) and pembrolizumab (NCT04847466) is currently launched in the United States. Therefore, integration of the CAR-NK cells and antitumor drugs is promising for further altering immunosuppressive tumor microenvironment (TME) and administrating the aggressiveness and the metastatic ability of the resistant tumors [117].

Conclusion and perspective
The heterogeneous NK cell population is advantaged immune cells with powerful cytotoxic activity and plays a unique role in both innate and adoptive immune responses, while the signatures could be eluded by tumor microenvironment [118,[140][141][142]. NK cells engineered with CAR expression (CAR-NK cells) have been celebrated as a landmark breakthrough of anti-tumor immunotherapy by discerning germline-encoded cell surface receptors between healthy and cancer tissues [143,144]. Differ from NK cells and CAR-T cells, the genetically modified NK (CAR-NK) cells have superiority in further augmenting the specificity and cytotoxicity of adoptive NK cells without causing adverse effects including GvHD, CRS or immune cell-associated neurotoxicity syndrome (ICANS) [5,60,143,145]. Thus, CAR-NK cells with favorable cytotoxicity, short lifespan and low manufacturing costs have been considered as promising alternatives to engineered CAR-T cells [143].
Despite the encouraging progress of CAR-NK cellbased immunotherapy, the discontented response-totoxicity ratio and the insufficiently broad spectrum of indications further limit the application of these therapies [4]. Moreover, compared to the updates of CAR-T-based immunotherapy, the number of clinical trials and the detailed information of CAR-NK cell infusion in patients, including the in vivo spatio-temporal metabolism and host factors in the microenvironment that reversely contribute to CAR-NK cells, have not been extensively investigated [60]. Generally, compared to CAR-T cells, CAR-NK cell-based adoptive immunotherapy is also restricted to the major limitations before large-scale practical application such as insufficient capacity in proliferation and activation in vivo and durability, together with the obstacle in preparation of CAR-NK cells (e.g., low genetic transfection efficiency, low proportion of NK cells in blood, and limited amplification efficiency) [133,146]. Conversely, CAR-NK cells have various merits over CAR-T cells due to their unique biological characteristics [117]. On the one hand, CAR-NK cells can be conveniently prepared from a wider range of autogenous and allogeneic sources without causing severe adverse reaction (e.g., aGVHD, CRS) by CAR-T-based implantation. On the other hand, CAR-NK cells are adequate for cell immunosurveillance dispense with pre-sensitization and thus have more flexible killing capacity upon both hematologic and solid tumor cells over CAR-T cells via both the CAR-dependent manner and CAR-independent intrinsic mechanisms, which thus provide alternative strategies for conquering tumor escape and varied adverse events (e.g., "on-target, off-tumor toxicity" during CAR-T application) [5,112,117,147].
Nevertheless, the success of CAR-NK cells in the administration of multiple malignancies provides proofof-principle for harnessing the immune system therapeutically, yet the short lifespan (2 weeks) and in vivo kinetics (e.g., proliferation rate, ageing) of CAR-NKs also narrows the therapeutic window and the resultant short duration of responses after infusion [122,148,149]. Therefore, the ex vivo expansion and activation of primary NK cells as well as the storage and shipping of NK cells for large-scale CAR-NK cell generation are prerequisites for ensuring the safety and effectiveness in vivo. For instance, a series of methodologies have been continuously developed for the persistence and activation of primary NK cells such as feeder cell stimulation (e.g., the irradiated PBMCs, K562-mb15-41BBL cells, EBV-LCLs) [150][151][152][153], cytokine cocktail (e.g., IL-2, IL15, 1 L-18) [14,35] and physicochemical irritation (e.g., bioreactors with an assorted bag) [154]. Of note, it is great important to improve the freezing and shipping conditions (e.g., refrigeration, liquid nitrogen, drikold) for sustaining primary NK cell vitality, which will finally help CAR-NK cells necessitate adaptation of cancer immunotherapy under GMP conditions [5,155].
Therefore, before large-scale application in cancer immunotherapy, a cohort of central issues in basic research and clinical practice of CAR-NK cells need to be improved. Firstly, many healthy tissues of the body also express cancer-associated surface antigens on tumor cells, which might cause potential off tumor or on target toxicity of CAR-NK cells. Current pioneering studies have suggested the engineering solution by inserting suicide genes or transducing genes encoding cytokines into the CAR-transduced effectors to prolong the in vivo persistence, respectively [45,118,156]. Secondly, the lessons learned from modified T cells (e.g., CAR-T, TCR-T) and NK cells in the administration of malignancies are worthy to be further exploited in CAR-NK cell-mediated cancer immunotherapy in future such as the optimization of large-scale NK cell-expansion approaches, antigen targets including the chimeric co-stimulatory converting receptors (CCCRs), structures with nanobodies and delivery efficiency as well as the selection of ideal patient populations in clinical trials [91,111,117]. Likewise, the potential tumor escape from CAR-NK cell cytotoxicity by shedding pivotal ligands and even via immunosuppressive tumor microenvironment should also take into consideration [14,140,157]. In particular, the pivotal signaling pathways and inhibitory checkpoints (e.g., CIS) that orchestrate CAR-NK cell "fitness" (e.g., effector function, survival and differentiation) and biofunction in tumor microenvironments have not been well defined [41,120,158]. Thirdly, despite CAR-NK cells are considered as "off-the-shelf " cancer immunotherapy, there's still a long way before the large-scale generation of clinical-grade products under good manufacturing practice (GMP) and convenient to universally save the lives of inpatients with standard supervision. For example, Gaddy and Broxmeyer reported the functional maturation subset (adult-like NK activity) and the phenotypic maturation subset (adult-like CD3 − CD56 + CD16 + or CD3 − CD56 + CD16 − phenotype) of UC-NK cells by IL-2, IL-12 or IL-15 stimulation [42]. As to the recommended parameters of cell products, the manufactured CAR-NK cells should contain mostly CD3 − CD56 + cells (≥90%), minimally CD3 + (≤0.2%) and CD14 + (≤5%) cells, together with the removal of endotoxin, mycoplasma and bacterial contaminations as well as contamination of cocultured cells (≤1%) [159,160]. Fourthly, the schedule of CAR-NK cell-based cancer immunotherapy, including the dosage, duration, kinetics and the concurrent interactions with endogenous immune cells as well as the underlying mechanisms of CAR-NK cell function, is awaiting to be optimized according to large-scale clinical trials [5,161]. For instance, the molecular understanding of the biofunction of the natural cytotoxicity receptors (NCRs) (e.g., NKp30, NKp44, NKp46) and multidimensional immune correlations (e.g., NKG2A + , CD8α + ) in cancer immunosurveillance, and in particular, the ontogenic development and maturational signals of NK cells is instrumental to exploring novel access points to combat malignancies [42,60,144,162].
Moreover, the comprehensive treatment composed of a plethora of methods, including traditional therapy (e.g., surgery, radiotherapy, chemotherapy), non-cellular immunotherapy (e.g., CTLA-4, mRNA vaccine) and cellular immunotherapy (e.g., CAR-T, TCR-T, γδT, ML-NK cells) as well as auxiliary methods (TriKEs, ROCK engagers, TriNKETs) and immune-checkpoint inhibitors (e.g., CTLA-4, PD-1/PD-L1), are under clinical investigation to augment the longevity and cytotoxicity of CAR-NK cells [146,163]. For example, a latest study upon combined approach suggested that antitumor activity and metabolic fitness of armored CAR-NK cells with IL-15 secretion could be enhanced by targeting a cytokine checkpoint, which represented an important milestone in the exploration of the nextgeneration cancer immunotherapy [41,117,164,165]. Another study conducted combined CAR-T infusion after NKG2D.ζ-NK cell administration and found improved anti-cancer activity and tumor infiltration [55]. Collectively, the pioneering preclinical and clinical studies have suggested the multifaceted opportunities and challenges of allogeneic CAR-NK cells, which are recognized as pivotal and promising "off-the-shelf " product in the next-generation cellular immunotherapies targeting recurrent and refractory malignancies.


Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

Introduction
MicroRNAs (miRNAs) are small single-stranded non-coding RNAs of approximately 21-22 nt long, which are used by various organisms to regulate growth, cellular homeostasis and development [1]. They mediate post-transcriptional gene silencing by binding to target mRNAs, repressing their translation and/or promoting their degradation. Recently, miRNAs have also been found to act on promoters and enhancers to modulate gene expression [2]. Abnormal expression of miRNAs is implicated in the pathogenesis of several human diseases, such as cancer, as well as neurological, cardiovascular and metabolic diseases [3,4], indicating that a precise dosage and regulation are crucial for homeostasis.
MiRNAs are generated from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex, which contains the RNase III enzyme DROSHA and its cofactor DGCR8, to generate precursors (pre-miRNAs) of~60-80 nt in length. Pre-miRNAs are then exported to the cytoplasm by Exportin 5 to be processed by the RNase III Dicer into mature miRNAs [1].
Most pri-miRNAs are structurally similar to mRNAs. They are transcribed by RNA polymerase II (Pol II), capped, spliced and polyadenalyted [5]. More recently, genome-wide a nuclear regulation mechanism. This regulation appears independent of the nucleotidic and amino acidic sequences of the smORF, but rather relies upon the presence or absence of a smORF. For Human pri-miR-497, Drosophila pri-miR-8 and pri-miR-14, their levels are sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events in this smORF-dependent regulation mechanism.
Thus, in this study we identified smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNA expression, in both Humans and flies.

smORF51 Regulates the Levels and Activity of Human primiR-155/miR-155
We have previously shown that the Human pri-miR-155 and pri-miR-497 transcripts contain translatable smORFs of 51 and 63 nt, respectively (Figures 1a and 2a) [37]. Since smORFs were shown to affect lncRNA stability by triggering co-translational RNA decay pathways [26], we investigated whether the translatable smORFs, present in pri-miR-155 and pri-miR-497, were also involved in the regulation of the pri-miRNA expression. To this end, we constructed vectors expressing the spliced pri-miR155 isoform with the wild type (WT) or the ATGs-mutated (MUT) smORF51. We previously showed that mutation of the three ATGs present within smORF51 into TAG abolishes its translatability [37]. RNA fold predictions showed no alteration of the MFE (minimum free energy) secondary structures between the WT and mutated pri-miR-155 ( Figure S1a). Transfection of the WT or mutant pri-miR-155 constructs in HeLa cells revealed that while the overexpression of both pri-miR constructs was detected at the RNA levels ( Figure S2a), mutant pri-miR-155 was expressed at lower levels than the WT construct ( Figure 1b). Since spliced pri-miR-155 can be used for miR-155 processing [44,45], we investigated whether smORF51 mutation would impact the production and activity of mature miR-155 by co-transfecting a luciferase sensor of miR-155 activity, together with a WT or mutant pri-miR-155 construct or a control vector (VEC). Luciferase reporter assays revealed that expression of both pri-miR-155 constructs repressed the miR-155 sensor (Figure 1c), indicating that both WT and mutant spliced pri-miR-155 constructs are processed into functional mature miR-155 in HeLa cells, in agreement with what was previously reported in B-cell lymphomas for the WT spliced pri-miR-155 [44,45]. However, the luciferase activity was significantly increased (~18%) with the mutant pri-miR-155 construct, compared to WT (Figure 1d), indicating a less efficient repressive capacity. Since we previously showed that overexpression of the smORF51encoded peptide, miPEP155, in HeLa cells has no impact on pri-miR-155 expression and on miR-155 activity [37], these results suggest that the smORF51 of pri-miR-155 positively regulates the expression of pri-miR155 in cis, consequently affecting miR-155 activity. Pri-miR-155 levels were normalized to EGFP (encoded in the pri-miRNA expression plasmids, and used to monitor transfection efficiency) and set to 1 for WT pri-miR-155 transfected cells. Data are means ± S.E.M. n = 4. (c,d) Relative activity of miR-155 processed from the WT or mutant pri-miR-155 constructs transfected in HeLa cells together with the luciferase miR-155 sensor. Cells were tested 48 h post-transfection for dual luciferase assays. (c) The luciferase activities of pri-miR-155 transfected cells were compared to that of control vector transfected cells (VEC), set to 100. (d) The relative luciferase activity of MUT pri-miR-155 was compared to that of WT pri-miR-155, set to 100. Data are means ± S.E.M. n = 10. * p < 0.05, ** p < 0.01, **** p < 0.0001. Figure 2. The smORF63 of Human pri-miR-497 regulates the expression levels and activity of pri-miR-497/miR-497. (a) Schematic structure of Human pri-miR-497 with the miR-497 (black box) and smORF63 (green box) indicated. The arrows in the pri-miR-497 delimit the sequences cloned and tested. (b) Relative expression levels of pri-miR-497 WT or ATG-mutated (MUT) or with the smORF63 replaced by the smORF51 from pri-miR-155 (ORF pri-miR-155) transfected in Hela cells and determined by qRT-PCR. The pri-miR-497 levels were determined and normalized as in Figure 1b. Data are means ± S.E.M. n = 4. (c) Relative expression levels of miR-497 processed from WT or MUT pri-miR-497 transfected into Hela cells and determined by qRT-PCR. The miR-497 levels were normalized as in Figure 1b. Data are means ± S.E.M. n = 7. (d,e) Relative activity of miR-497 processed from the WT or MUT pri-miR-497 constructs transfected in Hela cells together with the luciferase miR-497 sensor. Cells were tested 48 h post-transfection for dual luciferase assays. (d) The luciferase activities of pri-miR-497 transfected cells were compared to that of vector transfected cells (VEC), set to 100. (e) The relative luciferase activity of MUT pri-miR-497 was compared to that of WT pri-miR-497 set to 100. Data are means ± S.E.M. n = 8. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: not significant.

smORF63
Regulates the Levels and Activity of Human pri-miR-497/miR-497 We then tested a possible role of smORF63, present in the pri-miR-497 (Figure 2a), on the regulation of pri-miR-497 expression. For this, vectors expressing the pri-miR-497 transcript, spanning from the 5' end to 53 nt downstream of miR-195 (delineated by arrows in Figure 2a), with the WT or ATG/TGA-mutated smORF63 were generated. In this mutant transcript, translation of smORF63 is suppressed [37]. Transfection of the WT or mutant pri-miR-497 constructs in Hela cells shows that while the expression of both pri-miR constructs was detected at the RNA levels ( Figure S2b), the mutant pri-miR-497 levels were approximately 1.5-fold that of WT (Figure 2b). Quantification of the mature miR-497 levels revealed that both WT and mutant pri-miR-497 constructs are processed into mature miR-497 ( Figure S3); however, the processing of mutant pri-miR-497 yielded higher levels of mature miR-497 than WT (Figure 2c). Thus, mutation of smORF63 also affects miR-497 synthesis. In agreement with this, mutant pri-miR-497 was more efficient in repressing a luciferase sensor of miR-497 activity than its WT counterpart in luciferase assays (Figure 2d,e). Considering that miPEP497, encoded by smORF63, does not regulate the levels of its own pri-miRNA and the activity of the processed miR-497 [37], this result suggests that smORF63 acts in cis to inhibit the expression of pri-miR-497, thereby affecting miR-497 production and activity. This is not due to an effect on the secondary structure, since predicted MFE secondary structures show no difference between WT and mutant pri-miR-497 ( Figure S1b). We next tested whether the sequence of smORF63 was specifically required for this regulation by constructing a pri-miR-497 in which smORF63 was substituted by smORF51 of the pri-miR155. Transfection of this construct in Hela cells showed that pri-miR-497 with a smORF51 substitution behaved as WT pri-miR-497 (Figure 2b), indicating that this regulation does not depend on the nucleotide composition of the smORF, but rather on the presence of a smORF. Since the WT and mutant pri-miR-497 transcripts are expressed from the same promoters, this suggests a post-transcriptional mechanism.
Since the mutation of smORF63 increases both pri-miR-497 and miR-497 levels, and given that pri-miRNA processing takes place in the nucleus, this suggests that smORFs can regulate pri-miRNA expression in the nucleus. Moreover, the fact that this regulation is smORF-dependent suggests the involvement of ribosomes. This is reminiscent of previous studies showing that mammalian nonsense codons can be cis effectors of nuclear mRNA half-life, by reducing the abundance of nuclear mRNAs in a post-transcriptional decay mechanism involving translation by ribosomes [46][47][48][49]. To test whether translational events are involved in the regulation of pri-miR-497 by smORF63, cells transfected with the WT pri-miR-497 were treated with the translation inhibitor cycloheximide (CHX). CHX treatment for 1h induced the accumulation of both exogenous and endogenous pri-miR-497 (Figure 3a,b, respectively). These results suggest a smORF-dependent posttranscriptional mechanism involving translational events. One such mechanism could be NMD, because translation is necessary for NMD, and there is strong evidence for intranuclear NMD [50][51][52]. To test this hypothesis, we used NMDI14, a NMD inhibitor that disrupts the SMG7-UPF1 complex, a key component of the NMD pathway [53]. NMDI treatment caused the accumulation of CDKN1A mRNA, a bona fide NMD substrate [54] (Figure 3c), without affecting the levels of pri-miR-497, both exogenous and endogenous ( Figure 3d,e, respectively). This suggests that smORF63 regulates pri-miR-497 expression by an NMD-independent mechanism involving translational events.
Altogether, these results suggest that smORFs can act in cis to regulate positively or negatively the expression of pri-miRNAs/miRNAs in Humans. The CDKN1A mRNA or endogenous pri-miR-497 levels determined by qRT-PCR were normalized to actin (b) or GAPDH (c,e) and set to 1 for the untreated cells. * p < 0.05, *** p < 0.0005, ns: not significant.

Drosophila pri-miR-8 Contains a smORF Regulating Its Expression and Activity
We next investigated whether the smORF-dependent pri-miRNA/miRNA regulation identified above also occurs in other species. We chose to address this question in Drosophila melanogaster, first focusing on pri-miR-8 that was previously shown to contain a translatable smORF of 213 nt (Figure 4a) [36]. To test a possible role of the smORF213 in the regulation of pri-miR-8 expression, vectors expressing part of the pri-miR-8 transcript, spanning over pre-miR-8 (delineated by arrows in Figure 4a), with the WT or ATG/AGT-mutated smORF213, were generated. We previously showed that the mutant transcript is no longer able to translate the smORF213 [36]. Expression of the WT or mutant pri-miR-8 constructs in Drosophila S2 cells revealed that while the expression of both pri-miR constructs was detected ( Figure S4), the pri-miR-8 mutant accumulated at twice the levels of the WT construct ( Figure 4b). We then tested whether mutation of smORF213 affected miR-8 synthesis. Surprisingly, quantification of mature miR-8 levels indicated that, while both WT and mutant pri-miR-8 constructs are processed into mature miR-8, the mutant pri-miR-8 yielded lower levels of mature miR-8 ( Figure 4c). Accordingly, luciferase assays, using a luciferase sensor of miR-8 activity in S2 cells, confirmed that both WT and mutant pri-miR-8 constructs were processed into functional miR-8; however, WT pri-miR-8 exhibited more miR-8 activity than its mutant counterpart ( Figure 5a). Similar experiments were performed in vivo using a GFP miR-8 sensor expressed under the tubulin promoter (tub-GFP-miR8) in wing imaginal discs, in which miR-8 is functional [55]. Expression of WT or mutant pri-miR-8 under the patched (ptc) GAL4 promoter led to the repression of GFP in and outside the ptc domain (visualized with mCherry), indicating that miR-8 acts in a non-cell autonomous manner (Figure 5b, left panels). However, the area of the repressed domains was reduced with the mutant pri-miR-8 compared to WT (Figure 5b, right panel). These results were confirmed by generating flies overexpressing WT or mutant pri-miR-8 constructs in wings, using the wing driver line MS1096. As expected, overexpression of these pri-miR-8 constructs induced a "small wing" phenotype ( Figure 5c, left panel), as observed upon miR-8 overexpression [36,56]. However, quantifying wing size revealed a weaker activity of the mutant pri-miR-8 compared to WT (Figure 5c, right panel). Since we previously showed that miPEP8, encoded by smORF213, does not regulate the expression of pri-miR-8 and the activity of miR-8 both in S2 cells and in flies [36], our results suggest that smORF213 acts in cis to regulate the expression of pri-miR-8. Accordingly, overexpressing the WT or mutant pri-miR-8 in miR-8 expression domains, using a miR-8 GAL4 driver, revealed that mutant pri-miR-8 accumulated at higher levels than its WT counterpart and exhibited lower levels of mature miR-8 ( Figure S5). Similar to pri-miR-497, the coding sequence of smORF213 was not specifically required for this regulation. Indeed, while deleting the entire smORF213 (pri-miR-8 ∆ORF) had similar effects as mutating the ATG of this smORF, i.e., accumulation of pri-miR-8 (Figure 4b), replacing smORF213 by smORF225 from pri-miR-14 (pri-miR-8 ORF pri-miR-14) restored pri-miR-8 levels to that of WT (Figure 4b). Nevertheless, the secondary structure of pri-miR-8 ORF pri-miR-14 was substantially altered using RNA fold prediction ( Figure S6). This suggests that the differences in pri-miR-8 levels observed with the ATG-mutated and ∆ORF pri-miR-8 were not due to altered folding of their secondary structures ( Figure S6). Moreover, in all mutants of pri-miR-8, the refolding does not affect their pre-miR-8 secondary structure ( Figure S6). Together, these experiments suggest that the function of the smORF is independent of its amino acid sequence to fine-tune miR-8 levels. Since pri-miR-8 regulation requires the presence of a smORF, the involvement of translational events was tested in S2 cells transfected with WT pri-miR-8 treated or not with CHX. Upon CHX treatment, WT pri-miR-8 accumulated (Figure 6a), suggesting a mechanism requiring translation by ribosome. However, the regulatory mechanism triggered by the smORF appears different from that of pri-miR-497. Indeed, with the ATG-mutated pri-miR-8 construct, miR-8 expression was inversely correlated with pri-miR-8, suggesting that the processing of the pri-miRNA could be affected. This step in miRNA biogenesis is carried out in the nucleus by the microprocessor complex that contains the RNase III DROSHA. Depletion of DROSHA impairs pri-miRNA processing, thereby stabilizing pri-miRNA transcripts and increasing pri-miRNAs levels [57]. To test whether processing of the pri-miR-8 mutant is altered, WT or mutant pri-miR-8 transfected cells were treated with the transcription inhibitor actinomycin D for various times, and RNA decay was monitored. In these experiments, the mutant pri-miR8 was more stable than its WT counterpart (Figure 6b). Moreover, if processing of the mutant pri-miR-8 is altered, DROSHA depletion should only have a minor impact on its accumulation. To test this hypothesis, S2 cells were transfected with dsRNA against EGFP (control) or against drosha, and with WT or mutant pri-miR-8. Drosha dsRNA transfection resulted in efficient reduction of drosha mRNA (~75%) (Figure 6c). Confirming the results obtained previously (Figure 4b), the mutant pri-miR-8 accumulated to twice the levels of WT pri-miR-8 in EGFP dsRNA transfected cells (Figure 6d). However, while WT pri-miR-8 increased (~1.35 fold) in drosha depleted cells compared to control cells, no significant change in mutant pri-miR-8 levels (~1.06) was observed (Figure 6d). Quantification of mature miR-8 levels showed that mutant pri-miR-8 yielded lower levels of miR-8 than WT pri-miR-8 in EGFP dsRNA transfected cells (Figure 6e), as observed previously (Figure 4c). Nevertheless, upon drosha depletion, miR-8 levels processed from WT pri-miR-8 decreased more efficiently (2.3-fold) than from mutant pri-miR-8 (1.6-fold). Altogether, these results suggest that smORFs serve to fine tune pri-miRNA processing.

smORFs Regulate the Levels and Activity of Drosophila pri-miR-14/miR-14
We then tested whether the difference observed between human and fly is dependent on the species studied or the pri-miRNA considered. For this, we investigated whether Drosophila pri-miR-14 is also regulated by smORFs. The sequence of this pri-miRNA exhibits six small ORFs (Figure 7a) with, to date, no evidence of translation. To examine a possible role of smORFs on pri-miR-14/miR-14 expression, we constructed vectors expressing pri-miR-14 with the WT or all ATG-mutated smORFs, except for smORF6 that overlaps pre-miR-14 (Figure 7a). Transfection of these pri-miR-14 constructs in Drosophila S2 cells revealed that while the expression of both pri-miR constructs was detected ( Figure  S7) the ATGs-mutated pri-miR-14 construct (MUT) was expressed at higher levels (2.5-fold) than WT (Figure 7b) and yielded higher levels of mature miR-14 (Figure 7c). To characterize the potential translation of pri-miR-14, in vitro translation experiments were performed using constructs in which each smORF, except smORF1 that was excluded in the analysis because of its small size (27 nt), was fused to an HA tag. An efficient translation was observed only for smORF4 (225 nt, Figure 7d). ATG/TAA mutation of this smORF (MUT ORF4) behaved similarly to the ATGs-mutated construct (MUT), expressing higher levels of pri-miR-14 ( Figure 7e) and miR-14 (Figure 7f) than WT. The RNA fold prediction tool indicated no impact on pre-miR-14 secondary structure for the two pri-miR-14 mutants ( Figure S8). Importantly, overexpression of the smORF4-encoded peptide, miPEP14, in S2 cells had no impact on pri-miR-14 and miR-14 levels ( Figure S9), suggesting that smORF4 also regulates pri-miR-14/miR-14 expression in cis. We then tested whether smORFs impinge on miR14 activity by generating flies overexpressing WT or mutant pri-miR-14. We focused on the Drosophila wing, where miR-14 overexpression reduces wing size [58]. Despite being overexpressed approximately 20-fold ( Figure S7), WT pri-miR-14 transfection in S2 cells only caused a small increase of miR-14 levels compared to endogenous levels (NT) (Figure 7c). Accordingly, no significant variation of wing size was observed in flies overexpressing WT pri-miR-14 compared to control wings (white, w) (Figure 7g, right panel). However, wing size was significantly reduced in flies overexpressing the mutant pri-miR-14, highlighting a higher activity of this pri-miRNA compared to WT (Figure 7g), which is consistent with the increased levels of miR-14 processed from this mutant pri-miR-14 (Figure 7c,f). Thus, smORFs can impinge differently on pri-miRNA expression, and this is apparently unrelated to the species studied but rather is dependent on the pri-miRNA considered.
To test whether translational events are also involved in the regulation of pri-miR-14, as shown for Human pri-miR-497 and Drosophila pri-miR-8 (Figure 3a,b and Figure 6a), S2 cells were treated or not with CHX. CHX treatment caused an accumulation of endogenous pri-miR-14 (Figure 8a), suggesting again a smORF-dependent regulation mechanism requiring translation. Similar to pri-miR-497, NMD did not appear involved in this mechanism. Indeed, experiments performed using dsRNAs directed against upf1 (Figure 8b) or upf2 (Figure 8c), two NMD core components [59], showed no significant variation in endogenous pri-miR-14 levels when these NMD factors were depleted, compared to EGFP dsRNA used as control (Figure 8e). In contrast, transformer (tra) mRNA, a known NDM substrate [59] accumulated upon upf1 or upf2 depletion (Figure 8d), as expected. These results suggest that, similar to pri-miR-497, smORF4 regulates pri-miR-14 expression by an NMD-independent mechanism involving translation.
Altogether these results strongly suggest that smORFs can act in cis to fine-tune steady state level expression of pri-miRNAs/miRNAs, either positively or negatively, in Humans and flies.  of against Upf1 or Upf2. The Upf1, Upf2, tra and pri-miR-14 levels, determined by qRT-PCR, were normalized to tubulin and set to 1 for EGFP dsRNA transfected cells. Data are means ± S.E.M. n = 4. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Discussion
In this study, we have identified smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression both in Humans and Drosophila melanogaster and, to our knowledge, this is the first report of such a function for smORFs. Our results indicate that this function of smORFs is independent of their amino acid sequence. Previously, and in this study, we provided evidence that overexpression of smORF-encoded peptides has no impact on the expression and activity of their pri-miRNAs/miRNAs [36,37], excluding a trans effect of the smORFs in the regulation of pri-miRNAs/miRNAs. In contrast, depending on the pri-miRNA studied, smORFs differently influence the expression of pri-miRNAs. Indeed, smORFs have a positive effect on Human pri-miR-155 expression, whereas they negatively regulate Human pri-miR-497 and Drosophila pri-miR-14 expression. The fact that mutation of smORFs affects both the pri-miRNA and miRNA levels, and given that pri-miRNA processing, the first step of miRNA biogenesis, takes place in the nucleus, the suggestion is that smORFs regulate pri-miRNA levels in the nucleus. The mechanism by which smORF51 regulates the expression levels of pri-miR-155 is still unclear. However, for Human pri-miR-497 and Drosophila pri-miR-8, the substitution of the smORF by another one does not affect pri-miRNA expression levels, indicating that this regulation does not depend on the nucleotide sequence of the smORF, but rather relies upon the presence or absence of a smORF. For Human pri-miR-497 and Drosophila pri-miR-8 and pri-miR-14, their levels are sensitive to the ribosome-stalling drug cyclo-heximide, suggesting the involvement of translational events in this smORF-dependent regulation mechanism. This regulation likely occurs with other Drosophila pri-miRNAs, because among five further pri-miRNAs analyzed, the levels of four of them were also sensitive to CHX ( Figure S10). With the idea that smORFs, present within pri-miRNAs, could be considered by the RNA quality surveillance pathway as premature stop codons (PTCs) in the nucleus, we tested the involvement of NMD, an evolutionarily conserved translation-dependent mechanism that scans mRNAs for PTCs and destroys faulty transcripts. This hypothesis is consistent with several studies providing strong evidence for intranuclear NMD [50][51][52]. Moreover, although it is widely believed that translation occurs only in the cytoplasm, studies have shown that some translation occurs in the nucleus of mammalian cells [60][61][62]. In addition, nascent RNAs bearing premature stop codons (PTCs) are eliminated by a mechanism sensitive to a translation inhibitor, reinforcing the idea that nuclear ribosomes exert RNA surveillance by scanning newly made transcripts, with faulty transcripts being degraded by NMD [60]. In agreement with this idea, other studies have shown that eukaryotic cells can detect ORFs within the nucleus [63]. However, NMD inhibition (Human pri-miR-497) or dsRNA against key NMD components (Drosophila pri-miR-14) do not indicate an involvement of NMD in the smORF-dependent regulation of these pri-miRNAs. These results contrast with a study identifying Human miRNA host genes (MIRHGs) as NMD targets [26]. However, since these pri-miRNAs encode intronic miRNAs, which are processed from the excised introns, these results were interpreted as translation events of the smORFs present in the cytoplasmic spliced RNAs triggering NMD similarly to smORFs-bearing cytoplasmic lncRNAs [26].
Concerning the Drosophila pri-miR-8, ATG-mutation of the smORF213 increases pri-miR-8 levels but decreases miR-8 levels, suggesting that pri-miRNA processing is affected. In agreement with this, mutant pri-miR8 appeared more stable than its WT counterpart and Drosha depletion did not result in significant changes in mutant pri-miR-8 levels compared to WT. Since WT pri-miR-8 is sensitive to cycloheximide, this also suggests a link between ribosome and pri-miRNA processing. To date, such a link has never been reported in animals. Interestingly, STV1, a conserved ribosomal protein, binds pri-miRNAs to promote their interaction with the processing complex in Arabidopsis [64]. Additionally, a functional interaction has been identified between RNase III and the E. Coli ribosome [65], suggesting that a link between ribosomes and RNAse III enzymes is evolutionarily conserved. These results lead us to propose that ribosome interaction with the pri-miRNAs and DROSHA (a RNAse III enzyme) might facilitate pri-miRNA processing. Interestingly, a recent study reported an unconventional role of ribosomes in small-non coding RNA formation [66]. Indeed, ribosomes also mediate PIWI-interacting RNA (piRNA, a class of small noncoding RNA) biogenesis from long intergenic piRNA precursors in mice by first translating the uORFs of piRNA precursors, then translocating them into the uORF downstream regions where the ribosome-protected regions will become piRNAs after endonucleotic cleavage [66]. Montigny et al. 2021 [36] generated flies by knock-in strategy using a specific P landing platform to replace the entire miR-8 locus by the WT or the ATG-mutated smORF213 pri-miR-8 in Drosophila. In these transgenic flies, miR-8 was expressed at similar levels to the endogenous miR-8 [36]. Unfortunately, no difference in pri-miR-8 accumulation between the WT and mutant pri-miR-8 was detected in these flies ( Figure S11), in contrast to results obtained by overexpression of these two pri-miR-8 in Drosophila, revealing that mutant pri-miR-8 accumulates at higher levels than its WT counterpart ( Figure S5). This discrepancy can be explained by the fact that this regulation either occurs transiently or takes place only in a few miR-8 expressing cells or is too weak to be significantly detected on endogenous transcripts.
Importantly, this study indicates that smORFs embedded in pri-miRNAs may exhibit dual functions: first they encode functional peptides (called miPEPs for miRNA-encoded peptides) and second, they may act as cis-regulatory elements involved in the regulation of pri-miRNA/miRNA expression. Whereas in plants, all miPEPs discovered so far have been found to upregulate their miRNA directly by enhancing the transcription of their associated pri-miRNA [67], this function is apparently not conserved for human and fly miPEPs [36,37]. Instead, in animals the regulation of pri-miRNA/miRNA expression may be carried out by the smORFs themselves, although the precise mechanism still requires further investigation.
Altogether, this study adds a new layer of regulation of miRNA biogenesis. Given the prevalence of smORFs in Human and Drosophila pri-miRNAs [36,37], it will be very interesting to see how different smORFs influence the levels of different pri-miRNAs, and the proteins involved in this regulation.
ized to Snord47 or EGFP. Primers for stem loop RT and qPCR were synthesized by Eurofins Genomics (Ebersberg, Germany) and are listed in Table S1.
For fly experiments, total RNA extractions from young adult fruit flies (2-5 days) or S2 cells harvested 48 h post-transfection, RT and qPCR for quantifications of pri-miRNAs and coding genes were performed as previously described [36]. Exogenous pri-miRNA levels were normalized to GAL4 or GAPDH, endogenous pri-miRNAs or coding genes levels were normalized to tubulin or RP49. For quantification of mature miRNAs, a stem loop RT-qPCR procedure [69] was used using RT stem loop primers designed according to Kramer et al. 2011 [71], and the small RNA U14 was used as a reference gene for normalization. Primers for stem loop RT and qPCR were synthesized by Sigma and are listed in Table S2. QPCRs were performed on the LightCycler 480 Instument II (Roche Life Science, Penzberg, Germany) and the RNA abundance of the examined genes was calculated.

Dual Luciferase Reporter Assays
Hela cells were co-transfected with the luciferase sensors of miRNA activity (psiCHECK2) and either the empty vector or pri-miRNAs expressing vectors (pcDNA3 EGFP) as described previously [37]. S2 cells were transfected with the luciferase sensor of miR-8 activity pA-cLucR3'esg, the pAcLuc2p vector (constructed by inserting the Luc2P of pGL4.11 vector from Promega into the pAcGAL4 vector), the pAcGAL4 vector and either the empty vector or the pri-miRNAs expressing vectors (pUAS-Attb) as previously described [36]. Transfected cells were lysed 48h after transfection and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions, with a luminoskan and the Skanlt Tm software for microplate readers (Thermo Fisher Scientific, Waltham, MA, USA) for the human experiments and a Victor Nivo luminometer instrument (Perkin Elmer, Waltham MA, USA) for the fly experiments. The ratio Renilla luciferase activity (which quantifies the miRNA activity)/Firefly luciferase activity (which monitor the transfection efficiency) was calculated to indicate the activity of the reporter.

Immunoblotting
The smORF sequences cloned into the pF25A ICE T7 Flexi vector (Promega) were translated in vitro using TnT ® T7 Insect Cell Extract Protein Expression System (Promega). After addition of Laemmli buffer, translation products were separated on 4-20% SDS-PAGE gels (BioRad) and transferred to 0.1 µm nitrocellulose membrane (Amersham GE Healthcare, UK) according to Lauressergues et al. 2015 [72]. The membrane was blotted overnight at 4 • C with rabbit anti-HA (C29F4) from Cell Signalling Technology (Beverly, MA, USA), washed then incubated at room temperature with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology, CA, USA). After washes, detection was achieved with chemiluminescence detection reagent (Clarity Western ECL substrate, BioRad). Image acquisitions of immunoblots were performed with a ChemiDoc Touch imaging system (BioRad).

Fly Strains and Genetics
Drosophila stocks were maintained on standard cornmeal-yeast medium (Dutscher, Bernolsheim, France) and experiments were performed at 25 • C. UAS-pri-miR-8 and UAS-pri-miR-14 transgenic lines were inserted in attP86F site through PhiC31-mediated integration with injections performed by Bestgene Inc (Chino Hills, CA, USA). For analysis of miR-8 activity in imaginal wing discs, the ptc GAL4; tubulin GFP miR-8 sensor line was crossed either with the UAS mCherry or WT or ATG-mutated UAS pri-miR-8. For the experiments of wing phenotype, UAS-pri-miR-8 and UAS-pri-miR-14 transgenic lines were crossed with MS1096-GAL4 (BDSC:8860). For wing measurements, experiments were performed as previously described [36]. Wings or wing disc images were acquired on a Zeiss Axiozoom stereomicroscope (Zeiss, Germany). Measurements of wing size were performed using IMAGE J software (version 1.49u, Wayne Rasband, Research Services Branch of the NIH's National Institute of Mental Health, Bethesda, MA, USA).

Statistical Analyses
Statistical analyses were performed using GraphPad Prism software (version 6; GraphPad Software Inc., San Diego, CA, USA). Data are shown as means ± SEM and were considered statistically significant at p < 0.05 and n represents the number of experiments. For statistical analysis, the Mann-Whitney test or the one sample t-test were used. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, ns: non-significant.


Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis

A reliable qPCR experiment requires the selection of reference genes with a stable level of expression in a given experimental system. This study attempts to determine the reference genes (RGs) for the A. sativa–P. graminis experimental setup. We evaluated nine candidate reference genes in A. sativa (oat line Pg4 and the cultivar Kasztan) during compatible and incompatible interactions with different pathotypes of Puccinia graminis f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). We found that the most appropriate combination of RGs for RT-qPCR data normalization were HNR (heterogeneous nuclear ribonucleoprotein 27C) + EF1A (elongation factor 1-alpha) + EIF4A (eukaryotic initiation factor 4A-3). The worst candidates for normalization in this dataset were CYP (cyclophilin) and TUA (alpha tubulin). Identified reference genes are suitable candidates for the standardization of gene expression studies in the A. sativa–P. graminis interaction system and potentially other related pathogens. To date, this is the first report of RGs selection in this pathosystem.

years 3,4 . Annual oat production reaches 23 million tonnes, with Russia, Canada, Australia and Poland being the largest oat-producing countries 5 .
The global production of cereals is severely affected by rust diseases caused by a specialized group of fungi from the Pucciniales order 6 . This diverse and widely distributed group of plant pathogens with more than 7800 species may attack the most frequently sown cereals in the world including oat 7 . Stem rust caused by Puccinia graminis Pers. f. sp. avenae Eriks. & E. Henn. regularly occurs world wide and poses a great threat to the oat yield and quality. The use of fungicides is the main way to prevent stem rust development, however it is associated with high costs and is becoming increasingly opposed by consumers. Therefore, genetic resistance is one of the most accepted important modern breeding goals. The life cycle of P. graminis, involving sexual and asexual reproduction leads to the emergence of new rust pathotypes and causes high virulence dynamics and phenotypic diversity 8,9 . This efficient fungus adaptability causes rapid loss of effectiveness of current genetic stem rust resistance sources, hence there is urgent need to develop durable plant resistance immune to the pathogen variability.
Successful implementation of durable resistance strategies into plant breeding relies on the understanding of the processes involved in pathogen-host interactions. Accurate quantification and validation of plant genes involved in controlling the disease development is performed by reverse transcription quantitative real-time polymerase chain reaction. Identifying reference genes with stable expression levels will ensure obtaining repeatable and reliable RT-qPCR results. The study attempts to determine the reference genes for the A. sativa-P. graminis experimental setup. The obtained results will provide valuable data necessary for a comprehensive analysis of gene expression in oat.

Methods
Experimental material and rust inoculation. The study material consisted of oat line Pg4 derived from the Hajira cultivar with major gene resistance to stem rust caused by P. graminis f. sp. avenae 10 and the polish cultivar Kasztan (Dawid × CHD 1685/84) susceptible to stem rust infection 11 . Seeds of the study genotypes were grown in plug trays filled with a universal substrate containing peat for 10 days in a phytotron at 18 °C for a 16-h photo period.
Two P. graminis f. sp. avenae pathotypes (D19 2/7, Cz19 3/6, Table 1) were used with a virulence profile defined based on a set of 12 differential oat lines carrying a single stem rust resistance gene (Pg1, Pg2, Pg3, Pg4, Pg6, Pg8, Pg9, Pg10, Pg12, Pg13, Pg15, Pg16) 12 as well as oat lines 'Pg-a' , ' Alpha' (CI 9221), 'Omega' (CI 9139) 13 and 'Wisconsin X-1588-2' (CI 8457). Pathotypes were selected from a collection of single-pustule isolates derived from stem rust populations collected in Poland from 2017 to 2020 9 . Both pathotypes are virulent to 'Kasztan' . Pg4 line is resistant to Cz19 3/6 and susceptible to D19 2/7. Dried pathotypes were stored in 1.5-ml microfuge tubes at − 70 °C. Before inoculation, urediniospores were heat-shocked for 4 min at 42 °C and multiplied on leaf fragments of the susceptible oat cultivar Kasztan 11 using the host-pathogen method of Hsam et al. 14 , originally used for Blumeria graminis f. sp. avenae and modified by Paczos-Grzęda and Sowa 15 . Study was conducted on the first leaves of 10-day-old seedlings. Inoculation was performed in settling towers by spreading urediniospores on plant material at a density of ≈ 200 spores/cm 2 . Pg4 line was inoculated using both P. graminis pathotypes and 'Kasztan' was inoculated with D19 2/7. Glass slides were placed between the inoculated leaves to monitor the inoculation density. The settling towers were thoroughly cleaned with ethanol between inoculations with different isolates and the room was sprayed with water. Seedlings were incubated in a spore-proof growth chamber at 18  Two-stepRT-qPCR reactions. The reaction of reverse transcription was performed on 2 µg RNA using oligo d(T) 18 primer. The cDNA synthesis was performed with NG dART RT kit (EURx Ltd., Poland) in a final volume of 20 µl. The no-reverse transcription control (NRT) was also included in the analysis. Obtained cDNA was diluted and used as a template in the qPCR reactions.  16,17 . Primers for qPCR reactions were taken from previous studies 18,19 or designed with PrimerBLAST tool 20 as indicated in Table S1.
The qPCR reactions were conducted on QuantStudio™ 3 apparatus (Applied Biosystems, USA) using Power Track SYBR Green Master Mix (Thermo Fisher Scientific Inc., USA). The reaction mixture of 20 μL consisted of 20 ng of cDNA, 1 × qPCR Mix and 400 nM of each primer. The reactions were performed according to the following cycling program: 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. In order to confirm the amplification specificity melting curve analysis was performed after each run with continuous data collection from 60 to 95 °C. Additionally, the existence of single PCR products was further verified by the means of electrophoresis in 2% agarose gel stained with ethidium bromide. All reactions were conducted in three biological replicates with three technical replicates along with no template control (NTC). Standard curves were generated from a fivefold serial dilution of pooled cDNA. Only primer pairs showing amplification efficiency of 90-110%, slope between − 3.6 to − 3.1 and correlation coefficient (R 2 ) over 0.99 were used for further analysis.
Analysis of gene expression stability. The data obtained from RT-qPCR was analysed using a dedicated module from ThermoFisher Cloud (ThermoFisher Scientific). The Cq values were corrected for reaction efficiency. The expression stability of candidate RGs was assessed by geNorm 21 , NormFinder 22 , BestKeeper 23 and delta Ct method 24 . Overall final ranking was generated according to the method proposed by Velada et al. 25 . Expression analysis of target gene PAL (phenylalanine ammonia lyase) was carried out for RGs validation. The RT-qPCR reaction conditions were as described above. The relative expression level of the target gene was calculated using the 2 −∆∆Ct method. Statistical significance between untreated and inoculated data subgroups was calculated by Student's t test at P ≤ 0.05.

Results
The study was conducted on the plant material with a major resistance gene to stem rust (oat line Pg4 with Pg4 resistance gene) as well as genotype susceptible to rust infection (cultivar Kasztan). The use of stem rust races with defined virulence profiles allowed to stimulate susceptibility (oat line Pg4 and cultivar 'Kasztan' infected by D19 2/7 P. graminis pathotype) as well as resistance response (oat line Pg4 infected by Cz19 3/6 P. graminis pathotype) and observe compatible and incompatible interactions respectively. Studied samples were analysed according to the following experimental sets: full dataset, A. sativa-P. graminis compatible interaction, A. sativa-P. graminis incompatible interaction, Pg4 oat line, Kasztan cultivar.
Primers amplification efficiency and specificity. For reference genes selection, cDNA of tested oat genotypes was used in the qPCR. The primers' specificity was verified by qPCR melting curve analysis and evaluated on 2% agarose gel. Single peaks confirming the specificity of the amplification were generated on the dissociation curves obtained for each primer pair, no signal was detected in the NTC samples. Moreover, single qPCR product bands were observed on agarose gel (Figs. S1, S2). PCR amplification efficiency evaluated by a standard curve method with a pool of all the cDNAs varied from 96.41% (CYP) to 122.4% (TBP) and the R 2 based on linear regression ranged from 0.992 (HNR, TBP) to 1 (ARF). The primer pair for TBP was excluded from the analysis for exceeding efficiency criterion of 110% ( Table 2). The Tm values of the primer pairs varied from 83.75 °C (EF1A) to 92.13 °C (CYP), and the amplicon sizes were between 88 bp (EIF4A) and 158 bp (EF1A) ( Table S1). The raw quantification cycle (Cq) values were estimated for the determination of the gene expression levels. The mean Cq values for analysed samples ranged between 22.79 (ARF) and 28.89 (HNR) (Fig. S3).
RGs expression stability was assessed with four algorithms, geNorm, NormFinder, BestKeeper and ΔCt method. For each algorithm, RGs were ranked from the most to least stable. ΔCt results. According to ΔCt analysis based on average standard deviation (mean SD) HNR and EF1A were ranked as the most stable candidate RGs in the full analysed dataset. Similar results were obtained for compatible interaction samples as well as for Pg4 oat line and Kasztan cultivar samples. For the incompatible interaction subgroup EF1A and EIF4A were found to be the least variable RGs. The highest mean SD was reported for TUA regardless of the dataset (Table S2c).
geNorm results. GeNorm v3.4 software was used to rank the candidate RGs according to the stability measure (M-value) 21 . The lowest M value always presents the most stably expressed candidate gene with the threshold set at 1.5. All tested genes in analysed datasets showed expression stability below 0.93. When all the samples were combined, the M-value of ARF, EIF4A and EF1A was the lowest, and for TUA it was the highest. The results remained very similar when the M-value was measured for the compatible interaction dataset and Pg4. Within incompatible interaction subgroup and samples of cultivar Kasztan, EF1A and HNR were the most stable among the RGs tested. Out of all genes,TUA was found to be the worst performing RG in each analysed sample set (Table S2d).
NormFinder results. In NormFinder analysis, intra-and intergroup variation within subgroups of a dataset were calculated 22 . In this analysis, SV (stability value) of all tested RGs was below 0.68. The lowest SV characterizing the best candidates for qPCR data normalization was presented by HNR, EF1A and EIF4. However, they differed in order when groups were evaluated separately. In compatible interaction subgroup as well as Pg4 oat line, ARF can also be considered as exhibiting relatively high expression stability. The highest SVs representing high variation in expression was reported for TUA and CYP (Table S2e).

Determination of the optimal number of RGs for data normalization. The geNorm algorithm
allows the prediction of the optimal number of RGs that should be used for accurate qPCR data normalization by calculating pairwise variation (V n /V n+1 ). The V n /V n+1 value below 0.15 indicates that the inclusion of additional RG does not make any significant contribution to the data analysis. In this study,V 2 / 3 was below 0.15 for incompatible interaction subgroup as well as Pg4 indicating that the two best-performing RGs are enough for sufficient data normalization in these datasets (Fig. 1). If all the samples need to be analysed together, three reference genes are required for optimal normalization, because the V 3 /V 4 value was lower than 0.15. Similar results were obtained for compatible interaction samples as well as for Kasztan cultivar samples. According to the comprehensive rankings (Table 3) the most appropriate combination of RGs for full dataset as well as Kasztan cultivar was HNR + EF1A + EIF4A. In the compatible interaction subgroup, HNR + EF1A + ARF were found to be the least variable RGs. In the incompatible interaction subgroup and Pg4 oat line, a pair of best-performing RGs EF1A + EIF4A was found to be sufficient for obtaining reliable results. CYP was shown to be the worst candidate for normalization in the full dataset, Pg4 oat line and Kasztan cultivar. In the compatible and incompatible interaction subgroup, the utmost variation wasdisplayed by TUA .

Expression analysis of target gene for reference genes validation.
To validate the reliability of the selected RGs, the expression analysis of PAL gene was performed in experimental samples harvested at various time points post inoculation. For each dataset, both best and worst performing RGs were used to demonstrate how incorrect data normalization may affect obtained results.
When normalization was carried out for Pg4-P. graminis compatible and incompatible interaction with most stable RG sets (HNR + EF1A + EIF4A and HNR + EF1A + ARF), no significant difference in expression between untreated (0 hpi) and inoculated data subgroups were observed (Fig. S4). However, when TUA and CYP were used, a clear over estimation of transcript level was noticed. PAL expression level normalized against CYP was significantly differentat every time point.
Relative expression of the PAL gene in Kasztan cultivar-P. graminis incompatible interaction normalized with most stable RG sets was significantly different in 8, 24, and 96 hpi. When CYP was used for normalization, a significant difference was observed at every time point. Data normalized against TUA showed no significant difference in PAL expression level (Fig. 2).
Obtained data confirm substantial inaccuracyin the expression profileswhen RGs selection is not carried out properly.

Discussion
Identification of reference genes exhibiting stable expression has been an objective of several recent studies carried out in Avena sativa [26][27][28] and Avena fatua 18 . Neither of them, however, investigated the effect of fungal infection on candidate RGs expression. Therefore, the goal of the present study was to evaluate the set of ten potential RGs in terms of their expression stability in the plant-pathogen system consisting of A. sativa and P. graminis. www.nature.com/scientificreports/

Scientific Reports
In our study, we tested the candidate RGs stability during A. sativa-P. graminis compatible and incompatible interactions throughout six time points post-inoculation. While analyzing the full dataset we found that the most appropriate combination of RGs for RT-qPCR data normalization was HNR + EF1A + EIF4A. In the compatible interaction subgroup data normalization also required the use of three internal controls (HNR + EF1A + ARF), whereas in the incompatible interaction subgroup, just a pair of best-performing RGs (EF1A + EIF4A) was found to be sufficient for obtaining reliable results. Likewise, high expression stability of HNR and EIF4A across all tested A. sativa samples was reported by Yang et al. 18 . It is also worth mentioning, that HNR and ARF used for normalization separately in the gene expression experiment, showed comparable output to the results obtained by three RGs normalisation. This might be an alternative to consider for large experimental studies or under budget constraints.
Duan et al. 27 emphasized the effect of specific sample subsets on the proper RGs selection. In their study, the pair of best-performing RGs in salt-stressed oat samples included EF1A and TBP. However, in the sample set comprising of different tissues, TBP displayed medium expression stability. The highest-ranked RGs chosen in this subgroup were EF1A and PP2A. Similar conclusions were reported by Tajti et al. 28 who conducted expression analyses in leaves and roots of oat seedlings subjected to various abiotic stresses. The comprehensive rankings generated for specific datasets showed high variance in RGs order. In some cases, the same RG was ranked either  www.nature.com/scientificreports/ at first or last position, depending on the experimental dataset (e.g., GAPDH in salt-stressed leaves and in cold stressed leaves, respectively). In our study, sample subgroups consisted of more homogeneous material, therefore there is little variance in the order of RGs among different sample sets. In fact, in four out of five datasets the same three RGs were reported to have the most stable expression, that is HNR, EF1A and EIF4A. The above mentioned study by Duan et al. 27 revealed poor expression stability of commonly used RGs (GAPDH, ACT and TUA ) in oat specimens. This is in concordance with our results which demonstrated medium-to-low expression stability of GAPDH and TUA in the tested material. Likewise, GAPDH was reported as the least stable RG in developing oat seeds and endosperms in the study of Yang et al. 18 . Nonetheless, Wise et al. 29 in their study on methods of enhancing crown rust resistance in cultivated oat by application of plant defence activators selected GAPDH in combination with HSP70 as stable internal controls for RT-qPCR data normalization. Moreover, GAPDH was chosen as a stable endogenous control in the research on oat-crown rust interaction carried out by Montilla-Bascón et al. 30 . Worth mentioning, however, is the fact that the aforementioned study evaluated the stability of only four candidate RGs. If the larger panel of potential RGs had been tested, it is likely that different gene would have been selected as the internal control.
Experiments aiming at elucidation of the molecular basis of resistance should investigate both compatible and incompatible interactions between host and pathogen 31 . Different interaction types may induce various alternations to the host transcriptome, therefore validation of RGs stability in both of them is crucial for proper data analysis. There have been several reports assessing RGs stability in plant-pathogen systems involving compatibleand incompatible interactions. Albuquerque et al. 32 studied RGs expression stability in tomato during interaction with Ralstonia solanacearum isolates displaying contrasting virulence profiles. Interestingly, identified most stable RGs differed between compatible and incompatible interaction analysis groups (PDS + ACT vs TIP41 + EF1A, respectively). Research conducted by Monteiro et al. 33 in cultivated grapevine-Plasmopara viticola pathosystem also revealed the need of utilizing different sets of RGs for studying compatible and incompatible interactions. Here, we obtained more congruous results among compatible and incompatible interaction datasets, with only one out of the three best RGs being different.
The literature shows that resistance-associated genes expression differs during compatible and incompatible interactions 34 Those differently expressed genes might be employed in fungal disease resistance screening. However, in order to avoid erroneous data interpretation utilization of carefully selected RGs is required. Here, we propose the use of identified sets of RGs to be explored in the further investigations of A. sativa-P. graminis and potentially other cereal pathosystems.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.  Analysis was performed against two sets of best performing RGs (HNR + ARF + EF1A and HNR + EIF4A + EF1A) separately or together as well as against worst performing RGs (CYP and TUA ). Data are shown as mean ± SD. * indicate that the difference in expression between untreated (0 h) and inoculated data subgroups is significant at P ≤ 0.05 as determined by Student's t test.