Production of a Novel Multi-Epitope Peptide Vaccine against Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the prevalent cancers in the world with a high recurrence rate. In recent years, different researches have focused on designing efficient multi-epitope peptide vaccines against HCC. In designing these vaccines, over-expressed antigens in HCC patients, such as α- fetoprotein (AFP) and glypican-3 (GPC-3), have been employed. In our previous study, a multi-epitope peptide vaccine for HCC was designed by in-silico methods. The designed vaccine construct included the AFP, GPC-3, and aspartyl-β-hydroxylase (ASPH) as CytoLoxic T cell Lymphocytes (CTL), one epitope from Tetanus Toxin Fragment C (TTFrC) as Helper T cell Lymphocytes (HTL), and a segment of microbial heat shock protein (HSP70) peptide407-426 as an adjuvant. All the mentioned parts were connected by appropriate linkers. The aim of this study is the production of the designed vaccine. Methods: This research is experimental and was carried out in Fasa, Iran, in 2017. The designed vaccine construct gene was transformed to the Escherchia coli BL21 (DE3) strain and expressed in different isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations (0.6 and 1 mM), times (4, 6, 8, 16 hours), and temperatures (25 and 37 °C). Then, the expressed protein was analyzed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot methods. Results: The best conditions for protein expression were obtained in the Super Optimal Broth (SOB) medium at 37 °C after the induction of expression by 1 mM IPTG for six hour. Conclusion: The recombinant HCC vaccine was produced with a proper concentration.

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There is a limited number of designed multi-epitope peptide vaccines for hepatocellular carcinoma. In designing these vaccines, over-expressed antigens in hepatocellular carcinoma patients, such as α-fetoprotein, glypican-3, and aspartyl-βhydroxylase, have been considered. • Using selected epitopes by in silico methods in vaccine construct, instead of the whole antigen, leads to design new vaccines construct which their immunogenesity should be evaluated.

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In this study, a novel multi-epitope peptide vaccine construct against hepatocellular carcinoma was expressed and purified. The designed vaccine construct is predicted to have high antigenicity. • A segment of heat shock protein-70 was used as a natural adjuvant in the vaccine construct. The applied adjuvant has not been used in hepatocellular multiepitope vaccines up to now.

Introduction
Hepatocellular carcinoma (HCC) is the seventh most prevalent cancer in the world and the fourth leading cause of cancer mortality worldwide. Therapeutic choices against HCC are poor, and dependent on the cancer phase. For example, treatments such as tumor resection and liver transplantation are effective for patients in the primary phase of HCC. However, the chance of surgical recurrence is approximately 70% in the first five years. 1,2 Patients in the end stage of HCC are treated with chemotherapy and the drug sorafenib, while only 10% of patients survive more than three years.
Iran J Med Sci November 2022; Vol 47 No 6 559 Sorafenib is a multi-kinase inhibitor that improves patient rehabilitation. 3 Therefore, an effective therapeutic method against HCC is required. In recent years, researchers have used three main approaches to stimulate the immune response against HCC, including adoptive immunotherapy Appropriates Natural Killer (NK) and Cytokine-Induced Killer (CIK) cells, indirect immunological approaches including vaccines, and indirect nonimmunological strategies such as the antigencoding mRNA system. Vaccines, which belong to the indirect immunological strategies, are an effective strategy against tumor cells and are generally divided into three types, including pulsed dendritic cells (DCs), peptide-based vaccines, and DNA-based vaccines. Peptide-based vaccines might include Tumor-Associated Antigens (TAAs) or Tumor-Specific Antigens (TSAs) and could stimulate the adaptive immune system properly. 4 Multi-epitope peptide vaccines are used against pathogenic bacteria, viruses, and tumor cells.
Multi-epitope peptide vaccines, which consist of Cytotoxic T cell Lymphocytes (CTL) and Helper T cell Lymphocytes (HTL) epitopes and provoke humoral and adaptive immune responses, are considered as a beneficial approach for tumor immunotherapy. For designing an effective multiepitope peptide vaccine against cancer, CTL and HTL epitopes could be selected from TSAs or TAAs. 5 In HCC, several antigens, including α-fetoprotein (AFP), 6 glypican-3 (GPC-3), and aspartyl-β-hydroxylase (ASPH), 7 are known as TAAs. Therefore, they are proper targets for the selection of CTL epitopes. AFP is known as a fetal liver protein, and about 80% of HCC patients have AFP in their blood serum, while the level of AFP is 1-3 ng/mL in healthy people. Thus, serum assessment plays an important function in the diagnosis and therapy of HCC. 8 As mentioned, another TAA in HCC is GPC-3, which pertains to heparan sulfate proteoglycans and plays a critical role in cell growth, survival, and development. 9 GPC-3 is overexpressed in 50% of HCC cases, and not in healthy tissues, and hence, it is a perfect biomarker for the diagnosis of HCC. 10 ASPH is an ideal TAA for HCC immunotherapy and is a transmembrane protein that belongs to the α-ketoglutaratedependent dioxygenase family. 11 This protein is over-expressed in HCC and infrequently detected in healthy adult tissues. 12 Adjuvants are employed in the vaccine construct to effectively stimulate the immune response in the host and additionally reduce the needed dose of vaccine injection. 13 Heat shock proteins (HSPs) possess diverse functions in biological processes, such as protein folding and degradation, elimination of misfolded proteins, and protein transportation. 14 Moreover, HSPs have a significant role in stimulating the innate and adaptive immunity and support providing of antigens to T CD8 + cells via the Major Histocompatibility Complex class I (MHC-I) pathway. 15 Accordingly, HSP can serve as a powerful immunoadjuvant. 16 In previous studies, two tandem repeats of microbial HSP70 peptide 407-426 were used as an adjuvant, which improves DC maturation, induction of T CD8 + , and T-helper 1 cell responses. [17][18][19] In this research paper, a novel multi-epitope peptide vaccine construct against HCC, which was designed by bioinformatics tools in our previous study, 20 was cloned and expressed in Escherchia coli host and then purified. The vaccine construct carries epitopes of AFP, GPC-3, and ASPH in the role of CTL epitopes, Tetanus Toxin Fragment C (TTFrC) as HTL epitopes, and two tandem repeats of HSP70 407-426 in the guise of a natural immune-adjuvant.

Host Strain and Culture Media
This experimental research was carried out at Fasa University of Medical Sciences, Iran, in 2017. The ethics committee approval code for this research is IR.FUMS.REC.1397.116.
E. coli BL21 (DE3) strain is a Gram-negative straight rod, and an acceptable organism to represent recombinant proteins. This strain can lead to high-yield protein expression, because it is under the control of the T7 promoter. In this study, E. coli BL21 (DE3) strain was applied for transformation. Furthermore, the pET-23d expression vector carries a C-terminal and N-terminal His Tag coding sequence and possesses an ampicillin resistance marker. This vector was utilized for gene expression; and Super Optimal Broth (SOB) medium, including 20 g/L trypton, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mL 1 M KCl, and 10 mL 1 M MgCl 2 was applied as media culture for protein expression.

Designed Vaccine Construct
Using bioinformatics tools, the design of a novel multi-epitope peptide vaccine against HCC was carried out. 20 Several servers were used to increase the accuracy of the vaccine design, including IEDB at http://tools.immuneepitope. org/analyze/html/mhc_binding.html, that uses some methods for prediction of MHC-I binding peptides, including Stabilized Matrix Method (SMM), Average Relative Binding (ARB), and Artificial Neural Network (ANN). 21 ProPred-I at www.Imtech.res.in/raghava/propred1/, uses quantitative matrix in prediction of the MHC class-I peptide. 22 PAComplex at http:// PAcomplex.life.nctu.edu.tw, it is the initial server which predicts peptide-MHC and peptide-TCR interaction. 23 BCPRED at http://ailab-projects1. ist.psu.edu:8080/bcpred/predict.html, the server selected the linear B cell epitopes based on the new method of the subsequence kernel which was based on a data collection of 701 linear B-cell epitopes and 701 nonepitopes. 24 These servers were used for the prediction of various immunogenic epitopes, including HTL, CTL, and B cell epitopes. The selected epitopes were joined together by proper linkers. Finally, antigenic, allergenic, and solubility properties of the designed vaccine construct were evaluated using appropriate servers. More details were mentioned in our previous work. 20 The vaccine construct, which subtends CTL epitopes (AFP, GPC-3, ASPH), HTL epitopes (TTFrC), and immunoadjuvant (two tandem repeats of microbial HSP70 407-426 ), was cloned into the pET-23d vector (ZistEghtesad Mad, Iran).

Transformation
E. coli BL21 (DE3) competent cells were prepared and plasmid vector pET-23d was transformed into competent cells using the calcium chloride method. 25 The transformed cells were grown in a Laurie-broth (LB) (Miller, China) plate containing 100 µg/mL ampicillin (Jaberebn Hayyan, Iran) and incubated at 37 °C for 24 hours.

Expression of the Vaccine Protein
The culture media containing transformed E. coli was at 37 °C overnight. After that, 1 mL of the culture media was diluted in 5 mL of fresh culture in SOB media comprising of 100 µg/mL ampicillin (Jaberebn Hayyan, Iran) on the shaker incubator at 37 °C, and was shaken until an Optical Density (OD) of 0.6 was achieved at 600 nm after about five hours. Then, the media was induced via two different concentrations of Isopropyl β-D-1-thiogalactopyranoside (IPTG), including 0.6 and 1 mM, and different times, times, including 4, 6, 8, 16 hours, and two different temperatures (25 and 37 °C) were evaluated. Subsequently, the transformed cells were harvested via centrifugation at 10000 rpm for five minutes at room temperature, and the obtained pellets were stored at -20 °C for further research.

SDS-PAGE Analysis
The expression of the recombinant vaccine protein in several above-mentioned conditions was measured with Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), 26 where 4 λ pellet (harvested in the previous step) was blended in 10 λ sample buffer and 6 λ DW, and heated at 95 °C for five minutes and centrifuged at 15000 rpm for 10 minutes at 4 °C. In the next step, an amount of 10 λ of the sample was loaded on the SDS-PAGE gel (12%) (Bio-Rad-USA), the protein marker and the power supply was run on 100-120 V for about two hours. Following that, the gel was stained with Coomassie Brilliant Blue (Biobasic-Canada) solution for about 16 hours, and then de-stained.

Purification of the Vaccine Protein
The purification of the recombinant protein was carried out using Ni-NTA affinity column chromatography (QIAGEN Company, Germany) following denaturing conditions. The bacterial pellets were solved in the lysis buffer (pH=8, 7 M urea, 0.1 M NaH 2 Po 4 , 0.01 M Tris-Cl) and shaken for about 20 minutes. In the next step, the cells were disrupted via ultrasonication (sixtime six-second cycles at 4 °C). The suspension was centrifuged at 12000 ×g for 20 minutes at 4 °C, and the supernatant was decanted to the Ni-NTA column and centrifuged (two minutes at 890 ×g). The column was washed repeatedly via a wash buffer (pH=6.

Western Blot Test
To prove the expression and purification of the recombinant vaccine protein, the Western blot method was employed. 27 In this technique first, proteins were detached and transferred onto a polyvinylidenefluoride membrane (PVDF). In the next step, the PVDF paper was blocked with 5% skim milk for about 16 hours at 4 °C. Anti-his tag antibodies were inserted for two hours with agitation. Then, they were washed three times with tween 20 (0.1%) in phosphatebuffered saline (PBS). Finally, after adding a diaminobenzidine (DAB) solution (0.05%) with 30 µL hydrogen peroxidase (30%) for 20 sec, the purified protein became visible.

Vaccine Construct and Transformation
The designed vaccine containing immunogenic epitopes against HCC was cloned into a pET-23d expression vector. The expression vector was transformed inside the competent E. coli BL21 (DE3) strain, and then, the transformed E. Coli was grown on LB agar containing 100 µg/mL ampicillin (figure 1).

Expression of the Vaccine Protein
The recombinant bacteria were cultured in SOB media containing 100 µg/mL ampicillin. Bacteria were grown to reach the OD of 0.6 at 600 nm, the bacterial growth rate over time is shown in table 1. Then, different IPTG concentrations (0.6 and 1 mM) were administrated in different conditions, including several times (4, 6, 8, and 16 hours) and several temperatures (25 and 37 °C). The recombinant protein was verified in all mentioned conditions. The band of the vaccine protein was about 32.3 kDa, which was in accordance with the molecular weight of the designed construct as predicted by the bioinformatics tool. Furthermore, optimal protein expression was noticed in the SOB medium at 37 °C after expression induction by 1 mM IPTG for six hours. The results of the SDS-PAGE analysis are shown in figure 2.

Purification of the Protein
The recombinant protein had His-tag at the C-and N-terminal; therefore, purification was accomplished using Ni-NTA affinity column chromatography. The recombinant protein was purified by denaturing conditions and verified by SDS-PAGE ( figure 3).

Western Blot Test
The expression and purification of the designed multi-epitope peptide vaccine were evaluated by the Western blot analysis. As shown in figure 4, the purified protein was confirmed by the Western blot method.

Discussion
In this research, a novel multi-epitope peptide     vaccine was expressed in a bacterial host (E. coli BL21 (DE3) strain) using SOB and LB culture media and purifying it for HCC immunotherapy. The purified peptide vaccine was confirmed by the SDS-PAGE and Western blot techniques, and based on the obtained results, the molecular weight of the purified vaccine was 32.3 KDa.
HCC is a cancer with a high mortality rate. This malignancy is not usually diagnosed in the primary stages, because it is known as a heterogeneous disorder that includes several etiological agents; therefore, it is diagnosed in the advanced stages, when it is usually late for treatment. 19 At the advanced stages, only sorafenib and regorafenib drugs are effective for HCC, and other treatments, such as surgery, are not helpful. Thereby, effective treatment against HCC is needed. Recently, scientists have considered immunotherapeutic methods to treat HCC, and some of the clinical trials on HCC patients include cytokine relying therapies such as interferons, 28 cell-based trials such as dendritic cell vaccines, 29 antibodybased treatments including AFP antibodies, 30 and antigen-specific vaccines such as GPC-3 peptide-based vaccine. 31 In particular, researchers have recently studied multi-epitope peptide vaccines as a new immunotherapeutic approach against HCC. Various investigations have utilized bioinformatics tools to design beneficial multi-epitope peptide vaccines, and bioinformatics makes that process easier. 32 Multi-epitope peptide vaccines possess some advantages over the others. They are detected through several T-cell subsets due to the presence of various MHC-restricted epitopes, which can powerfully stimulate the humoral and cellular immune responses, as they consist of several B-cell, CTL, and HTL epitopes. The presence of adjuvant in their compound increases their immunogenicity. 33 In our previous study, a novel multi-epitope peptide vaccine was designed against HCC via bioinformatics tools. 20 The designed vaccine has several CTL epitopes (AFP, GPC-3, and ASPH), HTL epitopes (TTFrC), and a powerful natural adjuvant (two tandem repeats of microbial HSP70 407-426 ). All epitopes were chosen by several bioinformatics servers. Important properties of the designed vaccine construct, such as antigenicity, were checked by ANTIGEN pro at (http://scratch.proteomics.ics. uci.edu) and VaxiJen. (http://www.jenner.ac.uk/ VaxiJen), which showed that the designed vaccine possesses high antigenicity. Furthermore, based on the results of the ALgPred server (https://webs.iiitd.edu.in/raghava/algpred2/), the designed vaccine construct was not allergenic, and some physicochemical parameters, such as high half-life and stability, were verified by the ProtParam server (http://web.expasy.org/ protparam/). The linear and conformational B-cell epitopes and interferon-gamma-inducing epitopes were recognized in the vaccine construct, because provoking the humoral and cellular immune responses is essential in multiepitope peptide vaccines. 34 AFP is one of the most important antigens considered in vaccine development against HCC. 35 In 2018, Zhang tested AFP in the development of a dendritic cell-based vaccine for HCC. Adenoviralencoded synthetic AFP and different forms of AFP were investigated to activate the T CD8 + and T CD4 + cells. These results showed that the secreted form of AFP leads to enhanced T CD4 + and T CD8 + cells responses. 36 37 In recent years, new research has been conducted based on ASPH for the treatment of HCC. Greten and colleagues designed and analyzed the ASPH-DC vaccine ex vivo on a mouse model in 2019, and the results showed that the designed vaccine led to increased T-cell response and activated the pre-apoptosis mechanism. 38 Therefore, based on the literature review, we used a combination of the antigens (AFP, GPC-3, and ASPH) that are over-expressed in HCC patients, which are known as suitable targets for the design of peptide-based vaccines. Immunogenic epitopes were used in the vaccine construct instead of the whole antigens. In addition, we used two tandem repeats of HSP70 407-426 as adjuvans, which showed the potential to stimulate humoral and cellular responses effectively in previous research. HSP70 leads to the production of IFN-γ and IgG2a, which are effective in the Th1 response. 39 In this study, to join CTL epitopes, the SSL and HEYGAEALERAG motifs were used as linkers. The HEYGAEALERAG linker is a cutting target for the proteasomal machine. 34 Furthermore, HTL epitopes attached via KK linker and EAAAK linker were applied to connect the adjuvant, which is a rigid motif that leads to the increased stability and folding of the vaccine construct. 40 The next step for the development of the multi-epitope peptide vaccine is the prosperous cloning and expression of the vaccine construct in the proper expression vector. In this research, we utilized the expression vector pET-23d, which is an accepted vector for the production of recombinant proteins in bacterial host E. coli. 41 To find the optimal expression of the designed vaccine, the vaccine construct was expressed in different conditions, including various concentrations of IPTG (0.6, 1mM) at two different temperatures (25 and 37 °C) and four different times (4, 6, 8, and 24 hours). According to the SDS-PAGE results, the best conditions for optimal protein expression were in the SOB medium at 37 °C after expression induction by 1 mM IPTG for six hours. The expression and purification of the designed vaccine protein were confirmed by SDS-PAGE and Western blot techniques, which showed a single band with 32.3 KDa molecular weight. Thus, based on the obtained results, we believe that the purified vaccine protein can stimulate humoral and cellular immunity against HCC patients effectively.
Based on the bioinformatics analysis, the produced vaccine has proper characterizations, such as high antigenicity and stability. However, in order to confirm these results, it is necessary to evaluate its immunogenicity in animal models.

Conclusion
In the present research, a novel multi-epitope peptide vaccine against HCC was produced. The multi-epitope construct vaccine contained CTL epitopes selected from AFP, GPC-3, and ASPH antigens, HTL epitopes from TTFrC, and an efficient natural adjuvant (HSP70), which were joined together by appropriate linkers. The best conditions for the vaccine construct expression was found to be the SOB medium at 37 °C after expression induction by 1 mM IPTG for six hours. Hence, the purified peptide vaccine can potentially stimulate the immune system properly, although further in vitro and in vivo experimental investigations on the designed vaccine are truly necessary.


s2fieldsofstudy: Biology Medicine

text:
CircRNA ITCH: Insight Into Its Role and Clinical Application Prospect in Tumor and Non-Tumor Diseases

CircRNA E3 ubiquitin protein ligase (ITCH) (circRNA ITCH, circ-ITCH), a stable closed-loop RNA derived from the 20q11.22 region of chromosome 20, is a new circRNA discovered in the cytoplasm in recent decades. Studies have shown that it does not encode proteins, but regulates proteins expression at different levels. It is down-regulated in tumor diseases and is involved in a number of biological activities, including inhibiting cell proliferation, migration, invasion, and promoting apoptosis. It can also alter disease progression in non-tumor disease by affecting the cell cycle, inflammatory response, and critical proteins. Circ-ITCH also holds a lot of promise in terms of tumor and non-tumor clinical diagnosis, prognosis, and targeted therapy. As a result, in order to aid clinical research in the hunt for a new strategy for diagnosing and treating human diseases, this study describes the mechanism of circ-ITCH as well as its clinical implications.

INTRODUCTION
In brief, diseases were categorized into two groups: tumors and non-tumor disorders. Malignant tumors, for example, are a type of incurable polygenic disease that has claimed the lives of millions of individuals worldwide. According to GLOBOCAN 2020, there were 19.3 million new malignant tumor cases and 9.9 million deaths globally in 2020 (Sung et al., 2021). Although surgical resection and advanced therapeutic interventions have improved the 5-year survival rate in patients with earlystage GC, the prognosis for late-stage GC patients remains poor due to uncontrolled tumor cell growth and migration (Lasithiotakis et al., 2014). Non-tumor disorders, such as degenerative, metabolic, congenital, and inflammatory pathologies, make up the great bulk of all pathologies, aside from tumors. As a result, finding effective diagnostic biomarkers and treatment targets is crucial for disease fundamental research.
Benefiting from high-throughput sequencing, researchers can take a nuanced and complete picture of the transcriptome and genome of a species. RNA sequencing (RNA-seq) technology has become one of the important means of transcriptomic studies of high-throughput sequencing, which can discover all RNAs that a particular cell can transcribe in a certain functional state, mainly including mRNAs and non-coding RNAs, while avoiding detection using standard molecular techniques. CircRNAs, which were previously thought to be misspliced products, have lately been shown to have a range of biological regulatory activities owing to the development of RNA-seq (Memczak et al., 2013). Most circular RNAs (circRNAs) are mainly composed of one or more exons encoding known proteins. The 3' and 5' terminals of circRNAs are covalently bonded to form a closed-loop structure, unlike typical linear RNAs. It has no free terminal and is unaffected by RNA exonuclease, resulting in a more stable and difficult-to-degrade copy. CircRNAs is primarily involved in the following four processes: 1) sponging microRNAs (miRNAs) or long noncoding RNAs (lncRNAs) as a competing endogenous RNA (ceRNA); 2) binding RNA binding proteins (RBPs); 3) interfering with gene transcription and splicing regulation; and 4) translating protein/polypeptide (Kristensen et al., 2019). Maass et al. (2017) identified 71 differentially expressed circRNAs in 20 human clinical samples including tissues, blood, proving that circRNAs can express stably in vitro and are associated with multiple diseases, which has good potential for biomarker development.
As a member of the E3 ubiquitin protein ligase (ITCH) HECT family, ITCH can ubiquitinate phosphorylated disheveled-2 (Dvl2), promoting its degradation (Bernassola et al., 2008). Phosphorylated Dvl2 is the upstream target of activating βcatenin in the canonical Wnt pathway, therefore ITCH can block the canonical Wnt pathway, regulating cell cycle (Li et al., 2015). In addition, ITCH can regulate immune responses, epidermal keratinocyte differentiation and receptor trafficking/signaling . Previous studies have shown that in ITCH −/− mice, some signal proteins (such as Jun family members and Notch) are abnormally accumulated, seriously affecting the autoimmune phenotype (Parravicini et al., 2008). Jun and Notch are also transcription factors that control the maintenance of epidermal stem cells and the regulation of keratinocytes. The degradation of these proteins mediated by ITCH may play a regulatory role in skin biology (Candi et al., 2005), indicating that it has certain potential in radiotherapy protection. Moreover, Sundvall et al. (2008) emphasized the role of pruritus in regulating the endocytosis and protein stability of erbb-4, a receptor belonging to the epidermal growth factor receptor (EGFR)/ErbB family. Therefore, ITCH involves a variety of physiological and pathological regulation through different mechanisms, including regulating Wnt, Jun, Notch, MAPK signaling, immune cells differentiation and EGFR family. CircRNA E3 ubiquitin protein ligase (circRNA ITCH, hereinafter referred to as circ-ITCH), a stable closed-loop RNA with no protein coding ability and derived from the 20q11.22 region of chromosome 20, is a new circRNA discovered in recent decades (Bernassola et al., 2008;Memczak et al., 2013). The initial study reported that circ-ITCH came from exon 6-13 of E3 ubiquitin protein ligase (ITCH) encoding gene (Li et al., 2015), but then it became exon 7-14 in related studies without any explanation , which is a point that needs to be clarified in subsequent experiments. Combining the database (Circular RNA Interactome and circBase) and related literature search, we considered it derived from exons 6-13 ( Figure 1), while 7-14 was a writing error in correlative papers. Recently, studies have revealed that circ-ITCH is down-regulated in multiple tumor tissues, and regulates cell proliferation, migration, invasion and apoptosis of malignant tumor, indicating it might be an important tumor suppressor Ghafouri-Fard et al., 2021;Su et al., 2022). Additionally, the down-regulation can also be seen in the peripheral blood and exosomes of patients. Its low expression has some diagnostic relevance and is linked to negative clinical outcomes (such as tumor sizes, lymph node metastasis and distant metastasis). Furthermore, circ-ITCH plays an essential function in non-tumor illnesses. Understanding its function and mechanism could help clinical researchers discover novel strategies to diagnose and treat a variety of diseases earlier.

Regulating Canonical Wnt Signaling Pathway
2012). The Wnt/β-catenin signaling pathway has two pathways: the canonical pathway and the non-canonical pathway, with the conventional pathway being the more common (Wei et al., 2012;Muralidhar et al., 2019). Canonical signal pathways consist of the Wnt protein, Wnt receptor [frizzled family protein (FZD) and low density lipoprotein receptor associated protein-5/6 (LRP-5/ 6)], Dvl2, β-catenin protein and et al. (Muralidhar et al., 2019) Wnt protein interacts with the FZD receptor on the cell membrane surface in an autocrine or paracrine manner, then recruits LRP-5/6 to create a complex that activates intracellular Dvl2 protein via a phosphorylation cascade. Through its PDZ domain (one of Dvl2's domains), phosphorylated Dvl2 favorably regulates β-catenin protein, facilitating its entry into the nucleus as a transcriptional regulator and activating the expression of downstream target genes including cyclinD1 and c-Myc (Muralidhar et al., 2019).
Through targeting certain miRNAs, Circ-ITCH inhibits the function of matching miRNAs, particularly miRNAs that inhibit linear ITCH. Specifically, researchers showed that reporter gene assays in the presence of circ-ITCH demonstrated that the inhibitory effects of different miRNAs (including miR-7, miR-17, miR-20a, miR-22-3p, and miR-214) were dampened by the co-expression of circ-ITCH, consistent with the "sponge" hypothesis (Huang et al., 2015;Li et al., 2015;Wang et al., 2018). Among these miRNAs, the miR-7 and miR-214 are most important because they can share the binding sites with the 3′-UTR of circ-ITCH and its parental gene ITCH (Verduci et al., 2021). It is worth mentioning that, as one of the most conservative and oldest miRNAs, miR-7 plays different roles in different cancers and participates in many signal pathways involving differentiation, proliferation regulation, apoptosis and migration. In most tumors, its expression is downregulated because its main activity is to inhibit tumor by inhibiting cell proliferation and survival. However, in lung cancer and oral cancer, its expression is up-regulated as a carcinogen, which is consistent with the research on circ-ITCH (Kora´c et al., 2021). Since circ-ITCH and ITCH share the 3'-UTR of miR-7, they will produce competitive inhibition. When circ-ITCH is up-regulated, the remaining ITCH content in vivo will be up-regulated (Verduci et al., 2021). ITCH can identify and ubiquitinate a range of proteins, the most important of which is phosphorylated Dvl2 (Wei et al., 2012). It is well known that Dvl family proteins are mostly made up of Dvl1-3, with Dvl2 serving as a key scaffold in the canonical Wnt pathway, connecting upstream Wnt protein with downstream β-catenin protein (Wei et al., 2012). As a result, below is the whole regulator mechanism: via sponging miR-7, miR-17, miR-20a, miR-22-3p, and miR-214, circ-ITCH increases ITCH levels. While phosphorylated Dvl2 labeled by ITCH ubiquitin promoted its degradation and inhibited the canonical Wnt pathway. A summary of recent studies has found that in esophageal squamous cell carcinoma (ESCC), colorectal cancer (CRC), lung cancer (LC), three negative breast cancer (TNBC), prostate cancer (PCa), hepatocellular carcinoma (HCC) and gastric cancer (GC), circ-ITCH could up-regulate the expression of linear ITCH via sponging miR-7, miR-17 and miR-20a, thereby inhibiting the canonical Wnt pathway and further suppressing the activation of c-Myc and cyclinD1 (Wan et al., 2016;Wang S. et al., 2019;Li S. et al., 2020;Peng and Wang, 2020;Yang et al., 2020). As widely reported, c-Myc is an oncogene. Its aberrant activation frequently results in unrestricted cell proliferation and immortalization, promoting cell malignancy and tumorigenesis (Glöckner et al., 2002). CyclinD1 (also known as G1/S-specific cyclin D1), on the other hand, regulates the cell cycle and promotes cell proliferation, and is up-regulated in a number of malignancies  (Glöckner et al., 2002). In addition, Wang et al. (2018) also confirmed that circ-ITCH can sponge miR-22-3p and increase the expression of CBL in papillary thyroid cancer (PTC), inhibiting cell proliferation and invasion, increasing apoptosis, and repressing PTC progression. CBL is also a member of the E3 ubiquitin ligase family, which can ubiquitinate as well as label βcatenin to promote its destruction and so block the Wnt pathway (Shashar et al., 2016). In most tumors, the glucose transporter 1 (GLUT1) gene is overexpressed. By mediating glucose via the plasma membrane and increasing glucose absorption, it plays a vital function in the early stages of intracellular glucose metabolism and promotes tumor growth (Koch et al., 2015). ITCH can down-regulate the expression of GLUT1 in melanoma, reducing glucose uptake and tumor cell growth, according to Lin et al. (2021), but whether this regulation also involves the Wnt pathway needs to be confirmed in further experiments.

Regulating PI3K/Akt Signaling Pathways and MEK/Erk Cascade
In the progression of many malignancies, activation of the PI3K/ Akt pathway and MEK/Erk cascade have been confirmed. Following activation, PI3K activates the Akt protein, which subsequently enters the nucleus and regulates cell proliferation, invasion, migration, metabolic reprogramming, autophagy, and aging, potentially causing malignant tumors (Hermida et al., 2017). The MEK/Erk cascade interacts closely with the PI3K/Akt cascade and is involved in tumor development.
After activating signaling pathways, many phosphorylated Erk substrates have been shown to contribute to cell proliferation and invasion (Burotto et al., 2014). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a miR-7, miR-22, and miR-224 target that inhibits the PI3K/Akt cascade (Sadri Nahand et al., 2021). P21 is the downstream target of the PI3K/Akt cascade. To promote cell proliferation, activated Akt can phosphorylate p21, blocking its cell cycle arrest function . Circ-ITCH sponges these miRNAs to upregulate PTEN expression in bladder cancer (BCa) and OS, blocking the PI3K/Akt cascade, and up-regulating p21 protein to prevent tumor cell proliferation, migration, invasion and promote apoptosis (Yang et al., 2018;Ren et al., 2019). A recent vitro experimental investigation found that circ-ITCH might further up-regulate PTEN in nasopharyngeal cancer (NPC) via sponging miR-214 , indicating that it can prevent NPC progression by blocking the PI3K/Akt pathway. Ras p21 protein activator 1 (RASA1) is a regulator of Ras-GDP and GTP, which promotes apoptosis and inhibits angiogenesis, cell proliferation by inhibiting Ras/Raf/MEK/Erk signals cascade . RASA1 has been shown to be low expressed in a number of tumors, and miR-14 has been identified to mute it . Additionally, Hu et al. (2018) found that in ovarian cancer (OC), circ-ITCH upregulated RASA1 by sponging miR-145, blocking the PI3K/ Akt pathway and MEK/Erk cascade, therefore decreasing tumor cell malignancy. Yan et al. (2020) found a negative connection between circ-ITCH expression and lncRNA HULC expression in OC. Previous research has demonstrated that through down-regulating the miR-125a-3p level, lncRNA HULC can activate the PI3K/Akt/mTOR pathway, promoting the proliferation, migration, and invasion of OC cells (Chu et al., 2019). Therefore, circ-ITCH might compete with lncRNA HULC for miR-125a-3p binding, blocking the PI3K/Akt/mTOR pathway and so acting as an anti-tumor agent. However, the hypothesis needs to be confirmed by further experiments.
Published reports by two independent groups of researchers in 2019 and 2021 suggested that circ-ITCH expression decreased in patients' OS sample tissue, and that circ-ITCH hindered the proliferation, migration, and invasion of OS cells via sponging miR-22 and miR-524 (Ren et al., 2019;Zhou W. et al., 2021). But interestingly, in 2020, Li et al. (Li H. et al., 2020) discovered that the expression of circ-ITCH was up-regulated in U2OS and SJSA-1 cell lines, and enhanced the expression of epidermal growth factor receptor (EGFR) by reducing the level of tumor suppressor miR-7 in OS. Then, when EGFR is overexpressed, it activates the PI3K/Akt and MEK/Erk pathways, promoting OS development. This finding contradicts the findings of two previous investigations. However, there is a flaw in the experiment: it did not verify the degree of circ-ITCH expression in the OS tissue sample. It's possible that this is due to the heterogeneity of OS cell lines or the complexity of studying the regulatory network. Consequently, in the research of circ-ITCH in OS, more parameters should be explored.

Regulating Cell Cycle-Related Proteins
Programed cell death receptor 4 (PDCD4) is described as a tumor suppressor, which is often down-regulated in tumors, promoting tumor cell apoptosis and inhibiting its proliferation, invasion and metastasis (Yang et al., 2021). MiR-106b-5p and miR-421 are common upstream targeting miRNAs of PDCD4 and can inhibit its expression Yang et al., 2021). Circ-ITCH specifically targets miR-106b-5p and miR-421 in clear cell renal cell carcinoma (ccRCC) and oral squamous cell carcinoma (OSCC) to up-regulate PDCD4 expression and prevent tumor progression, respectively (Hao et al., 2020;Gao et al., 2021). RAS association domain family member 6 (RASSF6) inhibits cell growth and promotes apoptosis in a variety of tumors by interrupting the cell cycle (van der Weyden and Adams, 2007). In OS, circ-ITCH sponges miR-524 to up-regulate RASSF6, inducing OS cell death and limiting its proliferation, according to Zhou et al. (Zhou W. et al., 2021). SAM and SH3 domain containing protein 1 (SASH1) is a tumor-suppressive protein that can regulate cell apoptosis and proliferation (Burgess et al., 2020). Circ-ITCH can suppress glioma growth and invasion by up-regulating SASH1 by targeting miR-106a-5p . Cytoplasmic polyadenylation element binding protein 3 (CPEB3), a RNA binding protein, plays a tumor-suppressive role though regulating the expression of malignant transformationrelated genes through post-transcriptional control (Pichon et al., 2012). In HCC, circ-ITCH binds to miR-421 to prevent CPEB3 down-regulation and tumor growth (Zhao et al., 2021). MafF belonging to the Maf family, a basic leucine zipper (bZIP) transcription factor, has been found to have anti-tumor properties in HCC (Tsuchiya and Oura, 2018). By modulating the miR-224-5p/MAFF axis, Circ-ITCH can also decrease cell growth and increase apoptosis . Li et al. (2020b) found that circ-ITCH sponges miR-93-5p to up-regulate forkhead box K2 (FoxK2) and block tumor growth in cervical cancer (CC). Simultaneously, FoxK2 expression was dramatically reduced in CC tissues, and miR-93-5p mimic transfection further decreased FoxK2 expression in CC cell lines. FoxK2 deletion improved the capacity of cells transfected with miR-93-5p mimic to invade. Homeobox B13 (HOXB13), according to earlier research, inhibits the cell cycle by promoting the ubiquitination and degradation of cyclinD1 in a variety of malignancies (Hamid et al., 2014). Circ-ITCH restrains PCa cell proliferation, invasion, and migration via sponging miR-17-5p and boosting HOXB13 up-regulation, as well as promoting apoptosis (Wang X. et al., 2019). Furthermore, via targeting miR-197, circ-ITCH can attenuate PCa cell proliferation and increase apoptosis, however the underlying mechanism is uncertain . In melanoma, circ-ITCH suppresses cell proliferation and metastasis through sponging miR-660, a previously reported tumor-promoting miRNA, further upregulating transcription factor cellular promoter 2 (TFCP2) . TFCP2, as a cell cycle regulating molecule, mainly plays a tumor suppressor role in melanoma. Its role is mainly to positively regulate the DAPK transcription by binding to the promoter of the death associated protein kinase (DAPK) gene, a tumor suppressor that is silenced in many cancers (Kotarba et al., 2018). Besides, TFCP2 can positively regulate the transcription of p21 CIP1 , a well-known cell cycle inhibitor protein (Goto et al., 2016;Kotarba et al., 2018). In addition, previous studies have shown that Klotho can inhibit the IGF-1/insulin pathway and regulate the expression of Bax/Bcl-2, thereby inhibiting cell proliferation and promoting apoptosis in A549 cells (Chen et al., 2010). While recently Wang et al.  found that circ-ITCH boosted Klotho expression though acting as a miR-199a-5p sponge, thus, suggesting that the function of circ-ITCH in GC may involve cell cycle-related regulatory proteins. In short, recent studies have discovered that circ-ITCH regulates the expression level of a number of cell cycle-related proteins, as a ceRNA, to induce tumor cell apoptosis and limit tumor cell growth, thereby acting as an antitumor agent.

Regulating Epithelial Mesenchymal Transition Process
Epithelial mesenchymal transition (EMT) is a process that occurs in almost all forms of tumors and is linked to tumor incidence, invasion, metastasis, recurrence, and medication resistance (Iwatsuki et al., 2010). E-cadherin and vimentin are two crucial proteins that are frequently used as EMT indicators (Iwatsuki et al., 2010). E-cadherin, which is encoded by CDH1, is involved in EMT and is linked to tumor invasion and diffusion (Ye et al., 2012). Vimentin, in particular, promotes EMT, whereas E-cadherin opposes it. Lin et al. (2020) discovered that circ-ITCH inhibits EMT in OC by increasing CDH1 expression via sponging miR-106a. Guo et al. (2022) revealed that the tumor suppressor role of circ-ITCH in HCC is associated with regulating EMT progression through KEGG enrichment analysis, and its regulation function is associated with sponging miR-184. It is well known that Klotho-mediated regulation of cellular EMT is a way to regulate tumor progression . Therefore, by modulating the miR-199a-5p/Klotho axis, circ-ITCH can block EMT and delay tumor growth in GC . In addition, earlier research has demonstrated that the Wnt pathway is important for regulating EMT (Kumari et al., 2021). As a result, circ-ITCH's modulation of the Wnt pathway might have an impact on the downstream EMT process, but more research is needed to confirm this. In summary, circ-ITCH modulates downstream targets including the Wnt pathway, the PI3K/Akt cascade, the MEK/ Erk cascade, cell cycle-related proteins, and EMT process via sponging different miRNAs, performing an anti-tumor effect in a range of malignant tumors (Figure 2).

Bone Diseases
Osteoporosis is a systemic bone disease that causes decreased bone density and quality, disturbed bone microarchitecture, and increased bone fragility, all of which increase the risk of fracture (Compston et al., 2019). Zhong et al. (2021) demonstrated that compared to normal tissues circ-ITCH expression was downregulated in osteoporosis samples, implying that it may play a protective role in bone degenerative diseases. Specifically, circ-ITCH up-regulated the expression of YAP1 by sponging miR-214. YAP1 is a prominent downstream effector of the Hippo pathway, and its up-regulation can stimulate the differentiation of mesenchymal stem cells into osteoblasts, according to previous research (Lorthongpanich et al., 2019). Moreover, YAP1 stimulates osteogenesis though interacting with β-catenin in osteoblasts (Pan et al., 2018). Taken together, the study found that circ-ITCH might enhance osteogenic differentiation in osteoporosis and ameliorate osteoporosis symptoms in mice (Zhong et al., 2021). Similarly, circ-ITCH expression is upregulated during periodontal ligament stem cell (PDLSC) osteogenic differentiation and may trigger osteogenic differentiation though regulating MAPK pathway (Gu et al., 2017).
Intervertebral disc degeneration (IDD) is a type of degeneration that can cause a variety of minor or self-limiting symptoms. Spinal discomfort is currently thought to be mostly caused by IDD. Degradation of the extracellular matrix (ECM) and apoptosis of the nucleus pulposus (NP) cells are other key markers of IDD development . Recently, Zhang et al. (2021) discovered that circ-ITCH might sponge miR-17-5p/SOX4 signaling to positively regulate the activation of Wnt/β-catenin pathway in IDD, causing ECM degradation and NP cell apoptosis. However, this finding contradicts prior findings, particularly in tumor research, in that it activates Wnt/β-catenin pathway. After Wnt/β-catenin activation, Zhang et al. were unable to further illustrate the regulatory mechanism. Combined with earlier research (Zimmerman et al., 2013), we speculate that activating Wnt/β-catenin

Cardiac Diseases
CircRNAs have been linked to the development of a number of cardiac diseases, including atherosclerosis, myocardial damage, heart failure, and drug-induced cardiotoxicity, according to research (Min et al., 2021). The current emphasis of circ-ITCH research in cardiac diseases is ischaemia-reperfusion (I/ R) injury and doxorubicin-induced cardiotoxicity (DOXIC). In I/R damage, a substantial amount of H2O2 can be created, aggravating oxidative stress injury (Wu et al., 2018). H2O2 caused apoptosis in H9c2 rat cardiac cells and reduced viability, ATP levels, and circ-ITCH expression in a recent study . Furthermore, H2O2 treatment boosted the expression of Wnt3a, Wnt5a, and β-catenin . Conversely, in H2O2 pretreatment H9c2 cells, overexpression of circ-ITCH reduced apoptosis and Wnt/βcatenin expression, suggesting that its cardioprotective effect is linked to the inactivation of Wnt/β-catenin signaling pathway in I/R injury . Specifically, circ-ITCH reduced cardiomyocyte apoptosis in I/R injury by sponging miR-17-5p and then inactivating the Wnt/β-catenin signaling pathway . Doxorubicin is an effective chemotherapeutic agent, but doxorubicin-treated patients are prone to cardiac toxicity and subsequently develop congestive heart failure (Yeh and Bickford, 2009). The two primary pathogenic processes leading to the pathogenesis of doxycycline have been identified in DOXIC as oxidative stress and DNA damage (Zhang et al., 2012). Recently, Han et al. (2020) discovered that overexpressed circ-ITCH reduces doxorubicininduced oxidative stress and DNA damage in cells and mitochondria. They also discovered that via sponging miR-330-5p in DOXIC, circ-ITCH upregulated sirtuin 6 (SIRT6), Survivin, and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) . SIRT6 has been found to reduce oxidative stress by activating Nrf2 and SOD2 proteins, two important endogenous anti-oxidant defense molecules (Rezazadeh et al., 2019;Tian et al., 2019). In addition, SIRT6 could also ameliorate DNA damage via activating PARP1, a key DNA repair enzyme (Tian et al., 2019). Reportedly, Survivin could inhibit doxorubicin-induced myocardial apoptosis and fibrosis . Additionally, SERCA2a could catalyze the hydrolysis of ATP and enhance cardiac contractility by binding to calcium translocating from the cytosol to the lumen of the sarcoplasmic reticulum (Reilly et al., 2001). Thus, circ-ITCH can alleviate DOXIC and has good potential as a therapeutic target in DOXIC.

Diabetic Microangiopathy
Diabetic patients' long-term glucose management is suboptimal, which can lead to diabetic microvascular problems such as diabetic neuropathy, diabetic retinopathy (DR) and diabetic nephropathy (DN). One of the most prevalent microvascular consequences of diabetes, diabetic retinopathy (DR), is a chronic, progressive diabetes mellitus-induced leakage and occlusion of retinal micro-vessels, resulting in a series of fundus lesions. DR is a persistent microvascular inflammation and proliferative neovascularization of the retina (Semeraro et al., 2015). And there is an interaction between the two pathological process (Capitão and Soares, 2016). TNF-α has been shown to be an inflammatory factor that plays a major role in high-glucose environments, where it is linked to vascular inflammation, endothelial dysfunction, oxidative stress, and disruption of the blood-retinal barrier, and contributes to the progression of DR synergistically (Capitão and Soares, 2016). The major enzymes responsible for degrading the ECM, matrix metalloproteinase (MMPs), have been linked to inflammatory disorders and diabetes (Kowluru and Mishra, 2017). Among these MMPs, MMP-2 and MMP-9 were both significantly up-regulated in retinal cells under high glucose conditions (Giebel et al., 2005). By suppressing TNF-α, MMP-2 and MMP-9, Zhou et al. (Zhou L. et al., 2021) revealed that overexpression of circ-ITCH might prevent neovascularization and inflammation, hence delaying DR progression. DN refers to the microvascular consequences of diabetes mellitus, with microalbuminuria at its core, and renal impairment in a subgroup of patients. Long-term chronic inflammation has been linked to the advancement of DN in studies (Wada and Makino, 2013). Previous research has shown that overexpression of SIRT6 could stimulate M2 macrophage transformation, inhibit high glucose-induced mitochondrial dysfunction and cell apoptosis by activating AMPK, all of which help to reduce inflammation in DN (Fan et al., 2019;Ji et al., 2019). A recent study in diabetic mice produced with streptozotocin found that circ-ITCH reduced kidney inflammation and fibrosis through modulating the mir-33a-5p/SIRT6 axis . The role and mechanism of circ-ITCH in uncomplicated diabetes is also worth exploring, according to current research development.

Hirschsprung Disease
Hirschsprung's disease (HSCR) is caused by a lack of proliferation and migration of intestinal nerve cells (ENCC), which leads to the absence of peristalsis and colon defecation (Heanue and Pachnis, 2007). It then causes the proximal colon to expand and hypertrophy, eventually resulting in the formation of a megacolon. Rearranged during transfection (RET) has recently been identified as a major regulator of ENCC formation, and inactivating mutations in this gene could result in HSCR (Ohgami et al., 2021). Accumulating evidence indicates that circRNAs are dysregulated and play critical roles in the development of HSCR. Xia et al. (2022) revealed that circ-ITCH expression was dramatically reduced in HSCR tissues, and its overexpression greatly promoted the ability of proliferation and migration of 293T, SH-SY5Y cell lines. Mechanistically, circ-ITCH overexpression activated RET by sponging miR-146b-5p, thereby relieving HSCR progression (Xia et al., 2022).
All in all, in non-tumor diseases such as IDD, osteoporosis, I/R injury, DOXIC, DR, DN, HSCR, and others, circ-ITCH plays a more complex regulatory role. It can, for example, regulate distinct target proteins to cause different states of the same signal pathway. These findings imply that the expression level of circ-ITCH and its regulatory mechanism are disease-specific. The role and mechanism of circ-ITCH in non-tumor illnesses and PDLSC osteogenic development are shown in Table 2.

CLINICAL APPLICATION AND PERSPECTIVE
With the increasing popularity of RT-PCR, diagnosing a growing range of diseases has gotten easier. RT-PCR can also be used to determine the degree of circ-ITCH expression. It also shows that it is practical and convenient because it expresses consistently in sample tissues, peripheral blood, and exosomes from patients. As a result, RT-PCR can be employed in practice to detect the level of circ-ITCH expression in patients' sample tissues, peripheral blood, or exocrine, allowing for early diagnosis. The clinical significance of circ-ITCH in human diseases is shown in Table 3.

Diagnostic Biomarkers
Although surgery, chemoradiotherapy, targeted therapy, and the therapeutic outcome of tumor patients have all improved over time, overall survival and quality of life remain a serious problem for patients. Hence, it is critical to diagnose and treat patients as soon as possible. In recent years, scientists have experimented with many approaches to improve the detection and surveillance of early malignant tumors, including radiation, immunology, and biomarkers. Diagnostic biomarkers have received a lot of attention among them. Many circRNAs, notably circ-ITCH, have shown tremendous promise as diagnostic biomarkers in various investigations (Tang et al., 2019).
Many studies have anticipated and proven the clinical application potential of circ-ITCH as a diagnostic biomarker due to its down-regulation in a variety of malignancies . In GC, circ-ITCH in tissue and serum-derived exosomes were explored separately for their diagnostic value. Among them, the AUC for detecting circ-ITCH in tissues was 0.7055 (sensitivity: 52.71%, specificity: 74.55%); the AUC in serum-derived exosomes was 0.6538 (sensitivity: 42.42%, specificity: 90.91%) . In multiple myeloma (MM), the AUC was 0.809 (sensitivity: 59.8%, specificity: 80.0%) . In PCa, circ-ITCH showed higher diagnostic value (AUC = 0.812 (95% CI: 0.780-0.845)), and its low expression was linked to a higher probability of lymph node metastases (p = 0.047) and an advanced T stage (p = 0.002) . Furthermore, the expression of circ-ITCH has been linked to tumor size, tumor grade, TNM stage and clinical stage. Specifically, in OC, the expression of circ-ITCH was linked to tumor size (p = 0.0009) and clinical stage (p = 0.0021) (Lin et al., 2020); in TNBC, it was linked to tumor size (p = 0.016), lymphatic metastasis (p = 0.008) and clinical stage (p = 0.002) (Wang S. et al., 2019); in OSCC, it was correlated with clinical stage (p = 0.027) and lymphatic metastasis (p = 0.035) (Hao et al., 2020); in EOC, it was associated with tumor size (p = 0.005) and International Federation of Gynecology and Obstetrics (FIGO) stage (p < 0.001) (Luo et al., 2018a); in GC, it was correlated with tumor grade (p = 0.02), T stage (p = 0.0216) and lymphatic metastasis (p = 0.034) (Ghasemi et al., 2019;Peng and Wang, 2020;Wang et al., 2021); in NPC, it was correlated with lymphatic metastasis (p = 0.0021), clinical stage (p = 0.0028) and bone metastasis (p = 0.0285) ; in non-small cell lung cancer (NSCL), it was linked to tumor size (p < 0.001), lymphatic metastasis (p < 0.001) and clinical stage (p = 0.003) (Li et al., 2019); in MM, it was correlated with international staging system (ISS) stage (p = 0.036) . Guo et al. (2017), on the other hand, discovered that the single nucleotide polymorphisms rs10485505 and rs4911154 of circ-ITCH were strongly related with an elevated risk of HCC, suggesting that it might be employed as a biomarker for HCC susceptibility. Similarly, single nucleotide polymorphisms of rs4911154 of circ-ITCH could aggravate the malignant transformation from thyroid nodule (TN) to thyroid cancer (Guo et al., 2021). Furthermore, circ-ITCH's collaboration with established diagnostic indicators like carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) may also boost diagnostic power. All in all, these findings indicate that it has a wide range of diagnostic utility in a variety of tumors, even in cancer susceptibility prediction, since it's convenient and non-invasive.

Prognostic Biomarkers
With the rapid growth of incidence rate and mortality rate of malignant tumors, its overall prognosis will be the main determinant of global public health and life expectancy. Surgery is the most effective treatment for malignant tumors,  but recurrence and metastasis have a significant impact on the prognosis (Lasithiotakis et al., 2014). Recent studies have found that circ-ITCH is closely linked to clinicopathological characteristics and can be employed as a tumor prognostic biomarker, which will aid in tumor treatment. The expression of circ-ITCH is linked to the prognosis of a range of tumors, including HCC, EOC, PCa, BCa, OC, and so on, according to Kaplan-Meier survival analysis (Guo et al., 2017;Luo et al., 2018a;Yang et al., 2018;Wang S. et al., 2019;Wang X. et al., 2019;Huang et al., 2019;Lin et al., 2020). These findings imply that reduced circ-ITCH expression was associated with lower overall survival (OS) and disease-free survival (DFS). Specifically, decrease in circ-ITCH was associated with worse OS (p = 0.018) and DFS (p = 0.017) in MM patients ; in NSLC, its downregulation was correlated with worse OS (p = 0.006) and DFS (p = 0.001) (Li et al., 2019); in PCa, its down-regulation was correlated with worse OS and DFS (both p < 0.001) ; in EOC, its down-regulation was correlated with worse OS (p = 0.003) (Luo et al., 2018a  . Taken together, these results suggest its promising value as a prognostic biomarker.

Therapeutic Target
Many molecules and signal pathways may be appropriate for targeted therapy as our understanding of tumor development improves. Circ-ITCH is a promising therapeutic target since it has an anti-tumor impact that is connected to a range of substances and pathways, as evidenced by recent studies. Upregulation of circ-ITCH to inhibit proliferation, invasion and migration of HCC cells, for instance, is one of the mechanisms by which lidocaine treats HCC (Zhao et al., 2021). Besides that, it also has great potential in improving chemoresistance and side effects of chemotherapy. Circ-ITCH can decrease MM cell growth and improve MM cell chemosensitivity to bortezomib (BTZ) . Furthermore, circ-ITCH can also alleviate the symptom of DOXIC, suggesting that it can be used simultaneously with chemotherapeutic drugs to alleviate chemotherapeutic side effects . At present, noncoding RNA (ncRNA) therapy focuses primarily on alternative and inhibitory therapies (Ning et al., 2019). Circ-ITCH's alternative therapy is projected to play a significant role in tumor therapy since it suppresses tumor cell proliferation, increases apoptosis, and slows tumor growth by targeting a range of pathway molecules.

In Non-Tumor Diseases
According to recent research, circ-ITCH also has an important role in non-tumor tissue. These findings, together with circ-ITCH's stable expression in patient tissues and peripheral blood, as well as well-defined regulatory mechanisms, point to its potential as a biomarker. In Hepatitis C virus infection, for instance, circ-ITCH expression was positively correlated with liver enzymes AST, ALT (p < 0.001) and child grade. With AUC = 0.661 (sensitivity: 65 percent, specificity: 70 percent), circ-ITCH has diagnostic significance in plasma of Hepatitis C virus infection (Sharkawi et al., 2022). However, no studies have looked at the possibility of circ-ITCH as predictive biomarkers in non-tumor diseases, and this is currently a blank area of research. Circ-ITCH has been reported to play well-defined regulatory roles in bone illnesses, cardiac diseases, diabetic microangiopathy, and Hirschsprung disease, indicating that they could be therapeutically targeted. Specifically, circ-ITCH replacement therapy appears to have promise as a treatment for DOXIC. Moreover, further experiments with circ-ITCH replacement drugs are also worth studying.

CONCLUSION
The focus of this review is on how circ-ITCH, a circular RNA, regulates gene expression in the post-transcriptional stage by acting as a sponge for miRNAs, blocking them from binding to their target mRNAs. The role of circ-ITCH in cell proliferation, apoptosis, invasion, migration, and EMT regulation, as well as related signaling pathways, is then explored. Circ-ITCH's potential as a diagnostic and predictive biomarker in tumor and non-tumor diseases is then confirmed. Furthermore, circ-ITCH has a lot of potential in disease treatment because of its well-defined regulatory mechanism, notably in terms of enhancing chemosensitivity and reducing chemotherapy adverse effects in malignant tumor. These discoveries not only illuminate the molecular basis of circ-ITCH, but also pave the path for future clinical applications. Based on the current research, we put forward some promising future research directions. To begin, the up-regulated biomarker is more ideal for clinical detection, but the down-regulated index can still be employed as long as the critical value is evident. As a result, it is crucial to explore the critical value of circ-ITCH in both patients and healthy people. Then, researchers should increase the sample size and AUC detection as much as possible in order to achieve a more accurate association between circ-ITCH and other classic diagnostic markers (CEA, CA19-9) or prognostic indicators (OS, DFS) (Li et al., 2020c). Furthermore, given its critical role in regulating human diseases, more simulations of activators or carrier administration are needed to validate their in vitro and in vivo effects. While circ-ITCH is low expressed in a number of malignancies, it is unclear which tumor has greater specificity and accuracy about circ-ITCH, meriting additional investigation. At the same time, it is also worthwhile to explore the translational applications of circ-ITCH in chemotherapy because of its demonstrated potential in tumor chemotherapy, including chemo-sensitization and alleviation of side effects. Besides, due to the increase of ITCH concentration and regulating epidermal keratinocyte differentiation, the potential of circ-ITCH in decreasing radiotherapy injury also needs to be developed. Finally, it was recently shown that circRNAs are plentiful and stable in exosomes, that they can be produced under a variety of physiological and pathological conditions, and that they can be detected in circulation and urine (He et al., 2021), all of which require further exploration.


A new species of Pancratium Dill. ex L. (Amaryllidaceae) from Eastern Ghats of India

Pancratium venkaiahii is described as a new species from Eastern Ghats of India. It is closely allied to P. st-mariae. A comprehensive description with photo substantiation and comparison table are provided.

ISSN 0974-7907 (Online); ISSN 0974-7893 (Print) The genus Pancratium having unique characteristics like perennial herbaceous bulbous, linear lanceolate leaves, umbellate Inflorescence, large flowers with funnel shaped perianth, six stamens attached on the throat of the perianth with filiform filaments united below by a coronal membrane into a toothed or lobed cup, oblong or linear dorsifixed anthers, tricarpellary, syncarpous and trilocular, inferior ovary with 2-seriatein numerous ovules and filiform style.
During our botanical explorations in the Eastern Ghats of Andhra Pradesh, first author was collected an interesting species of Pancratium from hills of Vizianagaram District (Figure 1). It is resembles P. stmariae. After critical taxonomic assessment it was identified as a new species and herbarium specimen was deposited in Herbarium, Department of Botany, Andhra University (Image 2). Some of the bulbs were introduced into the College Campus Garden. A detailed description, comparison table (Table 1), and photographs were provided in this article (Image 1).

J TT
membranous spathe, apex acuminate; flowers without fragrance, with very short perianth tube (1.3cm long), perianth lobes long, filament, greenish white anthers pollen oval shape and long style.
Conservation status: Rare in the study area, quantification of the natural populations of this species is not known but this species is facing threats from grazing and anthropogenic activities. First author introduced this species into the College Campus Garden and observed that all individuals were acclimatized and flowered but there was no fruit set . It was observed that the leaves of this plant was fed by caterpillars in the campus and this species is host for some lily moths.
Ethnomedicine: Generally, bulbs are used as medicine for veterinary diseases and is called 'adavivulli'. Ethno medicinal information for human beings is still not known.
India has more than 50% native Pancratium species including current report, and detailed studies need to www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.


Characterization of the complete mitochondrial genome of the Lesser Spotted Woodpecker (Dryobates minor) and its phylogenetic position

Abstract In this study, we sequenced and assembled the complete mitochondrial genome of Dryobates minor by next-generation sequencing. The mitochondrial genome of Dryobates minor is 16,847 bp in length and consists of 13 protein-coding genes (PCGS), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and 1 control region (CR). The CG content of the mitochondrial genome is 47.46%. Only one overlap among the 13 protein-coding genes was found: ND4L/ND4. Phylogenetic analysis based on a combined mitochondrial gene dataset indicated that the mitochondrial genome of Dryobates minor exhibited a close relationship with that of Picoides pubescens.

The Lesser Spotted Woodpecker (Dryobates minor) (Linnaeus, 1758) plays an important role in both forest structure and relative position (Miranda and Pasinelli 2001) and is widely distributed in Asia and Europe and also occurs in North Africa (Fagan and Holmes 2006). It is the smallest European woodpecker, and it may at once be identified by the broad barring on the wings and narrower bars across the lower back. The D. minor and other woodpecker species distributions can often reflect the interplay between habitat availability and climate (Virkkala et al. 2005;Heikkinen et al. 2007;Luoto et al. 2007). Due to the loss of forest biodiversity and disruptions of ecosystem function, the population of D. minor has decreased in recent years (Chapin Iii et al. 2000;Kok et al. 2018;Orlikowska et al. 2020). In addition to a trend toward warming springs, a decline in the number of low productivity has become a widespread problem for the D. minor (Smith and Smith 2020).
The complete mitochondrial genome of the D. minor was sequenced using muscle tissue collected from an individual collected at Heilongjiang, China (127 34 0 E, 45 17 0 N). The specimen was deposited at College of Wildlife and Protected Area, Northeast Forestry University (Zhensheng Liu, zhenshengliu@163.com) under the voucher number XBZMN211120. Based on the high-throughput Illumina Hiseq X platform, total genomic DNA was sequenced. The raw data were assembled using MitoZ (Meng et al. 2019) building contig and scaffold sequences, and results were corrected for final mitochondrial sequences using pilon v1.18 (Walker et al. 2014). Subsequently, the mitogenome was annotated with the MITOS WebServer (Bernt et al. 2013). We used whole genome shotgun (WGS) sequecing with the Illumina HiSeq sequencing platform to construct a library of Dryobates minor DNA fragments. Following Sangster and Luksenburg (2021), we verified the identity of our mitogenome sequence of D. minor with reference sequences of three commonly used markers in avian systematics: NADH dehydrogenase subunit 2 (ND2, 1041 bp; 550 woodpeckers, incl. one D. minor), part of cytochrome c oxidase subunit I (COI, 696 bp; 470 woodpeckers, incl. eight D. minor), and cytochrome b (Cyt b, 1141 bp; 115 woodpeckers, incl. two D. minor). In each of these analyses, our sequence of D. minor clustered with the reference sequences of D. minor, indicating that our sample was correctly identified. The sequence was submitted to GenBank with the accession number OL597538.
We constructed a Maximum Likelihood (ML) phylogeny in MEGA7 (Kumar et al. 2016) with 2,000 bootstrap replicates (Tamura et al. 2011). We constructed a phylogenetic tree for 15 species, with Strix leptogrammica (Linnaeus, 1758) as the outgroup. The phylogenetic tree showed that D. minor was more closely related to Picoides pubescens (Linnaeus, 1766) than to species in the genus Dendrocopos (Figure 1). This agrees with the findings of Fuchs and Pons (2015) and supports the transfer of Lesser Spotted Woodpecker from the genus Dendrocopos to the genus Dryobates (Sangster et al. 2016;Sangster and Luksenburg 2021).The complete mitochondrial genome of D. minor present in this study will provide useful genetic data for further phylogenetic and evolutionary analysis for Piciformes.

Ethical approval
The muscle tissue of Lesser Woodpecker was extracted from the individual which died naturally in the wild not more than 3 days old. The first day of searching in Maorshan yielded no results, while the second yislded a bady. In the scenario, ethical clearance is not necessary.

Author contributions statement
We thank Professor Zhensheng Liu, Professor Liwei Teng, Dr. Junda Chen, Dr. Dehuai Meng and Xiaoyu Zhou who has been involved in the conception and design of the study and Dr. Yuhui Si, Dr. Meichen Yu for publishing the final approval of the version and Professor Zhensheng Liu and Professor Liwei Teng for collecting the samples and revising the paper critically for intellectual content. All authors agree to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of the NCBI at (https://www.ncbi.nlm.nih. gov/) under accession no. OL597538.1. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA781112, SRR16962851 and SAMN23235464, respectively.