AWA and ASH Homologous Sensing Genes of Meloidogyne incognita Contribute to the Tomato Infection Process

The AWA neurons of Caenorhabditis elegans mainly perceive volatile attractive odors, while the ASH neurons perceive pH, penetration, nociception, odor tropism, etc. The perceptual neurons of Meloidogyne incognita have been little studied. The number of infestations around and within tomato roots was significantly reduced after RNA interference for high-homology genes in AWA and ASH neurons compared between M. incognita and C. elegans. Through in situ hybridization, we further determined the expression and localization of the homologous genes Mi-odr-10 and Mi-gpa-6 in M. incognita. In this study, we found that M. incognita has neuronal sensing pathways similar to AWA and ASH perception of C. elegans for sensing chemical signals from tomato roots. Silencing the homologous genes in these pathways could affect the nematode perception and infestation of tomato root systems. The results contribute to elucidating the process of the plant host perception of M. incognita.

Introduction
Plant-parasitic nematodes are a class of seriously damaging pathogens that are exclusively parasitic in plants and have characteristics such as widespread parasitism, adaptability, and ease of transmission [1,2]. The second-stage juveniles (J2s) of the root-knot nematodes (RNKs) infect the roots of plants, followed by root-tissue protrusion to form root knots; the formation of root knots prevents the plant roots from absorbing and transporting water and nutrients and, due to nutrient deficiency, the plant grows short, declines in yield, withers, and dies [3,4]. Plant-parasitic nematodes cause an estimated USD 173 billion in agricultural losses annually [5].
Chemotaxis has long been considered the primary reason for nematode host localization. Nematode chemotaxis refers to the nematode s use of head-sensing organs to sense water-soluble, volatile compounds produced by roots or inter-root microorganisms and to move and locate plant roots in response to changes in compound concentration [6,7]. Plant roots secrete many signaling substances, and RNKs can recognize plant hosts by sensing the signaling substances secreted by the plant root system [8]. Murungi et al. confirmed that the tomato root extracts methyl salicylate and tridecane were highly attractive to Meloidogyne incognita [9]. The root secretion of lauric acid of Chrysanthemum coronarium was able to attract M. incognita at low concentrations [10]. Studying the chemotaxis of plant-parasitic nematodes to their hosts and interfering with their localization to host roots is a new strategy for nematode management and control [11].
Caenorhabditis elegans has a highly developed chemosensory system that relies on its elaborate olfactory system to survive in the soil, sensing signals related to food, danger, or other animals [12]. There are 12 pairs of chemosensory neurons in the head of C. elegans that detect the external environment, and these are symmetrically distributed on both sides of the head [13,14]. Volatile chemicals that can be detected by AWA and AWC neurons mainly include alcohols, ketones, esters, amines, organic acids, aromatics and heterocycles [15,16]. AWA receptor ODR-10 leads to the perception of volatile compounds diacetyl [17]. AWA neurons also need ODR-7 to sense most of the compounds, and the loss of odr-7 causes the impairment of AWA neurons olfactory sensation [18]. ASH neurons are mainly involved in the nematode avoidance response induced by high osmotic pressure, heavy metals, detergents, bitter alkaloids, acidic pH, and some organic smells [19]. Gene gpa-11 is expressed on ASH neurons. It regulates the response to octanol by regulating serotonin, and jointly regulates the avoidance of quinine by odr-3 and gpa-3 [20]. PLC family genes plc-1, egl-8, unc-13, elo-1 encode and regulate the phospholipase pathway [21]. The PLC pathway is activated to produce the second messenger inositol triphosphate (IP 3 ) and diacetylglycerol (DAG), activate protein kinase (PKC), and release intracellular Ca 2+ [22]. TPRV ion channels are expressed in the nerves of C. elegans and regulate olfactory response, mechanoreceptor response, and osmotic response of the nematodes. Gene osm-9 and ocr-2 encode cyclic adenonucleotide ion channels, which play a role in insect light transmission, vertebrate pain perception, non-neural pressure perception and osmotic pressure perception [23,24].
Compared to the rich chemosensory gene pool of C. elegans, the chemotaxis-related genes of root-knot nematodes are less studied. Lauric acid in root exudates of Chrysanthemum coronarium regulated the specific expression of Mi-flp-18 gene encoding FMRFamidelike peptide neural regulator in a concentration-dependent manner [10]. Shivakumara et al. found that the movement track of M. incognita J2s was abnormal, and the number of nematodes that tended to infect the root tip of the tomato plant was significantly reduced after RNA interference the homologues Mi-odr-1, Mi-odr-3, Mi-tax-2 and Mi-tax-4. In ISH analysis, Mi-odr-1 mRNA seems to be located in the cell cluster related to amphibious neurons and phasmids [25]. In this study, we further aimed homologous genes of the AWA and ASH neurons in M. incognita and explored the signaling pathways of the southern root-knot nematode that are involved in the sensing of the tomato root system and root-infestation ability. It is helpful to understand the process of root-parasitic nematodes perceiving plant hosts and provide a new target for the control of parasitic nematode.

Relative Expression of Homologous Gene on AWA and ASH Neurons after RNA Interference
After 20 h of RNA interference with homologous genes on AWA neurons, each homologous gene on AWA was repressed to different degrees. The relative expression of homologous genes Mi-unc-13, Mi-elo-1, Mi-odr-10, Mi-egl-8, Mi-plc-1, Mi-osm-9, Mi-odr-7 Mi-gpa-6, and Mi-gpa-11 were significantly down-regulated on AWA neurons compared to the wild type and control template ( Figure 1A). After 20 h of RNA interference with homologous genes on ASH neurons, the relative expression of Mi-gpa-11 and Mi-osm-9 were significantly down-regulated compared to the wild type and control template ( Figure 1B).

Motility of M. incognita after RNA Interference with the AWA and ASH Homologous Genes
There was no difference in the motility of M. incognita following RNA interference with the homologous genes Mi-unc-13, Mi-elo-1, Mi-odr-10, Mi-egl-8, Mi-plc-1, Mi-osm-9, Mi-gpa-6, and Mi-odr-7 compared to the wild type and control template Figure 2B-D. There was no difference in motility of M. incognita after RNA interference with the homologous genes Mi-gpa-11 and Mi-osm-9 on ASH neurons compared to the wild type Figure 2E,F. This indicates that RNA interference with the related genes does not affect chemotaxis by impairing motility.

Chemotaxis of M. incognita to Tomato Root after RNA Interference with the AWA and ASH Neurons' Homologous Genes
The receptor genes Mi-odr-10 and Mi-odr-7 were subjected to RNA interference, and the number of nematodes that came around the roots was significantly reduced ( Figure 3A,B). After RNA interference for four genes of the PLC pathway Mi-unc-13, Mi-elo-1, Mi-egl-8 and Mi-plc-1 in AWA neurons in AWA, the number of nematodes aggregated around the roots was also significantly reduced ( Figure 3A). In addition, RNA interference with the gene Mi-gpa-6 and Mi-osm-9 also reduced the number of nematodes around the roots ( Figure 3C). These results suggest that there is a sensory pathway similar to that for AWA neurons in the process of the root perception of tomatoes by M. incognita.
After RNA interference with the G-protein gene Mi-gpa-11 in ASH neurons, the number of nematodes around the roots was significantly reduced ( Figure 3D). Mi-osm-9 jointly encodes TPRV ion channels and is expressed in both AWA and ASH neurons. After the gene Mi-osm-9 was subjected to RNA interference, the chemotaxis of nematode to root was significantly reduced ( Figure 3D). These results suggest that there is a pathway similar to that involving ASH neurons in the process of tomato root perception by M. incognita.

Infectivity to Tomato Root of M. incognita after RNA Interference with AWA and ASH Neurons' Homologous Genes
Like the chemotaxis of M. incognita, after receptor genes Mi-odr-10 and Mi-odr-7, four PLC pathway genes Mi-plc-1, Mi-egl-8, Mi-unc-13 and Mi-elo-1, and genes Mi-gpa-6 and Mi-osm-9 in AWA neurons were RNA interfered, nematode-infected roots were significantly reduced ( Figure 4A-C). It is suggested that the AWA neurons sensing pathway was necessary for the infection of M. incognita.
Mi-gpa-11 and Mi-osm-9 in ASH neurons were subjected to RNA interference. After that, the number of infecting nematodes in roots was significantly reduced ( Figure 4D). These results suggest that the sensing pathway of ASH neurons is involved in the infection of M. incognita.s

In Situ Hybridization of Receptor Genes in Neurons of M. incognita
Two receptor genes, Mi-gpa-6 and Mi-odr-10, in the AWA neurons of M. incognita were analyzed by in situ hybridization. Through hybridization, color rendering, and microscopic observation, it was observed that the genes Mi-gpa-6 ( Figure 5A) and Mi-odr-10 ( Figure 5B) had hybridization signals at the head neurons of M. incognita.
Studies have shown that the chemical signal transduction of C. elegans depends on the G-protein-coupled receptor signal pathway. The cGMP signal pathway of the second messenger synthesized by guanylate cyclase is the cyclic nucleotide-gated CNG channel (TAX-2 and TAX-4), or the transient receptive potential TRPV channel (OSM-9 and OCR-2) mediated by polyunsaturated fatty acid PUFA [26,27]. Zhu et al. [28] studied how nematodes recognize attractive molecules indole and 2-ethylhexanol. The experimental results showed that the chemotaxis of indole and 2-ethylhexanol requires str-193 on AWC and str-7 encoded G-protein coupled receptors on AWA. In further genetic screening of downstream effectors of olfactory signal cascade, Gα subunit GSA-1, guanylyl cyclase ODR-1, DAF-11, and cGMP-gated channel TAX-2/TAX-4 are necessary for indole sensing, while the PLC pathway activated by TRPV channel OSM-9/OCR-2 and GPA-6 is responsible for detecting 2-ethylhexanol.
Shivakumara et al. [25] cloned four allelopathic genes (Mi-odr-1, Mi-odr-3, Mi-tax-2 and Mi-tax-4) homologous to C. elegans in M. incognita. The movement track of the M. incognita J2s was abnormal, and the number of nematodes tending and invading tomato roots was significantly reduced after RNA interference with these genes. Moreover, the response to the low-dose nematode pheromone ascoroside # 18 was weakened. The results showed that Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 played an important role in the perception of volatile and non-volatile compounds of M. incognita. Using qPCR analysis, the authors speculate that Mi-tax-2 and Mi-tax-4 may function downstream of Mi-odr-1 and Mi-odr-3 in the chemotaxis pathway of M. incognita. It is speculated that M. incognita perceives chemical gradients via one or more core chemosensory genes, such as Mi-odr-1 and Mi-odr-3, followed by Mi-tax-2 and Mi-tax-4, transducts signaling in the sensory organs, and selectively chemo-orients to specific cues. The gene odr-1 is a gene on AWB and AWC neurons, encoding membrane-bound guanylyl cyclase (GCY) [29]. Gene odr-3 regulates the production of cyclic nucleotides (cGMP) [30]. Gene tax-2 and tax-4 are involved in the activation of cyclic guanylate-gated channels [31].
Our study focused on allelopathic homologous genes similar to the genes in the AWA and ASH neurons of C. elegans. The odr-10 gene encodes a receptor that senses volatile compounds and the attractive compound diacetyl, on AWA. The sensitivity of an odr-10 mutant to the volatile attractant diacetyl was reduced by 100 times [28]. The receptor gene odr-7 encodes an essential receptor for AWA neurons. Studies have proven that AWA neurons need odr-7 to sense most compounds. Its role is to promote the differentiation of AWA neurons. The loss of odr-7 caused olfactory impairment in AWA neurons [32]. After RNA interference with the homologous genes Mi-odr-10, Mi-odr-7, Mi-plc-1, Mi-egl-8, Migpa-6, and Mi-osm-9 in AWA neurons, it was found that the chemotaxis and infection of M. incognita for tomato roots decreased significantly. Based on the background of C. elegans, it is speculated that the signaling pathway involving the root-knot nematode's AWA neurons for sensing tomato roots is as follows: the root signal binds to the receptor ODR-10; activates the G-protein GPA-6; activates the PLC pathway regulated by PLC-1/EGL-8 downstream; produces the second messengers diacylglycerol (DAG) and inositol triphosphate (IP 3 ); activates the protein kinase PKC, resulting in the production of polyunsaturated fatty acids (PUFAs); regulates OSM-9; and then generates signal transduction that opens the TPRV ion channel. As there are many kinds of tomato root signals, our study only preliminarily explored the roles of the genes Mi-odr-7 and Mi-odr-10 in sensing tomato root signals, but it is not clear what specific types of compounds from tomato roots could be sensed, or which signals can be regulated.
The signaling pathway by which C. elegans' ASH neurons sense the corresponding compounds is still imperfectly understood [33]. The corresponding receptor genes in ASH neurons have not been reported, and the genes that regulate the phospholipase pathway remain unknown, so the related signaling pathways of ASH neurons still need to be explored and improved. Upon RNA interference with homologous genes in ASH neurons, the chemotaxis and infectivity of M. incognita for tomato roots were observed to decrease significantly. It is speculated that the signaling pathway for the sensing of tomato roots by ASH-like neurons in M. incognita includes the following: the receptor activates the G-protein GPA-11, activates the phospholipase pathway to produce the second messenger, regulates OSM-9, and then opens the TPRV ion channel.
Shivakumara et al.'s [25] in situ hybridization experiment of four homologous genes Mi-odi-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 of the M. incognita, found that there were hybridization signals in the head and tail receptors of the M. incognita, indicating that the genes expressed in the head and tail of the nematode. Lans et al. [34] determined the location of the genes gpa-6, gpa-5, and gpa-13 on C. elegans by an immunostaining localization method, and all of them expressed in the head. For two homologous receptor genes Mi-odr-10 and Mi-gpa-6, we found that they both had hybridization signals at the head of M. incognita.
The allelopathic system of root parasitic nematodes is involved in many processes, such as migration, host recognition, host infection, and the establishment of permanent feeding sites in the host. At present, a variety of compounds related to the chemical perception of root parasitic nematodes have been reported, but the molecular mechanism of their allelopathy is less studied; in particular, the signal transduction pathway needs more in-depth research. This study confirmed that the neuronal pathways similar to those of the AWA and ASH of C. elegans are involved in the root-signal-sensing process of M. incognita. Based on the identification and location of root-knot nematodes at plant hosts, a new way to prevent root-knot nematodes from recognizing plants is suggested. Due to the complexity of the root system, the chemotaxis shown by M. incognita may be mediated by a variety of signaling substances. Further research is needed to determine the types of substances that are perceived by AWA and ASH, resulting in signal transduction.

Culture and Collection of M. incognita
A three-week-old tomato seedling (Bela, purchased from the Shandong Shengda seed industry) was transplanted to a pot (18 cm high and 8.5 cm in diameter). Sterilized humus, vermiculite and perlite (V:V:V, 7:2:10) were used as culture substrates. About 2000 J2s of M. incognita were inoculated on tomato roots [35]. The inoculated tomato seedlings were placed in greenhouse for about 50 days. The mature egg mass from tomato roots was picked and placed in a 200 µm mesh screen with sterile water. The M. incognita J2s that hatched in the incubator were filtered and collected in a 15 mL centrifuge tube and centrifuged at 4000 rpm for 5 min; the supernatant was discarded, and the nematode concentration was adjusted for analysis.

RNA Interference for Homologous Genes in AWA and ASH Neurons
BLAST protein comparison was performed for homologous genes related to the AWA and ASH neurons in C. elegans (https://wormbase.org//#012-34-5) (accessed on 12 November 2021) and Meloidogyne database (https://meloidogyne.inrae.fr/) (accessed on 12 November 2021) to select homologous genes with protein homology higher than 40%. See Table S1 for the comparison results of homologous genes between Meloidogyne incognita and Caenorhabditis elegans. See Table S2 for the comparison of protein homology between Meloidogyne incognita and Caenorhabditis elegans.
The conserved functional region of the candidate allelopathic homologous gene of M. incognita was searched in the NCBI database. According to the requirements of the in vitro transcription MEGA script RNAi Kit (AM1626, Thermo Fisher Scientific, Shanghai, China), the homologous gene primer was designed with Primer3, and the T7 promoter sequence was added at the 5 end of the primer. The qPCR primers were designed according to the experimental requirements. β-actin was used as an internal reference gene as previously reported. See Table S3 for the primer information of RNAi in Meloidogyne incognita.
The total RNA of M. incognita J2s was extracted with the RaPure Total RNA Micro Kit (R4012, Magen). The cDNA was synthesized according to the instructions of Prime Script II 1st strand cDNA synthesis Kit (Takara). Plasmids were extracted with the E.Z.N.A. Plasmid DNA Mini Kit II (OMEGA) and sequenced by Tsingke Biotechnology Co., Ltd (Beijing, China). The MEGAscript RNAi Kit (AM1626, Thermo Fisher Scientific-the RNAi kit is used to synthesize a large number of dsRNA for RNAi experiments in nonmammalian systems. The Control Template is a linear dsDNA fragment with opposing T7 promoters. It yields a 500 bp dsRNA product) was used for in vitro transcription, and the dsRNA concentration was adjusted to 1 mg/mL. A 1.5 mL RNase-free centrifuge tube was used to collect 200 µL (about 20,000 pieces) of newly-hatched M. incognita J2s; 10 µL of 5% m-dihydroxybenzene, 50 µL of spermidine (30 mM), 5 µL of 5% gelatin, and 100 µL of dsRNA were added, and M9 buffer (5.8 g of Na 2 HPO 4 ·7H 2 O, 3.0 g of KH 2 PO 4 and 5.0 g NaCl) was used to make up the volume to 500 µL. No dsRNA was added to the wild type, and dsRNA for genes was added to the gene-interference group. M. incognita J2s were immersed in the immersion system for 20 h, and then recovered with M9 buffer for 20 h. The in vitro immersion conditions are reported in the relevant literature [36]. Three replicates were established, and the test was repeated three times.
The total RNA of M. incognita J2s treated in the experiment was extracted using the RaPure Total RNA Micro Kit (R4012, Magen). The RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (RR047A, Takara). TB Green Premix Ex Taq (RR820A, TaKaRa) was used to detect the gene expression. The 2 −∆∆ Ct method was used to calculate the relative gene expression.

Detection of Motility of M. incognita after RNA Interference
Tris-MES glue was prepared (23 g of Tris-MES and 100 mL of sterile water). After the glue was fully dissolved, 2.5 mL was aspirated and added to a 3 cm plate. The plate was divided into two areas, A and B ( Figure 1A). About 200 RNAi-transfected M. incognita in zone B were stored in an incubator at 28 • C for 24 h. The numbers of nematodes in areas A and B were counted, and the mobility was calculated. Three replicates were established, and the test was repeated three times. Mobility (%) = (number of nematodes in area A/total number of nematodes in areas A and B) × 100%.

Detection of Chemotaxis and Infectivity of M. incognita for Tomato Roots after RNAi
Tomato seeds (selected cooperation 908, Taishu Seed Industry Co.,Ltd, Xi'an, China) were germinated to about 2 cm on filter paper for analysis. Tomato sprouts were added to the Pluronic F-127gel plate [37], and about 200 RNAi-transfected M. incognita J2s were added 1.5 cm away from the sprouts. The chemotaxis and infectivity of M. incognita for tomato roots after RNAi were observed under a microscope at 4, 8, 12, and 24 h after treatment. Three replicates were established, and the test was repeated three times. Then, the buds were soaked in 1% sodium hypochlorite for 30 min, washed with sterile water and boiled in acid fuchsin solution (3.5% acid fuchsin; 25% acetic acid) for 10 min, for counting the number of invaded nematodes.

In Situ Hybridization Experiment
The probes were labeled using the PCR DIG Probe Synthesis Kit (Roche) and amplified by two-step PCR. The first step was double-primer amplification. The PCR consisted of the following: 3 µL of 10 × PCR buffer, 5 µL of dNTPs, 1.5 µL each of the forward and reverse primers, 0.25 µL of the Ex Taq-enzyme, 2 µL of the cDNA template, and enough ddH 2 O to make up a total volume of 50 µL. The PCR conditions were 95 • C pre-denaturation for 4 min, 98 • C for 10 s, 52 • C for 20 s, and 72 • C for 1 min, for 35 cycles, followed by extension at 72 • C for 10 min. After the purification and recovery of the first-step amplification product as a template, the digoxin-labeled probe was obtained by single-primer amplification with the forward primer or reverse primer, respectively. The PCR solution was prepared as follows: 3 µL of 10× PCR buffer, 3 µL of the digoxin-labeled dNTP mix, 1.5 µL of the forward or reverse primer, 0.6 µL of Ex Taq, 2 µg of the DNA template, and enough ddH 2 O to make up the volume to 30 µL. The PCR conditions were as described above. The freshly hatched M. incognita J2s were fixed with paraformaldehyde at 4 • C for 24 h. The nematode was cut off with a surgical blade and then treated with protease K (1 mg/mL), methanol, and acetone precooled at −80 • C. Then, the nematodes were pre-hybridized by suspension in a hybridization solution. Finally, digoxin-labeled probes were added to hybridize at 40 • C for 16 h. After hybridization, the chromogenic solution was added to develop color overnight at 25 • C in the dark. Observations were made and photos were taken under a microscope (Nikon ECLIPSE, 80I, Nikon, Tokyo, Japan).

Data Processing
GraphPad Prism 8.0 was used to process all the experimental data. Two-way ANOVA and Tukey's multiple comparisons were used for signification analysis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens11111322/s1, Table S1: Comparison of homologous genes between Meloidogyne incognita and Caenorhabditis elegans; Table S2: Comparison of protein homology between Meloidogyne incognita and Caenorhabditis elegans; Table S3: Primer information of RNAi in Meloidogyne incognita.


Long non-coding RNA HOXB-AS1 is a prognostic marker and promotes hepatocellular carcinoma cells’ proliferation and invasion

Abstract Long non-coding RNAs (lncRNAs) are broadly transcribed in the genome of human and play critical roles in the progression of multiple diseases. Long non-coding HOXB cluster antisense RNA 1 (HOXB-AS1) is a tumor exciter in various cancers. This study aimed to investigate the involvement of HOXB-AS1 in hepatocellular carcinoma (HCC). In the following study, HOXB-AS1 was unveiled to be highly expressed in HCC tissues as opposed to normal tissues. Silencing of HOXB-AS1 led to the loss of proliferation, migration, and invasiveness of HCC cells, namely Hep3B and Huh7. Moreover, the data showed that expression levels of HOXB-AS1 contribute significantly to the patient’s survival rates. Otherwise, HOXB-AS1 levels in the serum of patients proved HOXB-AS1 as a biomarker for analysis and treatment of HCC. In summary, this study highlights HOXB-AS1 as key upregulated lncRNA in HCC which being an oncogene can cause proliferation and metastasis of HCC cells. The results also highlighted HOXB-AS1 as a promising biomarker for early diagnosis and prognosis of patients with HCC.

Introduction
Hepatocellular carcinoma (HCC) has become the most common subtype of liver cancer in the last decade, comprising 90% of primary liver cancers [1]. Clinical studies of HCC patients have demonstrated very low survival rates, accounting for more than 600,000 global deaths [2]. However, many information gaps elaborating the exact molecular mechanisms, protein alterations, and hereditary factors exist and need to be further investigated.
Long non-coding RNAs (lncRNAs) with >200 nucleotides have been demonstrated for their vital roles in physiological, developmental, and differential biological processes [3] in human diseases, particularly carcinogenesis [4,5]. LncRNAs can affect the normal molecular processes by binding different transcription factors, localizing cellular proteins to specific sites, facilitating the intermolecular interaction of various components, and differentially controlling and monitoring the expression of different genes at a particular stage in a tissue-specific manner [6,7]. Several transcriptome sequence platforms and microarray techniques have highlighted the abnormal and elevated levels of lncRNAs in HCC angiogenesis, metastasis, apoptosis, progression, and proliferation [8]. For example, Tsang et al. [9] showed aberrant expression of lncRNAs in 20 pairs of HCCs and respective nontumorous liver tissues. Likewise, lncRNA HULC performs an auto-regulatory loop with miR-372 causing the blockade of translational repression and leads to overexpressed HULC in HCC [10]. MALAT-1 interferes with splicing regulation of diverse pre-mRNAs and contributes to metastasis of HCC [11]. Likewise, lncRNA HEIH causes cell cycle arrest by inhibiting the expression of the p16 gene [12]. Similarly, overexpressed intergenic lncRNA HOTAIR has been involved in HCC metastasis [13]. HOXB-AS1 is a significant lncRNA which has been evaluated for its oncogenic role in multiple myeloma, endometrial cancer, and glioma [14][15][16]. Nonetheless, its potential in HCC is still unknown. Therefore, this study concentrated on deciphering the role of HOXB-AS1 in HCC invasion and proliferation. From the HCC samples, we identified that HOXB-AS1 is significantly upregulated. We further characterized the role of HOXB-AS1 in HCC development and potential as a prognostic marker.

Tissue samples
In total, 60 subjects with HCC tissues and matched adjacent normal liver tissues were listed. Patients who underwent surgical procedures in the affiliated hospital were chosen for the study. Following surgery, tumor tissues were immediately subjected to snap freeze in liquid nitrogen for subsequent experimental and analytical work.
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors' institutional review board or equivalent committee.

Cell lines and transfection experiment
Four human cell lines, including Huh7, BEL-7404, HCCLM3, and Hep3B, and human normal liver cells L02 were chosen for the analysis. Cell lines were procured from Cell Bank of Chinese Academy of Science (Shanghai) and subjected to resuspension in a cell culture medium containing Dulbecco's modified Eagle's medium (90%) and fetal bovine serum (10%; R&D Systems, USA). Typical cell culture conditions comprising 37°C, 95% humidity, and 5% CO 2 were maintained in an incubator [17]. Standard transfection was performed in pcDNA3.1, showing respect to HOXB-AS1 and HOXB-AS1 siRNAs, with a negative controls procured from RiboBio (China). Lipofectamine (LFN) 3000 reagent (Invitrogen, USA) was used for the transfection assay following the manufacturer's guidelines, and transfection efficiency was measured by qRT-PCR [18].

Extraction of RNA and qRT-PCR for quantitative gene expression
Total tissue RNA was retrieved with Trizol reagent (A33250, Invitrogen), focusing on the manufacturer's guidelines.

Cell counting kit-8 (CCK-8) bioassay to calculate cell proliferation
Cell viability was confirmed with the CCK-8 (Sigma-Aldrich) following the manufacturer's reference book. Cells were plated into 96-well plates (3 × 10 3 cells/well) and incubated for 0, 24, 48, 72, and 96 h. Following this, 10 µL of CCK-8 proliferation solution was subsequently added to each well at each point and further incubated for 1 h at 37°C. Cellular optical densities were measured at 450 nm by using a microplate reader (Bio-Rad, USA) [18].

Transwell assay to estimate cell migration and invasion
The migratory ability of the cells was assessed as narrated by Zhang et al. [20] by using a Transwell chamber with inserts of 8 mm pore sizes (Millipore, USA). Membranes without any coating were utilized for migration assay, whereas cell invasion was calculated with Matrigel-coated membranes. Briefly, 5 × 10 4 Huh7 and Hep3B cells containing DMEM (250 μL) were planted into the upper chamber following the addition of FBS (10%, R&D Systems, USA) into the lower chamber. Incubation was performed at 37°C for 36 h, and the remaining cells were removed gently with the help of a cotton swab. Invasive or migrating Huh7 and Hep3B cells moved to the low chamber, adhered to methanol (20 min) and subsequently stained with 0.1% CV (R&D Systems, USA). Following staining for 20 min, imaging was done with the help of a phase-contrast microscope, choosing five fields of vision (Olympus, Japan).

Statistical analysis
Experiments were conducted in three sets and presented as mean value ± deviations from the standard value (SD). The significant difference was computed by using SPSS 23.0 followed by Dunnett's test, ANOVA, and Pearson's Correlation test. Clinicopathological characteristics of the patients were analyzed using the chi-square test, whereas survival rates were determined with the Kaplan-Meier survival curve. Unless otherwise indicated, the data are presented as the mean value ± SD. *p < 0.05 was considered statistically significant.

HOXB-AS1 is highly expressed in HCC tissues and cells
The study was designed to estimate and analyze the high and low expression levels of HOXB-AS1 in HCC, and for this, 60 pairs of HCC tissues and corresponding healthy tissues were put to qRT-PCR. The results from qRT-PCR clearly showed highly upregulated levels of HOXB-AS1 (**p < 0.01) in HCC, as shown in Figure 1a. To get a clear picture, patients were compared for the correlation of their clinicopathological characteristics and corresponding HOXB-AS1 levels. Considering the median expression value of HOXB-AS1 in HCC tissue as a cut-off value, 60 HCC patients were subdivided into two categories, one as exalted expression (n = 30) and the other as low expression (n = 30) HOXB-AS1 group. Results of chi-square analysis demonstrated the significance (**p < 0.001) and a direct correlation between lymph node and distant metastasis, TNM stage, and tumor differentiation. Nevertheless, no positive correlation was observed for HOXB-AS1 expression between the tumor size and localization, age, and sex of the patients ( Table 1). Next the expression levels of HOXB-AS1 were analyzed in HCC cell lines, including Huh7, BEL-7404, HCCLM3, and Hep3B, and normal cell line L02 through qRT-PCR analysis. The results from PCR demonstrated highly expressed HOXB-AS1 levels in HCC cell lines as opposed to normal cells (**p < 0.01, Figure 1b). The highest expression of HOXB-AS1 was detected in Huh7 followed by Hep3B. Based on the highest correlated values, Huh7 and Hep3B were subjected to further analysis. was significantly shorter (p = 0.0435) than those in the low expression group (Figure 2b).

Low expression of HOXB-AS1 inhibited cell proliferation, invasiveness, and migration
After confirming the role of HOXB-AS1 in HCC development, it was further assessed for its proliferative, invasive, and migratory potential. To analyze the significance of HOXB-AS1 in HCC development, qRT-PCR was carried out to detect the effectiveness of HOXB-AS1 knockdown in Hep3B and Huh7 cells. Results demonstrated that relative expression of HOXB-AS1 significantly decreased (**p < 0.01) in both cell lines after transfection with si-HOXB-AS1#1 and si-HOXB-AS1#2 as compared to the negative control (si-NC) group (Figure 3a). Cell viability was confirmed through the CCK-8 method by monitoring the light absorption values at 0, 24, 48, and 72 h in both Hep3B and Huh7 cells. Results from the bioassay indicated that transfection with small interfering RNAs, si-HOXB-AS1#1 and si-HOXB-AS1#2, significantly  impacted the light absorption ability of the cells, and HOXB-AS1 knockdown markedly decreased the cell viability of both the cells (Figure 3b). The results were further confirmed by the Transwell test (without matrix adhesive EMC) to confirm the cellular migration of HOXB-AS1 knockouts. The data, in consistence with previous experiments, showed that knockdown of HOXB-AS1 with si-HOXB-AS1#1 and si-HOXB-AS1#2 significantly (**p < 0.01) decreased cell migration of both the Hep3B and Huh7 cells in comparison to si-NC (negative control group) as shown in Figure 3c. A subsequent Transwell test (matrix adhesive EMC) to check the invasiveness of the Hep3B and Huh7 cells was performed. Again, the results reinforced the previous observation that small interfering knockouts of HOXB-AS1 (si-HOXB-AS1#1 and si-HOXB-AS1#2) significantly (**p < 0.01) reduced the invasive ability of the transfected Hep3B and Huh7 cells when compared to the negative control (Figure 3d).

Upregulated serum HOXB-AS1 is tumorderived and acts as a potential diagnostic biomarker for HCC
After determining the oncogenic potential of HOXB-AS1 in HCC, the results were further subjected to serum analysis of HCC patients. When characterized by the relative expression levels of HOXB-AS1 in serum through qRT-PCR, the data indicated significantly elevated levels (**p < 0.01) of HOXB-AS1 in the serum of 60 HCC patients as compared to 46 normal individuals (Figure 4a). This suggests HOXB-AS1 as the potential prognostic and diagnostic biomarker of HCC patients. Finally, the ROC curve was utilized to analyze the diagnostic correctness and efficiency of serum HOXB-AS1. The data suggest high sensitivity and efficacy of serum diagnosis of HCC showing AUC > 0.7 (Figure 4d).

Discussion
Mounting research reports have highlighted the significance of lncRNAs, their aberrant expressions, elevated levels, and tumor inhibitor and promoter roles in several carcinomas [21,22]. By directly interfering with RNA, DNA, and proteins [14][15][16], the regulatory effects of several ncRNAs in many different groups of liver carcinogenesis are been recorded. For instance, lncRNA HOTTIP/ HOXA13 expression is correlated with disease progression in HCC patients [23]. LncRNA PCNA-AS1 is an antisense RNA and regulates the proliferation of cellular nuclear antigens [24]. RNA HNF1A-AS1 has been recognized as an autophagy promotor that sponges hsa-miR-30b-5p and acts as an oncogene in HCC [15]. Similarly, the significance of HOXB-AS1 has been explained as an oncogene in several tumors. For example, HOXB-AS1 has been evaluated for its upregulated levels in glioblastoma cells and is directly associated with patient's survival [25]. Elevated expression levels of HOXB-AS1 have been reported in myeloma and endometrial cancer [26,27]. However, its potential role in the proliferation and invasiveness of HCC is yet unknown. Thus, for the first time, our study revealed that HOXB-AS1 was significantly upregulated in HCC tissues, suggesting that it may be a key regulated gene with proliferative potential in HCC progression. Furthermore, knockdown of HOXB-AS1 reduces the proliferation of Hep3B and Huh7 cells and weakens their migration and invasion capabilities. This phenomenon is well-documented that suppression of several oncogenes' lncRNAs significantly decreases the invasiveness, proliferation, and migration of tumorous cells. For instance, silencing of HOXB-AS1, HOXB2, or HOXB3 can lead to inhibition of cell proliferation and apoptosis stimulation of glioblastoma [17]. Similarly, silencing of HOXB-AS1 in myeloma cells suppressed myeloma cells' proliferation and invasiveness [26]. Most importantly, this study showed the potential of serum analysis to detect HCC tumors by determining HOXB-AS1 levels in patient's serum. When HOXB-AS1 levels were reported, the serum of 60 HCC patients showed considerably higher HOXB-AS1 expression as compared to 46 normal individuals indicating it as a promising prognostic biomarker. Further, serum test efficiency was determined by ROC curve analysis which showed its high accuracy and sensitivity in early detection of HCC tumors. However, our article has some limitations that need to be explored indepth in future. For example, we will explore the downstream regulatory mechanism of HOXB-AS1, including its effect on cell phenotype. In addition, in the next study, we will consider adding animal experiments to make the clinical significance of HOXB-AS1 more credible. This study greatly expands our knowledge of HOXB-AS1 in regulating HCC progression function. We provide the first evidence that HOXB-AS1 functions as a critical player in regulating proliferation, migration, and invasion in Hep3B and Huh7 cells, laying the foundation for further clarifying the roles of HOXB-AS1 in HCC development. The results also highlighted HOXB-AS1 as a promising biomarker for the early diagnosis and prognosis of patients with HCC.
Funding information: Authors state no funding involved.
Author contributions: Yubin Chen designed the project and collected data. Na Wang analyzed the data. Liangqi Cao drafted the manuscript. Dawei Zhang did all the experiments, and Heping Peng involved in data collection and analysis. All the authors revised and corrected the manuscript. Ping Xue contributed equally.


Anti-Ovarian Cancer Conotoxins Identified from Conus Venom

Conotoxins constitute a treasury of drug resources and have attracted widespread attention. In order to explore biological candidates from the marine cone snail, we isolated and identified three novel conopeptides named as Vi14b, Vi002, Vi003, three conotoxin variants named as Mr3d.1, Mr3e.1, Tx3a.1, and three known conotoxins (Vi15a, Mr3.8 and TCP) from crude venoms of Conus virgo, Conus marmoreus and Conus texile. Mr3.8 (I-V, II-VI, III-IV) and Tx3a.1 (I-III, II-VI, IV-V) both showed a novel pattern of disulfide connectivity, different from that previously established for the µ- and ψ-conotoxins. Concerning the effect on voltage-gated sodium channels, Mr3e.1, Mr3.8, Tx3a.1, TCP inhibited Nav1.4 or Nav1.8 by 21.51~24.32% of currents at semi-activated state (TP2) at 10 μmol/L. Certain anti-ovarian cancer effects on ID-8 cells were exhibited by Tx3a.1, Mr3e.1 and Vi14b with IC50 values of 24.29 µM, 54.97 µM and 111.6 µM, respectively. This work highlights the role of conotoxin libraries in subsequent drug discovery for ovarian cancer treatment.

Introduction
When comparing with other types of cancer, ovarian cancer is the eleventh most common in women, the fifth-most common cause of cancer-related death in women, and the most fatal gynecologic malignant cancer for females [1][2][3]. Hidden deep and often known as a silent killer, ovarian cancer is frequently not diagnosed (over 70%) until the disease has advanced to the late stage, since its symptoms are vague and difficult to detect [4,5]. In the year 2022 alone, 19,880 new cases and 12,810 deaths related to ovarian cancer were estimated by the National Cancer Institute of the United States [3]. Treatment of ovarian cancer is generally performed by a combination of debulking surgery and subsequent chemotherapy with the increasing use of immunotherapy and targeted therapies such as bevacizumab and PARP (poly ADP-ribose polymerase) inhibitors [6][7][8]. Paclitaxel/carboplatin and doxorubicin/carboplatin regimens are often used as first-line chemotherapeutic agents [2]. However, failure of chemotherapy may occur due to drug resistance problems and toxicity [9]. Herein, alternative anti-ovarian cancer drug candidates are urgently needed for the treatment of ovarian cancer.
In order to explore biological candidates from marine cone snails, we isolated and identified three novel conopeptides named as Vi14b, Vi002, Vi003, three conotoxin variants named as Mr3d.1, Mr3e.1, Tx3a.1, and three known conotoxins (Vi15a, Mr3.8 and TCP) from crude venoms of Conus virgo, Conus marmoreus and Conus texile collected in the South China Sea. In this work, we reported their isolation, sequence identification, synthesis, disulfide connectivity recognition and biological evaluation.

Peptide Isolation and Sequencing
Crude venom powders weighing 4.1 mg, 18.9 mg and 11.6 mg were prepared from the venom dusts of C. virgo, C. marmoreus and C. texile snail specimens, respectively. Through systemic separation, three novel conopeptides named as Vi14b, Vi002, Vi003, three conotoxin variants named as Mr3d.1, Mr3e.1, Tx3a.1, and three known conotoxins (Vi15a, Mr3.8 and TCP) were isolated and identified from the crude venoms of these three species of cone snails (Table 1). * The amino acid "L" in the sequence represented "I/L", since MS/MS sequencing could not distinguish leucine (L) and isoleucine (I).
The alkylation result of the isolated conopeptides showed that none of them contained free thiol. The mass difference (4.03 Da, Supplemental Material A, Figure S1) between the intact and the reduced Vi14b indicated that it contained two pairs of disulfide bridges. The Edman degradation result (Supplemental Material B, Figure S2) indicated a sequence of QQM*GR*GGTGQII*NE*KT*HGK*V**. The matrix-assisted laser desorptionionization-time of flight-tandem mass (MALDI-TOF-MS/MS) spectrum of Vi14b was shown in Figure 1 24 , b 27 , y 3 -y 11 , y 13 -y 16 , y 18 , y 20 -y 27 ions, combined with the key fragment ions (Table 2), indicated that its primary sequence was QQMCGRCGGTGQIIK (Ac) NECKTCHGKK (Ac) VTK, which corresponded with the Edman degradation data. This novel sequence was named Vi14b, since it was the second conotoxin discovered from C. virgo whose cysteine arrangement pattern (C-C-C-C) was framework XIV. Thereinto, two lysines (Lys, K) at the positions 15 and 25 of Vi14b were acylated since the mass addition of Lys was 42.0319, while the Lys at the positions 19, 24 and 28 were not modified. Acylation of lysine is a common PTM (posttranslational modification) among conotoxins.
quence was QQMCGRCGGTGQIIK(Ac)NECKTCHGKK(Ac)VTK, which corresponded wit the Edman degradation data. This novel sequence was named Vi14b, since it was the sec ond conotoxin discovered from C. virgo whose cysteine arrangement pattern (C-C-C-C was framework XIV. Thereinto, two lysines (Lys, K) at the positions 15 and 25 of Vi14 were acylated since the mass addition of Lys was 42.0319, while the Lys at the position 19, 24 and 28 were not modified. Acylation of lysine is a common PTM (posttranslationa modification) among conotoxins.  Conotoxins Vi002 and Vi003 both contained no disulfide bridge since their molecula weights before and after TCEP reduction were the same. In MALDI−TOF−MS/MS spec trum of Vi002 (Figure 2), the consecutive ions b1−b3, a1−a9, y13−y15 and the crucial ions (b1 b14, b17, b21, y1, y3, y5, a17, a20, a22, a24), integrating with the key fragment ions (Table 3), explic itly revealed a novel sequence of LSSGATALSGVPRLTKPAGRLTTTTVAVAF. For se  * y b ion represented the y ion of the intact sequence fragment, while y a was the fragment ion whose C-terminal amino acid residue was decarboxylated.
Conotoxins Vi002 and Vi003 both contained no disulfide bridge since their molecular weights before and after TCEP reduction were the same. In MALDI-TOF-MS/MS spectrum of Vi002 (Figure 2), the consecutive ions b 1 -b 3 , a 1 -a 9 , y 13 -y 15 and the crucial ions (b 10 , b 14 , b 17 , b 21 , y 1 , y 3 , y 5 , a 17 , a 20 , a 22 , a 24 ), integrating with the key fragment ions (Table 3), explicitly revealed a novel sequence of LSSGATALSGVPRLTKPA-GRLTTTTVAVAF. For sequence identification of Vi003, the serial b 1 -b 7 , b 9 -b 11 , y 3 -y 4 , y 7y 9 , y 11 ions and the key fragment ions (KG m/z 186.1237, KGE m/z 287.1714, KGES m/z 402.1983, KGESL m/z 515.2824, KEGSLL m/z 628.3665, KEGSLLG m/z 685.3879) were observed in the tandem mass spectrum (Supplemental Material A, Figure S2), indicating a novel sequence of NTESTKGESLLGK. It was a pity that we did not obtain enough Vi002 and Vi003 for Edman experiments. The amino acid "L" in the sequences of Vi002 and Vi003 represented "I/L", since MS/MS sequencing could not distinguish leucine (L) and isoleucine (I). Herein, primary sequences of Vi002 and Vi003 were temporarily determined to be (I/L)SSGATA(I/L)SGVPR(I/L)TKPAGR(I/L)TTTTVAVAF and NTESTKGES(I/L)(I/L)GK, respectively.
NTESTKGESLLGK. It was a pity that we did not obtain enough Vi002 and Vi003 for Edman experiments. The amino acid "L" in the sequences of Vi002 and Vi003 represented "I/L", since MS/MS sequencing could not distinguish leucine (L) and isoleucine (I).
The mass differences between the reduced and unreduced conopeptides Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP were 6 Da ( Figure A1), respectively, indicating that these five conotoxins all contained three pairs of disulfide bridges. The secondary MS spectra of the five conopeptides were shown in Figures A4-A8 of Supplemental Material A, while  Vi15a contained four pairs of disulfide bonds since the mass addition after the TCEP reduction was 8 Da ( , which precisely determined the sequence to be DCTTCAGEECCGRCTCPW GDNCSCTEW-(nh 2 ), a previously found disulfide-rich conotoxin Vi15a from C. virgo [24].
The mass differences between the reduced and unreduced conopeptides Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP were 6 Da ( Figure S1 in Supplementary Materials A), respectively, indicating that these five conotoxins all contained three pairs of disulfide bridges. The secondary MS spectra of the five conopeptides were shown in Figures  in Supplementary Materials A) and the Edman degradation experiment (**HWNW*DHL* S****, Figure S5 in Supplementary Materials B). In Figure S7 in Supplementary Materials A, the serial y 7 -y 9 , y 11 -y 15 , b 5 -b 6 and b 8 -b 15 ions clearly confirmed the sequence to be CC-SWDVCDHPSCTCCG, which was a C-terminal carboxylated variant of a known conotoxin Tx3a (CCSWDVCDHPSCTCCG-nh 2 ) from C. texile [27,28]. Thus, it was named Tx3a.1. The sequence for Figure S8 in Supplementary Materials A was assigned NCPYCVVYCCP-PAYCEASGCRPP (a previously found conotoxin named TCP [27]) by successive b/y ions (y 1 -y 22 22 ). The Edman degradation result (N*PY*VVY***PAY*E* SG****, Figure S6 in Supplementary Materials B) also supported the above sequence deduced from MS/MS sequencing.
The existence of disulfide bonds is of great significance for stabilizing conotoxins' spatial structures, enhancing activity and improving selectivity to target receptors [30]. Different disulfide linkages (quantity or connectivity pattern) produce different three-dimensional structures, which lead to differences in activity and selectivity afterwards [30][31][32]. In this work, we isolated a novel two-disulfide-bonded conotoxin Vi14b, two novel disulfide-poor conotoxins Vi002 and Vi003, and five three-disulfide-bridged conotoxins Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP. Conotoxins Mr3.8 and TCP were previously discovered from Conus venom isolation or transcriptomic/proteomic analyses [25][26][27][28]33]. C.D. Poulter et al. synthesized Tx3a and characterized its disulfide connectivity as I-V, II-IV, III-VI. They found that Tx3a can lead to an excitation response by injecting it into mice [34]. TCP (textile convulsant peptide) was an O-superfamily convulsant peptide with a VI/VII Cys framework purified from C. texitle venom components that had induced dramatic and diverse symptoms in mice [28,35,36]. To date, no other record of biological activity and target for Mr3.8, Tx3a.1 and TCP has been reported.
Since the amount of the isolated peptides in this work was quite limited, in order to investigate the structure and activity of these disulfide-rich conotoxins, we synthesized conotoxins Vi14b, Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP using a one-step oxidation strategy. For every conotoxin, we individually performed the HPLC co-elution of the isolated peptide and its oxidative products in order to track the desired isomer that was consistent with the native peptide. Subsequently, the correct isomer was purified by HPLC system. The homogeneities between the synthetic peptide and the corresponding isolated one were verified by the HPLC co-elution experiment.

Disulfide Connectivity
Conotoxins Vi14b, Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP were synthesized using a one-step oxidation strategy. The disulfide connectivities of these folded peptides were unknown. Herein, their disulfide linkages were determined by MALDI-TOF-MS/MS analysis of their two-cysteine-alkylated products (product I) and the four-cysteine-alkylated products (product II) generated from the stepwise reduction and subsequent alkylation of these folded peptides (Figure 3 and Figure S9 in Supplementary Materials A).  The MS/MS spectrum of the reduced two-cysteine-alkylated product (product I, Figure 3) of Vi14b was shown in Figure 4. We used "C (IAM) " to represent the cysteine that was alkylated by IAM (Iodoacetamide) in Figures 4-6 and Figures S10-S16 in Supplementary Materials A. The mass differences of y 25 /y 24 and y 8 /y 7 were 160.0307 and 160.0306, respectively, while mass differences of y 22 /y 21 and y 11 /y 10 were both 103.0091, indicating that Cys-I and Cys-IV were alkylated while Cys-II and Cys-III were in their reduced form. It meant that a disulfide linkage existed between Cys-I and Cys-IV. Herein, the disulfide connectivity of Vi14b was characterized as I-IV, II-III. The MS/MS spectrum of the reduced two-cysteine-alkylated product (product I, F ure 3) of Vi14b was shown in Figure 4. We used "C(IAM)" to represent the cysteine that w alkylated by IAM (Iodoacetamide) in Figures 4-6 and Figures A10-A16. The mass diff ences of y25/y24 and y8/y7 were 160.0307 and 160.0306, respectively, while mass differen of y22/y21 and y11/y10 were both 103.0091, indicating that Cys-I and Cys-IV were alkyla while Cys-II and Cys-III were in their reduced form. It meant that a disulfide linkage isted between Cys-I and Cys-IV. Herein, the disulfide connectivity of Vi14b was char terized as I-IV, II-III.       For product I (Figure 3) of Mr3d.1, Cys at positions 2 and 10 (2-Cys and 10-Cys) were speculated to be IAM-labeled since the mass difference (160.0307) of y 13 /y 12 and b 10 /b 9 (or y 5 /y 4 ) was deduced from its MS/MS spectrum ( Figure 5). In the MS/MS spectrum of product II of Mr3d.1 (Figure 6), the mass difference (160.0307) of y 13 /y 12 , y 9 /y 8 , y 5 /y 4 (or b 10 /b 9 ) meant the Cys at positions 2, 7 and 10 were alkylated. The observation of ions y 14 /y 13  With regard to Tx3a.1, Cys-I and Cys-III were ascertained to be alkyl-marked in its product I due to the mass difference (160.03) of ions of y 10 /y 9 and y 15 /parent ion (m/z 1832.5632) ( Figure S14 I-V, II-IV,  III-VI and I-III, II-V, IV-VI are common among the conotoxins with three pairs of disulfide  bonds, while the patterns of I-V, II-VI, III-IV (such as Mr3.8) and I-III, II-VI, IV-V (such as  Tx3a.1) were rarely reported and different from that previously established for the µand ψ-conotoxins [37]. The novel connectivity patterns could possibly cause these kinds of conotoxins, such as Tx3a.1 and Mr3.8, to present specific biological activity.
alkyl-labeled. Thus, the disulfide connectivity of Tx3a.1 was assigned as I-III, II-VI, IV-V. Similarly, disulfide linkages of TCP were notarized as I-III, II-V, IV-VI because of ions of b12/b11 and y15/y14 were observed in the MS/MS spectrum of its product I ( Figure SA16) and ions of b2 (m/z 275.0809), y19/y18, y15/y14, y9/y8 were detected in MS/MS spectrum of its product II ( Figure A17).
Thus, the disulfide connectivities of conotoxins Vi14b, Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 and TCP were assigned as shown in Figure 7. Generally, linkage patterns of I-V, II-IV, III-VI and I-III, II-V, IV-VI are common among the conotoxins with three pairs of disulfide  bonds, while the patterns of I-V, II-VI, III-IV (such as Mr3.8) and I-III, II-VI, IV-V (such as  Tx3a.1) were rarely reported and different from that previously established for the µ-and ψ-conotoxins [37]. The novel connectivity patterns could possibly cause these kinds of conotoxins, such as Tx3a.1 and Mr3.8, to present specific biological activity.

Blockage on Voltage-Gated Sodium Channel
The Cys arrangement pattern of Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1 was framework III, while that of TCP was framework VI/VII. Frameworks III and VI/VII are common in µ-conotoxins, which generally target voltage-gated sodium channels (VGSC). Sodium channel subtypes can be classified as TTX-sensitive (Na v 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7), or TTX-resistant (Na v 1.5, 1.8, 1.9) channels upon their sensitivity to tetrodotoxin (TTX) [13]. Na v 1.4 is highly expressed in adult skeletal muscle and is associated with disorders such as myotonia, myasthenia and periodic paralysis [23]. A potent and selective Na v 1.4 blocker µ-CnIIIC (IC 50 1.3 nM, framework III) has been commercialized as an instant line relaxer called XEPTM-018 and been used in a facial cream (Lirikos TM , Amorepacific Corporation) for wrinkle smoothing [38]. Na v 1.8 is generally distributed in DRG (dorsal root ganglion) neurons and is considered to be an important target for chronic pain therapy [39]. µO-MrVIB (IC 50 326 nM for Na v 1.8, framework VI/VII) is a potential drug for neuropathic pain treatment, and its investigation has entered clinical phase II [40]. Here, we evaluated the effect of conotoxins Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1, TCP on Na v 1.4 and Na v 1.8 channels. The result (Table 4 and Figure 8) indicated that conotoxins Mr3e.1, Mr3.8, Tx3a.1, and TCP blocked Na v 1.4 or Na v 1.8 by slight inhibition on the current at the complete resting state (TP1), while they inhibited Na v 1.4 or Na v 1.8 by 21.51~24.32% of currents in a semi-activated state (TP2) at 10 µmol/L. Other tested conotoxins showed no current inhibition on Na v 1.4 or Na v 1.8. Activities on other subtypes of VGSC or other types of ion channels remain to be determined.

Tx3a.1, Mr3.8 and Vi14b Inhibited the Proliferation of ID8 Cells
To study the anticancer potential of conotoxins Vi14b, Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1, TCP, the cytotoxicity of these peptides against ID8 ovarian cancer cells was detected by MTT assay. We treated the cells with different concentrations (0, 5, 10, 20, 40, 80, 160 μM) of peptides for 72 h. These peptides showed significant inhibitory effect on ID8 cells at a low concentration of 5 μM compared with the control group, especially Tx3a.1. The cell inhibitory rates were 45.08%, 23.79% and 19.39% for Tx3a.1, Mr3.8 and Vi14b, respectively, at a concentration of 10 μM. A stronger effect was observed after exposure to higher concentrations of Tx3a.1, with a 50% reduction in cell viability at a concentration of 24.29 μM. Mr3.8 killed half the cells at a concentration of 54.97 μM. The IC50 of Vi14b was 111.6 μM. The data (Figure 9) were presented as mean ± SD of three independent experiments. These results indicated that the Tx3a.1, Mr3.8 and Vi14b exhibited strong anticancer effects on ID8 cells in vitro. It was the first report of conotoxins showing anti-ovarian cancer activity. The mechanism of their anti-ovarian cancer activity remains to be explored later. In this work, we identified and structurally and biologically characterized the anti-ovarian cancer conotoxins from Conus venom. The result delivered a message that conotoxins, a class of active polypeptides with diverse structures, could be a rich source of anti-ovarian cancer drug candidates. Herein, this study highlights the role of conotoxin library in subsequent drug discovery for ovarian cancer treatment.

Tx3a.1, Mr3.8 and Vi14b Inhibited the Proliferation of ID8 Cells
To study the anticancer potential of conotoxins Vi14b, Mr3d.1, Mr3e.1, Mr3.8, Tx3a.1, TCP, the cytotoxicity of these peptides against ID8 ovarian cancer cells was detected by MTT assay. We treated the cells with different concentrations (0, 5, 10, 20, 40, 80, 160 µM) of peptides for 72 h. These peptides showed significant inhibitory effect on ID8 cells at a low concentration of 5 µM compared with the control group, especially Tx3a.1. The cell inhibitory rates were 45.08%, 23.79% and 19.39% for Tx3a.1, Mr3.8 and Vi14b, respectively, at a concentration of 10 µM. A stronger effect was observed after exposure to higher concentrations of Tx3a.1, with a 50% reduction in cell viability at a concentration of 24.29 µM. Mr3.8 killed half the cells at a concentration of 54.97 µM. The IC 50 of Vi14b was 111.6 µM. The data ( Figure 9) were presented as mean ± SD of three independent experiments. These results indicated that the Tx3a.1, Mr3.8 and Vi14b exhibited strong anticancer effects on ID8 cells in vitro. It was the first report of conotoxins showing antiovarian cancer activity. The mechanism of their anti-ovarian cancer activity remains to be explored later. In this work, we identified and structurally and biologically characterized the anti-ovarian cancer conotoxins from Conus venom. The result delivered a message that conotoxins, a class of active polypeptides with diverse structures, could be a rich source of anti-ovarian cancer drug candidates. Herein, this study highlights the role of conotoxin library in subsequent drug discovery for ovarian cancer treatment.

Venom Preparation and Peptide Isolation
Cone snail specimens were collected from coastal waters near Sansha city in the South China Sea and were frozen at −80 °C for hours. Dr. Ying Fu identified the specimens. The venom duct of the snails was dissected, and the venom duct cut into small sections (about 1cm long). They were extracted with 60% acetonitrile aqueous solution at 4 °C for 24 h, then centrifuged at 8000 r/min for 10 min. The supernatant was pipetted and filtered with 0.22 µm organic phase pinhole filter, then 60% acetonitrile aqueous solution was added to the precipitate for secondary extraction. This was repeated twice and the filtrate combined to obtain the crude venom solution, which was subjected to lyophilization to

Venom Preparation and Peptide Isolation
Cone snail specimens were collected from coastal waters near Sansha city in the South China Sea and were frozen at −80 • C for hours. Dr. Ying Fu identified the specimens. The venom duct of the snails was dissected, and the venom duct cut into small sections (about 1cm long). They were extracted with 60% acetonitrile aqueous solution at 4 • C for 24 h, then centrifuged at 8000 r/min for 10 min. The supernatant was pipetted and filtered with 0.22 µm organic phase pinhole filter, then 60% acetonitrile aqueous solution was added to the precipitate for secondary extraction. This was repeated twice and the filtrate combined to obtain the crude venom solution, which was subjected to lyophilization to yield crude venom powder.
The venom powder was dissolved in 3% acetonitrile aqueous solution and filtrated. The venom solution was fractionated using a preparative high-performance liquid chromatography (Pre-HPLC) instrument (Waters, Milford, MA, USA) with a C 18 column (Vydac Grace, 10 µm, 22 mm × 250 mm). The preparative system was conducted by linear gradient elution with 5 to 90% solution B (90% acetonitrile aqueous solution with 0.1% TFA) for 60 min at an 8 mL/min flow rate, while solution A was double-distilled water (ddH 2 O) with 0.1% TFA. The fractionated components were lyophilized and redissolved in 10% acetonitrile aqueous solution, then were subjected to a HPLC system equipped with a C 18 column (Vydac Grace, 5 µm, 4.6 mm × 250 mm). The separation process was performed with mixed eluant (solution A and B) at a flow rate of 0.8 mL/min. Conopeptides were yielded from the venom fractions by a 20-min linear gradient program of 30% to 50% solution B (namely 70% to 50% solution A) detecting by ultraviolet (UV) absorption at 214 nm.
The molecular weights of the purified conopeptides were determined using an ultraperformance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-TQD-MS, Waters, USA) spectrometer with a BEH C 18 column (130 A, 1.7 µm, 2.1 mm × 100 mm). Conditions were set as follows: detection range m/z 400-1500, cone voltage 30 V, capillary voltage 3.5 kV, dissolution flow rate 1000 L/h, dissolution temperature 500 • C, ion source temperature 150 • C. The UPLC program was performed by gradient elution from 5 to 60% solution B for 6 min at a flow rate of 0.5 mL/min.

Peptide Sequencing
The isolated peptides were individually alkylated by iodoacetamide to confirm whether they contained any free thiol or not. In order to ascertain the number of disulfide bonds in these conopeptides, we totally reduced the peptides by TCEP and detected their mass differences between intact peptides and the reduced forms (Figure 3), respectively.
Then the purified conotoxins were totally reduced to obtain linear peptides before sequencing. Each peptide was dissolved in 20 µL 50% acetonitrile aqueous solution. Then, 10 µL 50 mmol/L TCEP and 10 µL NH 4 HCO 3 were added to the peptide solution, which was incubated at 25 • C for 30 min. The reaction was monitored by UPLC analysis at 214 nm. The molecular weight of each reduced peptide was determined by UPLC-TQD-MS over m/z 100-2000.
Sequence identification was achieved by MALDI-TOF (matrix-assisted laser desorption-ionization-time of flight) tandem mass spectrometer (Bruker, Ultraflextreme, Germany). The reduced linear peptides were dissolved in 50% acetonitrile aqueous solution. The matrix solution was prepared by dissolving 1 mg cyano-4-hydroxycinnamic acid (HCCA) in 100 µL standard solution (47.5 µL ddH 2 O, 50 µL acetonitrile, 2.5 µL trifluoroacetic acid). For sample loading, 1 µL of each reduced peptide solution was individually dropped onto different circles on the plate and then dried off. One µL of matrix solution was successively dropped onto the same position on the plate. The sample plate was then loaded into the spectrometer when the solution was completely dried off. FlexControl was used to acquire MS data, while FlexAnalysis was applied to mark the masses. Conditions were set as follows: laser frequency 1000 Hz, laser energy range 20-40%, ion source voltage 7.5 kV. The voltages for secondary mass detection were 19.00 kV for Lift 1 and 2.80 kV for Lift 2. For the sequence identification, ion masses and related fragmentation masses in the secondary mass spectrum of each conopeptide were matched by Mascot search (http://www.matrixscience.com/cgi/nph-mascot.exe?1, accessed from 1 December 2020 to 23 February 2022) against the NCBI and Swiss-prot protein database. Modifications involved in the search were as follows: amidation, deamidation, hydroxylation of proline and valine, oxidation of methionine, carboxylation of glutamic acid, cyclization of N-terminal glutamine (pyroglutamate), bromination of tryptophan, and sulfation of tyrosine.
An Edman degradation experiment was performed for sequence elucidation as well. The solution was vortexed until the reduced peptide powder was dissolved in 50 µL ddH 2 O. Then, 10 µL of the peptide solution was dropped onto the polyvinylidene fluoride (PVDF) membrane and dried off. The PVDF membrane was transferred onto a PPSQ-53A protein sequencer (Shimadzu, Japan). Each phenylthiohydantoin (PTH)-amino acid generated from every cycle of N-terminal degradation of the peptide was analyzed by HPLC system equipped with a Wakosil PTH-II column (Wako, 4.6 mm × 250 mm). For the mobile phase, 40% acetonitrile aqueous solution with less than 2.5% acetic acid was used. The column temperature was set to 40 • C, and the flow rate was 1.0 mL/min. The retention time of each (PTH)-amino acid was compared with that of the corresponding standard amino acid to identify the primary amino acid sequence of the isolated conopeptides.

Peptide Synthesis
The linear conopeptides were separately synthesized using Fmoc-SPPS (9-Fluorenylmethoxycarbonyl-peptide solid phase synthesis) chemistry [41]. The Fmoc-amino acids were purchased from Chengdu Chron Chemical Co., LTD. Amino acid ligation was operated on the Wang-resin with HBTU/DIEA used as the coupling reagent. Kaiser test regent was applied to detect the state of every coupling cycle. Piperidine/DMF (v/v 20:80) was used to remove the Fmoc groups. The cysteine residues were protected by triphenylmethyl (trt) groups, which were then removed along with the peptide release process. Cleavage cocktail (TFA/thioanisole/phenol/EDT/H 2 O 87.5:2.5:2.5:2.5) was used to release the linear peptide from the resin at room temperature for 2 h, filtrate to remove the resin, and the filtrate was treated with cold ether (0 • C) overnight to precipitate the crude peptide. The precipitation was washed with cold ether twice and lyophilized to obtain crude peptide power, which was subjected to Pre-HPLC separation to yield pure linear peptide.
The obtained linear conopeptide was subjected to one-step air oxidation in a 0.1 M Tris-HCl/1 mM EDTA buffer with GSH/GSSG (1 mmol/L/0.5 mmol/L) at 40 • C for 60 min. The linear peptide concentration ranged from 50 µM to 100 µM. The oxidative products were detected by LC-MS and subsequently purified by HPLC system with a 20-min linear gradient elution of 30% to 50% solution B at a 0.8 mL/min flow rate.

Disulfide Connectivity Recognition
The stepwise reduction and alkylation protocol were performed as reported [42]. The folded peptide (20-30 µg in 100 µL ddH 2 O containing 0.1% TFA) was partially reduced by mixing with an appropriate volume of 100 mM TCEP dissolved in citrate buffer (0.1 M, pH 3.0) to a final concentration of 6 mM TCEP. The mixture was incubated at 40 • C ranging from 5 min to 25 min for folded peptide to generate the corresponding one-disulfidereduced product and two-disulfide-reduced product. The reaction state was monitored by LC-MS to confirm that the exact partially reduced product was generated. Then the reaction mixture was blended with alkylation solution (9.24 mg iodoacetamide dissolved in 25 µL acetonitrile and 75 µL 1 mM pH 8.0 Tris-HCl buffer) to alkylate the free thiol groups of the partial-reduced products. The alkylation reaction was conducted at 40 • C for 15-45 min and monitored by LC-MS. Then the mixture was subjected to going through HPLC column (Vydac Grace, 5 µm, 4.6 mm × 250 mm) to terminate the reaction and purify the alkyl-labeled products. The obtained two-cysteine-alkylated and four-cysteinealkylated products were subsequently reduced by TCEP to break the remaining disulfide bonds to gain linear peptides, which were named as products I and II, respectively. Then the products I and II of the synthetic conopeptide were subjected to MALDI-TOF-MS/MS analysis to confirm which thiol groups were alkylated.

Patch Clamp Recording for Voltage-Gated Sodium Channel
Blocking activity of voltage-gated sodium channels was conducted using the wholecell patch clamp technique [43]. CHO cells were cultured in HAM'S/F-12 medium containing 10% fetal bovine serum. 6.5 × 10 3 cells were plated on coverslips, cultured in 24-well plates (final volume 500 µL). Patch clamp assays were performed after 18 h. Solution preparation involved intracellular fluid Na v -001-2 (50 mM CsCl, 10 mM NaCl, 10 mM HEPES, 60 mM CsF, 20 mM EGTA, adjusted to pH 7.2 with CsOH), extracellular fluid K-007-1 (140 mM NaCl, 3.5 mM KCl, 1 mM MgCl 2 •6H 2 O, 2 mM CaCl 2 •2H 2 O, 10 mM D-Glucose, 10 mM HEPES, 1.25 mM NaH 2 PO 4 •2H 2 O, adjusted to pH 7.4 with NaOH). The testing peptide was dissolved with DMSO to a concentration of 10 mM solution. The peptide solution was diluted to 10 µM using extracellular fluid. Tetrodotoxin (TTX, 1 µM) dissolved in ddH 2 O containing 0.1% acetic acid was used as a Na v 1.4 positive control, while Na v 1.8 positive control was prepared by dissolving 10 mg A-803467 [5-(4-chlorophenyl-N-(3, 5-dimethoxyphenyl) furan-2-carboxamide] with DMSO and diluting it to 10 µM by K-007-1). The microelectrode manipulator was operated under an inverted microscope (MP285, Sutter Instrument, Novato, CA, USA). The recording electrode was placed on the cell, and negative pressure was applied to form a GΩ seal. Then, fast capacitance compensation was performed and negative pressure applied until the cell membrane was sucked to form a whole-cell recording mode. The membrane voltage was clamped at −120 mV after whole-cell sealing. Cell-plated coverslips were placed in the recording bath in an inverted microscope. The testing peptide solution and the peptide-free extracellular fluid flowed through the recording bath from low to high concentration by gravity perfusion. Each peptide was reacted for 5 min before the next peptide was detected. The data were collected by EPC-10 amplifier (HEKA, Reutlingen, Baden Wurdenburg, Germany) and stored by PatchMaster software (HEKA, Reutlingen, Baden Wurdenburg, Germany).
The reaction rate on voltage-gated sodium channel were defined as Peak current conotoxin Peak current control , and the percentage of inhibition was determined as 1 − Peak current conotoxin Peak current control × 100%. Meanwhile, the mean and standard deviation were calculated through three parallel experiments. The voltage-gated sodium channel activity data was analyzed by PatchMaster (HEKA, Reutlingen, Germany) & IGOR Pro (Wave Metrics, Portland, OR, USA). In addition, data processing and graphing were performed by the software GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

Ovarian Cancer Cell Viability Assay
The MTT method was used to determine the cytotoxicity of the synthetic conotoxins [44]. Briefly, ID8 cells (4 × 10 3 cells/well) were seeded in 96-well plates. After 12 h incubation, the culture medium was removed and the cells were treated with different concentrations of peptides for 72 h. After adding 1 mg/mL MTT solution, the cells were cultured for another 4 h. Subsequently, 100 µL DMSO was added to dissolve the formazan. The absorbance was measured at 570 nm using a SpectraMax ® M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The IC 50 values were expressed as mean ± SD of individual samples and were representative of three independent experiments. The data were analyzed by unpaired two-tail t-test (Student's t-test). Values of P less than 0.05 were regarded as statistically significant. Calculation of IC 50 values and statistical analysis were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).