Melittin Inhibits the Growth of Hepatocellular Carcinoma Huh7 Cells by Downregulating LARS2 and ZNF19

Melittin Inhibits the Growth of Hepatocellular Carcinoma Huh7 Cells by Downregulating LARS2 and ZNF19 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Melittin Inhibits the Growth of Hepatocellular Carcinoma Huh7 Cells by Downregulating LARS2 and ZNF19 Yanli Zhang, Hainan Yang, Hui Liu, Hui Ye, Zhongming Ye, Xiang LV, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7154719/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Although surgery remains the most effective treatment, more than half of patients experience recurrent liver metastases within two years. This highlights the need for new therapeutic strategies driven by basic research. In this study, we used CCK-8 assays to evaluate the proliferation of liver cancer cells treated with melittin compared to controls. Proteomic sequencing was performed to identify differentially expressed proteins in Huh7 cells following melittin treatment. Two target genes, LARS2 and ZNF19 , were further investigated using High Content Screening (HCS) to assess their roles in cell proliferation. CCK-8 results showed that melittin significantly reduced the viability of Huh7 and HepG2 cells at 24 and 48 hours in a dose-dependent manner. Proteomic analysis identified 142 upregulated and 8 downregulated proteins, while phosphoproteomic analysis revealed 88 upregulated and 21 downregulated phosphoproteins in the melittin-treated group. HCS assays demonstrated that silencing LARS2 and ZNF19 significantly inhibited liver cancer cell proliferation. These findings suggest that melittin suppresses the growth of Huh7 cells partly through downregulating LARS2 and ZNF19 . Hepatocellular carcinoma Melittin High Content Screening Proteome sequencing Protein phosphorylation sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights 1) Melittin significantly reduced the viability of Huh7 and HepG2 cells. 2) Melittin suppressed liver cancer cell proliferation by downregulating LARS2 and ZNF19. 1. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors globally and accounts for the fifth most common cancer worldwide [ 1 , 2 ] . Although surgery is the most effective treatment for HCC, the recurrence-free 5-year survival rate after curative resection is low [ 3 ] . Previous evidence indicates that over half of these patients still develop recurrent liver metastases within 2 years, and the 5-year survival rate is approximately 20–50% [ 4 , 5 ] . Therefore, new treatments may rely on advances in basic research. Melittin is a water-soluble toxic peptide derived from the venom of the bee. It consists of 26 amino acids, featuring a hydrophobic N-terminal region (amino acids 1–20) and a hydrophilic C-terminal region (amino acids 21–26) containing a sequence of positively charged amino acids [ 6 ] . It also possesses anti-HCC effects, including inhibiting the proliferation, migration, and invasion of HCC cells, suppressing liver metastasis, and suppressing hepatic epithelial-to-mesenchymal transition [ 7 – 10 ] . However, the underlying mechanism is unknown. High Content Screening (HCS) has played a crucial role in the discovery of drug target genes. This research technique allows for simultaneous genetic manipulations, including interference, knockout, or overexpression, of at least 20 or even more coding genes or non-coding RNAs. By using high-content imaging instruments to scan plates rapidly for multiple days, researchers can observe and capture the proliferation status of cells after regulating different genes. This method enables the screening of genes associated with proliferation. Given the therapeutic potential of melittin in HepG2 and Huh7 cells, our study aimed to elucidate the biological mechanism underlying melittin-induced inhibition of cell growth in these cell lines. We treated the tumor cells with melittin and analyzed the protein sequencing results of the treatment group and the control group to identify differentially expressed genes. Further screening and validation were conducted using HCS test. 2. Materials and Methods 2.1 Melittin Melittin (C131H229N39O31, > 97% purity) was a product of Santa Cruz Biotechnology (Santa Cruz, CA, USA). It was dissolved in distilled water to make a stock solution and then stored at − 20℃ until use avoiding repeated freezing and thawing. 2.2 Cell Culture The human liver cancer cell lines, Huh7 and Hep G2, were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (all from Hyclone, Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere with 5% CO 2 . 2.3 CCK-8 assays CCK-8 assays were performed to assess the proliferative properties of the control group and the melittin treatment groups (0µg/ml, 5µg/ml, 10µg/ml, 15µg/ml, 20µg/ml) following the manufacturer's instructions. Cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well and incubated at 37°C for 24 or 48 hours. Subsequently, the optical density (OD) values at 450 nm wave length were measured using a Multiskan FC (Thermo Fisher Scientific, Inc.). The inhibition rate was calculated as follows: Inhibition rate = (1 - OD_treatment group / OD_negative control group) × 100%. 2.4 Protein Extraction Protein Extraction Using the SDT Lysis Method: Specifically, samples were mixed with an appropriate amount of SDT lysis buffer and subjected to a boiling water bath for 15 minutes. The mixture was then centrifuged at 14,000g for 15 minutes, and the supernatant was collected. Protein quantification was subsequently performed using the BCA method. 2.5 Cell Transfection Cell lines were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS) and transfected using Lipofectamine 2000 (Invitrogen). 2.6 RNA extraction and realtime quantitative PCR Total RNA was extracted using TRIzol reagent, and cDNA was synthesized with the PrimeScript™ RT Master Mix kit according to the manufacturer’s instructions. Quantitative real-time PCR was then carried out using the SYBR Premix Ex Taq kit (Takara, Shiga, Japan). GAPDH served as the internal control for normalization. All experiments were conducted in triplicate. Gene expression levels were calculated using the 2 − ΔΔCt method, a widely accepted approach for relative quantification in real-time PCR. 2.7 Western Blotting Analysis Cells were lysed on ice using cold RIPA lysis buffer (EpiZyme, China) supplemented with protease inhibitors. The extracted proteins were separated by 10% or 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated overnight at 4°C with primary antibodies against LARS2 (1:1000), ZNF19 (1:1000), and GAPDH (1:3000). After three washes with TBST, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 hours. Protein bands were visualized using an ECL detection kit (EpiZyme, China). All antibodies were purchased from ABclonal Technology Co., Ltd. 2.8 Statistical Analyses We conducted our statistical analysis using SPSS version 21 (IBM Corp., Armonk, NY, USA) and R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). To compare the differences between two groups, we applied the Wilcoxon rank-sum test. For comparisons among multiple groups, we used one-way ANOVA followed by Dunnett’s post hoc test. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1 Melittin Inhibits the Viability of Liver Cancer Cells in a CCK-8 Assay We first investigated the effect of melittin on the viability of liver cancer cells. Optical density (OD) values were obtained for both the control group and the melittin-treated group at varying concentrations (0 µg/ml, 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml). Results from the CCK8 test indicated that melittin significantly inhibited the viability of Huh7 and HepG2 cells at 24 and 48 hours. The inhibitory rates of melittin on Huh7 and HepG2 hepatoma cells increased in a dose-dependent manner ( Fig. 1 ) . 3.2 Proteome Signatures of Groups Between Melittin Treatment(10 µg/ml)and Control in Huh7 Liver Cancer Cells Based on previous research findings, we discovered that melittin exhibits a concentration-dependent inhibitory effect on liver cancer cells. In subsequent experiments, liver cancer cells (Huh7) were treated with 10 µg/ml melittin and compared to a control group of untreated parental liver cancer cells. Proteomics techniques were employed to further analyze the differentially expressed proteins between the two groups. To clarify the underlying reasons for the distinct protein expression between the melittin treatment group (ME) and the control (NC) group, we screened for proteins with expression differences between them. As a result, a volcano plot identified 142 upregulated and 8 downregulated proteins (Wilcoxon rank-sum test, cutoff ratio (ME/NC) > 2 or < 0.85, Fig. 2 A). Enrichment analysis of differentially expressed proteins between the ME and NC groups was conducted. Three duplicate samples were annotated above the heatmap, and the significantly upregulated or downregulated genes are marked in the figure ( Fig. 2 B ) . Pathway enrichment showed that the integral component of the membrane was the top category in Cellular Component, while ATP-dependent microtubule motor activity and growth factor activity were the top two categories in Molecular Function. Additionally, negative regulation of transporter activity was the main function in Biological Process ( Fig. 2 C ). 3.3 Phosphoproteome Signatures of Groups Treated with Melittin (10 µg/ml) Compared to Control in Huh7 Liver Cancer Cells In this study, after protein sequencing, we further conducted protein phosphorylation sequencing on melittin-treated Huh7 liver cancer cells and compared the results with the untreated control group. The results showed 88 upregulated and 21 downregulated proteins in the melittin-treated group compared to the control ( Fig. 3 A ) . Enrichment analysis of differentially expressed proteins between the ME and NC groups identified the significantly upregulated and downregulated proteins, which are marked in the figure ( Fig. 3 B ) . Pathway enrichment using their corresponding phosphoproteins showed that the integral component of the plasma membrane was the top category in Cellular Component. Protein domain specific binding and cell adhesive protein binding were the main functions in Molecular Function, while regulation of protein localization to the membrane was the main function in Biological Process ( Fig. 3 C ) . 3.4 Genes such as shLARS2 and shZNF19 Showed Proliferation Inhibition in the HCS test High Content Screening (HCS) is a technique that allows for simultaneous genetic manipulations, such as interference, knockout, or overexpression, of multiple coding genes or non-coding RNAs. This process is followed by functional phenotype detection at the cellular level to identify genes with significant functions. In this study, HCS technology was employed to screen 13 candidate genes at the cellular level in liver cancer cells (Huh7). Among the 13 genes tested, the shLARS2 and shZNF19 groups showed a fold change ≥ 1.5. These genes were identified as positive hits in this experiment (proliferation inhibition positive cell groups, Fig. 4 ). 3.5 Further Confirmed shLARS2 and shZNF19 Proliferation Inhibition Function in HCS test In subsequent experiments, three RNA interference targets were designed for each of the previously screened target genes (LARS2 and ZNF19). Three plasmids, each carrying a different target, were packaged into lentiviruses. Using viral infection technology, stable cell lines were constructed and subjected to further HCS screening. The results showed RNA interference target genes such as LARS2 and ZNF19 significantly inhibited the proliferation of liver cancer cells ( Fig. 5 ) . 3.6 In Vitro Experiments Demonstrate That Melittin Treatment Suppresses the Expression of LARS2 and ZNF19 To further confirm the association between melittin treatment and the expression of LARS2 and ZNF19, we conducted real-time quantitative PCR and Western blot analyses comparing the melittin-treated and control groups. The results showed that the expression levels of LARS2 and ZNF19 were significantly downregulated in the melittin-treated group. Moreover, the findings from both PCR and Western blot experiments were consistent ( Fig. 6 ) . These results suggest that melittin inhibits the growth of hepatocellular carcinoma Huh7 cells by downregulating LARS2 and ZNF19. 4. Discussion Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors, with high prevalence in Asia and Africa [ 11 ] . A similar trend has been observed in the United States and the United Kingdom, where the incidence of HCC has increased substantially over the past two decades [ 12 , 13 ] . In recent years, there have been significant advances in the treatment of liver cancer. However, the overall survival rate remains unsatisfactory [ 14 – 16 ] . Additionally, the molecular mechanism behind HCC metastasis remains unclear, and additional research is still required to explore this mechanism [ 17 ] . Previous studies have shown that melittin inhibits HCC cell viability and migration, and can also suppress the metastasis of liver cancer cells [ 18 , 19 ] . In this study, liver cancer cells were treated with melittin, and the proteomic profiles of these cells were compared with those of untreated liver cancer cells to identify proteins with differential expression between the two groups. Subsequent genes were selected based on literature screening. Previous studies employing probe technology have localized nine KOX zinc finger genes to specific regions on four human chromosomes. In situ hybridization of cDNA probes to metaphase chromosomes revealed that KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively [ 20 ] . Prior research has explored the role of KCMF1 in renal cell carcinoma, revealing a discrepancy in the formation of ubiquitin ligase and autophagosomes mediated by KCMF1, as well as in ionic concentration in tumor cells. This discrepancy may be one of the possible factors driving cancer evolution [ 21 ] . KCMF1 plays a crucial role in the proliferation, migration, and invasion of trophoblast cells, with a similar trend observed in human colon cancer stem cells. This suggests that KCMF1 and the 14-3-3σ protein may influence the proliferation and colony formation of these cells [ 22 , 23 ] . SPTLC1 suppresses cell growth, and previous evidence suggests a significant decrease in its expression in RCC tissues compared to non-tumor tissues. Similarly, other studies have indicated that decreased SPTLC1 expression is predictive of poorer outcomes in ccRCC patients [ 24 , 25 ] . Previous research has shown that LARS2 enhances the expression of E2F1, which mediates its effects on cell proliferation and apoptosis. This regulatory mechanism suggests that LARS2 could be a promising therapeutic target for treating Alzheimer's disease (AD) [ 26 , 27 ] . Previous research has identified TNFRSF21 as a potential target for necroptosis in osteosarcoma therapy. Other evidence suggests that the circ_TNFRSF21/miR-214-3p/CHI3L1 axis could serve as promising diagnostic markers or therapeutic targets for cutaneous squamous cell carcinoma (cSCC) [ 28 , 29 ] . Knockdown of TRAF5 was found to inhibit HCC cell viability, colony formation, migration, invasion, and survival. This study concludes that silencing TRAF5 enhances necroptosis in hepatocellular carcinoma by inhibiting LTBR-mediated NF-κB signaling [ 30 ] . Researchers have investigated the function of PATZ1 in patients with glioblastoma, revealing that low PATZ1 expression correlates with poor prognosis in these patients. Moreover, overexpression of PATZ1 inhibits glioma cell proliferation and induces apoptosis by activating intrinsic apoptotic pathways [ 31 ] . ELF3 has been implicated in gallbladder cancer, with in vivo experiments providing further evidence that reducing ELF3 expression enhances the gemcitabine sensitivity of GBC cells and prolongs the survival of mice with orthotopically xenografted tumors. The study concludes that ELF3 promotes gemcitabine resistance through the PKMYT1/CDK1 signaling pathway in gallbladder cancer [ 32 ] . Previous evidence indicates that Bclaf1 levels increase in hypoxia in a manner dependent on HIF-1α. Additionally, it identifies Bclaf1 as a novel positive regulator of HIF-1α in the hypoxic microenvironment [ 33 ] . High-content screening was employed to identify kinase inhibitors that can overcome venetoclax resistance in activated CLL cells. These results were further confirmed through protein sequencing in different groups [ 34 ] . In this study, we compared Huh7 liver cancer cells treated with Melittin to untreated cells using proteomic and phosphoproteomic sequencing techniques to identify differentially expressed proteins. Subsequent experiments on HCS test further validated these findings. Knocking down the target genes LARS2 and ZNF19 significantly inhibited the proliferation of liver cancer cells, suggesting that these genes play important roles in the proliferation of liver cancer cells and may be potential targets for liver cancer treatment. LARS2 variants have been implicated in Perrault syndrome. Evidence from muscle tissue in a child with reversible myopathy revealed a reduction in LARS2 expression and mitochondrial complex I levels, along with an atypical form of degeneration [ 35 ] . Additional studies have demonstrated an association between LARS2 mutations and premature ovarian insufficiency (POI). Silencing LARS2 was shown to suppress cell proliferation while promoting apoptosis in granulosa cells (GCs). Mechanistically, LARS2 knockdown led to mitochondrial dysfunction and an accumulation of reactive oxygen species (ROS) [ 26 ] . Further investigations have identified a role for YAP in enhancing mitochondrial oxidative phosphorylation (OXPHOS) by upregulating LARS2 transcription. As LARS2 function relies heavily on its substrate amino acid, leucine, a combination of a low-leucine diet and YAP inhibition synergistically impaired mitochondrial function in TI-Tregs, ultimately restricting tumor growth [ 36 ] . Declarations Ethical approval：Not applicable. Consent to participate：Not applicable. Consent to publish：Not applicable. Availability of data and materials The datasets analyzed for the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by The Scientific Research Program of Shanghai Pudong New Area Health Commission (the General Program) (grant No. PW2021A-49 and PW2024A-58). Authors’ Contributions Yanli Zhang, Hainan Yang, designed the experiments and wrote the manuscript. Hui Liu,Hui Ye, helped in reviewing, acquiring, analysis for the work.. Zhongming Ye, did the statistical analysis. Xiang LV, Ming Leirevised critically the manuscript for important intellectual content. All authors have read and approved the final manuscript. Acknowledgments Not applicable. Author Details 1 Department of Critical Care Medicine, Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine, 358 Datong Road, Pudong New District, Shanghai, 200137, China. 2Department of Critical Care Medicine, Shanghai Pudong New Area Gongli Hospital 219 Miaopu Road, Shanghai, 200135, China. References Ahn S, Hyeon J, Park CK: Metadherin is a prognostic predictor of hepatocellular carcinoma after curative hepatectomy. Gut Liver 2013, 7(2):206-212. El-Serag HB, Rudolph KL: Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007, 132(7):2557-2576. Maluccio M, Covey A: Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma. 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Wen Y, Zhou X, Lu M, He M, Tian Y, Liu L, Wang M, Tan W, Deng Y, Yang X et al : Bclaf1 promotes angiogenesis by regulating HIF-1α transcription in hepatocellular carcinoma. Oncogene 2019, 38(11):1845-1859. Oppermann S, Ylanko J, Shi Y, Hariharan S, Oakes CC, Brauer PM, Zúñiga-Pflücker JC, Leber B, Spaner DE, Andrews DW: High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells. Blood 2016, 128(7):934-947. Riley LG, Rudinger-Thirion J, Frugier M, Wilson M, Luig M, Alahakoon TI, Nixon CY, Kirk EP, Roscioli T, Lunke S et al : The expanding LARS2 phenotypic spectrum: HLASA, Perrault syndrome with leukodystrophy, and mitochondrial myopathy. Hum Mutat 2020, 41(8):1425-1434. Bai J, Yan M, Xu Y, Wang Y, Yao Y, Jin P, Zhang Y, Qu Y, Niu L, Li H: YAP enhances mitochondrial OXPHOS in tumor-infiltrating Treg through upregulating Lars2 on stiff matrix. J Immunother Cancer 2024, 12(11). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7154719","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504833892,"identity":"5b18f977-1cda-4b44-b666-890aed972120","order_by":0,"name":"Yanli Zhang","email":"","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Zhang","suffix":""},{"id":504833893,"identity":"d81ee530-4e89-4e95-bd7a-d1a983fdca18","order_by":1,"name":"Hainan Yang","email":"","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hainan","middleName":"","lastName":"Yang","suffix":""},{"id":504833894,"identity":"c433d157-b978-47af-9cf1-0ab3b2d699a2","order_by":2,"name":"Hui Liu","email":"","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Liu","suffix":""},{"id":504833898,"identity":"eef1bdf7-a615-4230-98dd-7701ca5739c8","order_by":3,"name":"Hui Ye","email":"","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Ye","suffix":""},{"id":504833899,"identity":"4fc7536b-0943-48f3-8f0b-fe18aa85a32c","order_by":4,"name":"Zhongming Ye","email":"","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhongming","middleName":"","lastName":"Ye","suffix":""},{"id":504833902,"identity":"bf2500c4-4998-4946-8930-1acadd30315e","order_by":5,"name":"Xiang LV","email":"","orcid":"","institution":"Shanghai municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"LV","suffix":""},{"id":504833903,"identity":"dcb97981-79f6-428b-ab37-65268091dc32","order_by":6,"name":"Ming Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDACZiB+wCDBwHCA+cCBDz+I1ZIA1sKWeHBmD7E2JYCIAzzGhznYiFCt2857+EVChQUD3+0zHw4z8DDI84sdwK/F7DBfmkXCGQkGyXO5Gw4XWDAYzpydQEgLj5lBYpsEg8EZ3g2HZ/AwJBjcJkrLP5AWngeHediI02L8ILEBrIWBaC1mDAnHgH45w2YADGQJIvxy/ozxhw81dQx8Z5gff/jww0aeX5qAFiBgA8YjQ30DhCNBUDkIMH8gStkoGAWjYBSMXAAA8ClEQg1M+vMAAAAASUVORK5CYII=","orcid":"","institution":"Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ming","middleName":"","lastName":"Lei","suffix":""}],"badges":[],"createdAt":"2025-07-18 06:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7154719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7154719/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89977928,"identity":"b61be7ad-5f0e-41a8-b2e5-5e1b8dd0c7ca","added_by":"auto","created_at":"2025-08-27 06:12:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53409,"visible":true,"origin":"","legend":"\u003cp\u003eBar charts depicting the inhibition rate of melittin on Huh7 and HepG2 liver cancer cells.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/3a78e65eba1663df8e652bbe.png"},{"id":89977931,"identity":"70dbd2a2-981f-4b0f-a216-a43374d48039","added_by":"auto","created_at":"2025-08-27 06:12:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189152,"visible":true,"origin":"","legend":"\u003cp\u003eProteome signatures of ME and control (NC). A, volcano plot showing 142 upregulated and 8 downregulated proteins in the ME compared to the NC. Test method, Wilcoxon rank-sum test. Cutoff, ratio (ME/control) \u0026gt;2 or \u0026lt;0.85. Blue and yellow dots represent significantly differential proteins in ME and control, respectively. B, enrichment analysis of differentially expressed proteins between the ME and NC. Three duplicate samples were annotated above the heatmap. The heatmap depicts the adjusted intensity of proteins with log2-transformation. C, the biological functions and pathway enrichment analysis of these differentially expressed proteins. Biological functions are included cellular component, molecular function, and biological process.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/87727ba80f40dcb34d81a2ae.png"},{"id":89977934,"identity":"97bd9455-1467-4b29-a7de-d43b854444d9","added_by":"auto","created_at":"2025-08-27 06:12:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":173414,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphoproteome Signatures of ME and NC. A, Volcano plot showing 88 upregulated and 21 downregulated proteins in the ME compared to the control. Test method: Wilcoxon rank-sum test. Cutoff: ratio (ME/control) \u0026gt;2 or \u0026lt;0.85. Green and red dots represent significantly differential proteins in ME and control, respectively. B, Enrichment analysis of differentially expressed proteins between the ME and NC groups. Three duplicate samples were annotated above the heatmap. The heatmap depicts the adjusted intensity of proteins with log2-transformation. C, Biological functions and pathway enrichment analysis of these differentially expressed proteins. Biological functions include cellular component, molecular function, and biological process.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/5b063428a57a5fe09a0abec8.png"},{"id":89977949,"identity":"9290cb20-2450-4951-9bea-8e02f7d9a5e7","added_by":"auto","created_at":"2025-08-27 06:12:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99999,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curve of Huh7 cells. A. Cell Number of Huh7 cells in the presence of knockdown or overexpression of 13 genes on the indicated days. B. Cell Number/fold of Huh7 cells in the presence of knockdown or overexpression of 13 genes on the indicated days. PC is the positive target virus, which the knockdown efficiency of target gene is more than 70%. Ctrl is the control group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/bd88d94c79b5349be9e3b067.png"},{"id":89977944,"identity":"53193264-3740-401c-9b07-a0af7187acf8","added_by":"auto","created_at":"2025-08-27 06:12:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94522,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curve of Huh7 cells. A. Cell Number of Huh7 cells treated with LARS2-RNAi, ZNF19-RNAi on the indicated days. B. Cell Number/fold of Huh7 cells treated with LARS2-RNAi, ZNF19-RNAi on the indicated days. PC is the positive target virus, which the knockdown efficiency of target gene is more than 70%. Ctrl is the control group. C. Cell Number of Huh7 cells treated with LARS2-RNAi (89357-1), ZNF19-RNAi (89362-1) on the indicated days. D. Cell Number/fold of Huh7 cells treated with LARS2-RNAi (89357-1), ZNF19-RNAi (89362-1) on the indicated days. PC is the positive target virus, which the knockdown efficiency of target gene is more than 70%. Ctrl is the control group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/b5d09b81f7e609166db5f635.png"},{"id":89979930,"identity":"740ac57f-a4cd-4a76-b5d4-2bb018ac73b9","added_by":"auto","created_at":"2025-08-27 06:20:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":73307,"visible":true,"origin":"","legend":"\u003cp\u003ePCR and Western blot analysis of LARS2 and ZNF19 expression in the Melittin-treated and control groups. Left. Relative mRNA expression levels of LARS2 and ZNF19 in the Melittin-treated and control groups. Right. Protein expression levels of LARS2 and ZNF19 in the Melittin-treated and control groups\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/517a4282124a2af9e727f5bd.png"},{"id":90967073,"identity":"55a9e965-3abf-45ca-a9e5-44c34e2f60bf","added_by":"auto","created_at":"2025-09-10 06:47:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1423672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/6282a125-20a1-4f98-a1ad-90003586a1f5.pdf"},{"id":89979927,"identity":"8d46bfce-47d3-4256-bb93-9ff1f1745486","added_by":"auto","created_at":"2025-08-27 06:20:49","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":866816,"visible":true,"origin":"","legend":"","description":"","filename":"Supportingmaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-7154719/v1/e536206ad1f0629c93d6770f.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Melittin Inhibits the Growth of Hepatocellular Carcinoma Huh7 Cells by Downregulating LARS2 and ZNF19","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1) Melittin significantly reduced the viability of Huh7 and HepG2 cells.\u003c/p\u003e\u003cp\u003e2) Melittin suppressed liver cancer cell proliferation by downregulating LARS2 and ZNF19.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors globally and accounts for the fifth most common cancer worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although surgery is the most effective treatment for HCC, the recurrence-free 5-year survival rate after curative resection is low\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Previous evidence indicates that over half of these patients still develop recurrent liver metastases within 2 years, and the 5-year survival rate is approximately 20\u0026ndash;50%\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, new treatments may rely on advances in basic research.\u003c/p\u003e\u003cp\u003eMelittin is a water-soluble toxic peptide derived from the venom of the bee. It consists of 26 amino acids, featuring a hydrophobic N-terminal region (amino acids 1\u0026ndash;20) and a hydrophilic C-terminal region (amino acids 21\u0026ndash;26) containing a sequence of positively charged amino acids\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. It also possesses anti-HCC effects, including inhibiting the proliferation, migration, and invasion of HCC cells, suppressing liver metastasis, and suppressing hepatic epithelial-to-mesenchymal transition\u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanism is unknown.\u003c/p\u003e\u003cp\u003eHigh Content Screening (HCS) has played a crucial role in the discovery of drug target genes. This research technique allows for simultaneous genetic manipulations, including interference, knockout, or overexpression, of at least 20 or even more coding genes or non-coding RNAs. By using high-content imaging instruments to scan plates rapidly for multiple days, researchers can observe and capture the proliferation status of cells after regulating different genes. This method enables the screening of genes associated with proliferation.\u003c/p\u003e\u003cp\u003eGiven the therapeutic potential of melittin in HepG2 and Huh7 cells, our study aimed to elucidate the biological mechanism underlying melittin-induced inhibition of cell growth in these cell lines. We treated the tumor cells with melittin and analyzed the protein sequencing results of the treatment group and the control group to identify differentially expressed genes. Further screening and validation were conducted using HCS test.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Melittin\u003c/h2\u003e\u003cp\u003eMelittin (C131H229N39O31, \u0026gt;\u0026thinsp;97% purity) was a product of Santa Cruz Biotechnology (Santa Cruz, CA, USA). It was dissolved in distilled water to make a stock solution and then stored at \u0026minus;\u0026thinsp;20℃ until use avoiding repeated freezing and thawing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell Culture\u003c/h2\u003e\u003cp\u003eThe human liver cancer cell lines, Huh7 and Hep G2, were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (all from Hyclone, Life Sciences, Logan, UT, USA) at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 CCK-8 assays\u003c/h2\u003e\u003cp\u003eCCK-8 assays were performed to assess the proliferative properties of the control group and the melittin treatment groups (0\u0026micro;g/ml, 5\u0026micro;g/ml, 10\u0026micro;g/ml, 15\u0026micro;g/ml, 20\u0026micro;g/ml) following the manufacturer's instructions. Cells were seeded into 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and incubated at 37\u0026deg;C for 24 or 48 hours. Subsequently, the optical density (OD) values at 450 nm wave length were measured using a Multiskan FC (Thermo Fisher Scientific, Inc.). The inhibition rate was calculated as follows: Inhibition rate = (1 - OD_treatment group / OD_negative control group) \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Protein Extraction\u003c/h2\u003e\u003cp\u003eProtein Extraction Using the SDT Lysis Method: Specifically, samples were mixed with an appropriate amount of SDT lysis buffer and subjected to a boiling water bath for 15 minutes. The mixture was then centrifuged at 14,000g for 15 minutes, and the supernatant was collected. Protein quantification was subsequently performed using the BCA method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell Transfection\u003c/h2\u003e\u003cp\u003eCell lines were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS) and transfected using Lipofectamine 2000 (Invitrogen).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RNA extraction and realtime quantitative PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using TRIzol reagent, and cDNA was synthesized with the PrimeScript\u0026trade; RT Master Mix kit according to the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR was then carried out using the SYBR Premix Ex Taq kit (Takara, Shiga, Japan). GAPDH served as the internal control for normalization. All experiments were conducted in triplicate. Gene expression levels were calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method, a widely accepted approach for relative quantification in real-time PCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western Blotting Analysis\u003c/h2\u003e\u003cp\u003eCells were lysed on ice using cold RIPA lysis buffer (EpiZyme, China) supplemented with protease inhibitors. The extracted proteins were separated by 10% or 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated overnight at 4\u0026deg;C with primary antibodies against LARS2 (1:1000), ZNF19 (1:1000), and GAPDH (1:3000). After three washes with TBST, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 hours. Protein bands were visualized using an ECL detection kit (EpiZyme, China). All antibodies were purchased from ABclonal Technology Co., Ltd.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical Analyses\u003c/h2\u003e\u003cp\u003eWe conducted our statistical analysis using SPSS version 21 (IBM Corp., Armonk, NY, USA) and R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). To compare the differences between two groups, we applied the Wilcoxon rank-sum test. For comparisons among multiple groups, we used one-way ANOVA followed by Dunnett\u0026rsquo;s post hoc test. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Melittin Inhibits the Viability of Liver Cancer Cells in a CCK-8 Assay\u003c/h2\u003e\u003cp\u003eWe first investigated the effect of melittin on the viability of liver cancer cells. Optical density (OD) values were obtained for both the control group and the melittin-treated group at varying concentrations (0 \u0026micro;g/ml, 5 \u0026micro;g/ml, 10 \u0026micro;g/ml, 15 \u0026micro;g/ml, 20 \u0026micro;g/ml). Results from the CCK8 test indicated that melittin significantly inhibited the viability of Huh7 and HepG2 cells at 24 and 48 hours. The inhibitory rates of melittin on Huh7 and HepG2 hepatoma cells increased in a dose-dependent manner \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Proteome Signatures of Groups Between Melittin Treatment(10 \u0026micro;g/ml)and Control in Huh7 Liver Cancer Cells\u003c/h2\u003e\u003cp\u003eBased on previous research findings, we discovered that melittin exhibits a concentration-dependent inhibitory effect on liver cancer cells. In subsequent experiments, liver cancer cells (Huh7) were treated with 10 \u0026micro;g/ml melittin and compared to a control group of untreated parental liver cancer cells. Proteomics techniques were employed to further analyze the differentially expressed proteins between the two groups.\u003c/p\u003e\u003cp\u003eTo clarify the underlying reasons for the distinct protein expression between the melittin treatment group (ME) and the control (NC) group, we screened for proteins with expression differences between them. As a result, a volcano plot identified 142 upregulated and 8 downregulated proteins (Wilcoxon rank-sum test, cutoff ratio (ME/NC)\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.85, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Enrichment analysis of differentially expressed proteins between the ME and NC groups was conducted. Three duplicate samples were annotated above the heatmap, and the significantly upregulated or downregulated genes are marked in the figure \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Pathway enrichment showed that the integral component of the membrane was the top category in Cellular Component, while ATP-dependent microtubule motor activity and growth factor activity were the top two categories in Molecular Function. Additionally, negative regulation of transporter activity was the main function in Biological Process \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Phosphoproteome Signatures of Groups Treated with Melittin (10 \u0026micro;g/ml) Compared to Control in Huh7 Liver Cancer Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, after protein sequencing, we further conducted protein phosphorylation sequencing on melittin-treated Huh7 liver cancer cells and compared the results with the untreated control group. The results showed 88 upregulated and 21 downregulated proteins in the melittin-treated group compared to the control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Enrichment analysis of differentially expressed proteins between the ME and NC groups identified the significantly upregulated and downregulated proteins, which are marked in the figure \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Pathway enrichment using their corresponding phosphoproteins showed that the integral component of the plasma membrane was the top category in Cellular Component. Protein domain specific binding and cell adhesive protein binding were the main functions in Molecular Function, while regulation of protein localization to the membrane was the main function in Biological Process \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Genes such as shLARS2 and shZNF19 Showed Proliferation Inhibition in the HCS test\u003c/h2\u003e\u003cp\u003eHigh Content Screening (HCS) is a technique that allows for simultaneous genetic manipulations, such as interference, knockout, or overexpression, of multiple coding genes or non-coding RNAs. This process is followed by functional phenotype detection at the cellular level to identify genes with significant functions. In this study, HCS technology was employed to screen 13 candidate genes at the cellular level in liver cancer cells (Huh7). Among the 13 genes tested, the shLARS2 and shZNF19 groups showed a fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5. These genes were identified as positive hits in this experiment (proliferation inhibition positive cell groups, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Further Confirmed shLARS2 and shZNF19 Proliferation Inhibition Function in HCS test\u003c/h2\u003e\u003cp\u003eIn subsequent experiments, three RNA interference targets were designed for each of the previously screened target genes (LARS2 and ZNF19). Three plasmids, each carrying a different target, were packaged into lentiviruses. Using viral infection technology, stable cell lines were constructed and subjected to further HCS screening. The results showed RNA interference target genes such as LARS2 and ZNF19 significantly inhibited the proliferation of liver cancer cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 In Vitro Experiments Demonstrate That Melittin Treatment Suppresses the Expression of LARS2 and ZNF19\u003c/h2\u003e\u003cp\u003eTo further confirm the association between melittin treatment and the expression of LARS2 and ZNF19, we conducted real-time quantitative PCR and Western blot analyses comparing the melittin-treated and control groups. The results showed that the expression levels of LARS2 and ZNF19 were significantly downregulated in the melittin-treated group. Moreover, the findings from both PCR and Western blot experiments were consistent \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results suggest that melittin inhibits the growth of hepatocellular carcinoma Huh7 cells by downregulating LARS2 and ZNF19.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors, with high prevalence in Asia and Africa\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. A similar trend has been observed in the United States and the United Kingdom, where the incidence of HCC has increased substantially over the past two decades\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In recent years, there have been significant advances in the treatment of liver cancer. However, the overall survival rate remains unsatisfactory\u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Additionally, the molecular mechanism behind HCC metastasis remains unclear, and additional research is still required to explore this mechanism\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that melittin inhibits HCC cell viability and migration, and can also suppress the metastasis of liver cancer cells\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, liver cancer cells were treated with melittin, and the proteomic profiles of these cells were compared with those of untreated liver cancer cells to identify proteins with differential expression between the two groups. Subsequent genes were selected based on literature screening. Previous studies employing probe technology have localized nine KOX zinc finger genes to specific regions on four human chromosomes. In situ hybridization of cDNA probes to metaphase chromosomes revealed that KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Prior research has explored the role of KCMF1 in renal cell carcinoma, revealing a discrepancy in the formation of ubiquitin ligase and autophagosomes mediated by KCMF1, as well as in ionic concentration in tumor cells. This discrepancy may be one of the possible factors driving cancer evolution\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. KCMF1 plays a crucial role in the proliferation, migration, and invasion of trophoblast cells, with a similar trend observed in human colon cancer stem cells. This suggests that KCMF1 and the 14-3-3σ protein may influence the proliferation and colony formation of these cells\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. SPTLC1 suppresses cell growth, and previous evidence suggests a significant decrease in its expression in RCC tissues compared to non-tumor tissues. Similarly, other studies have indicated that decreased SPTLC1 expression is predictive of poorer outcomes in ccRCC patients\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Previous research has shown that LARS2 enhances the expression of E2F1, which mediates its effects on cell proliferation and apoptosis. This regulatory mechanism suggests that LARS2 could be a promising therapeutic target for treating Alzheimer's disease (AD)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Previous research has identified TNFRSF21 as a potential target for necroptosis in osteosarcoma therapy. Other evidence suggests that the circ_TNFRSF21/miR-214-3p/CHI3L1 axis could serve as promising diagnostic markers or therapeutic targets for cutaneous squamous cell carcinoma (cSCC)\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Knockdown of TRAF5 was found to inhibit HCC cell viability, colony formation, migration, invasion, and survival. This study concludes that silencing TRAF5 enhances necroptosis in hepatocellular carcinoma by inhibiting LTBR-mediated NF-κB signaling\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Researchers have investigated the function of PATZ1 in patients with glioblastoma, revealing that low PATZ1 expression correlates with poor prognosis in these patients. Moreover, overexpression of PATZ1 inhibits glioma cell proliferation and induces apoptosis by activating intrinsic apoptotic pathways\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. ELF3 has been implicated in gallbladder cancer, with in vivo experiments providing further evidence that reducing ELF3 expression enhances the gemcitabine sensitivity of GBC cells and prolongs the survival of mice with orthotopically xenografted tumors. The study concludes that ELF3 promotes gemcitabine resistance through the PKMYT1/CDK1 signaling pathway in gallbladder cancer\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Previous evidence indicates that Bclaf1 levels increase in hypoxia in a manner dependent on HIF-1α. Additionally, it identifies Bclaf1 as a novel positive regulator of HIF-1α in the hypoxic microenvironment\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHigh-content screening was employed to identify kinase inhibitors that can overcome venetoclax resistance in activated CLL cells. These results were further confirmed through protein sequencing in different groups\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In this study, we compared Huh7 liver cancer cells treated with Melittin to untreated cells using proteomic and phosphoproteomic sequencing techniques to identify differentially expressed proteins. Subsequent experiments on HCS test further validated these findings. Knocking down the target genes LARS2 and ZNF19 significantly inhibited the proliferation of liver cancer cells, suggesting that these genes play important roles in the proliferation of liver cancer cells and may be potential targets for liver cancer treatment.\u003c/p\u003e\u003cp\u003eLARS2 variants have been implicated in Perrault syndrome. Evidence from muscle tissue in a child with reversible myopathy revealed a reduction in LARS2 expression and mitochondrial complex I levels, along with an atypical form of degeneration\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Additional studies have demonstrated an association between LARS2 mutations and premature ovarian insufficiency (POI). Silencing LARS2 was shown to suppress cell proliferation while promoting apoptosis in granulosa cells (GCs). Mechanistically, LARS2 knockdown led to mitochondrial dysfunction and an accumulation of reactive oxygen species (ROS)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Further investigations have identified a role for YAP in enhancing mitochondrial oxidative phosphorylation (OXPHOS) by upregulating LARS2 transcription. As LARS2 function relies heavily on its substrate amino acid, leucine, a combination of a low-leucine diet and YAP inhibition synergistically impaired mitochondrial function in TI-Tregs, ultimately restricting tumor growth\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical approval：Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent to participate：Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent to publish：Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed for the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The Scientific Research Program of Shanghai Pudong New Area Health Commission (the General Program) (grant No. PW2021A-49 and PW2024A-58).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanli Zhang, Hainan Yang, designed the experiments and wrote the manuscript. Hui Liu,Hui Ye, helped in reviewing, acquiring, analysis for the work.. Zhongming Ye, did the statistical analysis. Xiang LV, Ming Leirevised critically the manuscript for important intellectual content. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1\u0026nbsp;Department of Critical Care Medicine, Seventh People\u0026apos;s Hospital of Shanghai University of Traditional Chinese Medicine, 358 Datong Road, Pudong New District, Shanghai, 200137, China.\u003c/p\u003e\n\u003cp\u003e2Department of Critical Care Medicine, \u003cstrong\u003eShanghai Pudong New Area Gongli Hospital 219 Miaopu Road, Shanghai, 200135,\u0026nbsp;\u003c/strong\u003eChina.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhn S, Hyeon J, Park CK: Metadherin is a prognostic predictor of hepatocellular carcinoma after curative hepatectomy. \u003cem\u003eGut Liver\u0026nbsp;\u003c/em\u003e2013, 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LG, Rudinger-Thirion J, Frugier M, Wilson M, Luig M, Alahakoon TI, Nixon CY, Kirk EP, Roscioli T, Lunke S\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: The expanding LARS2 phenotypic spectrum: HLASA, Perrault syndrome with leukodystrophy, and mitochondrial myopathy. \u003cem\u003eHum Mutat\u0026nbsp;\u003c/em\u003e2020, 41(8):1425-1434.\u003c/li\u003e\n \u003cli\u003eBai J, Yan M, Xu Y, Wang Y, Yao Y, Jin P, Zhang Y, Qu Y, Niu L, Li H: YAP enhances mitochondrial OXPHOS in tumor-infiltrating Treg through upregulating Lars2 on stiff matrix. \u003cem\u003eJ Immunother Cancer\u0026nbsp;\u003c/em\u003e2024, 12(11).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, Melittin, High Content Screening, Proteome sequencing, Protein phosphorylation sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7154719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7154719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Although surgery remains the most effective treatment, more than half of patients experience recurrent liver metastases within two years. This highlights the need for new therapeutic strategies driven by basic research. In this study, we used CCK-8 assays to evaluate the proliferation of liver cancer cells treated with melittin compared to controls. Proteomic sequencing was performed to identify differentially expressed proteins in Huh7 cells following melittin treatment. Two target genes, \u003cem\u003eLARS2\u003c/em\u003e and \u003cem\u003eZNF19\u003c/em\u003e, were further investigated using High Content Screening (HCS) to assess their roles in cell proliferation. CCK-8 results showed that melittin significantly reduced the viability of Huh7 and HepG2 cells at 24 and 48 hours in a dose-dependent manner. Proteomic analysis identified 142 upregulated and 8 downregulated proteins, while phosphoproteomic analysis revealed 88 upregulated and 21 downregulated phosphoproteins in the melittin-treated group. HCS assays demonstrated that silencing \u003cem\u003eLARS2\u003c/em\u003e and \u003cem\u003eZNF19\u003c/em\u003e significantly inhibited liver cancer cell proliferation. These findings suggest that melittin suppresses the growth of Huh7 cells partly through downregulating \u003cem\u003eLARS2\u003c/em\u003eand \u003cem\u003eZNF19\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Melittin Inhibits the Growth of Hepatocellular Carcinoma Huh7 Cells by Downregulating LARS2 and ZNF19","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:12:44","doi":"10.21203/rs.3.rs-7154719/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5754c551-43c7-4e08-b4b0-147e0ecf4caf","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T06:38:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 06:12:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7154719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7154719","identity":"rs-7154719","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}