Ring Finger Protein 141 (RNF141) Mediates Resistance to Sorafenib in Hepatocellular Carcinoma and Its Mechanisms

Ring Finger Protein 141 (RNF141) Mediates Resistance to Sorafenib in Hepatocellular Carcinoma and Its Mechanisms | 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 Ring Finger Protein 141 (RNF141) Mediates Resistance to Sorafenib in Hepatocellular Carcinoma and Its Mechanisms Sheng-Xiong Chen, Xiao-Li Xie, Ting Liu, Xiao-Xu Jin, Jun Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4219096/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 OBJECTIVE: This study aims to investigate the expression of ring finger protein 141 (RNF141) in hepatocellular carcinoma, its role in sorafenib resistance, and its possible mechanism. MATERIALS AND METHODS: The expression of RNF141 in the cancer and corresponding para-cancerous liver tissues of patients with hepatocellular carcinoma was detected using Immunohistochemistry (IHC) staining and Western blot. The liver cancer cell line (SMMC7721) and the sorafenib-resistant liver cancer cell line (SMMC7721-S) were transfected with lentivirus to overexpress or silence RNF141, and the IC50 of sorafenib was then measured. Flow cytometry and TUNEL staining were used to detect changes in cell apoptosis before and after overexpression and silencing of RNF141. The levels of the proliferation marker protein, proliferating cell nuclear antigen (PCNA), and the apoptosis marker protein, Cleaved PARP, were detected using Western blot. Additionally, a tumor xenograft model was constructed by subcutaneously injecting RNF141-knockdown SMMC7721 and SMMC7721-S stable transfected strains into nude mice. The study observed and recorded the shape, size, and weight of tumors in each group. Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC) staining of PCNA were used to verify the effect of RNF141 on the efficacy of sorafenib in vivo. Finally, digital gene expression profiling (DGE) was used to further screen the signaling pathways involved in RNF141-mediated HCC resistance to sorafenib. RESULTS: The study found that the expression of RNF141 was significantly higher in hepatocellular carcinoma tissues compared to corresponding paracancerous tissues (P<0.01), as shown by IHC staining results and Western blot analysis. Hepatocellular carcinoma cell lines that overexpress and silence RNF141, as well as sorafenib-resistant hepatocellular carcinoma cell lines, were successfully constructed. Overexpression of RNF141 resulted in an increase in the IC50 value of sorafenib in hepatocellular carcinoma cells, as well as the ability to resist sorafenib-induced proliferation inhibition and apoptosis. Conversely, silencing RNF141 resulted in a decrease in the IC50 value of sorafenib, and further enhanced sorafenib-induced proliferation inhibition and apoptosis. The digital gene expression profiling results were analysed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) signalling pathway enrichment analysis, which revealed a significant enrichment of the proteasome signalling pathway. CONCLUSION: RNF141 may contribute to sorafenib resistance in hepatocellular carcinoma through the proteasome signaling pathway. hepatocellular carcinoma sorafenib RNF141 E3 ubiquitin ligase ring finger structural domains Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatocellular carcinoma (HCC) is the most common type of primary hepatic carcinoma (PHC) encountered in clinical practice. It is currently the fourth most common malignant tumour and the second leading cause of tumour-related deaths in China 1 . In Europe and the United States, alcoholic liver disease is the primary cause of hepatocellular carcinoma, whereas in China, the incidence of hepatitis B and hepatocellular carcinoma is significantly higher. In fact, over 80% of hepatocellular carcinoma cases in China are due to the transformation of chronic hepatitis B or hepatitis C. Hepatocellular carcinoma is an insidious disease that progresses rapidly. Unfortunately, most patients are diagnosed in the middle to late stages, resulting in a low resection rate, high recurrence rate, and poor prognosis 2 . Treatment options for HCC depend on the patient's overall condition, with surgery being the most effective option for achieving the best prognosis. Ultrasound-guided radiofrequency ablation, hepatic artery embolization chemotherapy, radiotherapy, immunotherapy, and targeted therapy are effective treatment options for patients who cannot undergo surgery or are inoperable 3 . Sorafenib tosylate, a multi-targeted antitumor drug with dual antitumor effects, was developed by Bayer Pharmaceuticals in Germany. Sorafenib is the first molecularly targeted agent approved for the treatment of advanced HCC in the United States. It has been shown to increase patient survival time. However, resistance to sorafenib remains a significant problem, which can affect the prognosis of hepatocellular carcinoma 4 , 5 . Currently, there is a lack of molecular markers that can predict the efficacy of sorafenib in HCC. Therefore, further research on genes related to drug resistance is necessary. Ring finger protein (RNF protein) is a class of proteins with a RING structural domain. Studies have found that various RNF proteins are involved in the pathological development of many diseases, particularly in the development of malignant tumors, which cannot be ignored 6 , 7 , 8 , 9 . Some studies have shown that RNF is also involved in tumor resistance to chemotherapy and targeted therapy through various signaling pathways 10 , 11 . Previous studies conducted by the group have shown that RNF141 plays a role in the development of colorectal cancer as a pro-carcinogenic protein. It promotes tumor proliferation, invasion, and metastasis while inhibiting apoptosis. Hepatocellular carcinoma is classified as a malignant tumor of the digestive system. Therefore, it is important to investigate the expression of RNF141 in hepatocellular carcinoma and its role in the resistance of sorafenib. This investigation may provide new ideas and directions for the clinical treatment of hepatocellular carcinoma in the future. Materials and Methods Human liver tissues We selected twenty patients who underwent surgery for hepatocellular carcinoma in the Second Hospital of Hebei Medical University between June 2019 and June 2021. None of the patients had received comprehensive treatment such as hepatic artery embolization, radiotherapy, or radiofrequency ablation, and no tumors from other sites were found. Pathologically confirmed surgical samples were obtained from patients with hepatocellular carcinoma, including both the carcinoma and paracancerous liver tissues. Western blot analysis and immunohistochemical staining were performed on both the carcinoma and paracancerous liver tissues. Informed consent was obtained from all enrolled patients for this study. The Ethics Committee of the Second Hospital of Hebei Medical University approved the study (review resolution no. 2021-R440). Cell culture and lentiviral transfection The SMMC7721 human hepatocellular carcinoma cell line was obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The sorafenib-resistant SMMC7721-S human hepatocellular carcinoma cell line was purchased from Shanghai Aolu Biotechnology Co. The cells were cultured in DMEM (Invitrogen) high glucose medium supplemented with 10% fetal bovine serum (FBS, HyClone), 50 units/ml penicillin, and 50 µg/ml streptomycin. The culture was maintained statically at 37°C, 5% CO2, and 95% O2 in a constant temperature incubator. The cells were used within 6 months after resuscitation and were verified to be mycoplasma-free by STR analysis. Quality checks were routinely performed for morphology and growth curves. To generate stable RNF141 overexpressing cell lines, we screened for efficient RNF141 knockdown siRNA based on the transfection effect of RNF141-siRNA. The valid siRNA sequences were then packaged into shRNA lentiviral vectors by Shanghai Gemma Pharmaceuticals Technology Co. LV16 vectors were used as shRNA lentiviral vectors, with the element order of U6-shRNA-Luciferase 17-Puro, and insertion sequences of GGCATCACGGATCATGAAT. Lentiviruses overexpressing RNF141 were used as LV5 vector with the component sequence of EF-1aF/GFP & Puro, and the insertion sequences of the control viral vectors were all TTCTCCGAACGTGTCACGT (irrelevant sequences). Protein blot analysis Protein expression was compared using protein blotting with a rabbit polyclonal antibody RNF141 (1:30, Santa Cruz, USA). The cells were inoculated in cell culture flasks and cultured in 6-well plates until they reached 70% confluency. Subsequently, cell protein extracts were prepared according to the indicated treatments. Protein blotting was performed using 80–150 µg of protein extracts. The proteins were detected using ECL chemiluminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ). β-actin (abways,)protein was used as a control for upsampling. The protein blotting experiments were repeated at least three times. Determination of half inhibitory concentration (IC50) of compounds The IC50 of sorafenib in hepatocellular carcinoma was determined using the Cell Counting Kit for Cell Proliferation/Toxicity (CCK8). To evaluate cell growth at different concentrations of sorafenib, cells in logarithmic growth phase were seeded in 96-well plates and incubated with varying concentrations (5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0 µM) of sorafenib for 48 hours. The optical density (OD) values were measured by incubating the cells with both the hepatocellular carcinoma cell line (SMMC 7721) and the hepatocellular carcinoma-resistant cell line (SMMC 7721-S), followed by the application of CCK8 reagent for one hour. Each experiment was performed in triplicate. Histological and immunohistochemical analysis The sections were stained using hematoxylin and eosin, and immunofluorescence. Immunohistochemistry (IHC) was performed as follows: slides were routinely deparaffinized, rehydrated for antigen repair, and endogenous peroxidase was blocked using 3% hydrogen peroxide. Subsequently, the slides were closed and incubated with the primary antibody against RNF141 (dilution 1:1000, Proteintech, Wuhan, China) overnight at 4°C. The following day, they were incubated with polymer horseradish peroxidase-coupled anti-rabbit secondary antibody. The sections were then stained with 3,3′-diaminobenzidine, counterstained with hematoxylin, dehydrated, and blocked. Flow and TUNEL staining for apoptosis detection The cells were analyzed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) to analyze early/late apoptosis or necrotic cell death. We also detected total cell apoptosis using Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL) assay (Beyotime, Shanghai, China). Statistical analysis Normally distributed data were presented as mean and standard deviation (SD), and non-normally distributed data were displayed as median with interquartile range. Student's t-test was used to analyse the difference between two groups, while one-way ANOVA was utilised for the difference among diverse groups. All statistical analyses were performed using SPSS software version 19.0 (IBM, Armonk, NY, USA). P-values less than 0.05 were considered statistically significant. Results RNF141 expression is up-regulated in HCC cells and tissue samples This study aimed to determine the role of RNF141 in HCC and clarify its expression in hepatocellular carcinoma. IHC staining of RNF141 was performed on 20 pairs of human hepatocellular carcinoma tissues and their paraneoplastic liver tissues. The results showed a significant increase in RNF141 expression in hepatocellular carcinoma tissues compared to paraneoplastic tissues. The expression was observed in both the cytoplasm and nucleus of the cells (Fig. 1 A). The study quantified the expression of RNF141 protein in cancer tissues and corresponding paracancerous liver tissues of 20 cases of hepatocellular carcinoma using Western blot. The results showed that RNF141 expression was significantly higher in HCC than in paracancerous tissues (Fig. 1 B). The relationship between RNF141 and clinicopathological features such as gender, age, tumour size, tumour grade and microvascular invasion in 20 patients with hepatocellular carcinoma was also analysed. The study found that the expression of RNF141 was not associated with basic patient characteristics, such as gender and age. However, it was positively correlated with tumour grade and the presence of microvascular invasion. There was no significant correlation with other clinical characteristics of hepatocellular carcinoma patients (Table 1 ). Table 1 Correlation between RNF141 expression level and clinicopathological characteristics in 20 HCC patients Clinical characteristics No. of patients RNF141 relative expression ( \(\stackrel{-}{\varvec{x}}\) ± s ) P value All cases 20 (100%) Gender 0.761 Male 14 (70%) 1.21 ± 0.17 Female 6 (30%) 1.12 ± 0.25 Age 0.572 ≥ 60 15(75%) 1.07 ± 0.24 <60 5(25%) 1.24 ± 0.18 Differentiation 5cm 14(70%) 6(30%) 1.24 ± 0.13 1.05 ± 0.36 * P <0.05 Evaluation of hepatocellular carcinoma cells (SMMC7721) and hepatocellular carcinoma drug-resistant cell line (SMMC7721-S) for sorafenib sensitivity The protein expression level of RNF141 was detected by Western blot. It was found that the expression level of RNF141 was significantly higher in the sorafenib-resistant hepatocellular carcinoma strain (SMMC7721-S) compared to the hepatocellular carcinoma cells (SMMC7721) (Fig. 2 A). The IC50 of sorafenib was also detected using CCK-8 in the hepatocellular carcinoma cell line (SMMC7721) and the sorafenib-resistant cell line (SMMC 7721-S) with values of 2.55 µM and 5.18 µM, respectively (Fig. 2 B). The results indicate that SMMC 7721-S cells, which are resistant to human hepatocellular carcinoma, exhibit greater sensitivity to sorafenib. Overexpression of RNF141 in hepatocellular carcinoma cells enhances their resistance to sorafenib We infected a hepatocellular carcinoma cell line (SMMC7721) with Lv-RNF141 lentivirus and screened it with puromycin for 2 weeks. The Western blot assay successfully overexpressed RNF141 in hepatocellular carcinoma cells (SMMC7721) (Fig. 3 A). The CCK8 assay showed that overexpression of RNF141 increased the IC50 value of hepatocellular carcinoma cells (SMMC7721) to sorafenib from 2.59µ M to 4.68µ M (Fig. 3 B). The expression of the proliferation marker protein PCNA in hepatocellular carcinoma cells (SMMC7721) was examined by Western blot. It was found that overexpression of RNF141 largely counteracted the lowering effect of sorafenib on PCNA (Fig. 3 C). This suggests that overexpression of RNF141 was resistant to the inhibitory proliferative effect of sorafenib. Flow cytometry was used to detect apoptosis. The results of TUNEL staining were consistent with this. Overexpression of RNF141 was found to reduce apoptosis in hepatocellular carcinoma cells and resist apoptosis caused by sorafenib (Fig. 3 D) (Fig. 3 E). This was further validated by Western blot detection of the expression of apoptosis marker protein cleaved PARP. It was discovered that overexpression of RNF141 reduced cleaved PARP expression and also prevented the increase in cleaved PARP expression caused by sorafenib (Fig. 3 F). These findings indicate that overexpression of RNF14 is linked to sorafenib resistance. Silencing RNF141 resulted in reduced sorafenib resistance in hepatocellular carcinoma cells To investigate the effect of RNF141 on sorafenib resistance in hepatocellular carcinoma, we constructed a lentiviral vector (Lv-shRNF141) using si-RNF141 sequence to silence the RNF141 gene in a hepatocellular carcinoma sorafenib-resistant cell line (SMMC7721-S). The Western blot assay demonstrated successful silencing of RNF141 expression in hepatocellular carcinoma sorafenib-resistant cells (SMMC7721-S) by Lv-shRNF141 (Fig. 4 A). The IC50 value of SMMC7721-S against sorafenib decreased from 5.25 µM to 4.17 µM after RNF141 gene silencing, as shown by the CCK8 assay (Fig. 4 B). The expression of the proliferation marker protein PCNA in hepatocellular carcinoma cells was also examined by Western blot. It was discovered that inhibiting RNF141 while administering sorafenib treatment resulted in even greater inhibition of PCNA expression by sorafenib (Fig. 4 C). Flow cytometry revealed that treatment with sorafenib induced significant apoptosis in hepatocellular carcinoma cells, and the number of apoptotic cells increased further after silencing RNF141 expression (Fig. 4 D) (Fig. 4 E). Based on these findings, we detected the expression of cleaved PARP, an apoptosis marker protein, by Western blot. Both sorafenib treatments increased cleaved PARP expression, and silencing RNF141 expression while applying sorafenib treatment further increased cleaved PARP expression (Fig. 4 F). These results suggest that silencing RNF141 reduces sorafenib resistance and enhances its efficacy. Silencing RNF141 enhances the anti-tumour effect of sorafenib in vivo To investigate the effect of RNF141 on the anti-tumour efficacy of sorafenib in vivo, we constructed a xenograft tumour model in nude mice using a stable lentivirus-infected sorafenib-resistant cell line of hepatocellular carcinoma with silenced RNF141 (SMMC7721-S). It was discovered that either sorafenib treatment alone or silencing of RNF141 expression could inhibit tumour growth. However, the most significant tumour-suppressive effect was observed when sorafenib treatment was combined with silencing of RNF141 expression (Fig. 5 A). The tumour volume and weight were measured and statistically analysed. The tumours of nude mice treated with silencing of RNF141 expression alone or application of sorafenib were reduced and lightened compared to the tumours of control nude mice. The most significant tumour suppression effect was observed with the combination of silencing of RNF141 expression and sorafenib treatment (Fig. 5 B, C). These results suggest that reducing RNF141 expression could decrease sorafenib drug resistance and enhance its anti-tumour effectiveness in nude mice. DGE-seq differential gene analysis, GO and KEGG pathway analysis Two DGE libraries (Lv-shNC + sorafenib, Lv-shRNF141 + sorafenib) were constructed for this study. Gene expression levels were quantitatively analyzed for each sample separately, and the distribution of gene expression levels in different samples is shown in box plots in Fig. 6 A. The study analysed the variability and correlation of each sample within and between groups. Correlation coefficients within and between groups of each sample were obtained based on the FPKM values of all genes in each RNA sample. The calculated correlation coefficients were displayed in the following heatmap (Fig. 6 B), which demonstrates the low variability of RNA samples between groups and the good reproducibility of the RNA samples within the groups. A total of 2417 genes with significant differences were detected in the comparison of the two groups, Lv-shNC + sorafenib and Lv-shRNF141 + sorafenib. Of these, 1250 genes were down-regulated and 1167 genes were up-regulated in the Lv-shRNF141 + sorafenib group (Fig. 6 C). The volcano plot in Fig. 6 D visualises the distribution of differentially expressed genes between the two groups, Lv-shNC + sorafenib and Lv-shRNF141 + sorafenib. The FPKM values of genes underwent mainstream hierarchical clustering analysis. The resulting heatmap clearly distinguished the Lv-shRNF141 + sorafenib group's gene expression patterns from those of the Lv-shNC + sorafenib group (Fig. 6 E). The 30 most significant GO classifications were selected from the results of GO enrichment analysis (Fig. 6 F). KEGG pathway enrichment was performed using padj < 0.05 as the threshold for significant enrichment. The resulting (Fig. 6 G) shows the KEGG signalling pathways with the most significant enrichment, represented by a histogram. Further exploration of RNF141 and its downstream signalling pathways in sorafenib resistance in hepatocellular carcinoma cells will follow. Discussion Primary liver cancer (PLC) is a malignant tumour of the gastrointestinal tract. The pathological types of PLC include hepatocellular carcinoma (HCC), cholangiocellular carcinoma, and mixed hepatocellular carcinoma 12 . HCC is the most common type in clinical practice, with chronic hepatitis B virus infection being the main cause of primary liver cancer in China. Primary liver cancer typically presents no noticeable symptoms in its early stages. Once symptoms do appear and the patient seeks medical attention, the cancer may have already progressed to an advanced stage, making surgical treatment impossible and resulting in a very poor prognosis 13 . For intermediate and advanced liver cancer, effective treatments include hepatic artery interventional embolisation chemotherapy, ultrasound and CT-guided radiofrequency ablation (RFA) of tumours combined with immuno-targeting. However, recurrence and metastasis of hepatocellular carcinoma are common after treatment, resulting in a 5-year survival rate of less than 30% 14,15 . Over the past decade, the introduction of targeted drug sorafenib for advanced liver cancer has benefited patients by delaying their survival 16 . In the last five years, research on targeted drugs for liver cancer has rapidly developed, resulting in the introduction of other first-line targeted drugs such as lenvatinib, and second-line drugs including regorafenib, cabozantinib, and ramucirumab 17 , 18 . However, most patients with advanced disease still have limited benefit, and drug resistance often develops. The ubiquitin-proteasome system regulates important physiological activities, including cell proliferation, differentiation, apoptosis, and DNA repair 19 . The E3 ubiquitin ligase (E3) mediates ubiquitin translocation, controlling the fate or subcellular localization of ubiquitinated proteins by precisely recognizing E2 ubiquitin-conjugating enzyme (E2) substrates 20 . The E3 gene is often disrupted in tumours, resulting in the malfunction of tumour suppressors and contributing to genetic or oncogenic cell transformation and tumour progression 21 . The Ring-finger (RNF) structural domain is a member of the E3 ubiquitin ligase protein family 22 . The Ring structural domain is present in over 700 proteins, but only a small proportion of them function as E3 ubiquitin ligases 23 . RNF141 is located on the short arm of human chromosome 11, specifically in region 1, band 5, sub-band 4. It contains six exons and encodes a ring finger protein 141 with a molecular weight of 26 kDa, which includes a ring finger structural domain 24 . Several RNF proteins have been found to play closely related roles in the pathological process of malignant tumour development 25 . RNF38, a member of the RNF protein family, is abundantly found in the human testis, and its dysfunction has been linked to various human disorders, particularly tumours 26 . Upregulation of RNF38 expression was found to promote cell invasion and metastasis in non-small cell lung cancer by inducing cell-epithelial-parenchymal transformation (EMT) 27 . Research has demonstrated that RNF2, a ubiquitination ligase, is expressed at higher levels in breast cancer tissues compared to paracancerous tissues 28 . Additionally, a study found that RNF24 mRNA expression was significantly elevated in hepatocellular carcinoma compared to normal liver tissues, and RNF24 protein was more highly expressed in hepatocellular carcinoma tissues than in corresponding normal liver tissues. According to a study, the high expression of RNF24 in hepatocellular carcinoma is closely related to the clinical stage and tumour grade of patients 29 . Previous studies have shown a close relationship between RNF and tumour cell proliferation, apoptosis, invasion, and metastasis 30 . The functions of RNF vary in different types of cancer, with some serving inhibitory and others serving promotional functions, depending on the ubiquitinated substrates of RNF 31 , 32 , 33 . Research has demonstrated that RNF2 expression is significantly increased in tumour cells of gastric cancer patients, and knockdown of RNF2 inhibits tumour cell viability by affecting the cell cycle 34 . In both in vivo and in vitro experiments, it was discovered that RNF220 is significantly upregulated in colorectal cancer 35 . Furthermore, cell scratch and plate clone formation experiments demonstrated that RNF220 promotes the proliferation, migration, and invasion of colorectal cancer cells. Subsequently, a subcutaneous xenograft tumour mouse model was established, which revealed that RNF220 promotes tumour growth in vivo 35 . This upregulation promoted invasion and metastasis of both types of cancer cells by increasing the epithelial mesenchyme. In hepatocellular carcinoma, RNF38 degraded neuroblastoma cells, which had a clear inhibitory effect on the TGF-β signalling pathway, through ubiquitylation 36 . The expression of RNF38 was upregulated in both hepatocellular carcinoma and NSCLC. In hepatocellular carcinoma, RNF38 degrades neuroblast differentiation-associated protein (AHNAK) through ubiquitination. AHNAK has a clear inhibitory effect on the TGF-β signalling pathway and can be used as an indicator of poor prognosis and recurrence of hepatocellular carcinoma 37 . Studies have reported that RNF93 plays a role as a tumour suppressor in the development of malignant tumours 38 . The expression of RNF93 is down-regulated in colon cancer tissues due to its interaction with cytoskeletal dynein proteins such as coronin 1B, cortical actin, and filament-binding LIM protein 1 through its B-box 2. Additionally, its PRY domain-mediated homology II causes an aggregation effect that is co-localized on focal adhesion protein. This significantly inhibits the migration of colon cancer cells and plays a role as a tumor suppressor in the occurrence and development of colon cancer 39 . It has been reported in the literature that the RNF family is closely associated with resistance to chemotherapeutic agents such as cisplatin, oxaliplatin, adriamycin, and targeted agents like cetuximab 40 , 41 . Sorafenib is a multikinase inhibitor that promotes apoptosis, attenuates angiogenesis, and inhibits tumour cell proliferation. It is currently an effective first-line treatment for advanced hepatocellular carcinoma (HCC). However, the development of sorafenib resistance is becoming increasingly common due to its targeting of multiple kinase pathways. Therefore, further research is needed to address this issue. Recent studies have shown that the development of sorafenib resistance in HCC and HCC progression involve epigenetics, transporter processes, regulated cell death, and the tumour microenvironment 42 . Sorafenib can activate pathways such as PI3K/Akt and JAK/STAT, EMT, and tumour hypoxia, leading to acquired resistance 43 . However, there have been relatively few studies conducted on the relationship between RNF and resistance to the targeted agent sorafenib. The study found that the expression level of RNF141 in hepatocellular carcinoma tissues was significantly higher than in the corresponding paraneoplastic liver tissues. High expression of RNF141 was closely related to the tumour grade of the patients and the presence or absence of microvascular invasion. These findings are consistent with previous studies on other RNF proteins of the same family. Additional ex vivo experiments demonstrated that overexpression of RNF141 hindered sorafenib-induced apoptosis and impeded cell proliferation. Conversely, silencing of RNF141 significantly improved the therapeutic efficacy of sorafenib. The xenograft tumour model also indicated that the combination of sorafenib and silencing of RNF141 significantly inhibited tumour growth compared to sorafenib alone or silencing of RNF141 treatment. The text describes the role of RNF141 in mediating resistance to sorafenib in hepatocellular carcinoma cells. Digital gene expression profiling in sorafenib-resistant SMMC7721-S cells with silencing of RNF141 revealed that RNF141 may also mediate the development of sorafenib resistance in hepatocellular carcinoma cells through the proteasome signalling pathway. In summary, this study found that RNF141 can contribute to resistance to sorafenib in hepatocellular carcinoma cells. Silencing RNF141 can enhance the efficacy of sorafenib. The molecular mechanism of RNF141 and sorafenib resistance in hepatocellular carcinoma was further elucidated. Further in-depth studies will be conducted to investigate whether RNF141 mediates sorafenib resistance in hepatocellular carcinoma cells through the proteasome signalling pathway. Sorafenib is the primary targeted therapeutic agent for patients with advanced hepatocellular carcinoma. Therefore, silencing RNF141 expression has the potential to become a new target for hepatocellular carcinoma treatment, improving the therapeutic effect of sorafenib on hepatocellular carcinoma. Conclusion sorafenib is the first-line treatment for patients with advanced HCC, However, resistance to sorafenib remains a significant problem, which can affect the prognosis of hepatocellular carcinoma.we found that RNF141 may contribute to sorafenib resistance in hepatocellular carcinoma through the proteasome signaling pathway. we proposed that the down-regulation of RNF141 is of great significance for the application of sorafenib in patients with advanced HCC. Declarations Conflict of interest The authors declare that they have no relevant financial or non-financial interests to disclose that are relevant to the content of this article. Ethical approval HCC tissues and matched adjacent tissues of patients were collected for protein analysis. Written informed consent was obtained from all patients and our study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University and all animal procedures were approved by the ethics committee of the Second Hospital of Hebei Medical University (approval letter No.: 2021-R440). Informed Consent and Consent to Participate Written informed consent has been obtained from patients. Funding Supported by Medical Science Research Project Plan of Hebei Province(NO.20220086) Acknowledgements I gratefully acknowledge the contribution of our research group for providing us the technical assistance and I am especially grateful to my master tutor Dr. Huiqing Jiang for his help in my study. Authors ， contribution Chen SX, Xie XL and Jiang HQconceived, designed the study. Chen SX, ie XL ,Liu T and Wang YJ and YJW performed most experiments, analyzed the data, wrote the manuscript and edited the paper. Xie XL and Jiang HQ helped to supervised the study. Jin XX,Hao ZJ helped to perform the experiments and analyzed the data. Xie XL and Jiang HQ helped to edited the paper. References Siegel RL, Miller KD, Fuchs HE, Jemal A（2022）Cancer statistics, 2022. CA Cancer J Clin72:7-33. https://doi.org/10.3322/caac.21708. Bruix J, Reig M, Sherman M（2016）Evidence-Based Diagnosis, Staging, and Treatment of Patients With Hepatocellular Carcinoma. 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Bioengineered 12:12060-12069. https://doi.org/: 10.1080/21655979.2021.2003664. Peng R, Zhang PF, Yang X, Wei CY, Huang XY, Cai JB, Lu JC, Gao C, Sun HX, Gao Q, Bai DS, Shi GM, Ke AW, Fan J（2019）Overexpression of RNF38 facilitates TGF-β signaling by Ubiquitinating and degrading AHNAK in hepatocellular carcinoma. J Exp Clin Cancer Res 38:113. https://doi.org/ 10.1186/s13046-019-1113-3. Xiong D, Zhu SQ, Wu YB, Jin C, Jiang JH, Liao YF, Long X, Wu HB, Xu JJ, Li JJ, Ding JY（2018）Ring finger protein 38 promote non-small cell lung cancer progression by endowing cell EMT phenotype. J Cancer 9:841-850. https://doi.org/10.7150/jca.23138. Cheng Y, Yan Z, Liu Y, Liang C, Xia H, Feng J, Zheng G, Luo H（2014）Analysis of DNA methylation patterns associated with the gastric cancer genome. Oncol Lett 7:1021-1026. https://doi.org/10.3892/ol.2014.1838. Lee OH, Lee J, Lee KH, Woo YM, Kang JH, Yoon HG, Bae SK, Songyang Z, Oh SH, Choi Y（2015）Role of the focal adhesion protein TRIM15 in colon cancer development. Biochim Biophys Acta 1853:409-21. https://doi.org/10.1016/j.bbamcr.2014.11.007. Long Y, Zhao Q, Huang Y（2021）RNF38 enhances 5-Fluorouracil resistance in colorectal cancer by activating the Wnt pathway.J BUON 26:1246-1251. Yang B, Ke W, Wan Y, Li T（2021）Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer. Biochem Biophys Res Commun 545:89-97. https://doi.org/10.1016/j.bbrc.2 021.01.046. Zhu YJ, Zheng B, Wang HY, Chen L（2017）New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta Pharmacol Sin 38:614-622. https://doi.org/10.1038/aps.2017.5. Niu L, Liu L, Yang S, Ren J, Lai PBS, Chen GG（2017）New insights into sorafenib resistance in hepatocellular carcinoma: Responsible mechanisms and promising strategies. Biochim Biophys Acta Rev Cancer 1868:564-570. https://doi.org/10.1016/j.bbcan.2017.10.002. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4219096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288613759,"identity":"08e58e15-ae1c-4aeb-9787-8cd34b579226","order_by":0,"name":"Sheng-Xiong Chen","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Xiong","middleName":"","lastName":"Chen","suffix":""},{"id":288613761,"identity":"4e76aa9e-7f4d-47b4-bb27-65e87b4f62c5","order_by":1,"name":"Xiao-Li Xie","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University, Hebei Institute of Gastroenterology","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Li","middleName":"","lastName":"Xie","suffix":""},{"id":288613763,"identity":"f892b246-b839-4df9-bdd0-698962b66874","order_by":2,"name":"Ting Liu","email":"","orcid":"","institution":"The First Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Liu","suffix":""},{"id":288613765,"identity":"54787585-07e4-4244-91ec-0a6be674de3c","order_by":3,"name":"Xiao-Xu Jin","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University, Hebei Institute of Gastroenterology","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Xu","middleName":"","lastName":"Jin","suffix":""},{"id":288613766,"identity":"96328586-0659-46f8-9559-b5c29211a426","order_by":4,"name":"Jun Wang","email":"","orcid":"","institution":"Shijiazhuang People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wang","suffix":""},{"id":288613768,"identity":"83602131-2ae5-46c4-aa44-10e9f96d9d00","order_by":5,"name":"Huiqing Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACxmYQ0cAgZ8DAAxFoIFaLMfFaYMoSNxCthbmd+dnDrzus07ez9x7dzMNgI7vhAPOzB/gdxmZuLHsmPXdnz7m02zwMacYbDrCZGxDwi5m0ZNvh3A03csyAWg4nbjjAwyaBXwv7N5CWdIP7b0Ba/hOjhcdM8mPb4QSDGzwgLQeI0lImzdiWbrjhTF7azTkGycYzD7OZ4dVi2H98m+TPNmt5g+Nnj914U2En23e8+Rl+LQ3AgOZhYIZyQUHFjEc5CMiDHPeDoLJRMApGwSgY0QAA38ZKgTXelZQAAAAASUVORK5CYII=","orcid":"","institution":"The Second Hospital of Hebei Medical University, Hebei Institute of Gastroenterology","correspondingAuthor":true,"prefix":"","firstName":"Huiqing","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-04-04 16:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4219096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4219096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54394308,"identity":"1020edc7-66c6-4e91-90d4-cad9907143ad","added_by":"auto","created_at":"2024-04-09 21:11:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":455790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of RNF14 in HCC and corresponding adjacent liver tissues \u003c/strong\u003e(A) Representive microscopic feature of HCC and adjacent non-tumor tissues in HE-stained liver sections of patient (magnification, ×200). Immunohistochemical staining (magnification, ×200) showing the expression and localization of RNF141 in liver tissues from HCC patients. (B)Protein expressions of RNF141 in tissues of HCC and corresponding adjacent liver. Date represented the means ± SD, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 \u003cem\u003evs.\u003c/em\u003e adjacent normal group.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/95518a5c267937acdb9d940c.jpg"},{"id":54394336,"identity":"c259eda7-cb4c-49c1-b7b6-5def5b63c378","added_by":"auto","created_at":"2024-04-09 21:11:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":218544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein expressions of RNF141 and the IC50 values of sorafenib in hepatoma SMMC7721-S and hepatoma SMMC7721cells.\u003c/strong\u003e(A)Wester nblot analysis showed the protein expression levels of RNF141 in hepatocarcinoma SMMC7721-S and hepatoma SMMC7721cells.(B) CCK-8 assays were used to detect the IC\u003csub\u003e50\u003c/sub\u003e values of sorafenib in 2 human HCC cells (SMMC7721,SMMC7721-S).Data are normalized to β-actin bar charts indicate the means ± SD from at a minimum of three consecutive independent trials, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs.\u003c/em\u003e indicated groups.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/25572e7a04406b3ea96299f9.jpg"},{"id":54394344,"identity":"87f3bb95-164a-4f39-a9ba-5d04e787737d","added_by":"auto","created_at":"2024-04-09 21:11:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":698801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of RNF141 in hepatocellular carcinoma cells enhances their resistance to sorafenib.\u003c/strong\u003e(A) Western blot analysis showed the protein expression levels of RNF141 (normalized to β-actin) in Lv-NC and Lv-RNF141 groups of SMMC7721 cells.(B) Effect of RNF141 overexpression on the sorafenib IC50 values in HCC SMMC7721 cells. (C) Western blot analysis showed PCNA protein expression in Lv-NC ± sorafenib and Lv-RNF141 ± sorafenib groups of hepatocarcinoma（SMMC7721）cells. (D) TUNEL staining (10×) of SMMC7721 cells in Lv-NC ± sorafenib and Lv-RNF141 ± sorafenib groups. (E) The apoptosis analysis was conducted by flow cytometry in Lv-NC ± sorafenib/Lv-RNF141 ± sorafenib groups of SMMC7721 cells. (F) Western blot analysis showed cleaved PARP protein expression in Lv-NC ± sorafenib/Lv-RNF141 ± sorafenib groups of SMMC7721 cells.Data were normalized to β-actin and measured by scanning densitometry. Bar charts represented the means ± SD from at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. indicated groups.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/5a880ff888fcfca58ef4ed8c.jpg"},{"id":54394303,"identity":"7219e65a-abb4-44b5-8e1f-4f15acc630c8","added_by":"auto","created_at":"2024-04-09 21:11:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":687158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing RNF141 resulted in reduced sorafenib resistance in hepatocellular carcinoma cells.\u003c/strong\u003e(A) Western blot analysis showed the protein expression levels of RNF141 in Lv-shNC and Lv-shRNF141 groups of HCC（SMMC7721-S）cells. (B) Effect of silencing RNF141 on the sorafenib IC50 values in HCC （SMMC7721-S） cells .(C) Western blot analysis showed PCNA protein expressions in Lv-shNC ± sorafenib and Lv-shRNF141 ± sorafenib groups of SMMC7721-S cells. (D) TUNEL staining (10×) of SMMC7721-S cells in Lv-shNC ± sorafenib and Lv-shRNF141 ± sorafenib groups. (E) The apoptosis analysis was conducted by flow cytometry in Lv-shNC ± sorafenib/Lv-shRNF141 ± sorafenib groups of SMMC7721-S cells. (F) Western blot analysis showed cleaved PARP protein expression in Lv-shNC ± sorafenib/Lv-shRNF141 ± sorafenib groups of SMMC7721-S cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. indicated groups.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/de2124864ecd94844a10db54.jpg"},{"id":54394279,"identity":"0c96fe58-3195-4145-9a1c-e7a56fc1bc67","added_by":"auto","created_at":"2024-04-09 21:11:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing RNF141 enhances the anti-tumour effect of sorafenib in vivo.\u003c/strong\u003e(A) (A) Morphologies of collected tumors in subcutaneous HCC SMMC7721-S xenografts in nude mice. (B) Curves of tumor growth. (C) Tumor weights were measured after collection in each group. Bar charts represented the means ± SD from at least three independent experiments, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. indicated groups.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/c71cb6400e52227ba7d708b3.jpg"},{"id":54394306,"identity":"b4e5c9bb-eb43-4ac3-9459-3a3a5933a9ca","added_by":"auto","created_at":"2024-04-09 21:11:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":660388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDGE-seq differential gene analysis, GO and KEGG pathway analysis.\u003c/strong\u003e(A) Box plot of the gene expression distribution, the abscissa in the figure is the sample name, and the ordinate is log2(FPKM+1). (B) Heatmap of the correlation between samples, the horizontal and vertical coordinates are the square of the correlation coefficient of each sample. (C) Statistical histogram of the number of differential genes in sample comparison combinations. green, blue and gray respectively indicate total, down-regulated and up-regulated differential genes, and the number on the column indicates the number of differential genes. (D) Volcano map of the differential gene, the abscissa is the log2FoldChange value, the ordinate is -log10padj, and the blue dotted line represents the threshold line of the differential gene screening criteria. (E) Cluster analysis of differentially expressed genes (DEGs). (F) The enrichment analysis of GO functions .(G) Pathway enrichment analysis displayed the top 20 signaling pathways in KEGG.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/5e9af784a6ab6452e5c683ee.jpg"},{"id":55264456,"identity":"2d216a23-655c-481b-8a16-98c93640b83a","added_by":"auto","created_at":"2024-04-25 01:44:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1282795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4219096/v1/b17e038a-9a56-49f3-bacd-19031f168dcc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ring Finger Protein 141 (RNF141) Mediates Resistance to Sorafenib in Hepatocellular Carcinoma and Its Mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is the most common type of primary hepatic carcinoma (PHC) encountered in clinical practice. It is currently the fourth most common malignant tumour and the second leading cause of tumour-related deaths in China \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In Europe and the United States, alcoholic liver disease is the primary cause of hepatocellular carcinoma, whereas in China, the incidence of hepatitis B and hepatocellular carcinoma is significantly higher. In fact, over 80% of hepatocellular carcinoma cases in China are due to the transformation of chronic hepatitis B or hepatitis C. Hepatocellular carcinoma is an insidious disease that progresses rapidly. Unfortunately, most patients are diagnosed in the middle to late stages, resulting in a low resection rate, high recurrence rate, and poor prognosis \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Treatment options for HCC depend on the patient's overall condition, with surgery being the most effective option for achieving the best prognosis. Ultrasound-guided radiofrequency ablation, hepatic artery embolization chemotherapy, radiotherapy, immunotherapy, and targeted therapy are effective treatment options for patients who cannot undergo surgery or are inoperable \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Sorafenib tosylate, a multi-targeted antitumor drug with dual antitumor effects, was developed by Bayer Pharmaceuticals in Germany. Sorafenib is the first molecularly targeted agent approved for the treatment of advanced HCC in the United States. It has been shown to increase patient survival time. However, resistance to sorafenib remains a significant problem, which can affect the prognosis of hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Currently, there is a lack of molecular markers that can predict the efficacy of sorafenib in HCC. Therefore, further research on genes related to drug resistance is necessary.\u003c/p\u003e \u003cp\u003eRing finger protein (RNF protein) is a class of proteins with a RING structural domain. Studies have found that various RNF proteins are involved in the pathological development of many diseases, particularly in the development of malignant tumors, which cannot be ignored \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Some studies have shown that RNF is also involved in tumor resistance to chemotherapy and targeted therapy through various signaling pathways \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previous studies conducted by the group have shown that RNF141 plays a role in the development of colorectal cancer as a pro-carcinogenic protein. It promotes tumor proliferation, invasion, and metastasis while inhibiting apoptosis. Hepatocellular carcinoma is classified as a malignant tumor of the digestive system. Therefore, it is important to investigate the expression of RNF141 in hepatocellular carcinoma and its role in the resistance of sorafenib. This investigation may provide new ideas and directions for the clinical treatment of hepatocellular carcinoma in the future.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman liver tissues\u003c/h2\u003e \u003cp\u003eWe selected twenty patients who underwent surgery for hepatocellular carcinoma in the Second Hospital of Hebei Medical University between June 2019 and June 2021. None of the patients had received comprehensive treatment such as hepatic artery embolization, radiotherapy, or radiofrequency ablation, and no tumors from other sites were found. Pathologically confirmed surgical samples were obtained from patients with hepatocellular carcinoma, including both the carcinoma and paracancerous liver tissues. Western blot analysis and immunohistochemical staining were performed on both the carcinoma and paracancerous liver tissues.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003ewas obtained from all enrolled patients for this study. The Ethics Committee of the Second Hospital of Hebei Medical University approved the study (review resolution no. 2021-R440).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and lentiviral transfection\u003c/h2\u003e \u003cp\u003e The SMMC7721 human hepatocellular carcinoma cell line was obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The sorafenib-resistant SMMC7721-S human hepatocellular carcinoma cell line was purchased from Shanghai Aolu Biotechnology Co.\u003c/p\u003e \u003cp\u003eThe cells were cultured in DMEM (Invitrogen) high glucose medium supplemented with 10% fetal bovine serum (FBS, HyClone), 50 units/ml penicillin, and 50 \u0026micro;g/ml streptomycin. The culture was maintained statically at 37\u0026deg;C, 5% CO2, and 95% O2 in a constant temperature incubator. The cells were used within 6 months after resuscitation and were verified to be mycoplasma-free by STR analysis. Quality checks were routinely performed for morphology and growth curves.\u003c/p\u003e \u003cp\u003eTo generate stable RNF141 overexpressing cell lines, we screened for efficient RNF141 knockdown siRNA based on the transfection effect of RNF141-siRNA. The valid siRNA sequences were then packaged into shRNA lentiviral vectors by Shanghai Gemma Pharmaceuticals Technology Co. LV16 vectors were used as shRNA lentiviral vectors, with the element order of U6-shRNA-Luciferase 17-Puro, and insertion sequences of GGCATCACGGATCATGAAT. Lentiviruses overexpressing RNF141 were used as LV5 vector with the component sequence of EF-1aF/GFP \u0026amp; Puro, and the insertion sequences of the control viral vectors were all TTCTCCGAACGTGTCACGT (irrelevant sequences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein blot analysis\u003c/h2\u003e \u003cp\u003eProtein expression was compared using protein blotting with a rabbit polyclonal antibody RNF141 (1:30, Santa Cruz, USA). The cells were inoculated in cell culture flasks and cultured in 6-well plates until they reached 70% confluency. Subsequently, cell protein extracts were prepared according to the indicated treatments. Protein blotting was performed using 80\u0026ndash;150 \u0026micro;g of protein extracts. The proteins were detected using ECL chemiluminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ). β-actin (abways,)protein was used as a control for upsampling. The protein blotting experiments were repeated at least three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of half inhibitory concentration (IC50) of compounds\u003c/h2\u003e \u003cp\u003eThe IC50 of sorafenib in hepatocellular carcinoma was determined using the Cell Counting Kit for Cell Proliferation/Toxicity (CCK8). To evaluate cell growth at different concentrations of sorafenib, cells in logarithmic growth phase were seeded in 96-well plates and incubated with varying concentrations (5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0 \u0026micro;M) of sorafenib for 48 hours. The optical density (OD) values were measured by incubating the cells with both the hepatocellular carcinoma cell line (SMMC 7721) and the hepatocellular carcinoma-resistant cell line (SMMC 7721-S), followed by the application of CCK8 reagent for one hour. Each experiment was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunohistochemical analysis\u003c/h2\u003e \u003cp\u003eThe sections were stained using hematoxylin and eosin, and immunofluorescence. Immunohistochemistry (IHC) was performed as follows: slides were routinely deparaffinized, rehydrated for antigen repair, and endogenous peroxidase was blocked using 3% hydrogen peroxide. Subsequently, the slides were closed and incubated with the primary antibody against RNF141 (dilution 1:1000, Proteintech, Wuhan, China) overnight at 4\u0026deg;C. The following day, they were incubated with polymer horseradish peroxidase-coupled anti-rabbit secondary antibody. The sections were then stained with 3,3\u0026prime;-diaminobenzidine, counterstained with hematoxylin, dehydrated, and blocked.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow and TUNEL staining for apoptosis detection\u003c/h2\u003e \u003cp\u003eThe cells were analyzed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) to analyze early/late apoptosis or necrotic cell death. We also detected total cell apoptosis using Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL) assay (Beyotime, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNormally distributed data were presented as mean and standard deviation (SD), and non-normally distributed data were displayed as median with interquartile range. Student's t-test was used to analyse the difference between two groups, while one-way ANOVA was utilised for the difference among diverse groups. All statistical analyses were performed using SPSS software version 19.0 (IBM, Armonk, NY, USA). P-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNF141 expression is up-regulated in HCC cells and tissue samples\u003c/h2\u003e \u003cp\u003eThis study aimed to determine the role of RNF141 in HCC and clarify its expression in hepatocellular carcinoma. IHC staining of RNF141 was performed on 20 pairs of human hepatocellular carcinoma tissues and their paraneoplastic liver tissues. The results showed a significant increase in RNF141 expression in hepatocellular carcinoma tissues compared to paraneoplastic tissues. The expression was observed in both the cytoplasm and nucleus of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The study quantified the expression of RNF141 protein in cancer tissues and corresponding paracancerous liver tissues of 20 cases of hepatocellular carcinoma using Western blot. The results showed that RNF141 expression was significantly higher in HCC than in paracancerous tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The relationship between RNF141 and clinicopathological features such as gender, age, tumour size, tumour grade and microvascular invasion in 20 patients with hepatocellular carcinoma was also analysed. The study found that the expression of RNF141 was not associated with basic patient characteristics, such as gender and age. However, it was positively correlated with tumour grade and the presence of microvascular invasion. There was no significant correlation with other clinical characteristics of hepatocellular carcinoma patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between RNF141 expression level and clinicopathological characteristics in 20 HCC patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRNF141 relative expression\u003c/p\u003e \u003cp\u003e(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{\\varvec{x}}\\)\u003c/span\u003e\u003c/span\u003e\u0026plusmn; s )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAll cases\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.761\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.21\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.12\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.572\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15(75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.07\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.24\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDifferentiation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;0.01\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI-II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (66.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.85\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII-IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (33.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.81\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMicrovascular invasion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.012\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (25.00%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.75\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(75.00%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.99\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0. 533\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;5cm\u003c/p\u003e \u003cp\u003e\u0026gt;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(70%)\u003c/p\u003e \u003cp\u003e6(30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003cp\u003e1.05\u0026thinsp;\u003cb\u003e\u0026plusmn;\u003c/b\u003e\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of hepatocellular carcinoma cells (SMMC7721) and hepatocellular carcinoma drug-resistant cell line (SMMC7721-S) for sorafenib sensitivity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe protein expression level of RNF141 was detected by Western blot. It was found that the expression level of RNF141 was significantly higher in the sorafenib-resistant hepatocellular carcinoma strain (SMMC7721-S) compared to the hepatocellular carcinoma cells (SMMC7721) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The IC50 of sorafenib was also detected using CCK-8 in the hepatocellular carcinoma cell line (SMMC7721) and the sorafenib-resistant cell line (SMMC 7721-S) with values of 2.55 \u0026micro;M and 5.18 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The results indicate that SMMC 7721-S cells, which are resistant to human hepatocellular carcinoma, exhibit greater sensitivity to sorafenib. \u003cb\u003eOverexpression of RNF141 in hepatocellular carcinoma cells enhances their resistance to sorafenib\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe infected a hepatocellular carcinoma cell line (SMMC7721) with Lv-RNF141 lentivirus and screened it with puromycin for 2 weeks. The Western blot assay successfully overexpressed RNF141 in hepatocellular carcinoma cells (SMMC7721) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The CCK8 assay showed that overexpression of RNF141 increased the IC50 value of hepatocellular carcinoma cells (SMMC7721) to sorafenib from 2.59\u0026micro; M to 4.68\u0026micro; M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The expression of the proliferation marker protein PCNA in hepatocellular carcinoma cells (SMMC7721) was examined by Western blot. It was found that overexpression of RNF141 largely counteracted the lowering effect of sorafenib on PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This suggests that overexpression of RNF141 was resistant to the inhibitory proliferative effect of sorafenib. Flow cytometry was used to detect apoptosis. The results of TUNEL staining were consistent with this. Overexpression of RNF141 was found to reduce apoptosis in hepatocellular carcinoma cells and resist apoptosis caused by sorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This was further validated by Western blot detection of the expression of apoptosis marker protein cleaved PARP. It was discovered that overexpression of RNF141 reduced cleaved PARP expression and also prevented the increase in cleaved PARP expression caused by sorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These findings indicate that overexpression of RNF14 is linked to sorafenib resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSilencing RNF141 resulted in reduced sorafenib resistance in hepatocellular carcinoma cells\u003c/h2\u003e \u003cp\u003eTo investigate the effect of RNF141 on sorafenib resistance in hepatocellular carcinoma, we constructed a lentiviral vector (Lv-shRNF141) using si-RNF141 sequence to silence the RNF141 gene in a hepatocellular carcinoma sorafenib-resistant cell line (SMMC7721-S). The Western blot assay demonstrated successful silencing of RNF141 expression in hepatocellular carcinoma sorafenib-resistant cells (SMMC7721-S) by Lv-shRNF141 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The IC50 value of SMMC7721-S against sorafenib decreased from 5.25 \u0026micro;M to 4.17 \u0026micro;M after RNF141 gene silencing, as shown by the CCK8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The expression of the proliferation marker protein PCNA in hepatocellular carcinoma cells was also examined by Western blot. It was discovered that inhibiting RNF141 while administering sorafenib treatment resulted in even greater inhibition of PCNA expression by sorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Flow cytometry revealed that treatment with sorafenib induced significant apoptosis in hepatocellular carcinoma cells, and the number of apoptotic cells increased further after silencing RNF141 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Based on these findings, we detected the expression of cleaved PARP, an apoptosis marker protein, by Western blot. Both sorafenib treatments increased cleaved PARP expression, and silencing RNF141 expression while applying sorafenib treatment further increased cleaved PARP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results suggest that silencing RNF141 reduces sorafenib resistance and enhances its efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSilencing RNF141 enhances the anti-tumour effect of sorafenib in vivo\u003c/h2\u003e \u003cp\u003eTo investigate the effect of RNF141 on the anti-tumour efficacy of sorafenib in vivo, we constructed a xenograft tumour model in nude mice using a stable lentivirus-infected sorafenib-resistant cell line of hepatocellular carcinoma with silenced RNF141 (SMMC7721-S). It was discovered that either sorafenib treatment alone or silencing of RNF141 expression could inhibit tumour growth. However, the most significant tumour-suppressive effect was observed when sorafenib treatment was combined with silencing of RNF141 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The tumour volume and weight were measured and statistically analysed. The tumours of nude mice treated with silencing of RNF141 expression alone or application of sorafenib were reduced and lightened compared to the tumours of control nude mice. The most significant tumour suppression effect was observed with the combination of silencing of RNF141 expression and sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). These results suggest that reducing RNF141 expression could decrease sorafenib drug resistance and enhance its anti-tumour effectiveness in nude mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDGE-seq differential gene analysis, GO and KEGG pathway analysis\u003c/h2\u003e \u003cp\u003eTwo DGE libraries (Lv-shNC\u0026thinsp;+\u0026thinsp;sorafenib, Lv-shRNF141\u0026thinsp;+\u0026thinsp;sorafenib) were constructed for this study. Gene expression levels were quantitatively analyzed for each sample separately, and the distribution of gene expression levels in different samples is shown in box plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. The study analysed the variability and correlation of each sample within and between groups. Correlation coefficients within and between groups of each sample were obtained based on the FPKM values of all genes in each RNA sample. The calculated correlation coefficients were displayed in the following heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), which demonstrates the low variability of RNA samples between groups and the good reproducibility of the RNA samples within the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 2417 genes with significant differences were detected in the comparison of the two groups, Lv-shNC\u0026thinsp;+\u0026thinsp;sorafenib and Lv-shRNF141\u0026thinsp;+\u0026thinsp;sorafenib. Of these, 1250 genes were down-regulated and 1167 genes were up-regulated in the Lv-shRNF141\u0026thinsp;+\u0026thinsp;sorafenib group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The volcano plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD visualises the distribution of differentially expressed genes between the two groups, Lv-shNC\u0026thinsp;+\u0026thinsp;sorafenib and Lv-shRNF141\u0026thinsp;+\u0026thinsp;sorafenib. The FPKM values of genes underwent mainstream hierarchical clustering analysis. The resulting heatmap clearly distinguished the Lv-shRNF141\u0026thinsp;+\u0026thinsp;sorafenib group's gene expression patterns from those of the Lv-shNC\u0026thinsp;+\u0026thinsp;sorafenib group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The 30 most significant GO classifications were selected from the results of GO enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). KEGG pathway enrichment was performed using padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the threshold for significant enrichment. The resulting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) shows the KEGG signalling pathways with the most significant enrichment, represented by a histogram. Further exploration of RNF141 and its downstream signalling pathways in sorafenib resistance in hepatocellular carcinoma cells will follow.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrimary liver cancer (PLC) is a malignant tumour of the gastrointestinal tract. The pathological types of PLC include hepatocellular carcinoma (HCC), cholangiocellular carcinoma, and mixed hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. HCC is the most common type in clinical practice, with chronic hepatitis B virus infection being the main cause of primary liver cancer in China. Primary liver cancer typically presents no noticeable symptoms in its early stages. Once symptoms do appear and the patient seeks medical attention, the cancer may have already progressed to an advanced stage, making surgical treatment impossible and resulting in a very poor prognosis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For intermediate and advanced liver cancer, effective treatments include hepatic artery interventional embolisation chemotherapy, ultrasound and CT-guided radiofrequency ablation (RFA) of tumours combined with immuno-targeting. However, recurrence and metastasis of hepatocellular carcinoma are common after treatment, resulting in a 5-year survival rate of less than 30% \u003csup\u003e14,15\u003c/sup\u003e. Over the past decade, the introduction of targeted drug sorafenib for advanced liver cancer has benefited patients by delaying their survival \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the last five years, research on targeted drugs for liver cancer has rapidly developed, resulting in the introduction of other first-line targeted drugs such as lenvatinib, and second-line drugs including regorafenib, cabozantinib, and ramucirumab \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, most patients with advanced disease still have limited benefit, and drug resistance often develops.\u003c/p\u003e \u003cp\u003eThe ubiquitin-proteasome system regulates important physiological activities, including cell proliferation, differentiation, apoptosis, and DNA repair \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The E3 ubiquitin ligase (E3) mediates ubiquitin translocation, controlling the fate or subcellular localization of ubiquitinated proteins by precisely recognizing E2 ubiquitin-conjugating enzyme (E2) substrates \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The E3 gene is often disrupted in tumours, resulting in the malfunction of tumour suppressors and contributing to genetic or oncogenic cell transformation and tumour progression \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The Ring-finger (RNF) structural domain is a member of the E3 ubiquitin ligase protein family \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The Ring structural domain is present in over 700 proteins, but only a small proportion of them function as E3 ubiquitin ligases \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. RNF141 is located on the short arm of human chromosome 11, specifically in region 1, band 5, sub-band 4. It contains six exons and encodes a ring finger protein 141 with a molecular weight of 26 kDa, which includes a ring finger structural domain \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Several RNF proteins have been found to play closely related roles in the pathological process of malignant tumour development \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. RNF38, a member of the RNF protein family, is abundantly found in the human testis, and its dysfunction has been linked to various human disorders, particularly tumours \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Upregulation of RNF38 expression was found to promote cell invasion and metastasis in non-small cell lung cancer by inducing cell-epithelial-parenchymal transformation (EMT) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Research has demonstrated that RNF2, a ubiquitination ligase, is expressed at higher levels in breast cancer tissues compared to paracancerous tissues \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, a study found that RNF24 mRNA expression was significantly elevated in hepatocellular carcinoma compared to normal liver tissues, and RNF24 protein was more highly expressed in hepatocellular carcinoma tissues than in corresponding normal liver tissues. According to a study, the high expression of RNF24 in hepatocellular carcinoma is closely related to the clinical stage and tumour grade of patients \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have shown a close relationship between RNF and tumour cell proliferation, apoptosis, invasion, and metastasis \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The functions of RNF vary in different types of cancer, with some serving inhibitory and others serving promotional functions, depending on the ubiquitinated substrates of RNF \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Research has demonstrated that RNF2 expression is significantly increased in tumour cells of gastric cancer patients, and knockdown of RNF2 inhibits tumour cell viability by affecting the cell cycle \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In both in vivo and in vitro experiments, it was discovered that RNF220 is significantly upregulated in colorectal cancer \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Furthermore, cell scratch and plate clone formation experiments demonstrated that RNF220 promotes the proliferation, migration, and invasion of colorectal cancer cells. Subsequently, a subcutaneous xenograft tumour mouse model was established, which revealed that RNF220 promotes tumour growth in vivo \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This upregulation promoted invasion and metastasis of both types of cancer cells by increasing the epithelial mesenchyme. In hepatocellular carcinoma, RNF38 degraded neuroblastoma cells, which had a clear inhibitory effect on the TGF-β signalling pathway, through ubiquitylation \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The expression of RNF38 was upregulated in both hepatocellular carcinoma and NSCLC. In hepatocellular carcinoma, RNF38 degrades neuroblast differentiation-associated protein (AHNAK) through ubiquitination. AHNAK has a clear inhibitory effect on the TGF-β signalling pathway and can be used as an indicator of poor prognosis and recurrence of hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Studies have reported that RNF93 plays a role as a tumour suppressor in the development of malignant tumours \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The expression of RNF93 is down-regulated in colon cancer tissues due to its interaction with cytoskeletal dynein proteins such as coronin 1B, cortical actin, and filament-binding LIM protein 1 through its B-box 2. Additionally, its PRY domain-mediated homology II causes an aggregation effect that is co-localized on focal adhesion protein. This significantly inhibits the migration of colon cancer cells and plays a role as a tumor suppressor in the occurrence and development of colon cancer \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It has been reported in the literature that the RNF family is closely associated with resistance to chemotherapeutic agents such as cisplatin, oxaliplatin, adriamycin, and targeted agents like cetuximab \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSorafenib is a multikinase inhibitor that promotes apoptosis, attenuates angiogenesis, and inhibits tumour cell proliferation. It is currently an effective first-line treatment for advanced hepatocellular carcinoma (HCC). However, the development of sorafenib resistance is becoming increasingly common due to its targeting of multiple kinase pathways. Therefore, further research is needed to address this issue. Recent studies have shown that the development of sorafenib resistance in HCC and HCC progression involve epigenetics, transporter processes, regulated cell death, and the tumour microenvironment \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Sorafenib can activate pathways such as PI3K/Akt and JAK/STAT, EMT, and tumour hypoxia, leading to acquired resistance \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. However, there have been relatively few studies conducted on the relationship between RNF and resistance to the targeted agent sorafenib.\u003c/p\u003e \u003cp\u003eThe study found that the expression level of RNF141 in hepatocellular carcinoma tissues was significantly higher than in the corresponding paraneoplastic liver tissues. High expression of RNF141 was closely related to the tumour grade of the patients and the presence or absence of microvascular invasion. These findings are consistent with previous studies on other RNF proteins of the same family. Additional ex vivo experiments demonstrated that overexpression of RNF141 hindered sorafenib-induced apoptosis and impeded cell proliferation. Conversely, silencing of RNF141 significantly improved the therapeutic efficacy of sorafenib. The xenograft tumour model also indicated that the combination of sorafenib and silencing of RNF141 significantly inhibited tumour growth compared to sorafenib alone or silencing of RNF141 treatment. The text describes the role of RNF141 in mediating resistance to sorafenib in hepatocellular carcinoma cells. Digital gene expression profiling in sorafenib-resistant SMMC7721-S cells with silencing of RNF141 revealed that RNF141 may also mediate the development of sorafenib resistance in hepatocellular carcinoma cells through the proteasome signalling pathway.\u003c/p\u003e \u003cp\u003eIn summary, this study found that RNF141 can contribute to resistance to sorafenib in hepatocellular carcinoma cells. Silencing RNF141 can enhance the efficacy of sorafenib. The molecular mechanism of RNF141 and sorafenib resistance in hepatocellular carcinoma was further elucidated. Further in-depth studies will be conducted to investigate whether RNF141 mediates sorafenib resistance in hepatocellular carcinoma cells through the proteasome signalling pathway. Sorafenib is the primary targeted therapeutic agent for patients with advanced hepatocellular carcinoma. Therefore, silencing RNF141 expression has the potential to become a new target for hepatocellular carcinoma treatment, improving the therapeutic effect of sorafenib on hepatocellular carcinoma.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003esorafenib is the first-line treatment for patients with advanced HCC, However, resistance to sorafenib remains a significant problem, which can affect the prognosis of hepatocellular carcinoma.we found that RNF141 may contribute to sorafenib resistance in hepatocellular carcinoma through the proteasome signaling pathway. we proposed that the down-regulation of RNF141 is of great significance for the application of sorafenib in patients with advanced HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no relevant financial or non-financial interests to disclose that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCC tissues and matched adjacent tissues of patients were collected for protein analysis. Written informed consent was obtained from all patients and our study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University and all animal procedures were approved by the ethics committee of the Second Hospital of Hebei Medical University (approval letter No.: 2021-R440).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent has been obtained from patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by Medical Science Research Project Plan of Hebei Province(NO.20220086)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI gratefully acknowledge the contribution of our research group for providing us the technical assistance and I am especially grateful to my master tutor Dr. Huiqing Jiang for his help in my study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e，\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChen SX, Xie XL and Jiang HQconceived, designed the study. Chen SX, ie XL ,Liu T and Wang YJ and YJW performed most experiments, analyzed the data, wrote the manuscript and edited the paper. Xie XL and Jiang HQ helped to supervised the study. Jin XX,Hao ZJ helped to perform the experiments and analyzed the data. Xie XL and Jiang HQ helped to edited the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A（2022）Cancer statistics, 2022. CA Cancer J Clin72:7-33. https://doi.org/10.3322/caac.21708.\u003c/li\u003e\n\u003cli\u003eBruix J, Reig M, Sherman M（2016）Evidence-Based Diagnosis, Staging, and Treatment of Patients With Hepatocellular Carcinoma. 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Biochim Biophys Acta Rev Cancer 1868:564-570. https://doi.org/10.1016/j.bbcan.2017.10.002.\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, sorafenib, RNF141, E3 ubiquitin ligase, ring finger structural domains","lastPublishedDoi":"10.21203/rs.3.rs-4219096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4219096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eOBJECTIVE:\u003c/strong\u003e \u0026nbsp;This study aims to investigate the expression of ring finger protein 141 (RNF141) in hepatocellular carcinoma, its role in sorafenib resistance, and its possible mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMATERIALS AND METHODS:\u003c/strong\u003e \u0026nbsp;The expression of RNF141 in the cancer and corresponding para-cancerous liver tissues of patients with hepatocellular carcinoma was detected using Immunohistochemistry (IHC) staining and Western blot. The liver cancer cell line (SMMC7721) and the sorafenib-resistant liver cancer cell line (SMMC7721-S) were transfected with lentivirus to overexpress or silence RNF141, and the IC50 of sorafenib was then measured. Flow cytometry and TUNEL staining were used to detect changes in cell apoptosis before and after overexpression and silencing of RNF141. The levels of the proliferation marker protein, proliferating cell nuclear antigen (PCNA), and the apoptosis marker protein, Cleaved PARP, were detected using Western blot. Additionally, a tumor xenograft model was constructed by subcutaneously injecting RNF141-knockdown SMMC7721 and SMMC7721-S stable transfected strains into nude mice. The study observed and recorded the shape, size, and weight of tumors in each group. Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC) staining of PCNA were used to verify the effect of RNF141 on the efficacy of sorafenib in vivo. Finally, digital gene expression profiling (DGE) was used to further screen the signaling pathways involved in RNF141-mediated HCC resistance to sorafenib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS:\u003c/strong\u003e The study found that the expression of RNF141 was significantly higher in hepatocellular carcinoma tissues compared to corresponding paracancerous tissues (P\u0026lt;0.01), as shown by IHC staining results and Western blot analysis. Hepatocellular carcinoma cell lines that overexpress and silence RNF141, as well as sorafenib-resistant hepatocellular carcinoma cell lines, were successfully constructed. Overexpression of RNF141 resulted in an increase in the IC50 value of sorafenib in hepatocellular carcinoma cells, as well as the ability to resist sorafenib-induced proliferation inhibition and apoptosis. Conversely, silencing RNF141 resulted in a decrease in the IC50 value of sorafenib, and further enhanced sorafenib-induced proliferation inhibition and apoptosis. The digital gene expression profiling results were analysed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) signalling pathway enrichment analysis, which revealed a significant enrichment of the proteasome signalling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION:\u003c/strong\u003e RNF141 may contribute to sorafenib resistance in hepatocellular carcinoma through the proteasome signaling pathway.\u003c/p\u003e","manuscriptTitle":"Ring Finger Protein 141 (RNF141) Mediates Resistance to Sorafenib in Hepatocellular Carcinoma and Its Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 21:10:44","doi":"10.21203/rs.3.rs-4219096/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":"61b32b1b-5383-4bd5-bb88-a5791bd75ab3","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-20T21:10:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-09 21:10:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4219096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4219096","identity":"rs-4219096","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}