Clinical Significance And Molecular Mechanism Of MiR-423-5p And HNRNPA1 In Hepatocellular Carcinoma

Chapter I Clinical significance and molecular mechanism of miR-423-5p in hepatocellular carcinomaBackground: Hepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system with a low 5-year survival rate.At present,the treatment of HCC is mainly surgical resection,but there are still a large number of patients with poor prognosis.Therefore,Improving the early diagnosis and prognosis of HCC and exploring more effective treatment methods are currently urgent problems to be solved.MicroRNAs(miRNAs)play a key role in the progression of many malignancies.Abnormal expression of miRNAs has significant clinical significance,which can regulate the process of tumor progression,and has great potential for improving tumor targeted therapy.In this study,we analyzed the miRNAs differential expression profiles of HCC in the GEO database.Finally,mir-423 was screened out,which has a significantly increased expression level in HCC,and further predicted its potential target gene TGFBR3.Bioinformatics analysis combined with literature reports,this study speculates that miR-423 may play its role in HCC by regulating the TGFBR3/PI3K/AKT pathway.The research results of this project will enrich the types of functional miRNAs in HCC,provide a new biomarker and potential therapeutic target for this disease,and provide new ideas and new methods for the treatment of HCC.Objective: This study evaluated the diagnostic and prognostic value of miR-423-5p and TGFBR3 through clinical studies.The biological functions and molecular mechanisms of miR-423-5p and TGFBR3 in the malignant progression of HCC were explored by combining bioinformatics analysis.Method: The miRNA-423-5p expressing abnormality in HCC was screened from the GEO database,and predict its target gene was TGFBR3.The FunRich software analyzed the PI3K/AKT signaling pathways involved in miR-423-5p and TGFBR3.Bioinformatics analyzed the expression levels and clinical significance of miR-423-5p and TGFBR3 in HCC-related data of GEO and TCGA databases.The expression relationship analysis and GSEA analysis of miR-423-5p and TGFBR3 in HCC were performed using LinkedOmics database.The PPI network of TGFBR3 protein was constructed by STRING database,and the KEGG signaling pathway involved in TGFBR3 was also analyzed.The bioinformatics analysis results were validated by retrospectively collecting tissue samples and clinical prognostic information of 132 patients with HCC.Quantitative real-time PCR(qRT-PCR)was used to evaluate miR-423-5p and TGFBR3 expression level in tissues of 132 HCC patients.The clinical significance of miR-423-5p and TGFBR3 was evaluated by receiver operating characteristic(ROC)analysis and Kaplan-Meier survival curve analysis and multivariate Cox regression analysis.In vitro cell experiments were performed to investigate the effects of miR-423-5p and TGFBR3 on cell behavior in hepatocarcinoma cells: cell proliferation was detected by MTT assay,cell migration and invasion were detected by Transwell chamber assay,cell apoptosis was detected by flow cytometry,and in vivo experiments were constructed.Tumor xenograft models were used to examine the effects of miR-423-5p and TGFBR3 on HCC tumor growth in vivo.Dual luciferase reporter assays and Western blotting assays were used to analyze the expression-directed regulatory relationship between miR-423-5p and TGFBR3 in HCC.Western blotting was performed to assess the key protein expression levels involved in the PI3K/AKT pathway in molecular mechanisms.The MTT assay,Transwell chamber assay,flow cytometry,etc.were used to detect whether miR-423-5p and TGFBR3 affect cell biological behavior through the PI3K/AKT pathway in HCC cells.Result: By analyzing and screening the four GEO data sets,we looked for the key gene miR-423 in HCC,and miR-423 was always highly expressed in the GEO database(GSE22058)/TCGA database.The results of Kaplan Meier-plotter database showed that the prognosis of patients with high expression of miR-423 was poor.The high expression of miR-423 in GSE20594 dataset was significantly correlated with BCLC staging and vascular invasion in HCC patients.The results of LinkedOmics database indicated that miR-423 expression in TCGA was significantly correlated with age,population,pathological grade,tumor T stage,tumor N stage,and overall survival prognosis of HCC patients(n=375).The results of GSEA analysis showed that miR-423 may be involved in the development of cell cycle,DNA replication,cancer-associated miRNA,p53 signaling pathway and other biological activities related to tumor development.The expression of miR-423-5p was significantly up-regulated in HCC tissues and HCC cell lines.miR-423-5p can be used as a diagnostic biomarker to distinguish between HCC tissues and non-cancerous tissues.The high expression of miR-423-5p was associated with the clinicopathological features of HCC patients and was significantly associated with the poor survival prognosis of HCC patients.High expression of miR-423-5p in HCC cells promotes cell proliferation,migration,invasion and inhibition of apoptosis in vitro,and promotes HCC tumor growth in vivo.The GEPIA,Kaplan Meier-plotter and CCLE database results show that TGFBR3 was lowly expressed in different human tumors and also associated with poor prognosis of various tumors.PPI network analysis enriched the TGFBR3 protein involved in the KEGG pathway associated with cancer development and progression.Bioinformatics analysis predicted that TGFBR3 might be a miR-423-5p target gene in HCC,and their expression relationships are negatively correlated in the LinkedOmics database.FunRich software analysis revealed that the PI3K/AKT signaling pathway may be involved in miR-423-5p and TGFBR3.Low expression of TGFBR3 was significantly associated with clinicopathological features and poor survival prognosis in patients with HCC.Overexpression of TGFBR3 in hepatocarcinoma cells can inhibit proliferation,migration,and promote apoptosis in vitro,and inhibit tumor growth in vivo.Dual luciferase reporter assay and Western blotting experiments demonstrated that TGFBR3 was a target gene of miR-423-5p in HCC,and overexpression of miR-423-5p can inhibit the expression of TGFBR3.High expression of miR-423-5p activates the PI3K/AKT signaling pathway and affects the biological activities of HCC cells.TGFBR3 can antagonize the biological function of miR-423-5p and the activation of PI3K/AKT signaling pathway by miR-423-5p in HCC.Conclusion: Abnormal expression of miR-423-5p and TGFBR3 can be used as candidate biomarkers for diagnosis and prognosis of HCC patients.miR-425-5p promotes tumor progression by activating TGFBR3-mediated PI3K/AKT signaling pathway.Chapter II Prognostic Value and Preliminary Molecular Mechanism of HNRNPA1 in Hepatitis B Virus-Associated Hepatocellular CarcinomaBackground: The high rate of chronic hepatitis B virus(HBV)infection in the Chinese population makes China's hepatocellular carcinoma(HCC)account for 55% of the world's new annual cases,making it one of the highest incidence areas of HCC in the world.However,the prognosis of surgical resection and liver transplantation still does not achieve the desired results.According to clinical data,approximately 30% of HCC patients have a negative AFP.Therefore,the discovery of other sensitive tumor biochemical markers and study its molecular mechanism is particularly important for patients with HCC.Previous studies have shown that HNRNPA1 was highly expressed in various human cancers and involved in the regulation of tumor development.There were relatively few reports on the functional role of HNRNPA1 in HBVassociated HCC.This study combined bioinformatics analysis to study the relationship between HNRNPA1 and HBV positive HCC(HBV-HCC)prognosis,and to explore the molecular mechanism of HNRNPA1 in HBV-HCC.we hope to provide a new therapeutic targets and prevention ideas for early intervention and personalized treatment of HCC.Objective: To study the clinical value and potential molecular mechanism of HNRNPA1 in HBV-HCC.Methods: This study first analyzed the expression of HNRNPA1 gene in HCC tissues and normal liver tissues using GEPIA database,GEO database and TCGA database,and predicted the relationship between HNRNPA1 expression and prognosis of HCC.The expression of HNRNPA1 was verified by reverse transcription-quantitative polymerase chain reaction(RTq PCR)and immunohistochemistry(IHC)to determine the relationship between HNRNPA1 expression and prognosis of HCC.HNRNPA1 co-expressed genes were screened from Linked Omics and c Bio Portal databases,GO and KEGG analyses were performed on HNRNPA1 co-expressed genes in DAVID 6.7 database,HNRNPA1 protein-protein interaction network(PPI network)was constructed in STRING database,and visualized by Cytoscape software.Finally,the relationship between HNRNPA1 and mi R-22 and EGFR signaling pathways in HBV-HCC was analyzed by dual luciferase reporter assay and Western blotting.Results: By analyzing data from databases such as GEPIA,GEO,and TCGA,HNRNPA1 expression was found to be significantly higher in HBV-positive HCC samples,and these results were supported by RT-q PCR and IHC validation.Survival analysis in the GSE14520 dataset and 151 HBV-HCC patients showed a significant reduction in overall survival(OS)in patients with high HNRNPA1 expression.HNRNPA1,which was highly expressed in the GSE14520 dataset,has a certain prognostic significance for HBV-HCC patients.In addition,Both GO and KEGG analyses demonstrated that HNRNPA1 co-expressed genes were involved in ribonucleoprotein complex biogenesis and assembly,RNA processing,translation,RNA splicing,etc.The PPI network shows that HNRNPA1 was co-expressed with SNRPD1,SNRPD2,POLR2 H and RBMX.Furthermore,Gene set enrichment analysis revealed that HNRNPA1 may regulate HCC progression by influencing the WNT signaling pathway and cell cycle,etc.Finally,the dual luciferase reporter assay confirmed that HNRNPA1 was a direct target gene of mi R-22,which is characterized by decreased luciferase activity and decreased HNRNPA1 expression in cells with overexpressing mi R-22.Western blotting results indicated that HNRNPA1 may play an oncogene role in HBV-associated HCC through EGFR signaling pathway.Conclusion: High expression of HNRNPA1 in HCC tissues was associated with HCC recurrence.Up-regulation of HNRNPA1 expression was associated with poor prognosis in patients with HBV-related HCC resection and can be used as a biomarker for prognosis in patients with HBV-associated HCC.Proteins such as SNRPD1,SNRPD2,POLR2 H and RBMX were positively correlated with HNRNPA1 protein expression,and there may be direct interactions.In HBV-associated HCC,HNRNPA1 is a direct negative regulatory target gene of mi R-22,and HNRNPA1 may act as an oncogene through EGFR signaling pathway,which may be a potential therapeutic target for HBV-related HCC patients.