Gene Expression Profile Analysis Of HCC Based On TCGA Database And Study On Expression Pattern And Mechanism Of ARHGEF39 In HCC

HCC(Hepatocellular carcinoma,HCC)is the world’s third largest cancer death because it is attributable to a variety of genetic mutations and epigenetic changes.The hepatitis b virus(HBV)infection,alcohol,aflatoxin,obesity and diabetes are other risk factors for HCC.Although there are many new methods for the diagnosis and treatment of liver cancer,the prognosis is still poor due to high recurrence and metastasis rates.An important reason for poor prognosis is the lack of an accurate prognosis grading system for HCC and an effective early HCC screening method.Therefore,it is reasonable to evaluate the value of potential biomarkers in the diagnosis and treatment of HCC.The mRNA expression profile of liver cancer tissues was analyzed based on the TCGA database.The mRNA expression data related to the prognosis of liver cancer were screened in the TCGA database,and then transcriptomic sequencing gene analysis was performed on the HCC samples in our hospital to find the differentially expressed genes that may be related to the prognosis.Then we focused on ARHGEF39 as the key gene in the follow-up and verified it.ARHGEF39,also known as C9orf100,is a member of the human Dbl family of guanine nucleotide exchange factors(RhoGEFs)which are important activators of Rho family small GTPases.The overexpression of ARHGEF39 has also been identified in various human malignancies,including lung cancer,gastric cancer,and HCC.Together these data suggest that ARHGEF39 might be frequently upregulated in different cancer type and associated with cancer progression.Previously,it was reported that the mRNA level of ARHGEF39 in HCC samples was significantly upregulated compared to that of adjacent non-tumor tissues.Increased expression of ARHGEF39 mRNA was positively correlated with the number of nodules and with serum alpha-fetoprotein(AFP)levels.However,we still have little understanding of the expression and prognostic relevance of ARHGEF39 protein in HCC.The objective of this research was to thoroughly analyze ARHGEF39 protein expression patterns and its potential role as diagnostic and prognostic biomarkers in HCC.ARHGEF39 expression level of HCC in The Cancer Genome Atlas(TCGA)dataset was analyzed.Quantitative realtime polymerase chain reaction and immunohistochemistry were employed to determine ARHGEF39 mRNA and Protein levels in our own study collected HCC tissues and matched non-cancerous tissues.Moreover,the association of ARHGEF3 9 expression with the clinicopathological factors and prognosis of HCC were investigated.The level of ARHGEF39 in HCC tissues was significantly higher than that in adjacent normal tissues(P<0.05)from TCGA database.High level of ARHGEF39 was a significant prognostic factor of poor overall survival(OS)(TCGA,P=0.006).Consistently,the expression levels of ARHGEF39 mRNA and protein in HCC specimens were significantly higher than those in adjacent liver specimens(P<0.05)from our cohort.The ARHGEF39 expression in carcinoma tissues revealed a positive correlation with the levels of serum alpha-fetoprotein.The over-expression of ARHGEF39 protein significantly correlates with advanced TNM stage.Further analysis revealed that high ARHGEF39 level was significantly associated with poor OS(P<0.001)and short disease-free survival(DFS)(P<0.001).Cox multivariate analysis indicated that ARHGEF39 was an independent,unfavorable prognostic factor(P=0.000)of OS and DFS.Further on the basis of the previous part of the study,We investigated the effect of ARHGEF39 on the growth and metastasis of hepatocellular carcinoma and its potential molecular mechanism,Long non-coding RNA(LncRNA)is defined as RNA molecules more than 200 nucleotides in length and without the protein-coding function.There is increasing evidence that lncRNA is involved in various regulatory processes of tumor cells,including differentiation,proliferation,apoptosis,metastasis,and stem cell characteristics.MiRNAs are a class of evolutionarily conserved short non-coding RNAs that can bind to the 3’ untranslated region(UTR)of mRNA for post-transcriptional regulation.MiRNAs have also been reported to play a role in a variety of cell processes including metastasis,apoptosis,proliferation,and differentiation.Growing evidence indicates that LncRNAs act as competing endogenous RNAs(ceRNAs)that bind with microRNAs(miRNAs)and regulate their downstream functions.According to bioinformatics analysis,we speculate that ARHGEF39 might promote HCC progression by competing with LINC00470 for the active site of mir-4500,The results suggested that LINC00470 functions as a competing endogenous RNA to regulate ARHGEF39 expression by sponging miR-4500 in HCC;In addition,rescue experiments showed that mir-4500 inhibitor could eliminate the effect of LINC00470 on inhibiting the progression of HCC cells.Therefore,we demonstrated that LINC00470 can be used as the ceRNA of mir-4500 to promote the progression of HCC by regulating the expression of ARHGEF39,which provides a potential therapeutic target for the treatment of HCC patients.Part 1:Analysis and screening of hepatocellular carcinoma related genes based on TCGA databaseObjective:Hepatocellular carcinoma(HCC)is a leading cause of cancer-related death.The aim of this study was to identify underlying hub genes and dysregulated pathways associated with the development of HCC using bioinformatics analysis.Methods:1.In this study,we systematically investigated the TCGA whole-transcriptome sequencing data of hepatocellular carcinoma(HCC)by comparing the global gene expression profiles between tumors and their corresponding nontumorous liver tissue.2.Differentially expressed protein-coding genes were subjected to transcriptome sequencing in 5 pairs of liver cancer tissue and matched adjacent non-cancerous tissue.3.We screened out the DEGs from the two platforms and by selecting the intersection of these two platforms,we identified the common DEGs in the sequencing data from different laboratories.4.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed,5.The candidate genes were validated using qPCR on our cohort of 19 pairs of human HCCs.6.Furthermore,The prognostic significance of these genes was evaluated in TCGA(The Cancer Genome Atlas)on patients with hepatocellular carcinoma(HCC).Reselts:1.According to the TCGA database,a total of 1588 differentially expressed geneswere screened out,including 1520 upregulated genes and 68 downregulated genes;Transcriptome sequencing Differentially expressed genes(DEGs)and their expression patterns using mRNA collected from tissue samples,A total of 1281 differentially expressed genes were screened between tumor and adjacent non-neoplastic liver tissue,including 873 upregulated genes and 408 downregulated genes.2.DEGs of each profile were obtained and screened.The intersection of each DEGs was obtained by Venn analysis,Finally,we obtained 341 upregulated and 3 1 downregulated DEGs in HCC.3.A Gene Ontology enrichment analysis showed that condensed nuclear chromosome outer kinetochore,Ndc80 complex,and chromosome passenger complex were significantly enriched in the intersection of DEGs.KEGG pathway analysis showed enrichment in gene DNA replication,cell cycle and other pathways4.Through our analysis,NT5DC2,ARHGEF39,SPSB2,SLC26A6 and SLC52A2 were identified as candidate genes for further study in human HCC samples.5.these 5 genes above mentioned were successfully validated using qPCR on our cohort of 19 pairs of human HCCs.6.The prognostic power of these genes was corroborated in the TCGA database and by Kaplan-Meier plotter(KM-plotter)analysis.Conclusion:This study provides useful information on the transcriptomic landscape and molecular mechanism of hepatocarcinogenesis for development of new biomarkers and further in-depth characterization.Part 2:Expression of Rho Guanine Nucleotide Exchange F actor 39(ARHGEF39)and Its Prognostic Significance in Hepatocellular CarcinomaObjective:Previous studies have reported that ARHGEF39 might be frequently upregulated in different cancer types and relevant to cancer progression.However,the expression pattern and clinicopathological features of ARHGEF39 in patients with hepatocellular carcinoma(HCC)needs further exploration.Methods:1.Data-mining from public databases and our own transcriptome data to study,ARHGEF39 expression level of HCC in The Cancer Genome Atlas(TCGA)dataset was analyzed.Association between ARHGEF39 level in hepatocellular carcinoma(HCC)tissues and prognosis of patients was analyzed by using data downloaded from The Cancer Genome Atlas(TCGA)database.2.The expression of ARHGEF39 in HCC and its adjacent tissues were verified by gene and protein levels,respectively3.Moreover,the association of ARHGEF39 expression with the clinicopathological factors and prognosis of HCC were investigated.Results:1.The level of ARHGEF39 in HCC tissues was significantly higher than that in adj acent normal tissues(P<0.05)from TCGA database.High level of ARHGEF39 was a significant prognostic factor of poor overall survival(OS)(TCGA,P=0.006).2.Consistently,the expression levels of ARHGEF39 mRNA and protein in HCC specimens were significantly higher than those in adj acent liver specimens(P<0.05)from our cohort.3.We investigated the correlations between the ARHGEF39 and the clinicopathological characteristics of patients and found that ARHGEF39 were significantly associated with TNM stage(P=0.040,χ2=4.654)and Serum alpha-fetoprotein(AFP)level(P=0.002,9.597)4.Further analysis revealed that high ARHGEF39 level was significantly associated with poor OS(P<0.001)and short disease-free survival(DFS)(P<0.001).Cox multivariate analysis indicated that ARHGEF39 was an independent,unfavorable prognostic factor(P=0.000)of OS and DFS.Conclusions:ARHGEF39 might act as an oncogene in the progression of HCC and might serve as a promising potential prognostic indicator and therapeutic target for HCC.Part 3:ARHGEF39 promotes hepatocellular carcinoma progression through competing with LINC00470 for miR-4500 biding sitesObjective:The aim of the present study was to identify the function of ARHGEF39 and its transcriptional regulatory mechanism in hepatocellular carcinoma(HCC).methods:1.The human hepatoma cell lines Hep3B and HepG2 were cultured according to standard methods.RNA levels of ARHGEF39,mir-4500 and LINC00470 were detected by qRT-PCR.2.Protein levels of ARHGEF39 and other related genes were detected by western blot.3.MTT assay and clonal assay were used to determine the cell viability4.Wound healing and Transwell assay were used to analyze the changes in cell migration and invasion.5.Caspase 3/7 activity assay and Nucleosome ELISA assay were used to examine the changes in cell apoptosis.6.Xenograft tumor in nude mice was used to evaluate the growth a of HCC cells in vivo.7.Luciferase reporter assay verified the direct interaction between miR-4500 and ARHGEF39 or LINC004708.RNA pull-down assay revealed a direct interaction between miR-4500 and ARHGEF39 or LINC00470Results:1.Down-regulation of ARHGEF39 expression Inhibits Proliferation,Invasion and Metastasis,and Induces Apoptosis in HCC cell lines.2.ARHGEF39 inhibition reduced tumor growth in vivo.3.ARHGEF39 is a direct downstream target of miR-4500 in HCC cells,ARHGEF39 was negatively regulated by mir-4500 in HCC cells.As a target protein of miR-4500,the expression level of ARHGEF39 can be inhibited by the over-expression of miR-4500,indicating a negative regulatory relationship between ARHGEF39 and miR-4500 in HCC cell lines.4.We provide evidence that LINC00470 acted as a "sponge" of miR-4500 to regulate the expression of ARHGEF39.5.ARHGEF39 is Regulated by LINC00470/miR-4500 Signal Axis and Promotes HCC Cell Progression and Metastasis.Conclusion:LINC00470 promotes the expression of ARHGEF39 through the adsorption of mir-4500 to regulate the malignant process of liver cancer;ARHGEF39,LINC00470,miR-4500 can be used as a potential target for the clinical diagnosis and treatment of liver cancer.