Gene Expression Profiles Analysis On Hepatocellular Carcinoma And Gallbladder Carcinoma; Study On The Single Nucleotide Polymorphism Of Genes In Hepatocellular Carcinoma

Part 1 Study on the single nucleotide polymorphism of AFP andHNF1A genes in hepatocellular carcinomaSingle nucleotide polymorphism(SNP) is one of DNA variant at the genome, occurring in the encoding and non-encoding region, which is closely related to the development and progression of the diseases. The aim of this study was to search for the association of SNPs located in AFP and HNF1 A genes with HCC susceptibility and the underlying regulation mechanism. 1058 cases samples from Chinese Han population were recruited and genotyped using polymerase chain reaction–ligation detection reaction(PCR-LDR) method. AFP gene SNP loci rs4018, rs6834059 and rs4024, as well as the HNF1 A gene SNP site rs73539 were included in this study. A case control study was designed to evaluate the association between these SNP sites and HBV infection, HBV related HCC. Wild-type and mutant-type Luciferase reporter gene for rs4024 and rs735396 were constructed, and the activities of these vectors were detected. Moreover, the effect of rs4024 and rs735396 polymorphisim on transcriptional regulation was further validated by EMSA assay in vitro. Results: rs6834059 and rs4024 of AFP gene was statistically correlated with patients with or without cirrhosis and TNM stage, respectively. The frequencies of genotype GG of rs6834059 were significantly higher in patients with cirrhosis than those without cirrhosis, while the frequencies of genotype GG of rs4024 were significantly higher in patients with later TNM stage than those with early TNM stage. There was a significant correlation between the rs735396 polymorphism and the susceptibility to HCC related HBV, and the patients with GG genotype was connected with higher risk of HCC. Luciferase reporter gene assay showed that the GG genotypes of rs4204 and rs735396 exhibited a higher transcriptional activities. In addition, EMSA showed that there were some difference in binding to nuclear extract between genotype GG and AA of rs4204. Conclusion: AFP and HNF1 A gene polymorphisms were significantly correlated with the susceptibility and disease progression of HCC. Further functional studies revealed the roles of those SNPs in gene transcriptional regulation.Part 2 Comparative analysis of gene expression profiles ofhepatocellular carcinoma and cirrhosisLiver cirrhosis is an important risk factor for hepatocellular carcinoma(HCC), and it tends to progress to HCC and eventually leads to death. The aim of this study was to find out the Hub genes involved in the evolution of cirrhosis to HCC, and the role of these genes in the disease process by comparing the gene expression data between HCC and cirrhosis. We downloaded 5 datasets about HCC and cirrhosis from the datebase of Gene Expression Omnibus(GEO). Gene expression data of HCC and normal liver tissue samples was combined as dataset HCC, while the cirrhosis tissue samples data was defined as dataset C. A weighted gene coexpression network was constructed for dataset HCC with package WGCNA of R. Modules were identified by cluster analysis with package flash Clust and dynamic Tree Cut. Hub genes were screened out by calculating connectivity. Functional annotations were given for the hub genes in module yellow and module-specific genes with DAVID(Database for Annotation, Visualization and Integration Discovery, http://david.abcc.ncifcrf.gov/) and functional annotation networks were visualized with Cytoscape. Results: After exclusion of outlier samples, 394 HCC samples and 29 normal samples were included in dataset HCC, while 215 cirrhosis samples were included in dataset C. A total of 6 modules were identified in the weighted gene coexpression network of dataset HCC: blue, brown, turquoise, green, red and yellow. Module blue, brown and turquoise had high preservation between dataset HCC and C, while module yellow exhibited lowest preservation. Genes in these modules have been found to be associated with transcription, mitosis, cation transportation, cation homeostasis, secretion and regulation of cyclase activity. Several hub genes of module yellow were cytokines, such as CCL22 and IL19, which might play important roles in the development of HCC. Conclusion: Gene expression profiles of HCC were compared with those of cirrhosis and many critical genes were identified, which might contribute to the progression of HCC. Further functional studies of these genes could improve the understanding about the pathogenesis of HCC.Part 3 Study on RNA-Seq of the gallbladder carcinomaGallbladder carcinoma is a relatively rare malignant tumor, which has the characteristics of high degree of malignancy, easy to early metastasis, difficult to early detection, not sensitive to chemotherapy drugs and so on. The purpose of this study was to explore the effect of differentially expressed genes between cancer and adjacent tissues on the formation and development of gallbladder cancer, and to find new therapeutic targets of Gallbladder carcinoma. First, 16 DEGs from RNA sequencing(RNA-Seq) data in the previous study from our team were selected. Then, these DEGs were validated in a cohort of 35 patients with gallbladder cancer by real-time fluorescence quantitative PCR(Real-time PCR). Moreover, the correlation between the differentially expressed genes and clinical characteristics was evaluated. Results: The expression of 12 DEGs in validated group was consistent with those in the RNA-Seq data. The expression level of BIRC5, TK1, TNNT1 and MMP9 is related to the prognosis of gallbladder carcinoma after surgery. There was a significant correlation between the expression of BIRC5 gene and TNM(tumor-node-metastasis) stage. In addition, we also found that there was a significant correlation between the levels of serum CA19-9 and TNNT1, MMP9 and CLIC3. Survival analysis showed that the patients with high expression of TK1 and MMP9 was associated with worse clinical outcome. Conclusion: In present study, it is found that several DEGs might play important role in molecular mechanism of gallbladder cancer. These DEGs not only have the potential to be a biomarker for the diagnosis and prognosis of gallbladder cancer, but also to find new ways to treat cancer in the futher.