Screen Hepatocellular Carcinoma Association Copy Number Variation And Genes By Genome Wide Single Nucleotide Array

Part one Screen Hepatocellular Carcinoma Association Copy Number Variation by Genome Wide Single Nucleotide ArrayObjective:To search genome-wide recurrent CNV that could provide a genetic etiology mechanistic for HCC patients.Methods:1.We performed analysis of CNV using the Affymetrix SNP 500K arrays on 50 case (HbsAg positive with HCC) and 50 control (HbsAg positive with no HCC) peripheral blood samples.2. Five of the obtained CNV regions were verified in 282 cases (HbsAg positive with HCC) and 278 controls (HbsAg positive without HCC) peripheral blood using SYBR Green real-time PCR.Results:1.We identified 13 CNV regions,10 regions for copy number gain and 3 regions for copy number loss.2. Compare with database of DGVs and database of UCSC,4 novel regions are detected (gain of 2q14.3,6q16.1,8q23.2,17q22); 6 regions overlapped with the Database of Genomic Variants; 2 regions have been reported by some inherited disease in UCSC; 1 regions have been reported both in UCSC and DGVs.3. The three most frequent genomic alterations regions included gain of chromosome 2p12 (64%),5q34 (60%) as well as loss of 15q23 (62%).4. Five random selected CNV regions were verified by SYBR Green real-time PCR and they were all verified the result.5. Additionally, we also identified 14 genes associated HCC,5 new genes were first reported have association with HCC.Conclusions:Our study suggested that CNV might be potentially important for the HCC variation and might be used as a genetic marker to locate genes associated with HCC in Chinese population. Part two Analysis the relationship and functional classification among HCC related genes by networkObjective:To investigate the relationship and functional classification among HCC related genesMethods:Using GO, MAS3.0, DAVID, and STRING these four internet web to analysis the function of the 14 genes that associated with CNV and HCC, and detect these genes'signaling pathway, functional classification, protein networks, and interaction with other genes, thus to predict the probably action mode and pathway of these 14 genes in HCC.Results:The results showed the 14 genes involved in phosphor protein, plasma membrane part, sequence variant, alternative splicing, transcription, splice variant, plasma membrane, extracellular region, signal peptide, signal, mutagenesis site, disease mutation, plasma membrane part, transcription regulator activity, disulfide bond, signal, signal peptide, polymorphism, splice variant, glycoprotein. STRING analysis found two crucial nodes in the network:GABRG2, KIF2B were related to cancer.Conclusions:14 CNV associated genes may play an important role in HCC. Part three The relationship among NOVA1 gene, copy number variation segment of 14q12 and Hepatocellular carcinomaObjective:To investigate the relationship among copy number variation segment in 14q12, associated gene Neuro-oncological ventral antigen 1 (NOVA1) and the HCC.Methods:1. we use the SYBR Green real-time quantitative PCR in 282 case (HbsAg positive with HCC) and 278 control (HbsAg positive without HCC) peripheral blood samples to verify the copy number gain region in 14q12, which was detected by Affymetrix 500K SNP array; 2.Detect the NOVA1 protein expression in 120 HCC tissues by immunohistochemistry method; 3. Detect the copy number gain region of 14q12 and NOVA1 protein expression in 40 patients (HbsAg positive with HCC).Results:1.14q12 copy number variation region gain in 282 cases (HbsAg positive with HCC), comparing with the 278 control (HbsAg positive with no HCC) there is a statistical significance.2. Compared with the nomal tissue, NOVA1 protein expression was significantly increased in HCC tissues, and locus mainly in the nucleus.3. Both 14q12 copy number variation region and NOVA1 protein expression were detected in 40 HCC cases'tissues, and the result show that patients with 14q12 copy number amplification in their tissues the NOVA1 protein were also highly expressed, there is a statistical significance.Conclusions:Copy number gain region in 14q12 is associated with HCC, and its related gene NOVA1 is a tumor related genes, may play an important role in HCC. Part four Effection of NOVA1 gene's over expression on the human Hepatocellular carcinoma cellsObjective:To detect the effections of NOVA1 protein's over expression on the human hepatocellular carcinoma cells.Methods:At first, we detect NOVA1 protein's expression in six hepatocellular carcinoma cells and one normal liver cell by using reverse Transcription-Polymerase Chain Reaction (RT-PCR), to select the cell that has low NOVA1 protein expression; then followed by construction of NOVA1 gene over-expression plasmid, and transfect SMMC-7721 and 97L cells, which has low expression of NOVA1 and finally, by detecting the cell growth curve and apoptosis to study the NOVA1's possible functions.Results:1. The hepatocellular carcinoma cells SMMC-7721 and MHCC97L have low NOVA1 protein espression.2. In the hepatocellular carcinoma cells SMMC-7721 and MHCC97L which have low NOVA1 protein espression, NOVA1 protein over expression makes the cells apoptosis decreased, has longer cell cycle as well.Conclusions:NOVA1 gene over expression could make a prograss on biological behavior of hepatocellular carcinoma cells. NOVA1 is a HCC related genes, may have an important role in the occurrence of liver cancer. Part five Effection of NOVA1 gene's knockout on the human Hepatocellular carcinoma cellsObjective:To detect the effections of NOVA1 gene's knockout on the human hepatocellular carcinoma cells.Methods:At first, we detect NOVA1 protein's expression in six hepatocellular carcinoma cells and one normal liver cell by using reverse Transcription-Polymerase Chain Reaction (RT-PCR), to select the cell that has high NOVA1 protein expression; then followed by construction of NOVA1 gene's knockout plasmid, and transfect HepG2 and MHCC97H cells, which has high expression of NOVA1 and finally, by detecting the cell growth curve and apoptosis to study the NOVA1's possible functions.Results:1. Hepatocellular carcinoma cells HepG2 and MHCC97H have high NOVA1 protein espression.2. In the hepatocellular carcinoma cells HepG2 and MHCC97H, NOVA1 gene's knockout makes the cells apoptosis increased, has short cell cycle as well.Conclusions:NOVA1 gene's knockout could have contribution to hepatocellular carcinoma cells'biological behavior. NOVA1 is a HCC related genes, may have an important role in the occurrence of liver cancer.