| In the world, the incidence of hepatic cellular carcinoma (HCC) is 6th of male cancer and 11th of femal. In South Africa and Southeast Asia, it's the second common malignancy and it's third common malignancy in China. The incidence of hepatocellular carcinoma in an upward trend, suggesting that the emergence of new carcinogenic factor. The annual global add new liver 260,000 cases, of which 42.5% in China, hepatocellular carcinoma mortality for PLC was 20.4 per 10,000. At present, the incidence of hepatic cellular carcinoma (HCC) is third in the malignant tumors in China which seriously damage the health of people in our country.Molecular biology studies show that the incidence of HCC is a complex process which is composed of multi-gene participation, multi-step, multi-stage and the interaction of factors both in vivo and in vitro. Previous study of HCC was restricted to individual gene or a few genes, and was short in the comprehensive and systematic knowledge aimed directly at numerous genes involved in HCC, which restricted the study of pathogenesis, diagnosis, and treatment of HCC. Therefore, it is of great importance to systematically study the molecular events in the development of HCC.In recent years, the methods of cloning differentially expressed genes are constantly emerging. With the combination of gene microarray technology, it becomes one of the methods to separate differentially expressed genes simply and quickly, which has been successfully applied in the research of genes that express differentially in gastrointestinal tumors, genital system tumors, cardiovascular diseases, et al. In this dissertation we applied cluster analysis to study the results of cDNA microarray of rat's HCC using bioinformatics methods and screened highly expressed genes .We identified the expression of the target genes of interest in human HCC tissues in multiple levels. Combining with clinical data, we studied the relationship between gene expression pattern and clinical prognosis. Then we suppressed the expression of target genes in human HCC cells in vitro by RNAi to explore their roles in the process of apoptosis.Methods1 The results Rat Expression Array 230 2.0, the cDNA microarray of rat HCC were analyzed by bioinformatics methods.2 RT-PCR, Real-time quantitative PCR and immunohistochemistry were performed to identify the expression of Alphab Crystallin(CRYAB) gene in human HCC tissues.3 The survival analysis of the clinical data of HCC was done by Kaplan-Meier Method. COX's Proportional Hazard Model was applied for single and multi-factor analysis.4 RNAi interference vectors were constructed and transfected into BEL7402 cell line by liposomes. The efficiency of transfection was analyzed by immuocytochemistry and immunofluorescence. The expression changes of CRYAB mRNA and protein in BEL7402 cell line were observed by RT-PCR and Western Blot.5 The cell proliferation activity was measured by MTT assay. Annexin V/7-AAD double staining and PI staining were performed to detect the apoptosis rate in BEL7402 cells after transfection.1 The differentially expressed genes in rat HCC were observed by the preliminary andsecondary analysis of the gene microarray results, in which there were 1227 genes up-regulated and 1015 genes down-regulated. Of all the up-regulated genes, there are 60 genes that the difference degree was more than 4.96 biological functions and 48 biological pathways were obtained using Cluster Analysis.2 AlphaB-Crystallin gene(CRYAB) was one of the overexpressed genes screened. Its probe ID was No. 1370026, and its difference degree was 4.3. No reports of CRYAB gene were seen in the human HCC.3 CRYAB gene was overexpressed in human HCC, which had been proved by the results of RT -PCR, Real-time quantitative PCR(Difference rate is 4.913) and immunohistochemistry(positive rate is 42.9%). This confirmed the results of gene microarray forecasted4 Combining the clinical follow-up data of 98 HCC patients with immunohistochemical results, the survival analysis showed that postoperative life span between CRYAB positive expressed group and CRYAB negative expressed group was of significant difference(P=0.0410). The survival rate of CRYAB negative expressed group was at a stable level, while that of CRAYAB positive expressed group constantly descended 18 months after operation. Multi-factor analysis revealed that CRYAB(P=0.007) and vein thrombosis(P=0.037) were the independent prognostic factors of HCC, individually.5 pRNAT- RNAi-CRYAB interference vectors were constructed by reforming PRNAT- -U6 vectors and were transfected into BEL7402 cell line by liposomes. The effects of transfection and interference were identified by RT-PCR,Western Blot, immuocytochemistry and immunofluorescence. 6 The results of MTT show that, after48h, the inhibition rate of cell proliferation activity of inhibiting group is 0.322%±0.0082, and control group is 0.13%±0.0356%, respectively. Annexin V/7-AAD double staining and PI staining showed that the cell apoptosis rate in early stage of inhibiting group is 5.74%±0.275%, Negative control group is 3.51%±0.179%, normal control group is 3.16%±0.185%. These results, combining with other related data, proved that CRYAB gene possessed the function of inhibiting cell apopotosis.1 The overexpression of CRYAB gene in human HCC can be used as a diagnostic indicator of clinical prognosis2 The overexpression of CRYAB gene may be related to the malignant transformation of hepatic cells and the metastasis and invasion of HCC cells3 pRNAT-RNAi-CRYAB interference vectors can effectively interfere the expression of CRYAB gene in HCC cells in vitro.4 CRYAB gene can inhibit apoptosis of HCC cells. |
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