The Study Of Roles Of Oncogene K-ras In Human Gastric Cancer And Its Molecular Mechanisms

Gastric cancer is one of the most common malignancies with high mortality in the world, which has a high incidence in China and seriously endangers the health of the Chinese people. The cause is that the exact molecular mechanism responsible for the occurrence and development of gastric cancer are not quite clear. Therefore, it is very important to identify etiology and pathogenesis, which may lead to find valuable diagnostic markers and provide a theoretical basis for the development of new treatment strategies.K-ras, a member of Ras family, is initially impact on human cancer. K-Ras protein plays a key role in tumorgenesis and development through regulating its downstream signaling protein involved in cell cycle and is closely related to cell survive, proliferation, migration and angiogenesis in tumor. Wild-type k-ras protein is activated by the upstream EGFR leading to short-term activation, followed by rapid inactivation. Activation/inactivation of k-ras are controlled in normal condition. Mutant protein with abnormal protein function is active without EGFR activation and the effect is not controlled, resulting in the continued proliferation of tumor and so on.The molecular mechanisms leading to malignant tumors of k-ras mutations is not yet clear and require further study.ObjectiveTo detect k-ras mutations in gastric cancer and investigate its roles in the development, metastasis and invasion of gastric cancer so as to look for new theraputical targets for gastric cancer.Methods(1) Exonland exon2of k-ras gene was amplified by polymerase chain reaction (PCR) taken Gastric cancer genomic DNA as template. K-ras mutations of gastric cancer cell lines were detected by gene sequencing.(2) Identification of k-ras plasmid (mutant and wild type) and negative control plasmid (pQCXIH) was detected by using recombinant techniques such as bacterial transformation, plasmid extraction, restriction enzyme digestion, gel electrophoresis, gene sequencing and other methods. K-ras plasmid was transfected into gastric carcinoma cell line SGC7901with liposome. Expression of k-ras protein and mRNA in the transfected and parental SGC7901cells was examined by Western blotting and RT-PCR, respectively.(3) k-ras plasmid (mutant and wild type) and negative control plasmid were transfected into human gastric cancer cell lines SGC7901and MKN45. The expression of Fascinl was examined by using Western blotting and RT-PCR.(4) k-ras plasmid (mutant and wild type) and negative control plasmid were transfected into human gastric cancer cell lines SGC7901and MKN45. The expression of Snail and Slug were examined by using Western blotting and RT-PCR.(5) k-ras shRNA and negative control plasmid were transfected into human gastric cancer cell line BGC823. The expression of Snail, Slug and Fascin1were examined by using Western blotting and RT-PCR.(6) k-ras plasmid (mutant and wild type) and negative control plasmid was transfected into human gastric cancer cell lines SGC7901and MKN45. The biological effects of gastric cancer cells such as invasion and metastasis compared to normal were examined by transwell assays.(7) k-ras shRNA and negative control plasmid were transfected into human gastric cancer cell line BGC823. The biological effects of gastric cancer cells such as invasion and metastasis compared to normal were examined by transwell assays.Results(1) Codon12,13and61of k-ras oncogene were no mutations in human gastric cancer cell lines SGC7901, MKN45, MKN28, BGC823and HGC-27. Codon12of k-ras oncogene in human gastric cancer cell line AGS was mutation.(2) Mutation of k-ras promotes cell migration and invasion in gastric cancer.(3) As compared with SGC7901and MKN45, the expression of snail and slug was significantly increased but no difference in the expression of Fascinl in mutation over-expression group.(4) K-ras shRNA inhibit the gastric cell migration and invasion in gastric cancer.(5) When compared to BGC823, the expression of snail and slug were down regulated but no difference in the expression of Fascinl in k-ras shRNA group.Conclusion(1)K-ras mutations occur in some gastric cancer cell lines.(2)K-ras mutations promote invasion and metastasis of gastric cancer.(3)K-ras mediated gastric cancer invasion and metastasis through the regulation of snail and slug expression.(4)There was no correlation found between k-ras and fascinl.