| Background and objective: Gastric carcinoma, GC is the third largest disease which cause cancer.Althoughthere are various treatments for gastric carcinoma, technology has improved, but the clinical prognosis is still poor, mortality is high.After radical surgery, postoperative recurrence and transfer rate remains as high as 40-80%, most patients with gastric cancer 5-year survival rate is just 20-30%.Tumor recurrence?metastasis and chemotherapy drug resistance is the main reason for the patients with poor prognosis. And hypoxia may induce tumor metastasis and chemotherapy drug resistance. Epithelial and mesenchymal transition (EMT) is considered as a transfer starting.This study contains high-throughput sequencing, cells experimental and clinical specimens to explore c-MET mediated transformation of epithelial and mesenchymal acquired chemotherapy for gastric cancer, the role and mechanism of drug resistanceMethods: Select SGC7901 agastric cancer cell line as the research object, establish OXA resistance of SGC7901 / OXA cell lines. Observing the cell morphology,compare the expression of epithelial and mesenchymal marks level and the expression of EMT related transcription factors ZEB1, doing invasion and migration experiment,to explore acquired drug resistance of EMT process;Cell proliferation and apoptosis experimental to test drug-resistant cell sensitivity of OXA; shRNA ZEB1 expression,compare more interference group and the control group cell morphology, EMT process,invasion and migration ability to change and its effects on the sensitivity of OXA;Detecting HGF expression, c-MET expression and amplification in the two groups of cell lines,;Respectively using c-MET inhibitors, c-MET shRNA to inhibit or reduce the c-MET expression,tnen observing the cell morphology, compare the expression of epithelial and stromal marks level, the expression of EMT related transcription factors ZEB1and the influence of c-MET on gastric cancer cell lines of acquired drug resistance EMT process;Collecting 76 cases of failure of oxaliplatin chemotherapy in patients with gastric cancer before and after treatment ,detecting express of HGF, c-MET, EMT markers, and the change of c-MET amplification,combing with clinical data to analysis c-MET, and the relationship between EMT markers and chemotherapy curative effect;Using Illumina sequencing platform with a standard experimental operation for total RNA extraction, build sample library and high throughput sequencing in cell line 6; After sequencing data, using standard quality to filter the raw data, through the combination of grouping samples to compare KEGG database, based on the analysis of biological information, such as resistant cells specific expression genes and participate in the regulation of micrornas, IcRNA and circular RNA regulatory elements;Using Cytoscape software such as the expression of regulatory elements to construct a total network diagram;Then by real-time fluorescence quantitative RT-pcr and Western blot methods to determination differences in gene expression in drug resistant cell lines and parental cell lines.Acqured mirroRNA-ceRNA-mRNA resistance regulation network space.Results: 1. Using the method of increasing concentration to establish gastric cancer drug-resistant cell line SGC-7901 / L-OH, which remain the stable resistance,providing a good experimental model.2 SGC-7901 / L-OHP did happen EMT process, and c-MET of high expression in drug-resistant cell lines, so c-MET involved in SGC-7901 / L-OHP of EMT.3c- MET siRNA can inhibit gastric cancer SGC-7901 / L-OHP'EMT process, c -MET is characteristic and highly suggestive of MET may be affected by EMT involved in gastric cancer resistant cells of L-oxaliplatin into the sensitivity of the OHP.4. Through standard Illumina process sequencing, we can get no less than 10G original data in LOP7901 and SGC-7901 samples ,Q20 ratio greater than 95%, Q30 ratio greater than 90%. Accordance with the requirements of the sequencing. After analysing of biological information:there are 61139 mRNA, 30048 lncRNA, 1269 circRNA.5. Through differentially expressed genes in the 2 groups were compared after homogenization,we can received 2340 differentially expressed regulatory elements,which up-regulation 926, down-regulation 1414.There are 2570 differenced genes to,which up-regulation 1161 genes, down-regulation 1409 genes.6. Based on the grouping expression differences gene expression and regulation component annotation and clustering analysis, we received 309 GO up-regulation mechanism,205 down-regulation mechanism; up-regulation 30 KEGG pathways,down-regulation 20 pathways .Search gastric cancer, drug resistance and oxaliplatin as keywords,we can eventually get MARK?Norch pathways may be one of the main pathway to induce drug-resistanance.7. Through the collaborative control components and differentially expressed genes,we got around HGF gene, GDF15 genetic and HMCN2 gene expression space regulation network, the network may be the main regulatory mechanism of drug resistance of gastric cancer cells.8. E cadherin, N-cadherin, Vimentin and C-MET express differently in SGC-7901 /L-OHP, E-cadherin express decreased with N-cadherin, Vimentin , C-MET and enhancement, which has certain contact with formation and metastasis and drug resistance of tumor.Conclusion: 1. Using the method of increasing concentration to establish the gastric cancer drug resistance cell line SGC-7901 / L-OHP, remains the stable resistance,providing a good experimental model in vitro studies.2. C-MET expresses highly in SGC-7901 / LOHP cell line, and C-MET involved in gastric cancer cells in the process of drug-resistant of EMT.3.By high-throughput sequencing ,we can find MARK and Norch pathways as the core mechanism of gene expression changes.4. Based on the cooperative control components and differentially expressed genes expression analysis, we got around HGF gene, GDF15 genetic and HMCN2 gene expression space regulation network, providing precise intervention in gastric cancer drug resistance problems with molecular targets. |
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