| Background:The incidence of gastric cancer in China in a variety of malignant tumors in the first place,the second highest mortality rate.MiRNAs has a great relationship with the development of tumor.In different tumors,miRNA is differentially expressed and has correlation with tumor diagnosis and classification.It indicates that it may become an important biomarker for tumor initiation and development,clear treatment target and correct prognosis.Studies have shown that miR-214 is abnormal in many tumors.We speculate that miR-214 is associated with the occurrence,development and prognosis of gastric cancer.It is expected to become a biomarker for predicting gastric cancer progression,metastasis,therapeutic effect and prognosis.the doctoral thesis is composed of four parts:Part 1 Expression of miR214 in gastric carcinoma and its relationship with clinicopathology and prognosisAims:To detect the expression of miR-214 in gastric cancer tissue and paracancerous tissue,and analyze the relationship between miR-214 and clinicopathological parameters and explore the relationship between miR-214 and prognosis of gastric cancer.Materials and methods:100 patients with gastric cancer admitted to our hospital were selected.The expression of miR-214 in 100 gastric cancer tissues and the adjacent tissues of cancer was detected by RT-PCR.The clinical data and follow-up information were collected.The relationship between miR-214 and prognosis of gastric cancer patients was analyzed.Results:The expression level of miRNA-214 in gastric cancer tissues was significantly higher than that of normal tissues adjacent to cancer(p<0.05).It was found that the survival time of patients with low miR-214 expression was longer than that of high expression patients(P<0.001).The single factors on the prognosis of patients with gastric cancer analysis results show that the neural invasion,Borromann classification,TNM stage and miR-214 expression were all factors affecting the prognosis of patients with gastric carcinoma(P<0.05),but the multivariate analysis showed that TNM stage was the independent prognostic factors(P>0.05).Conclusion:The expression of miR-214 is up-regulated in gastric cancer tissue and is more obvious in metastatic gastric cancer.The expression of miR-214 in gastric cancer is correlated with tumor staging and survival time.Detection of miR-214 in tissues may serve as a target gene for evaluating prognosis in patients with gastric cancer.The detection of miR-214 may be the target gene for evaluating the clinical prognosis of gastric cancer patients.Part 2 Study on the biological function of miR-214 in gastric cancer cellsAims:To explore the function significance of miR214 in the proliferation,apoptosis,migration on gastric cancer.Materials and methods:SNU16 and SNU5 was infected with miR-214 inhibitor,mimics,mimics-NC or inhibitor-NC,and the cell proliferation,cell migration,cell invasion and apoptosis were detected in the infection group,NC group and inhibition group,respectively,and the effect of miR-214 on the biological function of gastric cancer cells was analyzed.Stably overexpressing miR-214 or control were injected subcutaneously into the dorsal flnk of mice.At the end of experiments,the mice were sacrified and tumors were dissected and weighed.Results:CCK-8 assay showed that cell proliferation was significantly faster in mimics-miR-214 group in the mimics-NC group(P<0.05),miR-214 inhibitor of cell proliferation was significantly weaker than that of miR-214 inhibitor-NC group(P<0.05);scratch test results showed that when gastric cancer cell transfected into mimics-miR214,cell scratch healing rate in 36h was significantly higher than that of NC group(P<0.05),the inhibitor group was significantly lower than NC group(P<0.05);Transwell assay results showed that cell number overexpression of miR-214 cells through the membrane was significantly higher than in group NC(P<0.05),and inhibition of miR-214 cells through membrane was obviously less than group NC(P<0.05);through flow cytometric analysis detection,compared with NC group,expression of miR-214 and the down-regulation of miR-214 cell apoptosis rate had no significant change(P>0.05).miR-214 promotes tumor growth in nude mice.Conclusion:The overexpression of MiR-214 significantly promotes the proliferation,migration and invasion of gastric cancer cells,and the expression of miR-214 has no effect on cell apoptosis.Part 3 Bioinformatic analysis of mir214 target gene and function of TET3 in gastric cancerAims:To investigate mir214 target gene and the role of TET3 gene expression in the progress of gastric cancer.Materials and methods:mir214 target genes were predicted with stabase 2.0.Correlation analysis was done between mir214 and TET3 based on TCGA database.Prognostic values for TET3 gene were performed by Kaplan-meier plotter,including overall survival and post progression survival.415 gastric carcinoma patients were were analyzed with GSEA enrichment analysis.A protein-protein interaction(PPI)network was established with STRING 10.5.SNU16 cells stably overexpressing TET3 or control were injected subcutaneously into the dorsal flnk of mice.At the end of experiments,the mice were sacrified and tumors were dissected and weighed.Results:mir214 target genes were predicted with stabase 2.0 showed that TET3 is one of target genes of mir214.Correlation analysis of mir2:14 and TET3 indicated that they have negative correlation.The KMplot results revealed that TET3 resulted in significant overall survival rates and post progression survival(P<0.05).GSEA analysis of TET3 between these two groups enriched cancer related pathway.Protein and protein interaction analysis showed that TET3and related genes were enriched in tumor related signaling pathways,which was highly correlated with tumor proliferation,migration and invasion.It is further indicated that TET3 is associated with the progression of gastric cancer.Overexpression of TET3 inhibits tumor growth in nude mice.Conclusion:Low expression of TET3 promotes the development of gastric cancer.Part 4 miR-214 regulates the expression of TET3 at the translational levelAims:To explore the mechanism of microRNA214 and TET3,and illuminate their cellular functions in the process of proliferation,migration and invasion in gastric cancer.Materials and methods:The TET3 expression levels were detected by qRT-PCR and western blot in SNU-16 and SNU5 cells transfected with miR-214 inhibitor,mimics,mimics-NC and inhibitor-NC.To characterize whether TET3 induced cell biological behaviors could be mediated by miR-214,we performed co-transfection of pcDNA-TET3 plasmid and miR-214 mimics in SNU-16 and SNU5 cells.The cell proliferation,cell migration,cell invasion and apoptosis were detected in above groups,respectively.The pGL3 vectors containing wild type or mutant putative miR-214 binding site in human TET3 3’UTR were synthesized.For the luciferase reporter assay,SNU-16 and SNU5 cells were co-transfected with miR-214 mimics,mimics-control and Luc-TET3 wild,Luc-TET3mutResults:SNU-16 and SNU5 cells were transfected with miR-214 mimics or control vector to establish miR-214 overexpression cell lines,and an increase in miR214 and decrease in TET3 expression was verifid by qRT-PCR and western blot.SNU-16 and SNU5 cells were transfected with miR-214 inhibitor or control vector to establish miR-214 lowexpression cell lines,and a decrease in miR-214 and increase in TET3 expression was verifid by qRT-PCR and western blot.Co-transfection experiment showed that,TET3 was an important mediator responsible for the effects of miR-214 on gastric cancer cell migration and proliferation.Overexpression of miR-214 resulted in a 2-fold decrease of luciferase activity of WT TET3 3’UTR as compared to the miR-control.Whereas the reduction of the hcifcrase activity with mutant TET3 3’UTR was not observed.Conclusion:miR-214 regulates the expression of TET3at the translational level to regulate proliferation and migration capability in gastric cancer. |
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