A Study For The Role Of MiR-511/1297/596in Lung Adenocarcinoma

In this research we discussed the effect of miRNA in lung adenocarcinoma. The research was divided into two major parts. Part one studied the effects of miR-511and miR-1297on lung adenocarcinoma by regulating TRIB2gene expression. Part two studied the influence mechanism of miR-596on lung adenocarcinoma occurrence, development and migration on cellular level.1. miR-511/1297suppress the growth of adenocarcinoma cells by regulating TRIB2gene expression(1) The regulation of miR-511/1297on TRIB2geneExpression of TRIB2in lung adenocarcinoma tissue was detected by immunohistochemistry. Results displayed that the expression of TRIB2gene in lung adenocarcinoma was significantly higher than in the normal tissue. TRIB2is anticipated to be important in lung adenocarcinoma occurrence and development as an oncogene. TRIB2related miRNA (miR-511/1297) was forecasted and synthetized. miR-511/1297cotransfected A549cells with pcDNA-GFP-TRIB2-3’UTR respectively. GFP expression was detected by fluorescence microscope and FACS. The results showed that GFP in miRNA transfected groups were significantly lower than that in control group. miR-511/1297were further proved to be able to repress the expression of TRIB2protein through Western blotting. Results showed that miR-511/1297can regulate the expression of TRIB2. In addition, expression of C/EBPa was elevated after transfected with miR-511/1297by Western blotting, which indicated TRIB2may play important role in lung adenocarcinoma occurrence by affecting C/EBPa signal factor.(2) Effects of miR-511/1297on lung adenocarcinoma cells proliferation and apoptosisOn cellular level, after48hours miR-511/1297transfecting A549cells, both of the two miRNAs expression were elevated tested by Real-time PCR. A549cells proliferation inhibition ratio significantly increased after transfected with miR-511/1297detected by MTT. Cell nucleus in miR-511/1297transfected groups appeared obvious apoptotic morphological characteristic by Hochest33258staining. Furthermore, Annexin V-FITC/PI dual-dye was adopted to detect cell apoptosis rate. Results showed that the apoptosis rate of miR-511/1297groups significantly increased. All above indicate that miR-511/1297can apparently inhibit adenocarcinoma A549cells proliferation and promote cells apoptosis.(3) miR-511/1297suppress the growth of A549cells in nude mouseOn individual level, the tumour growth rates of miR-511/1297groups were obviously slower than that in control group. Likewise, the tumors size and quality were less than control group. The results certify that miR-511/1297can also inhibit lung adenocarcinoma occurrence and development on individual level. 2. Effects of miR-596on lung adenocarcinoma occurrence and metastasis by targeting MTSS1gene(1) Regulation of MTSS1gene expression by miR-596MTSS1gene3’UTR has been specificity amplified then cloned into pcDNA-GFP vector to construct pcDNA-GFP-MTSS1-3’UTR. miR-596cotransfected A549cells with pcDNA-GFP-MTSS1-3’UTR. After48hours cultivation, GFP expression in miR-596group significantly decreased compared to control group detected by fluorescence microscope and FACS, which indicated that miR-596could regulate MTSS1gene3’UTR. siRNAs specific to MTSS1were designed and synthetized. miR-596and siRNAs were transfected into A549cells and Western blotting was adopted to further testify the regulatory function. At the same time, the expression of phosphorylated Src in529locus significantly increased after transfected with miR-596detected by western blotting, which indicated that MTSS1gene may play important role in lung adenocarcinoma occurrence by affecting phosphorylation of Src Kinase.(2) Effects of miR-596on lung adenocarcinoma cellsAfter miR-596transfecting A549cells, Cells proliferation inhibition ratio significantly increased detected by MTT. The result of Annexin V-FITC/PI dual-dye showed that apoptosis rate of miR-596group significantly increased as well. Meanwhile, cells migration was detected by Transwell migration assay and the result showed that cells migration abilities of miR-596group significantly reduced. The results certify that miR-596can inhibit the proliferation and migration of lung adenocarcinoma cells and also can promote cells apoptosis.Sum up two parts of researches above, TRIB2and MTSS1may play important roles in lung adenocarcinoma occurrence as oncogenes. There are miRNA regulation sites in TRIB2and MTSS13’UTR. miRNA further affect downstream signal factors expression by regulating TRIB2and MTSS1and thereby play inhibitory effects on A549cell proliferation.