The Expression And Function Study Of MicrRNA-10b In Non-small Cell Lung Cancer

Objective:More and more research shows that miRNA plays an important role in the development process of lung cancer. We use real-time PCR to detect the different expression of miR-10b in non-small cell lung cancer and adjacent normal tissues in this study, we observe the effection of cell growth, invasion and metastasis by overexpressing miR-10b in lung cancer cell lines, and the prediction and validation of the target gene of miR-10b,to explore the role of miR-10b in the development of non-small cell lung cancer and it’s mechanism. Methods:1, Detecte the different expression of miR-10b in non-small cell lung cancer and and adjacent normal tissues by RT-PCR.2, Detecte the expression of miR-10b in A549cells and95-C cells by RT-PCR. A549cells and95-C cells were randomly allocated into3groups:blank group, empty vector transfected group and miR-10b expression plasmid transfected group. Under the induction of LipofectamineTM2000, the recombinant was transfected into95-C. After transfection, transfection effciency was observed by fluorescence microscope. miR-10b expression level was detected by RT-PCR, The change of cell proliferation was detected by cell proliferation assay; The change of cell cycle and apoptosis were detected by flow cytometry, the change of invasive ability of cell were detected by Transwell experiment, the change of migration ability of cells were detected by the wound stratch assay in each group.3, Using bioinformatics methods to predict the target genes of miR-10b, and use the luciferase report gene experiment and Real-time PCR and Westblotto validate Results:1, miR-10b expression is higher in non-small cell lung cancer than the adjacent normal tissues in the40cases, the difference was statistically significant (P<0.05), and the miR-10b expression in the group with lymph node metastasis is higher than the group without lymph node metastasis, the difference was statistically significant (P<0.05).2, There has relative high expression of miR-10b in A549cells and95-C cells (P<0.05), compared with the blank control group and the empty vector transfection group, the cell proliferation rate was obviously increased, the cell apoptosis rate decreased, the cell invasion and migration ability enhanced in the miR-10b plasmid transfection group after transfection, the difference was statistically significant(P<0.05). Predicted by bioinformatics software, we filter out the KLF4as miR-10b target genes. We constructed two KLF4fragment of the3’UTR luciferase reporter gene vector, cotransfected plasmid miR-10b, and KLF4-3’UTR-wt reporter vectors,the luciferase activity was reduced35%; cotransfected miR-10b plasmid vector and prediction target point mutation KLF4-3’UTR-mut, the luciferase activity did not change significantly. The expression of KLF4mRNA decreased in the miR-10b expression plasmid transfected group, but the difference had no statistically significant (P>0.05); the expression of KLF4protein decreased after transfection in the miR-10b expression plasmid transfected group, the difference was statistically significant (P<0.05);. Conclusion:1, The expression of miR-10b is higher in non-small cell lung cancer than in the adjacent normal tissues,and associated with the metastasis of lymph node;2, miR-10b can promote the proliferation,reduce the apoptosis,enhance the invasion and migration ability of A549and95-C.3, KLF4is the direct target of miR-10b, and miR-10b might through down-regulating KLF4to promote the proliferation and enhance the invasion and migration of NSCLC.