The Research Of Molecular Mechanism On MiR-25 Involved In Lung Cancer Metastasis And Invasion

Objective: To investigate the relative expression of mi R-25 in the plasma of non-small cell lung cancer(NSCLC)patients and NSCLC cell lines,and further explore the mechanism of miR-25 involvement in the invasion and metastasis of NSCLC.Methods: 1.We collected the plasma of NSCLC patients and healthy group with contrast and detected the expression of miR-25 with fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR);2.Fluorescent quantitative RT-PCR method was applied for detection the expression of miR-25 in normal lung epithelial cell BEAS-2B and six NSCLC cell lines;3.After transfecting pSIF-GFP-miR-25 or miR-25 inhibitor into A549 and H1299 cells with the lipofectamine method,wound healing assay and transwell invasion model were performed to determine the effect of miR-25 on the migration and mobility ability of NSCLC cell lines;4.Three bioinformatics prediction websites(Miranda,PicTar and TargetScan)were used to predict the potential mRNA targets of miR-25,and Western blot method was used to measure the protein expression level of mRNA targets in BEAS-2B,A549 and H1299 cells;5.Dual-luciferase reporter assay was performed to identify the potential relationship between miR-25 and mRNA targets;6.We transfected pSIF-GFP-miR-25 or miR-25 inhibitor into A549 and H1299 cells,and measured the protein expression level of mRNA targets with Western blot method.Results: 1.The relative expression of miR-25 in the plasma of NSCLC patients was significantly higher than that in healthy controls,and correlated with metastasis of lung cancer;2.The expression of miR-25 in several NSCLC cell lines was higher than that of normal lung epithelial cells;3.Over-expression of miR-25 could promote migration and invasion of NSCLC cell lines,while NSCLC cell lines transfected with mi R-25 inhibitor showed the opposite effect;4.Bioinformatics analysis predicts that SGPP1 and LATS2 may be two potential targets of miR-25.SGPP1 and LATS2 played a role in tumor suppressor genes in a large number of literatures,at the same time,the result of western blot analysis showed that SGPP1 and LATS2 protein expression level in A549 and H1299 cells lower than those in BEAS-2B cells;5.We confirmed that miR-25 down-regulated SGPP1 and LATS2 expression through binding to the 3’UTR of SGPP1 and LATS2 mRNA by the luciferase reporter assays;6.After over-expressing miR-25,protein expression of SGPP1 and LATS2 in A549 and H1299 cells decreased,while inhibit mi R-25 expression,protein expression in cells increased,which indicate that SGPP1 and LATS2 gene expression was negatively regulated by miR-25 post-transcriptionally,once again confirmed that SGPP1 and LATS2 were the target genes of mi R-25.Conclusion: We confirmed that miR-25 expression in the plasma of NSCLC patients was higher than that in healthy controls,and the expression level of miR-25 was correlated with the TNM stages of cancer.The expression of mi R-25 in several NSCLC cell lines was higher than that of normal lung epithelial cells.Over-expression of miR-25 could promote migration and invasion of NSCLC cell lines.We have further predicted and verified that SGPP1 and LATS2 were target genes of miR-25 and speculated that mi R-25 is possible to participate in invasion and migration of lung cancer by targeting SGPP1 and LATS2,and play an important role in the process of NSCLC.