The Role Of MiR-154-3p In Metastasis Of Non-Small Cell Lung Cancer

Objective: To investigate the expression of micro RNA-154-3p(miR-154-3p) in non-small cell lung cancer(NSCLC) tissues and cell lines, and explore the roles and functional mechanism of miR-154-3p in metastasis progression of NSCLC.Methods: The quantitative Real-Time PCR(qRT-PCR) was used to determine the expression of miR-154-3p in NSCLC tissues(N=21), metastasis NSCLC tissues(N=6), cell lines(A549/ H292/ H1975/ 95D/ SPC-A-1) compared to the paracarcinoma tissues(N=21), non-metastasis NSCLC tissues(N=4) and the human embryo lung cell(WI38) respectively. Negative control RNA(NC) or miR-154-3p mimics was transiently transfected into A549 and H292 cells, then the effects of miR-154-3p on migration and invasion of NSCLC cells were tested by wound healing assay and Transwell invasion assay. The expressions of EMT related proteins E-Cadherin/ZO-1(epithelial marker) and Vimentin/ N-Cadherin(mesenchymal marker) were detected by Western blot. Micro RNA bioinformatic softwares including Pic Tar, miRBD, miRanda and DIANA-micro T were used to predict the probable targets of miR-154-3p. The predicted top100 genes of every software were selected for Venn diagram analysis, and the interested target gene was further validated by Dual-Luciferase Reporter Assay and Western blot. The expression of target gene in NSCLC cell lines was determined by q RT-PCR and Western blot. Immunochemistry was used to detect the expression of the taget gene in normal lung tissues and NSCLC tissues.Results: The expression of miR-154-3p was significantly lower in NSCLC tissues, metastasis NSCLC tissues and NSCLC cell lines than the respective controls. Overexpression of miR-154-3p attenuated the migration and invasion abilities of both A549 and H292 cells. Western blot analysis showed E-cadherin was upregulated in A549 and H292 cells, while Vimentin was downregulated only in A549 cells and no obvious change observed in H292 cells. The Venn diagram showed the common targets of miR-154-3p. Dual-Luciferase Reporter Assay and Western blot revealed that miR-154-3p could bind to BMI1 3'UTR directly and regulate BMI1 expression negatively. q RT-PCR and Western blot results showed BMI1 was upregulated in NSCLC cell lines which confirmed in NSCLC tissues by Immunochemistry.Conclusion: miR-154-3p is expressed lower in NSCLC tissues and cell lines. miR-154-3p could inhibit the migration and invasion of NSCLC cells and block the EMT progression. BMI1, as one target of miR-154-3p is expressed higher in NSCLC cell lines and tissues than the normal ones.