The Role Of MiR-512-3p In NSCLC Proliferation And Metastasis

Lung cancer is one of the most common malignant tumors in the world, and non-small cell lung cancer (NSCLC) accounts for about 80% of these cases, while small cell lung cancer accounts for the last 20%. And the most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.MicroRNAs are a class of 18～25-nucleotide non-coding small RNA, and function as negative regulator of gene expression by binding with their target mRNAs by completely or not completely complementary base pairing. Recent research indicated that, miRNAs play their roles in many biology processes, including cell proliferation, migration, apotosis, differentiation, EMT and so on. Aberrant expression of microRNAs is closed related with cancer. It is reported that microRNAs may function as a novel class of oncogenes or tumor suppressor genes. And microRNAs have important roles in the process, diagnose, treatment and prognosis of lung cancer.In this article, we investigated the role of miR-512-3p in NSCLC and its regulatory mechanism. At first, we detected the expressions of microRNAs in NSCLC cell line A549 cells treated with RA or not using microRNA array, and found that the expression of several microRNAs changed, including miR-512-3p. In order to verify the result of microRNA array, we detected the expression of miR-512-3p in A549 and H1299 cell lines treated with RA using RT-PCR, and miR-512-3p showed a high level, which was consistent with the microRNA array. Then we studied the role of miR-512-3p in NSCLC cell lines. Overexpression of miR-512-3p by transient transfection inhibited the abilities of adhesion, migration and invasion of A549 and H1299 cell lines, but had no significant effects on cell proliferation. What’s more, we seleted the H1299 cells stably expressing miR-512, and they showed the poor abilities to adhere, to migrate and to invade compared with the control H1299 cells stably expressing GFP, and the capacity of anchorage-independent growth was decreased. In addition, miR-512-3p inhibitor can increase the capacity of adhesion, migration and invasion of H1299 cells stably expressing miR-512, which indicated that the effects of miR-512-3p on cell adhesion, migration and invation can be reversed by miR-512-3p inhibitor.In order to study the regulatory mechanism of miR-512-3p in NSCLC, we predicted the potential target genes of miR-512-3p using bio informatics, and choosed DOCK3 as a candidate. We proved that DOCK3 was definitely regulated by miR-512-3p using dual luciferase assay and western blot. Knockdown of DOCK3 by DOCK3 siRNA could decrease the abilities to adhere, to migrate and to invade of A549 and H1299 cell lines, which was consistent with the downregulation of DOCK3 by miR-512-3p. What’s more, transfection of DOCK3 siRNA into H1299 cells stably expressing miR-512 reversed the effects of miR-512-3p inhibitor on cell adhesion, migration and invasion. All of the above results indicated that the effects of miR-512-3p on cell adsion, migration and invasion may rely on its regulation on DOCK3.In summary, we showed miR-512-3p inhibited cell adhesion, migration and invasion of NSCLC cell lines A549 and H1299. DOCK3 was a target gene of miR-512-3p, and mediated these roles of miR-512-3p.Tumor microenvironment has been illustrated to exert an important role in tumor progression and treatment response. Potential clinical implications suggest that the molecular state of normal cells adjacent to cancer cells has prognostic value. AXL, a receptor tyrosine kinase (RTK), had been reported to be able to increase the invasion in lung adenocarcinoma (LAD) cell lines. However, no comparison was made between tumors and normal/adjacent normal tissues. We performed meta-analysis of 21 microarray datasets on LAD (including 1060 LAD and normal/adjacent normal tissues) and found that the expression of AXL was significantly overexpressed in adjacent normal tissues (Z=-7.21, P<0.0001), but not in normal tissues (Z=-1.62, P=0.106), compared with tumors. Tissue microarray showed that the protein expression level of AXL was significantly down-regulated in tumors (P=0.003) or in stage IB subgroup (P=0.028), compared with the adjacent normal tissues. Our study suggested that AXL expression was significantly overexpressed in adjacent normal tissues compared with LADs. The clinical significance of the phenomena needs further investigation.