MiRNA-182 Promotes NSCLC Cell Proliferation, Invasion And Migration By Regulating RECK

Background: Lung cancer remains one of the leading causes of tumor-related death worldwide. It is predicted that about 600000 individuals die of lung cancer in china every year. This high mortality is caused by poor prognosis, which is affected by the late presentation of disease, tumor heterogeneities within histological subtypes, and the limited understanding of lung cancer biology. Non-small cell lung cancer(NSCLC) accounts for approximately 80% of all lung cancer cases. Therapeutic decisions and prognosis in NSCLC have been mainly based on performance status and tumor stage, but rarely on molecular analysis. It is important to find new markers for targeted therapy. MicroRNAs(miRNAs) play key roles in fundamental cellular activities such as cell growth, differentiation, proliferation, and apoptosis. Studies in the past decades have shown that abnormal expression of mi RNAs in human cancers is tightly associated with tumor promotion and inhibition. An increasing number of miRNAs were shown to be involved in progression and prognosis of NSCLC, including mi R-128, miR-195, miR-483, mi R-451, and mi R-630. In our previous study, we found that many miRNAs, including miR-182, were dysregulated in lung cancer cell lines. These findings indicate that MIR-182 plays key roles in lung cancer progression.Objective:The present study aimed to evaluate the role of mi R-182 in NSCLC and the underlying mechanism, to identify the target gene(RECK) of miR-182, and to provide a new clue for exploring the role and the mechanism of mi R-182 in NSCLC carcinogenesis. It offers new insight into selecting diagnostic and prognostic biomarkers as potential therapeutic targets.Methods1. qRT-PCR was employed to quantify the expression level of mi R-182 in NSCLC cell lines, plasma of thirty healthy individuals and fifty NSCLC patients, and to quantify the expression level of mi R-182 in matched tissues of 30 patients. By integrating the clinic data, we analyzed the correlation between the expression level of miR-182 and histology differentiation, histology and TNM stages.2. Cell proliferation, invasion and migration of A549 and H1299 were performed by overexpression of miR-182 in vitro. Luciferase vectors(pMIR-RECK), which contain the binding site of mi R-182 to RECK 3’UTR, were constructed. The predicted target gene(RECK) of mi R-182 was identified via luciferase activation assays. The mRNA or protein expression of RECK was detected via qRT-PCR or Western blot, respectively. qRT-PCR was employed to quantify the expression level of RECK in matched tissues of 30 patients. The correlation between the expression level of miR-182 and that of RECK in NSCLC tissues was analyzed.Results1. Mi R-182 expression in NSCLC cell lines(549, H1299, PC9 and NCI-H460) was significantly higher than that in MRC-5(P<0.05). Mi R-182 was increased in NSCLC tissues and cell lines and was correlated with metastatic capacity in NSCLC tissues( P<0.05).2. Mi R-182 expression in plasma of 50 NSCLC patients was significantly higher than that of 30 healthy individuals(P<0.05).3. MiR-182 was frequently increased in plasma of NSCLC patients with lymph node metastasis or TNM stage(III or IV)(P<0.05), and mi R-182 was not correlated with patients’ age, gender and histology differentiation(P>0.05).4. In NSCLC cell lines, ectopic overexpression of mi R-182 promoted cell proliferation, invasion and migration in vitro.5. The luciferase activity in the experimental group was significantly decreased compared with that in the control group(P<0.05), confirming that miR-182 mimics repressed the luciferase expression of the p MIR-RECK UTR, and suggesting that RECK was a target gene of miR-182.6. MiR-182 mimics significantly decreased the Mrna(P<0.05) and protein expression levels of RECK, and MiR-182 inhibitors significantly increased the m RNA(P<0.05) and protein expression levels of RECK in the A549 cells.7. The average level of RECK m RNA was significantly elevated in NSCLC tissues compared with that in the corresponding normal tissues, and RECK m RNA level was inversely correlated with mi R-182 level(r=-0.482, P<0.01).Conclusion1. MiR-182 expression in plasma of NSCLC patients is significantly higher than that of healthy individuals. It suggests that miR-182 can be used as a NSCLC biomarker. MiR-182 in plasma of NSCLC patients is correlated with lymph node metastasis or TNM stage(III or IV).2.MiR-182 promotes cell proliferation, invasion and migration in vitro. RECK is identified to be a target gene of mi R-182. RECK mRNA level is inversely correlated with miR-182 level. This suggests that miR-182(oncomir) promotes tumor growth and metastasis in NSCLC by regulating the RECK signaling pathway and provides a novel clue for investigating the role and the mechanism of miR-182 in NSCLC carcinogenesis.