The Function Of MicroRNA-449A In Lung Cancer Cells And Its Underlying Mechanisms

Lung cancer is the leading cause of cancer deaths worldwide, and has a rapidly increasing incidence and mortality in our country. Five-year survival rate of lung cancer is only20%. A major reason for poor outcomes in the lung cancer patients is that have spread beyond the primary site at the time of diagnosis. MicroRNAs (miRNAs) are endogenous, small noncoding RNAs about22nt long that are found in eukaryotes, and can regulate the translation or degradation of messenger RNA (mRNA) through pairing with complementary nucleotide sequences in the3’-untranslated region (3’-UTR) of target mRNA. miRNA negative regulates the gene expression in a posttranscriptional manner. To date, miRNAs have been involved in a wide range of cellular processes, such as development, cellular proliferation, differentiation, and apoptosis. Recently, growing evidence has indicated that deregulation of miRNAs contributes to tumorigenesis. miRNAs can function as oncogenes or tumor suppressors involved in cancer developmemt.Dysregulation of microRNA-449a (miR-449a) has been detected in various types of human cancers. However, the biological function of miR-449a in lung tumorigenesis remains largely unclear. In the present study, we aimed to identify the expression and pathophysiologic significance of miR-449a in lung cancer. Using quantitative RT-PCR (qRT-PCR), we analyzed the expression of miR-449a in156human lung cancer tissues (predominantly adenocarcinoma) and lung cancer cell lines. Our results showed that compared with paired normal tissues, miR-449a expression was significantly downregulated in156lung cancer tissues (p<0.001) and the lung cancer cell lines examined. We also investigated whether miR-449a downregulation is related to clinicopathological features of lung cancer patients, including sex, age, smoking history, histologic type, recurrence and death.χ2tests revealed a significant relationship between low miR-449a expression and patient sex, recurrence and death. To determine whether the expression of mature miR-449a was related to the prognosis of lung cancer patients, miR-449a was used for further survival analysis. Kaplan-Meier survival analysis revealed that lung cancer patients with reduced miR-449a expression had shorter survival than did patients with high miR-449a expression (p=0.019by log-rank test). The transient introduction of miR-449a into lung cancer cells caused cell cycle arrest, cell senescence, and cell apoptosis in vitro. miR-449a also suppressed tumor formation in vivo in nude mice. Further studies (Luciferase reporter assay, qRT-PCR, western-blot) revealed that overexpression of miR-449a significantly suppressed the luciferase activity of reporter plasmids containing the3’-untranslated sequence of E2F3, and also decreased E2F3mRNA and protein levels. Moreover, introduction of miR-449a significantly repressed the transactivation of E2F3. These results suggested that E2F3is a direct target of miR-449a. Moreover, silencing of E2F3by siRNA also inhibited cell proliferation and induced a senescent phenotype in lung cancer cells. E2F3overexpression rescued the suppression of cell viability caused by miR-449a. Overexpression of miR-449a also significantly decreased protein levels of CCND1and CDK.6. Taken together, our results suggest that miR-449a plays an important role in the tumorigenesis of lung cancer and might be a predictor of cancer recurrence and survival in lung cancer patients.It might have the potential therapeutic applications.