miR-150, p53 protein and relevant miRNAs consist of a regulatory network in NSCLC tumorigenesis

摘要

microRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs. Tumor protein p53, a transcriptional factor, plays an important role in the progression of tumorigenesis. miR-150 was the only miRNA predicted to target 3′-UTR of p53 by TargetScan. In order to investigate the function of miR-150, p53 and relevant miRNAs in non-small cell lung cancer (NSCLC), we constructed two expression vectors of p53 (pcDNA3.1-p53 and pcDNA3.1-p53-3′-UTR) and two report vectors (pGL3-p53-3′-UTR and pGL3-p53-3′-mUTR). The activity of luciferase transfected with miR-150 mimics was lower by 30% when compared to that of the miRNA-negative control (miRNA-NC). Moreover, the p53 protein was downregulated by at least 50% when miR-150 mimics were cotransfected with pcDNA3.1-p53- 3′-UTR when compared to miRNA-NC. We also determined the expression of miR-150 and p53 in NSCLC patient tissue samples. The expression of miR-150 in T2 stage tissue samples was higher than that in T1 stage tissue samples. The corresponding target gene p53 was correlated with miR-150 expression. In the present study, we further analyzed the cell cycle distribution. The cells transfected with pcDNA3.1-p53 were significantly arrested in the G1 phase when compared to the control cells. When miR-150 mimics were cotransfected with pcDNA3.1-p53-3′-UTR, the percentage of cells in the G1 phase was significantly lower by 4% when compared to miRNA-NC. To identify miRNAs that are regulated by the p53 protein, qRT-PCR was performed after pcDNA3.1-p53 transfection. miR-34a, miR-184, miR-181a and miR-148 were upregulated significantly. However, there was no distinct difference in the expression of miR-10a, miR-182 and miR-34c. Our results showed that miR-150 targets the 3′-UTR of p53, and p53 protein promotes the expression of miRNAs which affect cell cycle progression. These findings suggest that miR-150, p53 protein and relevant miRNAs are members of a regulatory network in NSCLC tumorigenesis.