Targeted p21WAF1/CIP1 activation by miR-1236 inhibits cell proliferation and correlates with favorable survival in renal cell carcinoma

Abstract

BackgroundmicroRNA’s function to silence gene expression by targeting 3′UTR regions has been widely studied. And microRNAs, similar to small double-stranded RNAs, have also been implicated to gene activation triggered by promoter-targeted, which is known as RNA activation (RNAa). p21WAF1/CIP1 (p21), as a key negative regulator of cell proliferation, is frequently down-regulated in various cancers, making it an ideal target for RNAa-based activation to restore its tumor suppressor function in renal cell carcinoma (RCC). MethodsReal-time polymerase chain reaction was used to identify the expression of miR-1236 and p21 in RCC cell lines and clinical specimens. Protein expression and signaling pathway modulation were validated through Western blot analysis, whereas p21, direct target of miR-1236, was validated by using chromatin immunoprecipitation assay. The Kaplan-Meier method and the log-rank test were used to calculate overall survival. The CellTiter 96® AQueous One Solution Cell Proliferation Assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine assays were used to evaluate the effect of miR-1236 on RCC cell proliferation. ResultsIn this study, we found that miR-1236 and p21 were both significantly down-regulated in RCC tissues and cell lines. Combined low expression of both miR-1236 and p21 emerged as an independent prognostic factor for poor clinical survival in 45 patients with RCC. Chromatin immunoprecipitation assay in the human RCC cell lines 786-O and ACHN showed that miR-1236 directly targeted the p21 promoter and that its activation may provide a plausible link between the positive correlation of miR-1236 and p21 in RCC specimens. Functional experiments showed that increased miR-1236 expression inhibited cell proliferation, and decreased CDK4/6 and cyclin D1 expression. Furthermore, knockdown of p21 using small interfering RNA reversed the antiproliferation function of miR-1236, whereas silencing the p21 expression attenuated the function of miR-1236 in RCC cells. ConclusionOur findings provide insight into the specific RNAa of miR-1236–targeted p21 promoter activation in suppressing RCC cell proliferation. Considering the poor prognostic outcomes associated with reduced miR-1236 and p21 expression, our data imply that these factors likely play important antitumor effects in RCC progression.