Targeted p21(WAF1/CIP1) activation by miR-1236 inhibits cell proliferation and correlates with favorable survival in renal cell carcinoma

Background: microRNA'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). p21(WAF1/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).

Methods: Real-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.

Results: In 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.

Conclusion: Our 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.