MicroRNA-99a induces G1-phase cell cycle arrest and suppresses tumorigenicity in renal cell carcinoma

Li Cui,Renfang Xu,Yaojun Zhou,Zhong Xue,Qianqian Shi,Wei Xia,Hua Zhou,Xianlin Xu,Hu Zhao,Xiaozhou He,Zinong Tian,Yunjie Cao,Tao Ding,Bo Zhang,Lu Zheng

doi:10.1186/1471-2407-12-546

Abstract

BackgroundA growing body of evidence suggests that microRNAs (miRNAs) play an important role in cancer diagnosis and therapy. MicroRNA-99a (miR-99a), a potential tumor suppressor, is downregulated in several human malignancies. The expression and function of miR-99a, however, have not been investigated in human renal cell carcinoma (RCC) so far. We therefore examined the expression of miR-99a in RCC cell lines and tissues, and assessed the impact of miR-99a on the tumorigenesis of RCC.MethodsMiR-99a levels in 40 pairs of RCC and matched adjacent non-tumor tissues were assessed by real-time quantitative Reverse Transcription PCR (qRT-PCR). The RCC cell lines 786-O and OS-RC-2 were transfected with miR-99a mimics to restore the expression of miR-99a. The effects of miR-99a were then assessed by cell proliferation, cell cycle, transwell, and colony formation assay. A murine xenograft model of RCC was used to confirm the effect of miR-99a on tumorigenicity in vivo. Potential target genes were identified by western blotting and luciferase reporter assay.ResultsWe found that miR-99a was remarkably downregulated in RCC and low expression level of miR-99a was correlated with poor survival of RCC patients. Restoration of miR-99a dramatically suppressed RCC cells growth, clonability, migration and invasion as well as induced G1-phase cell cycle arrest in vitro. Moreover, intratumoral delivery of miR-99a could inhibit tumor growth in murine xenograft models of human RCC. In addition, we also fond that mammalian target of rapamycin (mTOR) was a direct target of miR-99a in RCC cells. Furthermore, siRNA-mediated knockdown of mTOR partially phenocopied the effect of miR-99a overexpression, suggesting that the tumor suppressive role of miR-99a may be mediated primarily through mTOR regulation.ConclusionsCollectively, these results demonstrate for the first time, to our knowledge, that deregulation of miR-99a is involved in the etiology of RCC partially via direct targeting mTOR pathway, which suggests that miR-99a may offer an attractive new target for diagnostic and therapeutic intervention in RCC.

Highlights

A growing body of evidence suggests that microRNAs play an important role in cancer diagnosis and therapy
Results miR-99a is downregulated and correlates with overall survival in renal cell carcinoma To identify the expression of miR-99a in RCC, we firstly performed real-time quantitative Reverse Transcription PCR (qRT-PCR) using the renal cell line HK-2 and RCC cell lines 786–0 and OS-RC-2 and found that miR-99a expression in RCC cell lines (786–0 and OS-RC-2) was significantly lower than that in HK-2 (Figure 1A)
These findings showed a direct interaction between miR-99a and mammalian target of rapamycin (mTOR) mRNA in RCC cell lines

Summary

Introduction

A growing body of evidence suggests that microRNAs (miRNAs) play an important role in cancer diagnosis and therapy. Numerous studies have shown aberrant expression of miRNAs in human cancers [9], including RCC [10], some of which function as tumor suppressor genes or oncogenes [11]. Due to their tissue- and disease-specific expression patterns and tremendous regulatory potential, miRNAs are being identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools [12].

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