Cancer Letters

Cancer Letters

Volume 374, Issue 1, 28 April 2016, Pages 107-116
Cancer Letters

Original Articles
miR-206 functions as a novel cell cycle regulator and tumor suppressor in clear-cell renal cell carcinoma

https://doi.org/10.1016/j.canlet.2016.01.032Get rights and content

Highlights

  • We reviewed and experimentally analyzed the currently available miRNA expression profiles data of ccRCC.

  • miR-206 was identified as one of the most critical tumor-suppressing microRNAs in ccRCC.

  • miR-206 inhibited ccRCC cell proliferation through inducing cell cycle arrest by directly targeting cell cycle related gene CDK4, CDK9 and CCND1.

Abstract

Purpose

In this study we tried to systematically investigate the tumor suppressing microRNAs in ccRCC.

Materials and methods

The MTS cell viability and colony formation assay were used to systematically detect the tumor suppressing ability of down-regulated miRNAs in ccRCC. Then miR-206 expression was detected by RT-qPCR and in situ hybridization in ccRCC cell lines and clinical samples. Oligonucleotides were used to overexpress or down-regulate miR-206. MTS cell viability, EdU cell proliferation, colony formation assay, flow cytometry, Xenograft subcutaneously and orthotopic implantations were done to examine tumor suppressing effects of miR-206 in vitro and in vivo. Luciferase assay was performed to verify the precise target of miR-206.

Results

We reviewed and experimentally analyzed the currently available miRNA expression profiles data of ccRCC and identified miR-206 as one of the most critical tumor-suppressing microRNAs in ccRCC. In addition, miR-206 inhibited ccRCC cell proliferation through inducing cell cycle arrest by directly targeting cell cycle related gene CDK4, CDK9 and CCND1.

Conclusions

All these results suggested that miR-206 functioned as a novel cell cycle regulator and tumor suppressor in ccRCC and could be considered as a potential target for ccRCC therapy.

Introduction

Renal cell carcinoma (RCC) accounts for 3% of adult malignancies and is the most lethal urological malignancy, with about 65,150 new cases and 13,680 deaths estimated for 2013 in the United States [1]. RCC can be histologically classified into several subtypes, among which Clear-cell renal cell carcinoma (ccRCC) is the most common, accounting for 70–80% of all [2]. Apart from surgical therapies, current targeted therapies have improved survival in patients with advanced disease but complete response occurs rarely [3]. Therefore, increased understanding of the underlying molecular biology of RCC is necessary to identify the clinical and molecular phenotype of response and resistance, and to uncover novel targets [4].

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that suppress gene expression at the post-transcriptional level by blocking mRNA translation or degrading target mRNAs [5]. Increased studies have extended the function of miRNAs to both physiological and pathological conditions, including cancer [6]. Most current clinical trials are for the use of microRNAs as biomarkers for patient stratification, prognosis, and drug efficacy [7]. In addition to that, microRNAs is now under investigation as potential therapeutic agents for cancer. The first microRNA-based therapy specifically for cancer is MRX34: a synthetic miR-34a mimic loaded in liposomal nanoparticles, which is now is in a phase I clinical trial (NCT01829971) for primary liver cancer and liver metastases [8].

It is demonstrated that a pool of miRNAs is as well involved in RCC through modulating their target genes implicated in multiple biological processes. For example, oncogenic miR-185 which has been found to be significantly up-regulated in RCC and anti-correlated with the tumor suppressor gene PTEN [9]. Epithelial-mesenchymal transition-related microRNA-200s regulate molecular targets and pathways in renal cell carcinoma [10]. MicroRNA-218 inhibits cell migration and invasion in renal cell carcinoma through targeting caveolin-2 involved in focal adhesion pathway [11]. RCC is known to be characterized by the loss of the VHL gene. VHL-regulated miR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal cell carcinoma [12].

Unfortunately, the underlying molecular biology of microRNAs in ccRCC pathogenesis remains largely unknown. So in this study we tried to systematically investigate the tumor suppressing microRNAs in ccRCC.

Section snippets

Clinical sample preparation

A total of 41 paired clear cell renal cell carcinoma and corresponding noncancerous tissues (NCT) were obtained sequentially from patients undergoing radical nephrectomy from the period of 2010–2014. The basic clinical characteristics of these patients were listed in Table S1. Corresponding noncancerous tissues were acquired at least 5 cm away from the tumor site. Tissues specimens were snap frozen in liquid nitrogen before protein and RNA extraction. The study protocol was approved by the

Screening for the novel tumor suppressing miRNA in ccRCC

We previously performed a comprehensive meta-analysis of miRNA expression profiles in RCC and identified a down-regulated miRNAs panel including 38 miRNAs that reported in at least three expression profiling studies [18]. Based on that, we selected 23 miRNAs to perform further experimental analysis, while other 15 miRNAs which had been reported functions in RCC elsewhere were excluded (Table 1).

To investigate the tumor suppressing effect of these 23 down-regulated miRNAs, we first transfected

Discussion

In our previously performed comprehensive meta-analysis of miRNA expression profiles in RCC, we identified a down-regulated miRNAs signature including 38 miRNAs that reported in at least three miRNA expression profiling studies. Some of these down-regulated miRNAs have been suggested as tumor suppressors in RCC, such as miR-200/141/429 family [19], [20], [21], miR-204 [12], [22] and miR-138 [23]. In this study, we focused on miR-206 because it had the greatest proliferation inhibitory effect on

Conclusions

In this study, we reviewed and analyzed the currently available miRNA expression profiles data of ccRCC and identified miR-206 as one of the most critical tumor-suppressing microRNA in ccRCC. In addition, miR-206 inhibited ccRCC cell proliferation through inducing cell cycle arrest by directly targeting cell cycle related gene CDK4, CDK9 and Cyclin D1. All these results suggested that miR-206 functioned as a novel cell cycle regulator and tumor suppressor in ccRCC and could be considered as a

Conflict of interest

The authors have declared no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31372562, 81170650, 81270788, 81470935, 81402098, 81402105, 81402087), the National Major Scientific and Technological Special Project for Significant New Drugs Development (2012ZX09303018), the Chenguang Program of Wuhan Science and Technology Bereau (2013072304010833, 2015070404010199), and The National High Technology Research and Development Program 863 (2014AA020607), and the Natural Science Foundation of Hubei

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