MiR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

Silvia Galardi,Neri Mercatelli,Ezio Giorda,Simone Massalini,Giovanni Vanni Frajese,Silvia Anna Ciafre,Maria Giulia Farace

doi:10.1074/jbc.m701805200

Abstract

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation.

Highlights

Among factors whose misregulation was tightly linked to prostate cancer (PCa) progression, the cyclin-dependent kinase inhibitor p27Kip1 is a well established marker of poor prognosis as it was shown that absent or decreased p27Kip1 expression is associated with high tumor grade and poor prognosis of PCa and of several other human cancers [13,14,15,16,17,18]
In this work we describe the differential expression of two microRNAs, miR-221 and miR-222, encoded in tandem from a gene cluster located on chromosome X, in three human prostate carcinoma cell lines, the androgen-independent, strongly aggressive PC3 cell line, the androgen-responsive 22Rv1, and the androgen dependent, slowly growing LNCaP, which represent models of distinct stages of prostate carcinoma progression
We show that p27Kip1 expression in the three PCa cell lines inversely correlates with that of miR-221/miR-222, and that the ectopic overexpression of miR-221 or both microRNAs in LNCaP, where they are normally almost undetectable, has deep consequences on the proliferation rate and the cell cycle phase distribution

Summary

Introduction

This study, performed by Northern blot analysis, revealed that the microRNAs miR-221 and miR-222 show a differential expression: they are detectable in PC3 cells, deriving from a distal metastasis of an androgen-independent, highly aggressive tumor, whereas they are almost absent in LNCaP, derived from a local lymph node metastasis of an androgen-dependent, slowly growing carcinoma (Fig. 1A).

Results

Conclusion