Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF.

Yue Yu,Zhenya Zhang,Ling Li,Yoshio Kato,Joanna Groden,Sunil C Kaul,Zeenia Kaul,Ran Gao,Renu Wadhwa

doi:10.1038/srep30185

Abstract

Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2′-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16INK4A, pRB, HDM2 and p21WAF1.

Highlights

Have been shown to be either regulated by miRs or involve them for their activities to implement control on cell proliferation[18,19,20,21,22]
We report that miR-335 possesses tumor suppression function, mediated, at least in part, by targeting CARF that in turn regulates several cell cycle monitoring proteins including p16INK4, pRb, p53 and p21WAF1
A retroviral vector constituting two long terminal repeat (LTR) promoters at 5′and 3′ends of GFP gene was generated in a way that the random integration of this vector in the genome would result into (i) expression of GFP; detected by green fluorescence and (ii) its integration site-dependent arbitrary manipulation of the host cell genome

Summary

Introduction

Have been shown to be either regulated by miRs or involve them for their activities to implement control on cell proliferation[18,19,20,21,22]. Several epigenetic drugs (DNA methyltransferases and histone deacetylase inhibitors) that induce senescence in cancer cells have been in practice in conventional chemotherapy Their impact on the miRs and cancer progression remains largely unknown[24]. Based on the fact that more than 98% of the human genomic DNA constitutes protein-noncoding sequences, such random integration of the vector in genome was expected to induce a large variety of miRNAs (referred to as arbitrary miR library). Cells expressing such random library of miRs were subjected to 5-Aza-dC induced senescence for 3–5 days. We report that miR-335 possesses tumor suppression function, mediated, at least in part, by targeting CARF that in turn regulates several cell cycle monitoring proteins including p16INK4, pRb, p53 and p21WAF1

Objectives

Methods

Results