Coordinate Suppression of ERBB2 and ERBB3 by Enforced Expression of Micro-RNA miR-125a or miR-125b

Gary K Scott,Andrei Goga,Dipa Bhaumik,Crystal E Berger,Christopher S Sullivan,Christopher C Benz

doi:10.1074/jbc.m609383200

Abstract

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

Highlights

Active areas of research as their role in such processes as development, differentiation, and apoptosis comes into focus [1,2,3,4]
More than 400 human miRNAs have been identified to date with up to ϳ1000 miRNAs postulated to exist on the basis of bioinformatics analyses, leading to the speculation that a majority of human mRNAs are subject to miRNA regulation (9 –10). miRNAs are initially excised from a primary RNA transcript in the nucleus by the nuclear RNase III endonucleases Drosha working in cooperation with DGCR8 [4, 11]
Targeting of a mRNA by a miRNA remains a poorly defined process, it appears to be largely mediated by complementarity between nucleotides 2 to 5, occasionally referred to as the “seed sequences,” with a target element in the transcript 3Ј-UTR [1,2,3, 9, 12]

Summary

Introduction

Active areas of research as their role in such processes as development, differentiation, and apoptosis comes into focus [1,2,3,4]. A human breast cancer cell line, SKBR3, dependent upon endogenous ERBB2 amplification and receptor overexpression for its growth and survival, and the non-transformed and ERBB2-independent human mammary epithelial cell line, MCF10A, were retrovirally infected and selected for viral integration by puromycin selection.

Results

Conclusion