Expression of the lncRNA Maternally Expressed Gene 3 (MEG3) Contributes to the Control of Lung Cancer Cell Proliferation by the Rb Pathway

Elizabeth Long,Susan M. Dougherty,Lindsey Reynolds,Aamir Ahmad,Brian F. Clem,William W. Lockwood,Traci L. Kruer,Tanya de Silva

doi:10.1371/journal.pone.0166363

Elizabeth Long, Susan M. Dougherty + Show 6 more

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https://doi.org/10.1371/journal.pone.0166363

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Journal: PLOS ONE	Publication Date: Nov 10, 2016
Citations: 64	License type: CC BY 4.0

Affiliation: University of Louisville, University of British Columbia

Abstract

Maternally expressed gene 3 (MEG3, mouse homolog Gtl2) encodes a long noncoding RNA (lncRNA) that is expressed in many normal tissues, but is suppressed in various cancer cell lines and tumors, suggesting it plays a functional role as a tumor suppressor. Hypermethylation has been shown to contribute to this loss of expression. We now demonstrate that MEG3 expression is regulated by the retinoblastoma protein (Rb) pathway and correlates with a change in cell proliferation. Microarray analysis of mouse embryonic fibroblasts (MEFs) isolated from mice with genetic deletion of all three Rb family members (TKO) revealed a significant silencing of Gtl2/MEG3 expression compared to WT MEFs, and re-expression of Gtl2/MEG3 caused decrease in cell proliferation and increased apoptosis. MEG3 levels also were suppressed in A549 lung cancer cells compared with normal human bronchial epithelial (NHBE) cells, and, similar to the TKO cells, re-constitution of MEG3 led to a decrease in cell proliferation and elevated apoptosis. Activation of pRb by treatment of A549 and SK-MES-1 cells with palbociclib, a CDK4/6 inhibitor, increased the expression of MEG3 in a dose-dependent manner, while knockdown of pRb/p107 attenuated this effect. In addition, expression of phosphorylation-deficient mutant of pRb increased MEG3 levels in both lung cancer cell types. Treatment of these cells with palbociclib also decreased the expression of pRb-regulated DNA methyltransferase 1 (DNMT1), while conversely, knockdown of DNMT1 resulted in increased expression of MEG3. As gene methylation has been suggested for MEG3 regulation, we found that palbociclib resulted in decreased methylation of the MEG3 locus similar to that observed with 5-aza-deoxycytidine. Anti-sense oligonucleotide silencing of drug-induced MEG3 expression in A549 and SK-MES-1 cells partially rescued the palbociclib-mediated decrease in cell proliferation, while analysis of the TCGA database revealed decreased MEG3 expression in human lung tumors harboring a disrupted RB pathway. Together, these data suggest that disruption of the pRb-DNMT1 pathway leads to a decrease in MEG3 expression, thereby contributing to the pro-proliferative state of certain cancer cells.

Highlights

The retinoblastoma tumor suppressor pathway is disrupted in many human cancers. pRb regulates cell cycle progression and proliferation by binding to the E2F family of transcription factors and repressing their activity
Microarray analysis comparing WT mouse embryonic fibroblasts (MEFs) and MEFs isolated from mice genetically deleted of all three retinoblastoma protein (Rb) family members (Rb-1, Rbl1 and Rbl2) [triple knock-out (TKO)] revealed that Gtl2 expression is significantly decreased in TKO MEFs compared to WT MEFs (76-fold decrease, p = 4x10-13)
Our studies have identified a novel mechanism by which pRb controls cellular proliferation via increased expression of the long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3)

Summary

Introduction

The retinoblastoma tumor suppressor (pRb) pathway is disrupted in many human cancers. pRb regulates cell cycle progression and proliferation by binding to the E2F family of transcription factors and repressing their activity. PRb regulates cell cycle progression and proliferation by binding to the E2F family of transcription factors and repressing their activity. For cell cycle progression to occur, pRb must be inactivated through phosphorylation by cyclin dependent kinases (CDKs), CDK4 and CDK6 [1]. This phosphorylation causes pRb to dissociate from the E2F transcription factors, allowing transcription of genes required for S-phase and G2/M progression. The lncRNA maternally expressed gene 3 (MEG3) has been shown to function as a tumor suppressor and inhibit cell proliferation in a number of human cancer cell lines [2,3,4,5,6,7]. The mechanism for this is unclear, MEG3 has been shown to inhibit cell proliferation by both activation of p53, and by targeting the TGF-β pathway through direct chromatin interactions [3, 7, 8]

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