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

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.