Relationship of miR-101 and targeted epigenetic silencing of EzH2 on cell growth and invasiveness in ovarian cancer cells.

Abstract

e15538 Background: Enhancer of zeste homolog 2 (EzH2), the catalytic subunit of Polycomb group (PcG) of the chromatin modifying complex, is abnormally elevated in several cancers. High levels of EzH2 correlate strongly with high-grade malignancies that are invasive, poorly differentiated and of advanced stage at presentation. miR-101 belongs to a family of micro-RNAs (miRNAs) that are short non-coding RNA sequences involved in epigenetic regulation of gene expression. miR-101 has been shown to bind to EzH2 mRNA inhibiting protein translation. Though recent reports demonstrate significant down regulation of microRNA-101 (miR-101) in several solid tumors suggesting that it might serve as a solid tumor signature, its role in ovarian cancer is not well defined. Methods: MDAH-2774 and SKoV3 cell lines were transfected with miR-101 expression plasmid (miR-101 sequence is as follows: 5' UAC AGU ACU GUG AUA ACU GAA G 3') using effectene (Qiagen Inc.,Valencia,CA). Standard protocols were followed for western blot analysis of EzH2 levels after EzH2 knockdown with miR-101 plasmid transfection. Triplicate growth curves were generated using Cell Counting Kit-8 (CCK-8) (Dojindo, Gaithersburg, MD). Eight week-old CB17/ICr-SCID mice were injected with 3 ×106 MDAH-2774 cells that were transfected with miR-101 plasmid. Results: miR-101 achieved more than 70% knockdown of the EzH2 protein 48 hrs after transfection in both SKoV3 and MDAH-2774 cell lines, demonstrating efficient inhibition of EzH2. Further, significant reduction in cell proliferation in both MDAH-2774 and SKoV3 cell lines was observed in response to EzH2 knock down. MDAH-2774 cells transfected with miR-101 showed 15% less growth than the control on day 3 after transfection, versus 48% for SKOV3 cells. EzH2 knockdown in CB17/ICr-SCID mice was associated with marked reduction in palpable tumor generation by MDAH-2774 cells, indicating that EzH2 promotes the growth of MDAH-2774 cell line in vivo. Conclusions: The results indicate that miR-101 mediated epigenetic silencing of EzH2 may be a potential therapeutic strategy for ovarian cancer. No significant financial relationships to disclose.