Abstract
subunit of the chromatin-modifying complex of proteins often known as Polycomb group (PcG). EzH2 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. Recently, miR-101 has been shown to interact with EzH2 mRNA and inhibit protein translation. Though recent reports demonstrate significant down regulation of miR101 in several solid tumors suggesting that it might serve as a solid tumor signature, its role in pancreatic cancer in targeting EzH2 is not understood. Methods: PANC-1 and MiaPaca-2 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 miR-101 plasmid transfection. Triplicate growth curves were generated using Cell Counting Kit-8 (CCK-8, Dojindo, Gaithersburg, MD). Standard protocols were followed for colony formation assays, apoptosis and cell cycle phase determination. Eight week-old CB17/ICr-SCID mice were injected with 3 million PANC-1 cells subcutaneously that were transfected with miR-101 plasmid. Results: Transfection of miR-101 resulted in more than 80% knockdown of the EzH2 protein in both the cell lines. Furthermore, we show that the down regulation of miR-101 in turn increases the expression of EzH2. Significant reduction in cell proliferation, colony formation was observed in response to miR101 expression. MiR101 transfected cells showed marked reduction in palpable tumor generation by PANC1 cells, indicating its anti-oncogenic activity of In Vivo. Conclusion: MiR-101 inhibits cell proliferation, colony formation and ability of PANC-1 cells to generate palpable tumors. We conclude that miR-101 may be a potent tumor suppressor in pancreatic adeno-carcinoma by virtue of its repression of EZH2.
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