Reprogramming Using microRNA-302 Improves Drug Sensitivity in Hepatocellular Carcinoma Cells

Background: Although studies have shown that Oct4, Sox2, Klf4, and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs, the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors, including us, have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein, we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells.

Conclusions: miR-302–mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation, which caused H3K4 methylation and c-Myc repression.

Methods: Among three miRNAs (miR-200c, miR-302s, and miR-369s) that were previously presented for miRNA-mediated reprogramming, miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302, the cells were incubated in ES medium for 3 weeks and then characterized.

Results: iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression, low proliferation, high apoptosis, low epithelial-mesenchymal transition marker expression (N-cadherin, TGFBR2), and sensitization to drugs. Several miRNAs were changed (e.g., low oncomiR miR-21, high miR-29b). cMyc was decreased, and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP, FABP4, and ALB). AOF2 (also known as LSD1 or KDM1), one of the targets for miR-302, was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres, including cMyc down-regulation and H3K4 methylation. In drug-resistant cells, sensitization was achieved through miR-302-mediated reprogramming.