Kim Jihye, Jiang Jinmai, Badawi Mohamed, Schmittgen Thomas D
College of Pharmacy, The Ohio State University, Columbus, OH, United States.
College of Pharmacy, University of Florida, Gainesville, FL, United States.
Biochem Biophys Res Commun. 2017 Jun 3;487(3):709-715. doi: 10.1016/j.bbrc.2017.04.121. Epub 2017 Apr 23.
CD44 and miR-221 are upregulated in hepatocellular carcinoma (HCC) cell lines and tumors, however a connection between the two has not been identified. As the expression of miR-221 directly correlated with CD44 in HCC cells, we hypothesized that miR-221 may directly or indirectly regulate CD44 expression. Inhibition of miR-221 with antisense in Sk-Hep-1 or SNU-449 cell lines reduced CD44 protein expression while miR-221 mimic increased CD44 protein levels. miR-221 antisense did not alter the CD44 mRNA levels in Sk-Hep-1 or SNU-449 cells suggesting that regulation of CD44 protein occurs post transcriptionally. To discover miRNAs that may be involved in the miR-221 regulation of CD44, we performed miRNA profiling in SNU-449 cells treated with anti-miR-221. Several miRNAs were increased with miR-221 inhibition including miR-708-5p, a miRNA that targets CD44. As miR-221 targets several regulators of the PI3K-AKT-mTOR pathway and a link between this pathway and CD44 has been previously shown in prostate cancer, we considered miR-221 regulation of CD44 may be through this pathway. Inhibition of miR-221 reduced p-4EBP1, a downstream effector of the PI3K-AKT-mTOR pathway. Likewise, inhibiting the PI3K-AKT-mTOR pathway with the ATP-competitive mTOR inhibitor PP242 reduced CD44 protein in SNU-423 and SNU-449 cells without altering CD44 mRNA levels.
CD44和miR-221在肝癌（HCC）细胞系和肿瘤中表达上调，但二者之间的联系尚未明确。由于miR-221在肝癌细胞中的表达与CD44直接相关，我们推测miR-221可能直接或间接调节CD44的表达。在Sk-Hep-1或SNU-449细胞系中用反义寡核苷酸抑制miR-221可降低CD44蛋白表达，而miR-221模拟物则增加CD44蛋白水平。miR-221反义寡核苷酸未改变Sk-Hep-1或SNU-449细胞中CD44的mRNA水平，提示CD44蛋白的调节发生在转录后。为了发现可能参与miR-221对CD44调节的微小RNA（miRNA），我们在用抗miR-221处理的SNU-449细胞中进行了miRNA谱分析。几种miRNA在miR-221抑制后表达增加，包括靶向CD44的miR-708-5p。由于miR-221靶向PI3K-AKT-mTOR通路的多个调节因子，且该通路与CD44之间的联系先前已在前列腺癌中得到证实，我们认为miR-221对CD44的调节可能通过该通路。抑制miR-221可降低PI3K-AKT-mTOR通路的下游效应分子p-4EBP1。同样，用ATP竞争性mTOR抑制剂PP242抑制PI3K-AKT-mTOR通路可降低SNU-423和SNU-449细胞中CD44蛋白水平，而不改变CD44 mRNA水平。
