Fig 8.

Regulation of miR-181 expression by Twist in HCC stem cells. (A) The schematic diagrams of miR-181 genes showing the Twist1 binding e-boxes are located in 5′-promoter regions of miR-181a-1/miR-181b-1. (B) Real-time PCR confirmed reduced expression of mature miR-181a in HSC-SR and HSC-DF cells in Twist silencing group when compared with expression in their respective controls. (C, D) Total RNAs were isolated using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit and real-time PCR assay was carried out using Taqman MicroRNA Assay kit. Increased miR-181a and miR-181b binding with Twist were observed in HCC cancer stem cells under self-renewal or differentiation condition. *P < 0.05 when compared with respective controls. (E, F) Overexpression of miR-181a and miR-181b inhibited RASSF1A, TIMP3 and NLK protein expressions in HSCs. Representative immunoblots (E) and quantitative data (F, mean ± S.E.) from four separate blots are shown. *P < 0.05 relative to expression in controls.