Fig 6.

MicroRNA let-7 is involved in the HCC stem cell resistance to chemotherapy. (A, B) To validate the efficacy of the anti-let-7 inhibitor, HCC stem cells plated (2 × 10⁶ cells/well) in six-well plates were transfected with 1 μg of pRL-TK let-7a or let-7b (firefly luciferase construct), 1 μg of pRL-TK (Renilla luciferase construct), and either anti-let-7a (A), anti-let-7b (B) or control inhibitor. Luciferase assays were performed 48 hrs after transfection. The anti-let-7a (anti-let-7b) inhibitor directly blocked the effect of endogenous let-7a (let-7b) on the luciferase reporter. (C) siRNA to IL-6 or control was trans-fected with 1 μg of pRL-TK let-7a or let-7b (firefly luciferase construct), 1 μg of pRL-TK (Renilla luciferase construct). Dual-luciferase assay has demonstrated that silencing IL-6 significantly increased the miRNA expressions of let-7 family. (D, E) Human hepatocytes and HCC stem cells were seeded in a 96-well plate and incubated with different concentrations of sorafenib. The MTS assays were performed 72 hrs after treatment. The data were expressed as percentage of control group. The IC₅₀ values were calculated with Excel XLFit program and marked in the middle of the box. (D) Illustrates the detailed IC₅₀ analysis in different cell lines with anti-let-7a treatment whereas E exemplified IC₅₀ results expressed as the mean ± S.E. of eight different experiments. Silencing of let-7a significantly sensitizes HCC cancer stem cells to sorafenib treatment. (F, G) The expression of SOCS1 and downstream kinase is regulated by let-7a. Western blots of cell lysates were performed and sequentially probed with antibodies against SOCS1, Caspase-3, phospho and total Stat3, and tubulin as a loading control in HepG2 or HSC-SR cells transfected with let-7a mimics or inhibitors with related controls. Representative immunoblots (F) and quantitative data (G, mean ± S.E.) from four separate blots are shown.