Functional analysis of microRNAs in human hepatocellular cancer stem cells.
Bottom Line: Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion.Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion.In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy.
Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.Show MeSH
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Mentions: We next evaluated the effect of let-7a and let-7b inhibition on the response to chemotherapy in vitro. The effects of the anti-let-7a and anti-let-7b were assessed by cotransfecting with the pRL-Tk let-7a and let-7b firefly luciferase constructs that contains two let-7a and let-7b sites in the 3′-UTR of the firefly luciferase reporter. The significant increases in luciferase activity confirmed the efficacy of anti-let-7a and let-7b under the conditions used for our studies (Fig. 6A and B). Meanwhile, introduction of siRNA to IL-6 in HSC cells also up-regulates the luciferase activity, suggested IL-6 directly enhanced let-7a and let-7b expressions (Fig. 6C). Cytotoxicity in response to diverse agents such as sorafenib and doxorubicin was enhanced by pre-incubation with antisense inhibitors to let-7a and let-7b. IC50s to all two reagents have significantly reduced in response to chemotherapy in HSC cells pre-incubated with anti-let-7a when compared with a control inhibitor (Fig. 6D and E). Similarly, an increase in activated caspase-3 expression, the predicted target gene of let-7 family members, was noted in response to anti-let-7a in Western blots from sorafenib-treated HSC cells. Furthermore, anti-let-7a treatment in HCC CSCs has successfully increased SOCS1 expression, another predicted target gene of let-7a, and subsequently reduced Stat3 activity (Fig. 6F). Thus, inhibition of let-7a and let-7b increases chemotherapy-induced apoptosis in HCC CSCs, which may be involved in enhanced expression of Caspase-3 as well as SOCS1-regulated Stat3 pathway.
Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.