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Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

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.

View Article: PubMed Central - PubMed

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.

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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.
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fig06: 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.

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.


Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

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.
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Related In: Results  -  Collection

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fig06: 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.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus