A Study On STAT3 Activity Contributes To Sorafenib Resistance In Hepatocellular Carcinoma Cells

ObjectiveResearch on STAT3 activity contributes to sorafenib drug resistance in hepatocellular carcinoma cells. Method1. Constructing STAT3 gene overexpression recombinant plasmid p Lv-STAT3 and knock-down recombinant plasmid p Lv-sh STAT3, Hep3B-STAT3 cell line of STAT3 gene overexpression and SK-Hep1-sh STAT3 and Huh7-sh STAT3 cell lines of STAT3 gene knockdown were obtained by virus packaging and infection.2. The expression of endogenous and sorafenib treatment of Hep3 B, SK-Hep1, Huh7 cell lines STAT3, p-STAT3(Y705),p-STAT3(S727), Mcl-1 and Cyclin D1 were detected by Western Blot.3. The death of Hep3 B,SK-Hep1,Huh7 cell lines and Hep3 B cells of STAT3 gene overexpression and Vector after sorafenib treatement were record by microscope.4. The cell proliferation and viability were detected by CCK-8 kits.5. The death of Huh7 cells of STAT3 gene knockdown and Vecter after sorafenib treatement were detected by ab115347 plus Hoechst33342.6. Using AO/EB plus Hoechst33342 cell staining kits to detect the death of Huh7 cells after combination sorafenib with p-STAT3(Y705) inhibitor treatement. Result1. Hep3B-STAT3 cell line of STAT3 gene overexpression were constructed successfully, the expression of total STAT3, STAT3 phosphorylation were markedly improved in Hep3B-STAT3 cells. Meanwhile, SK-Hep1-sh STAT3 and Huh7-sh STAT3 cell lines of STAT3 gene knockdown were constructed successfully, the expression of total STAT3, STAT3 phosphorylation were significantly reduced in SK-Hep1-sh STAT3 and Huh7-sh STAT3 cells.2. In the same sorafenib treatment, the death of Hep3 B cells was markedly higher than SK-Hep1 and Huh7 cells’.3. Endogenous STAT3,p-STAT3(S727) of Hep3 B, SK-Hep1 and Huh7 cells were no obviously difference, but p-STAT3(Y705) of SK-Hep1 and Huh7 cells was markedly higher than Hep3 B cells’. The expression of p-STAT3(Y705), p-STAT3(S727) of SK-Hep1 and Huh7 cells were reduced after sorafenib treatment.4. By contrast, the cell growth and sorafenib resistence of Hep3 B of STAT3 gene overexpression was greatly improved. Meanwhile, the death of Hep3B-STAT3 cells was markedly reduced.5. By contrast, the growth and sorafenib resistence of SK-Hep1 and Huh7 of STAT3 gene knockdown were dramatically decreased. Meanwhile, the death of SK-Hep1-sh STAT3 and Huh7-sh STAT3 cells was markedly increased.6. Compared with sorafenib or p-STAT3(Y705) inhibitor alone, combination sorafenib with p-STAT3(Y705) significantly increased cell death rate of Huh7.7. The expression of Mcl-1,Cyclin D1 were enhanced in Hep3B-STAT3 cells, while decreased in Huh7-sh STAT3 cells.The expression of Mcl-1 and Cyclin D1 were reduced by sorafenib in a time and dose-dependent manner. Conclusion1. Up-regulation STAT3 gene significantly promotes Hep3 B cells proliferation and resistence to sorafenib, however, knockdown of STAT3 gene markedly decreases SK-Hep1 and Huh7 cells proliferation and increases sensitivity to sorafenib.2. Combination sorafenib with p-STAT3(Y705) inhibitor increases cell death of Huh7.3. The differential of sorafnib resistence in Hep3 B, SK-Hep1 and Huh7 is mediated by differential p-STAT3(Y705), leading to differential expression of Mcl-1. In conclusion, STAT3 gene is a key factor in sorafenib drug resistence of Hepatocellular carcinoma cells.