Effects Of Isovitexin On Stemness And MiR-34a-5p Expression In Hepatocellular Carcinoma Cancer Stem-like Cells Derived From SK-Hep-1 Cells

Objective:To illuminate whether isovitexin(ISOV),an effective component of Fructus Viticis total flavonoids,can regulate miR-34a-5p expression,and promote apoptosis as well as reduce the number selectively,thereby,inhibiting the stemness of liver cancer stem-like cells(LCSLCs)derived from SK-Hep-1 cells.Methods:First,SK-Hep-1 cells were cultured in vitro.The SK-Hep-1cell line-derived spheroid cells(SK-SC)were obtained by sphere formation culture method and used as a model of SK-Hep-1 derived LCSLCs.Sphere formation and clonogenic assays,flow cytometry as well as RT-qPCR were used to analyze the sphere formation and colony formation rate,the CD44~+cell populations and the mRNA expression of ALDH1,ABCG2 and NANOG in SK-Hep-1 and SK-SC,respectively.Comparison of SK-Hep-1 and SK-SC histone/DNA fragment,Sub-G1populations and the protein expression of Bcl-2,Bax and Mcl-1 using ELISA,flow cytometry and Western blot,respectively.RT-qPCR was used to detect miR-34a-5p expression in L-02 cells,SK-Hep-1 cells and SK-SC.Second,SK-SC were treated ISOV with different concentrations(1.0,3.0,10.0?M)for 24 h,and were analyzed the SK-SC stemness,cell apoptosis,expression of Bcl-2,Bax,Mcl-1 proteins and miR-34a-5p.Third,miR-34a-5p mimic or inhibitor were conducted to transfect SK-SC or SK-Hep-1 cells,then determined the effect of miR-34a-5p on SK-SC stemness,apoptosis and expression of Bax,Bcl-2 and Mcl-1 proteins.Finally,miR-34a-5p mimic or inhibitor were combined with ISOV to treat SK-SC or SK-Hep-1 cells,respectively,then detected the miR-34a-5p expression,stemness,apoptosis and the protein expression of Bax,Bcl-2 and Mcl-1.Results:Our results demonstrated that SK-SC displayed higher stemness and resistance to apoptosis,as well as reduced miR-34a-5p levels compared with SK-Hep-1 cells.ISOV suppressed sphere and colony formation,and decreased CD44~+cell populations.In addition,ABCG2,ALDH1,and NANOG mRNA levels were decreased by ISOV treatment,while there was a concomitant increase in miR-34a-5p levels.With regarding as apoptosis-related proteins,ISOV increased Bax protein levels,and reduced Bcl-2 and Mcl-1 protein levels in SK-SC.Overexpression of miR-34a-5p significantly inhibited the stemness of SK-SC,regulated the expressions of apoptosis-related proteins to promote apoptosis.Downregulation of miR-34a-5p facilitated the stemness and resistance to apoptosis in SK-Hep-1 cells.Importantly,there was a cooperative effect when miR-34a-5p was overexpressed in the presence of ISOV in SK-SC.Down-regulation of miR-34a-5p attenuated the above effects of ISOV on SK-Hep-1 cells.Conclusion:1.ISOV can upregulate miR-34a-5p expression,induce apoptosis and suppress the stemness of LCSLCs derived from SK-Hep-1cells.2.ISOV suppresses the stemness at least partly by regulating apoptosis-related proteins to promote apoptotic cell death via upregulating miR-34a-5p expression in LCSLCs derived from SK-Hep-1cells.