Pien Tze Huang Suppresses The Growth Of Hepatocellular Carcinoma Cells Via Modulating MiRNA Regulatory Of Cancer Stem Cells

Objective:Assess the effect of PZH treatment on cell growth of Hepatocellular carcinoma(HCC)cells and explore the underlying mechanisms.Enrich the cancer stem cells(CSC)of HCC by spheroid formation assay and screen the different expressed miRNAs between spheroids of HepG2 and its parental cells.We further evaluate the effect of PZH on growth and tumorigenic potential of CSC in HCC and investigate the effect of PZH treatment of on expression of CSC associated miRNAs.Methods:1.HepG2 and BEL-7702 cells were treated with various concentrations of PZH(0-0.75 mg/mL),and the cell morphology was observed by microscopy.The cell viability was determined by MTT assay.Colony formation assay was performed to determine the cell survival.PI staining and FACS were used to detect the cell cycle progression.Hoechststaining was used to investigate the cell apoptosis.Western-bloting was performed to detect the expression of Bax,Bcl-2,CyclinDl,CDK4,OCT4 and SOX2.2.HepG2 cells were cultured in regular completely medium in 6 well plates or FBS freed cancer stem cell specific medium in ultra-low attachment(ULA)of 6 well plates respectively.Microscopy was performed to observe the cell morphology.RT-PCR was used to determine the expression of OCT4.FASC was used to analyze the percentage of CD 133 and CD90 positive cells.miRNA array was performed to screen the different expressed miRNAs.Q-PCR analysis was used to confirm the different expressed miRNA for the miRNA array.3.CSC of HCC was enriched by spheroid assay and treated with various concentrations of PZH,the cell viability was determined by cell number counting after trypan blue staining.PI staining followed by FACS analysis were used to determine the cell cycle progression.Hoechst staining was used to observe the cell apoptosis.The spheroid formation assay and tumorigenicity assay were performed to analyze the tumorigenic potential of CSC in HCC in vitro and in vivo.Western-blot analysis was used to determine the expression of Bax,Bcl-2,CyclinD1,CDK4,OCT4,SOX2 and p21.Moreover,Q-PCR analysis was performed to determine the expression of miR-483-5p and its target gene CDKN1A/p21.Results:1.PZH treatment significantly inhibited the cell growth and decreased the cell viability of HepG2 and BEL-7402 cells.Moreover,PZH treatment significantly suppressed the cell survival and arrested cell cycle transition from G0/G1 phase to S phase.Furthermore,PZH treatment induced cell apoptosis of HepG2 and BEL-7402 cells.Western-bloting indicated that PZH treatment significantly decreased the ratio of Bcl-2/Bax and down-regulated the expression of cell cycle related genes CyclinDl,CDK4.In addition,PZH treatment significantly down-regulated the expression of OCT4 and SOX2,which had been proved to be two of bio-markers for CSC.2.FBS freed CSC specific medium with ultra-low attachment(ULA)in 6 well plates was used to enrich the CSC from HepG2 cells.Moreover,we confirmed that the expression of sternness gene OCT4 was up-regulated and the percentage of CD 133 and CD90 positive cells were significantly increased in the spheroids of HepG2 cells,comparing with HepG2 cells.Take together,the enriched HepG2 spheroid cells exhibited the phenotype of CSC of HCC.Moreover,using miRNA array,we identify 234 miRNAs had been up-regulated and 218 miRNAs had been down-regulated in the spheroids of HepG2 cells(cut off>1.5,P value<0.05).The data from the miRNA array indicated that the expression of miR-483-5p and miR-582-5p were significantly up-regulated in CSC cells of HCC,which had been confirmed by Q-PCR analysis.3.PZH treatment obviously decreased the cell viability of HepG2 spheroid cells.Furthermore,PZH treatment significantly arrested the cell cycle on transition from G0/G1 phase to S phase and induced cell apoptosis of HepG2 spheroid cells.Moreover,PZH treatment suppressed the spheroids formation and the tumor-formation ability of spheroids of HepG2 cells in vitro and in vivo.In addition,PZH treatment significantly decreased the ratio of Bcl-2/Bax and down-regulated the expression of cell cycle related genes CyclinDl,CDK4 of HepG2 spheroid cells.In addition,PZH treatment significantly down-regulated the expression of OCT4 and SOX2 of HepG2 spheroid cells.We also found that the expression of miR-483-5p was significantly down-regulated in spheroids of HepG2 cells after PZH treatment,while up-regulated the expression of its target gene CDKN1A/p21 on both mRNA and protein levels.Conclusion:PZH treatment significantly inhibited the growth of HCC by inhibiting cell proliferation and inducing cell apoptosis,which may be mediated by decreasing the ratio of Bcl-2/Bax,down-regulating the expression of CyclinD1 and CDK4.We enriched the CSC of HCC by spheroid formation assay,abnormal expression of miRNA(including miR-483-5p and miR-582-5p)maybe involved in regulatory tumorigenic potential of CSC in HCC.Moreover,PZH treatment significantly suppressed the cell growth and tumorigenic potential of HepG2 spheroids cells in vitro and in vivo,and by regulating the ratio of Bcl-2/Bax,the expression CyclinD1,CDK4,SOX4,OCT4,miR-483-5p and its target gene CDKN1A/p21 maybe the underlying mechanism.