FTVF Inhibits Characteristics Of MHCC97H-derived HCSCs By Interrupting The Positive Feedback Loop Of DNMT1 And E2F1 By Upregulating MiR-342-3p

Objective:To explore and explain the molecular mechanisms by which Fructus Viticis total flavonoids(FVTF)up-regulates miR-342-3p to target inhibition of DNMT1 and E2F1,interrupts the positive feedback loop of DNMT1 and E2F1,and inhibits the characteristics of hepatocellular carcinoma(HCC)cancer stem cells(CSCs)derived from MHCC97 H cell line(HCSCs).Methods:Human HCC MHCC97H cells were cultured in vitro.HCSCs derived from MHCC97H cells were obtained by immunomagnetic microsphere sorting and spheroid formation culture.Then,MHCC97 H cells or their HCSCs were transfected with miR-342-3p mimics or inhibitors,and DNMT1 or E2F1 genes were knocked down or overexpressed.Subsequently,DNMT1 activity and mRNA expression,miR-342-3p expression,E2F1 protein expression as well as HCSCs characteristics of MHCC97 H cells or their HCSCs [including:hepatocellular carcinoma cancer sphere formation ability,colony formation ability,cancer stem cell markers(CD133,CD44 and ALDH1) and pluripotency transcription factor(Nanog,Bmi1 and Sox2)protein expressions ] were comparatively detected.Methylation specific PCR(MSP)and Dual-Luciferase reporter assay were used to identify the interaction of miR-342-3p with DNMT1 and E2F1 in MHCC97 H cells or their HCSCs.Subsequently,the effects of FVTF on DNMT1 activity and mRNA expression,miR-342-3p expression,E2F1 protein expression and characteristics of MHCC97H-derived HCSCs were systematically studied.We utilized FVTF combined with transfection of miR-342-3p mimics or inhibitors,with the loss of function,gain of function and rescue of function experiments based on knockdown or overexpress of DNMT1 or E2F1 genes,as well as with MSP and Dual-Luciferase reporter assay to explain the molecular mechanism underlying effects of FVTF.Results:Compared with MHCC97H cells,DNMT1 activity and mRNA expression were increased,miR-342-3p expression was decreased,E2F1 protein expression level was increased,and HCSCs characteristics were enhanced in MHCC97H-derived HCSCs.In MHCC97 H cells,transfection of miR-342-3p inhibitor or overexpression of DNMT1 could elevate DNMT1 activity and mRNA expression,reduce miR-342-3p expression level,increase E2F1 protein expression level and enhance HCSCs characteristics.Overexpression of E2F1 effectively inhibited DNMT1 activity and mRNA expression,but did not affect miR-342-3p expression in MHCC97 H cells.In MHCC97H-derived HCSCs,transfection of miR-342-3p mimics or knockdown of DNMT1 significantly inhibited the DNMT1 activity and mRNA expression,Increased miR-342-3p expression level,down-regulated the expression of E2F1 protein and inhibited the characteristics of HCSCs.Although knockdown of E2F1 could inhibit DNMT1 activity and mRNA expression of MHCC97H-derived HCSCs,it could not change the expression level of miR-342-3p.In addition,the results by MSP showed that the methylation level of miR-342-3p promoter in MHCC97H-derived HCSCs was higher than that in MHCC97 H cells.Dual-Luciferase reporter assay confirmed that DNMT1 and E2F1 are the direct target genes of miR-342-3p.In experiments on the effects of drug,the findings showed that FVTF reduced DNMT1 activity and mRNA expression,up-regulated miR-342-3p expression,decreased E2F1 protein amount,and inhibited the characteristics of HCSCs in MHCC97H-derived HCSCs,in a dose-dependent manner.Mechanistically,miR-342-3p mimics or knockdown of DNMT1 cooperated with FVTF to reduce the DNMT1 activity and mRNA expression,up-regulate the expression of miR-342-3p,decrease the expression level of E2F1 protein,and inhibit the characteristics of HCSCs in MHCC97H-derived HCSCs.However,knockdown of E2F1 does not affect the up-regulation of miR-342-3p expression by FVTF.Conversely,miR-342-3p inhibitors or overexpression of DNMT1 could almost eliminate the effects of FVTF,which reduces DNMT1 activity and mRNA expression up-regulates miR-342-3p expression,down-regulates E2F1 protein expression,and inhibits the characteristics of HCSCs in MHCC97 H cells.Exceptively,overexpression of E2F1 has no effect on the up-regulation of miR-342-3p by FVTF.Finally,we found that FVTF(10 μg/m L)resulted in demethylation of miR-342-3p promoter,and cooperated with miR-342-3p to reduce luciferase activities of DNMT1 mRNA 3’UTR and E2F1 mRNA 3’UTR sequences in MHCC97H-derived HCSCS.Conclusions:1.Abnormal activation of DNMT1,epigenetic silencing of miR-342-3p,up-regulation of E2F1 expression by derepression,up-regulation of DNMT1 by E2F1 form a positive feedback loop of DNMT1 and E2F1 to promote and maintain HCSCs characteristics of MHCC97 H cells.2.FVTF inhibits HCSCs characteristics by targeting for up-regulating miR-342-3p,thereby,inhibitions of DNMT1 and E2F1 expressions by interrupting the positive feedback loop of DNMT1 and E2F1 in MHCC97H-derived HCSCs.