| Studies have showed that the epigenetic modulation plays an important role in promoting and maintaining the characteristics of cancer stem cells(CSCs).However,the epigenetic mechanism of promotion and maintaince for CSC characteristics and drug action in hepatocellular carcinoma(HCC)remain not complete clarity.As our preliminary results shown,the DNMT1 activity is enhanced and miR-34a-5p level is down-regulated in liver cancer stem cells(LCSCs)derived from SMMC-7721 cell line,compared with the parental cells.In pharmacological screening experiments of antitumor activity,Fructus Viticis total flavonoids(FVTF)could effectively inhibit the LCSCs sphere-forming capability,reduce DNMT1 activity and up-regulate miR-34a-5p level.We also found that miR-34a-5p binds to the 3’untranslated region of FoxM1 using computer prediction.Based on the above evidences,we hypothesize that the DNMT1/miR-34a-5p/FoxM1axis plays an important role in promoting the characteristics of LCSCs and FVTF inhibits the characteristics of LCSCs by inhibiting the activity and expression of DNMT1,up-regulating expression of miR-34a-5p,directly targeted inhibiting FoxM1.In order to verify the aforementioned hypothesis,SMMC-7721 cell line derived-CD133~+liver cancer spheres were used as LCSCs in here.ELISA,Western blot,quantitative reverse transcription-PCR(qRT-PCR)and methylation-specific PCR(MSP)were used to determine the activity,protein and mRNA expression of DNMT1,expression and promoter methylation level of miR-34a-5p in SMMC-7721 cells and LCSCs,respectively.For evaluating in vitro LCSCs characteristics,sphere-forming and agar colony-forming assay,flow cytometry analysis,Western blot and qRT-PCR were used to detect sphere-formation and colony formation rate,CD133~+cell population,and protein and/or mRNA expression levels of CSC markers(CD133,CD44 and ALDH1)and pluripotency transcription factor(Bmi1,Sox2 and Oct4)in SMMC-7721cells and LCSCs,respectively.For evaluating in vivo LCSCs characteristics,tumor growth of xenograft models in nude mice and the protein expression of CD133 and CD44 in LCSCs were detected.For confirming that DNMT1 constitutive activation promotes characteristics of LCSCs and DNMT1 inhibition-mediated FVTF inhibition of characteristics in LCSCs,DNMT1 was down-knocked or over-expressed using decitabine(DAC)and DNMT1 shRNA or DNMT1 cDNA.We respectively used miR-34a-5p mimics and inhibitors to detect the effects that miR-34a-5p down-regulation promotes characteristics of LCSCs and miR-34a-5p up-regulation mediates inhibition of characteristics in LCSCs by FVTF.After identification of FoxM1 as a miR-34a target,we examined whether overexpression of FoxM1 promotes characteristics of LCSCs,FVTF inhibits characteristics of LCSCs by inhibition of FoxM1using luciferase reporter assay,FoxM1 knockdown or over-expression,DAC or thiostrepton(THI)treatment,respectively.In an extended study,spheres derived from MHCC97H cell line were used as liver cancer stem cell-like cells(LCSLCs).The phenotype and function of the DNMT1/miR-34a-5p/FoxM1 axis in MHCC97H-derived LCSLCs and the pharmacological effects of FVTF have been established.To identify the role of DNMT1/miR-34a-5p/FoxM1 signal axis in human HCC genesis and prognosis,HCC patient data from The Cancer Genome Atlas(TCGA)cohort and gene expression data series(GSE)dataset were analyzed.The results showed that SMMC-7721 derived-LCSCs exhibited elevated DNMT1 activity and expression,under-expressed miR-34a-5p,activated FoxM1 and enhanced in vitro and in vivo characteristics of LCSCs.The treatment of DAC or knockdown of DNMT1 inhibited DNMT1 activity and expression,up-regulated miR-34a-5p expression,decreased miR-34a-5p promoter methylation levels,and attenuated in vitro characteristics of LCSCs in LCSCs.Meanwhile,the xenograft tumor growth of LCSCs in nude mouse model was inhibited and the protein expression of DNMT1 was decreased as well.Over-expression of DNMT1 enhanced DNMT1 activity and expression,down-regulated miR-34a-5p expression,increased miR-34a-5p promoter methylation levels,and enhanced in vitro characteristics of LCSCs in SMMC-7721cells.MiR-34a-5p mimic significantly up-regulated expression of miR-34a-5p,attenuated in vitro characteristics of LCSCs and inhibited the xenograft tumor growth,accompanied by the increased expression level of miR-34a-5p.However,the activity and protein and mRNA expression of DNMT1 was not affected in LCSCs.MiR-34-5p inhibitor decreased miR-34a-5p expression and increased in vitro characteristics of LCSCs in SMMC-7721 cells,without affecting the activity and the protein and mRNA expression of DNMT1.In addition,miR-34a-5p mimic rescued the effects that over-expressed DNMT1 inhibits the expression of miR-34a-5p and enhances in vitro characteristics of LCSCs in SMMC-7721 cells.Luciferase reporter gene assay confirmed that FoxM1 is a direct functional target of miR-34a-5p in SMMC-7721 cells;FoxM1 knockdown or overexpression can mimic or antagonize the inhibition effects of miR-34a-5p on characteristics of LCSCs.FVTF inhibited the activity and mRNA expression of DNMT1,up-regulated miR-34a-5p expression,decreased miR-34a-5p promoter methylation level,down-regulated FoxM1 expression and attenuated characteristics of LCSCs in vitro,in a concentration-dependent manner.Meanwhile,xenograft tumor growth in nude mice was inhibited,accompanied by decreased expression of DNMT1 protein and increased expression of miR-34a-5p.DAC,miR-34a-5p mimic and THI cooperated with FVTF to inhibit characteristics of LCSCs in vitro and in vivo;reduce DNMT1 activity and mRNA expression,up-regulate miR-34a-5p expression,down-regulate FoxM1 expression,respectively.However,miR-34a-5p mimic did not affect the activity and mRNA expression of DNMT1,and THI did not affect the activity and mRNA expression of DNMT1 and expression of miR-34a-5p.Over-expression of DNMT1,miR-34a-5p inhibitor and FoxM1 over-expression could antagonize the effects that FVTF suppresses in vitro characteristics of LCSCs,DNMT1activity and mRNA expression,up-regulates miR-34a-5p expression,down-regulates FoxM1 expression in SMMC-7721 cells,respectively.However,the activity and mRNA expression of DNMT1 were not affected by treatment with miR-34a-5p inhibitor;the activity and mRNA expression of DNMT1 and miR-34a expression was not affected by FoxM1 over-expression.In the MHCC97H cell line,LCSLCs exhibited activation of DNMT1,under-expression of miR-34a-5p,overexpression of FoxM1 and enhanced characteristics of LCSLCs,these phenotypes could effectively attenuate by FVTF.DNMT1 and FoxM1 transcription levels were increased,miR-34a-5p level was reduced in HCC compared to paired para-carcinoma tissue,and DNMT1 and FoxM1 transcription levels were negatively correlated with miR-34a-5p level,which were confirmed by data from TCGA and GEO HCC cohorts.A Kaplan–Meier analysis showed that HCC patients in TCGA cohort with high DNMT1 and FoxM1 transcription levels had shorter overall than those with low DNMT1 and FoxM1 expression.In summary,we can gain the conclusions by integrating the above results as follows:1.Constitutive activation of DNMT1 epigenetic silencing miR-34a-5p expression leading to up-regulation of FoxM1 by disinhibition plays an important role in the promotion and maintenance of LCSCs characteristics.2.FVTF possessed the inhibitory effects of characteristics of LCSCs.Its mechanism is involved in inhibition of the activity and expression of DNMT1 and up-regulation of miR-34a-5p expression,targeted for inhibition of FoxM1 expression. |
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