| Objective: To explore whether all-trans retinoic acid(ATRA)reverses epithelial-mesenchymal transition(EMT)of hepatocellular carcinoma Hepa1-6 cells in vitro,and investigate its regulatary mechanism on miRNA-200 family and the downstream target genes,providing new strategies to the clinical treatment of hepatocellular carcinoma.Methods: In the first part,the Hepa1-6 cells were treated with DMEM containing different concentrations of ATRA(0,0.1,1.0,10.0 ?mol/L).Trypan blue,Crystal Violet Staining and Colony formation assay were used to assess cell proliferation and colony formation at indicated time points.Cell apoptosis was examined by Hoechst Staining.The parallel migration ability was detected by Wound Healing assay.Transwell assay was used to evaluate the cell ability of vertical migration and invasion.The mRNA expression of mature hepatic markers ALB(albumin),CK18(cytokeratin 18),TAT(tyrosine aminotransferase),ApoB(Apo lipoprotein B)and that of hepatocarcinoma marker AFP(alpha fetoprotein)was detected by real-time PCR,as well as the protein expression of AFP,ALB,CK18 was detected by western blot and immunofluorescence staining.Indocyanine green(ICG)uptake and Periodic Acid Schiff(PAS)staining were performed to assess the mature function of induced Hepa1-6 cells.The mRNA expression of Epithelial marker E-cadherin and interstitial marker N-cadherin,sail,twist,vimentin were detected by real-time PCR.In the second part,the optimum concentration of ATRA was used to induce Hepa1-6 cells,the mRNA expression of all miRNA200 family members was detected by real-time PCR in order to screen out significantly differently expressed miR200 moleclues.miRNA antagomir was used to specificly suppress the biological effect of corresponding miR200 members.The Hepa1-6 cells were divided into four groups mainly: a.control group;b.ATRA group;c.miRNA antagomir Negative Control(NC)+ATRA group;d.miR-200 s antagomir +ATRA group.MTT assay and Colony formation assay were used to assess cell proliferation and colony formation.Annexin V-FITC/PI staining assay was used to measure apoptotic cells by flow cytometry.The parallel and vertical migration abilities of cells were detected by Wound Healing assay and Transwell assay,respectively.The mRNA expression of ALB and CK18 was detected by Real-time PCR.Transwell invasion assay was used to assess the invasion ability.ICG uptake and PAS staining were performed to assess the mature function of different treated Hepa1-6 cells cells.After 3 days of ATRA treatment,transcription factor microarray was carried out to detect the activity of 345 different transcription factors;bioinformatics analysis was performed to access the target genes related to different transcription factors that asscioated with miR-200 family.Then the expression of the target genes was verified by real-time PCR and western blot.Results: In the first part,compared with control group,the proliferation,migration and invasion capacity of ATRA treated Hepa1-6 cells were obviously inhibited(p<0.05),and the apoptosis rates increased significantly.The mRNA expression of mature hepatic markers ALB,CK18,TAT,ApoB increased and that of hepatocarcinoma marker AFP decreased,the regulation of protein levels showed similar trend to mRNA levels.At 7th day after ATRA induction,Hepa1-6 cells showed comparable indocyanine green(ICG)uptake and glycogen storage function to that of the control.The mRNA expression of mesenchymal markers(N-cadherin,sail,twist and vimentin)was decreased and epithelial marker E-cadherin increaslingly expressed.Furthermore,the role of ATRA was dose-dependently strengthened with the increased concentrations of ATRA treatment,and the optimum concentration of ATRA was 10?mol/L.In the second part,1)after treatment with 10?mol/L ATRA,mRNA expression of miR200a-3p,miR200c-3p and miR141-3p were obviously up-regulated.2)Consistent with the results of first part,compared with control group,the proliferation,colony formation,parallel and vertical migration and invasion abilities of Hepa1-6 cells in ATRA group was obviously inhibited(p<0.05),the apoptosis rate and mRNA expression of ALB,CK18 were increased(p<0.05),even ICG uptake and PAS staining positive cells significantly were enhanced(p<0.05).Whereas,compared with NC +ATRA group,the proliferation,parallel migration and invasion abilities of hepa1-6 cells in mi R-200a/200c/141-3p antagomir +ATRA groups were enhanced(p<0.05),even reached the level of control in miR-200a/200c-3p antagomir +ATRA groups(p<0.05 compared with miR-141-3p antagomir +ATRA group).But,colony formation of three group showed no significant difference with NC+ATRA group;the apoptosis rate was all decreased in three groups(p<0.05).However,the vertical migration rate was increased,mRNA expression of ALB,CK18 were downregulated,the ICG uptake and PAS staining positive cells were decreased only in mi R-200a/200c-3p antagomir +ATRA groups compared with NC+ATRA group(p<0.05).MiR-141-3p antagomir could not inhibit the increased abilities of vertical migration,differentiation and maturation in ATRA induced Hepa1-6 cells.3)Result of transcription factor microarray showed ATRA treatment upregelated transcription activity of 8 transcription factors and down-regulated transcription activity of 35 transcription factors.Bioinformatics analysis results exhibited that Jun,Pouf1,Ets1,zeb1 and klf12 genes might be regulated by mi R-200a-3p,200c-3p,and 141-3p which corresponded to AP2,AREB1,Brn-3,Ets1/PEA3,and Fra-1/JUN transcription factors.Other miR-200 subtypes appeared no correlation with discrepant transcription factors.Furthermore,mRNA expression of Fra-1,Pouf1,Etv4 and zeb1 was significantly decreased and that of Klf12 was increased after treatment with ATRA.In addition,after treatment of mi R-200a/200c-3p antagomir,the protein expression of Fra-1,Pouf1,Etv4 and zeb1 was enhanced compared with miRNA negative control group(p<0.05),and Klf12 protein showed no significant change.Conclusion: ATRA can inhibit the growth,migration,and invasion of Hepa1-6 cells(hepatocellular carcinoma cells in mice),even promote its apoptosis and induce differentiation and mature in a dose-dependent manner by reversing the epithelial mesenchymal transition.ATRA may reverse EMT of Hepa1-6 cells by regulating miR-200a/200c/141-3p.miR-200a/200c-3p/141-3p were all involved in the effect of inhibiting proliferation,parallel migration,invasion and promoting apoptosis induced by ATRA.In addition,miR-200a-3p and miR-200c-3p might be mediated ATRA inhibiting cell vertical migration and promoting differentiation and maturation of hepatocellular carcinoma cells.Furthermore,reversal EMT of ATRA on Hepa1-6 cells by regulating miR-200 s may be related to Jun,Pouf1,Etv4 and zeb1. |
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