Histone Deacetylases Up-regulate CCAAT Enhancer Binding Protein α Expression Through Regulation Of MiR-124 And MiR-25

OBJECTIVE CCAAT enhancer binding protein α(C/EBPα),as an important transcription factor involved in cell proliferation,differentiation and metabolism,was up-regulated in primary hepatocellular carcinoma(HCC)and predicted poorer prognosis.In this study,we aims to explore how histone deacetylases(HDACs)up-regulated C/EBPα in HCC.METHOD:(1)HDAC inhibitor(TSA,SAHA,PXD101)or solvent control were used to investigate the effect of HDAC on C/EBPα expression in Huh7 cells and Hep3 B cells which having high level of C/EBP expression.Real-time quantitative polymerase chain reaction(RT-PCR)and western blotting assay were used to detect the change of C/EBPα expression.(2)The protein expressions of HDAC1,HDAC2,C/EBPα in liver cancer tissues and adjacent normal tissues of patients with hepatocellular carcinoma were detected by immunohistochemical method in a HCC tissue microarray,and their expressions were correlated using chi-square analysis.(3)On the one hand,the pGL3 luciferase vectors containing different ranges of C/EBP 5’UTR promoter sequences were constructed and transfectedseparately into Huh7 cells.Then HDAC inhibitor(TSA)was used to treat Huh7 cells and luciferase intensity was detected by luciferase assay to investigate whether HDAC acted on the 5’ UTR end of C/EBPα.On the other hand,the pmiRGLO luciferase reporter vector containing C/EBPα 3’UTR sequences was constructed and transfected into Huh7 cells.The luciferase intensity was measured after treatment with HDAC inhibitor TSA or SAHA.(4)Through literature review and the use of bioinformatics software Targetscan and miRwalk,possible miRNAs involved in C/EBPα regulation were screened.Then Huh7 cells were treated with HDAC inhibitor SAHA in a time course,to observe whether the selected miRNA was regulated by HDAC inhibitor and whether their changes were correlated with that of C/EBPα.Furthermore,Huh7 hepatoma cells were transfected with specific siRNA targeting HDAC1 and HDAC2 together with control siRNA(nc-siRNA),in order to study the effect of HDAC inhibition on miRNA content and C/EBPαmRNA.(5)The synthetic miR-124 inhibitor or miR-25 inhibitors together with the control miRNA inhibitors were transfected separately into Huh7 cells before treatment with different doses of HDAC inhibitor(SAHA)or solvent control,in order to determine whether HDAC regulate C/EBPα expression through miRNA.Furthermore,the predicted miR-124 binding site and/or miR-25 binding site were mutated in the C/EBPα 3’ UTR region and then cloned into the pmiRGLO luciferase reporter vector.After HDAC inhibitor SAHA treatment,the luciferase intensities from cells with different vectors were observed,in order to determine whether HDAC inhibitors induced miRNA act directly on the C/EBPα 3’ UTR region.(6)Total RNA was extracted from 21 pairs of liver cancer tissues and adjacent normal liver tissues.The expression of miRNA-124 and miRNA-25 were determined by quantitative RT-PCR and compared between liver cancer tissues and adjacent normal liver tissues were observed.(7)miR-124 and miR-25 mimics and control group(NC miRNA)were transfected into Huh7 HCC cells to observe whether miR-124 and miR-25 could affect the growth and proliferation of HCC cells.Cells were treated with 3-(4,5-dimethylthiazoli-2)-2,5-diphenyltetrazolium bromide(MTT)kit to observe cell activity and the expression of C/EBPα was observed by western blot.RESULTS(1)RT-PCR and Western blotting results showed that after treatment with HDAC inhibitors,the mRNA and protein expression of C/EBPαdecreased in a dose-dependent manner in both Hep3 B and Huh7 cells.(2)The correlation analyses found that C/EBPα protein was correlated with HDAC1 and HDAC2 protein contents in HCC tissues(P<0.05).(3)The dual-luciferase reporting system showed that HDAC inhibitor TSA treatment could increase C/EBPα promoter transcription activity by the use of pGL3 vectors containing different ranges of C/EBPα 5 ’UTR promoter regions.In contrast,TSA treatment impaired C/EBPα mRNA stability as shown by the dual-luciferase reporting assays with C/EBPα 3 ’UTR regions.(4)The qRT-PCR results showed that HDAC inhibitors induced miR-124 and miR-25 expression(P<0.05),which was associated with the decrease of C/EBPα mRNA.But the other miRNAs(e.g.miR-381)were not consistent with the changes of C/EBPα mRNA.Furthermore,those HCC cells silenced with HDAC1 or HDAC2 by specific siRNA showed consistent increase of miR-124 and miR-25 expression and decrease of C/EBPα.(5)After transfection of synthetic miR-124 inhibitor to Huh7 cells,the C/EBPα mRNA expression was increased(P < 0.05)but HDAC inhibitor treatment failed to decrease C/EBPα mRNA expression.Consistently,synthetic miR-25 inhibitor had similar results as miR-124 inhibitor.Furthermore,the miR-124 binding sites and miR-25 binding sites were mutated in the C/EBPα 3’UTR region and then cloned into pmirGLO vector respectively.The dual luciferase assays showed that mutation of miR-124 binding sites,or miR-25 binding sites or both blocked HDAC inhibitor induced C/EBPα instability.(6)The quantitative RT-PCR of human primary HCC tissues results showed that both miR-124 and miR-25 expression were lower in the cancer tissues,compared with adjacent normal liver tissues(P<0.05).(7)Finally,transfection of miR-25 mimics and miR-124 mimics into Huh7 cells inhibited cell proliferation,as revealed by the MTT assay.Western blotting results showed that these treatments decreased C/EBPα protein expression.Similarly,HDAC inhibitor decreased cell proliferation as well.CONCLUSION(1)In HCC cells,HDAC inhibition by inhibitors or specific siRNA could reduce C/EBPα mRNA expression in a dose-dependent manner.(2)HDAC does not directly act on C/EBPα 5’UTR,thus affecting the expression of C/EBPα.(3)Instead,HDAC inhibition induced miR-124 and miR-25 expression;the two miRNA acted on C/EBPα 3’UTR and consequently impaired C/EBPα mRNA instability and expression reduction.(4)miR-124,miR-25 and HDAC inhibitors decreased cell proliferation of HCC cells.