The Regulatory Mechanism And Diagnostic Value Of MiR-26a In Hepatocellular Carcinoma

Part I miR-26a is Repressed by EZH2-mediated H3K27 Trimethylation within the miR-26a PromoterObjective:To investigate the regulatory mechanism of miR-26a from an epigenetic perspective.Methods:QRT-PCR and Western blot were used to investigated the expression levels of miR-26a and EZH2 in HCC cell lines (HepG2 and SMMC7721) and a normal liver cell line (LO2). EZH2 overexpression and silence plasmids were constructed, and then transfected into HCC cells or LO2 cells to upregulate or downregulate EZH2 expression. Subsequently, the expression level of miR-26a was determined by qRT-PCR. Luciferase reporter assay and chromatin immunoprecipitation assay were used to investigate whether miR-26a was repressed by EZH2-mediated H3K27me3 within the miR-26a promoter.Results:The expression of miR-26a was downregulated while EZH2 was upregulated in HCC cell lines. Knockdown of EZH2 increased the expressions of miR-26a, CTDSPL and CTDSP2, while overexpression of EZH2 downregulated miR-26a, CTDSPL and CTDSP2 expressions. Knockdown of EZH2 increased miR-26a promoter activity. EZH2 and H3K27me3 were enriched at miR-26a promoter. Knockdown of EZH2 decreased the binding of EZH2 and the enrichment of H3K27me3 on the miR-26a promoter.Conclusions:miR-26a is repressed by EZH2-mediated H3K27 trimethylation within the miR-26a promoter.Part Ⅱ Identification of EZH2 As a Direct Target of miR-26aObjective:To investigate whether EZH2 is a direct target of miR-26a in HCC.Methods:miR-26a mimics or Control mimics were transfected into HepG2 and SMMC7721 cells, qRT-PCR and Western blot were used to determine the mRNA and protein expressions of EZH2. Luciferase reporter assay was performed to determine whether miR-26a could bind to the 3’UTR of EZH2 mRNA.Results:Ectopic expression of miR-26a significantly decreased both mRNA and protein levels of EZH2 in a dose-dependent manner. miR-26a but not control mimics significantly decreased the luciferase activity of EZH2-3’UTR-WT reporter, while EZH2-3’UTR-MUT reporter was not affected by miR-26a.Conclusions:EZH2 is a direct target of miR-26a in HCC cells.Part Ⅲ A Double-negative Feedback Loop between EZH2 and miR-26a Regulates HCC Cell GrowthObjective:To confirm the existence of the double-negative feedback loop between EZH2 and miR-26a in HCC cells, and investigate the effect of the feedback loop on HCC cell growth.Methods:HCC cells were transfected with exogenous miR-26a mimics, and the expressions of endogenous CTDSPL, CTDSP2 and E-cadherin were detected. HepG2 and SMMC7721 cells were transfected with pcDNA3.1 or pcDNA3.1-miR-26a, or cotransfected with pcDNA3.1-miR-26a and pcDNA3.1-EZH2, and then CCK8 and colony formation assays were performed. Similarly, HepG2 and SMMC7721 cells were transfected with sh-control or sh-EZH2, and then CCK8 and colony formation assays were performed.Results:Compared with control mimics, miR-26a mimics increased the mRNA expressions of CTDSPL, CTDSP2 and E-cadherin in both HepG2 and SMMC7721 cells. Ectopic expression of EZH2 abrogated miR-26a induction of CTDSPL, CTDSP2 and E-cadherin. Similar to miR-26a restoration, knockdown of EZH2 induced cell growth inhibition, and overexpression of EZH2 rescued the growth inhibition effect of miR-26a in HepG2 and SMMC7721 cells.Conclusions:EZH2 and miR-26a form a double-negative feedback loop regulating HCC cell growth.Part IV Serum miR-26a As Potential Biomarker of Hepatocellular CarcinomaObjective:This study aimed to investigate the expressions of serum miR-21, miR-26a and miR-101 in hepatocellular carcinoma (HCC) and their diagnostic value.Methods:Serum levels of miR-21, miR-26a and miR-101 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in 52 HCC patients,42 chronic hepatitis (CH) patients and 43 healthy controls. ROC curve analysis was performed to evaluate the diagnostic value. Clinical parameters were collected.Results:Serum level of miR-21 was higher while miR-26a and miR-101 were significantly lower in HCC patients than those in healthy controls (p<0.05, p<0.001 and p<0.05, respectively). Serum levels of miR-26a and miR-101 were significantly lower in HCC patients than those in CH patients (p<0.001 and p<0.05). ROC curve analyses revealed that miR-21, miR-26a and miR-101 could differentiate HCC patients from healthy controls, the area under ROC curve (AUC) were 0.621 (67.4% sensitivity and 55.8% specificity),0.754 (51.9% sensitivity and 95.2% specificity) and 0.631 (47.1% sensitivity and 81% specificity), respectively. Combination of miRNAs and Alpha-Fetoprotein (AFP) yielded an AUC of 0.914 with 87.0% sensitivity and 78.0% specificity. miR-26a and miR-101 had diagnostic potential for differentiating HCC from CH with AUC of 0.762 (75% sensitivity and 70% specificity) and 0.623 (54.9% sensitivity and 76.9% specificity). Combination of miR-26a, miR-101 and AFP yielded an improved AUC than AFP alone (0.854 vs 0.683). Notably, miR-26a could differentiate small-tumors HCC (<3cm) from CH with an AUC of 0.753 (80% sensitivity and 62.5% specificity).Conclusions:Serum miR-21, miR-26a and miR-101 are deregulated in HCC and can serve as potential biomarkers. Combination of these miRNAs and AFP provide a better detection than AFP alone. Serum miR-26a is a promising biomarker for early detection of HCC.