Screening And Functional Analysis Of Serum MiRNAs Which Differentional Expression In Patients With HBV-related Hepatocellular Carcinoma

Objective:To screen serum miRNAs which differentional expression in patients with hepatits B virus (HBV)-related hepatocellular carcinoma (HCC), and to evaluated the diagnostic value of the miRNAs as novel markers for HBV-related HCC and the important role and mechanism of the miRNAs in tumorigenesis and progression.Methods:1. To screen the miRNAs which are differential expression in serum between HBV-related HCC patients and healthy controls, miR-96, miR-18a, miR-10b, miR-125a and miR-378were selected based on previous reports that which are differential expression between HCC tissue and adjacent non-HCC livers, and were measured using real-time qRT-PCR on a training set of serum samples consisting of15HBV-related HCC patients and15healthy controls.2. The up-regulated expressed miR-18a in cancer serum was further validated in an independent large-scale set of serum from86HCC patients and45healthy controls by using real-time qPCR. Finally, serum samples from30patients with HBV-related chronic hepatitis or cirrhosis were also included to examine the specificity of the makers.3. To construct recombinant lentivirus vectors with over-expressing miR-378by gene cloning and packed by lentivirus.4. To determine the impact of miR-378(which down-regulated expressing in serum in patients with HBV-related HCC) on HCC cell proliferation, we established HepG2and HepG2.2.15(later was transfected with HBV and stably expressing HBV) transfectants stably expressing miR-378using lentivirus infection. The cell proliferation assays and colony formation assays were performed.5. Bioinformation softwares were used to predict the targets of miR-378. The targets were validated by luciferase reporter assay. Western blot analysis was used to monitor the expression level of the endogenous targets of miR-378in miR-378-overexpressing HepG2and HepG2.2.15.Results:1. Five miRNAs were quantified by real-time qRT-PCR in serum from15patients with HBV-related HCC and15healthy controls. We found that the miR-18a levels was significantly up-regulated(p=0.003, Mann-Whitney test), while miR-378was down-regulated in HCC serum when compared to controls (p=0.044). No significant difference was observed in the levels of miR-96, miR-10b, and miR-125a between HCC patients and healthy controls (p>0.05).2. We confirmed miR-18a for further validated in an independent large-scale set of serum. Similar trend of the results and significant differences were obtained in the validation set of86HCC patients and45healthy controls with the levels of serum miR-18a being significantly elevated in patients than in controls (P<0.0001, Mann-Whitney test). Receiver-operator characteristic (ROC) curve analyses were performed in pooled sets(101HBV-related HCC patients and60healthy controls).We found that serum levels of miR-18a discriminated HCC patients from healthy controls, with an area under the curve (AUC) of ROC of0.881[95%confidence interval (CI),0.829-0.933] At the cut-off value of1.765, the sensitivity was86.1%and the specificity was75.0%. Furthermore, serum miR-18a were significantly elevation in HCC when compared to those in30patients with HBV-related chronic hepatitis or cirrhosis (p<0.0001). ROC curve analyses revealed that serum miR-18a levels were useful markers for discriminating patients with HCC from chronic hepatitis or cirrhosis. It yielded an AUC of ROC curve of0.775(95%CI:0.6810.869). At the cut-off values of1.796, the sensitivity and specificity were77.2%and70.0%, respectively.3. Recombinant lentiviral vector expressing miR-378was successfully established and was confirmed by PCR and DNA sequencing. The constructed PGCSIL-miR-378lentiviral vector was detected by both immunofluorescence microscopy and expressed in293T cells.4. A cell proliferation assay and colony formation assays were performed on the HepG2.2.15and HepG2Cells after transfected with miR-378. The result revealed that up-regulated expression of miR-378can significantly inhibited HCC cell proliferation and colony formation when compared with Lenti-vector controls (P<0.01).5. Med19and IGF1R were predicted as the potential targets of miR-378by bioinformation softwares. The results of luciferase reporter assay revealed that, for Med19, the luciferase activity was decreased by9.7%in miR-378group, which co-transfected HEK293cell with miR-378and Med193’-UTR, when compared with negative control group, p>0.05.However, for IGF1R, the luciferase activity was significantly decreased by41.8%in miR-378group, which co-transfected HEK293cell with miR-378and IGF1R3’-UTR, when compared with negative control group, p<0.01. Furthermore, the expressing level of IGF1R protein was significantly decreased in miR-378-overexpressing HCC cells was verifyed by western-blot assays. IGF1R was verified as direct target of miR-378.Conclusion:1. Serum miR-18a is elevated in patients with HBV-related HCC and can be a novel noninvasive biomarker to distinguish HBV-related HCC from HBV-related chronic hepatitis or cirrhosis and healthy control.2. Serum miR-378is down-regulated expression in patients with HBV-related HCC. MiR-378suppresses tumor growth via directly targeting IGF1R in HCC. Down-regulated expression of miR-378caused by HBV infection might be one of the molecular mechanism of HBV-related HCC carcinogenesis and progression.