Experimental Study On Metastasis-related Mir-185 And Mir-19 In Hepatocellular Carcinoma Initial Experimental Study On Function Of Has-mir-19

Objective: we would make validation of these miRNAs in an independent cohort of HCC in early stage in order to be significant biomarkers for early stage HCC. Then,we analyzed the differential expressions of mir-19 and mir-185 in hepatocellular carcinoma in different metasitasis potential HCC cell line by quantitative real time PCR. We carried out further analyses mir-19 to determine whether it is functionally linked to HCC metastasis.Methods: We tested mir-19 and mir-185 in recurrence or non-recurrence specimens from radical resection of 95 patients with HBV-related early stage HCC taking miRNAs asssays by Taqman MGB probe-labeled quantitive RT-PCR. Then, statistic methods were used to analyze the relative expression and prognostic function according to the clinical follow-ups. A qRT-PCR method was established and using special looped-primers and Taqman MGB quencher to detect the relative expression level of mir-19 and mir-`185 and differernt metasitasis potential HCC cell line. Ectopic expression or inhibiting of mir-19 was carried out in HCC cell lines to assess its influence on cell growth, migration, and invasion.Results: In early stage HCC ,we identified two miRNAs(mir-19 and mir-185) which had a higher expression in patients who relapsed during the follow-up period in tumors(P<0.05). ROC Curve was taken in the calculation of cut point for mir-19 and mir-185. All patients were divided into high expression level-low recurrence group and low expression level-high recurrence group, Kaplan-Meier survival analysis uncovered a remarkable longer overall and time to recurrence(OS and TTR) in higher mir-185, and mir-19 was only significantly associated with TTR(P<0.05).High expresson of mir-19 and mir-185 in tumor correlated with good prognosis by univariate analysis as well as multivariate analysisi(P<0.05), to be indentified as independent protective factor for recurrence. Mir-19 and mir-185 were significantly expressed between high metastasis cell lines (MHCC97L and LM3) and low metastasis cell lines(HepG2 and 7402), which showed lower expression in the former. Functional studies indicated that up-regulating expression of mir-19 significantly inhibits HCC cell migration and cell growth. In-silico analysis revealed CREB3L4, PDE4A, JAM2, RUNX2, et al as potential targets of mir-19.Conclusion: mir-19 and mir-185 were indentified as independent protective factor for recurrence. These results suggest that mir-19 may suppress HCC metastasis partially through targeting CREB3L4, PDE4A, JAM2, RUNX2 and et al.