MiR-709 Promotes Hepatocellular Carcinoma Cells Proliferation,Invasion And Metastasis By Down-regulating GPC5 Expression

Hepatocellular carcinoma(HCC)is the fifth most frequent cancer and the third cause of cancer-related mortality worldwide,with a mortality rate almost equal to its incidence.In spite of recent advances in diagnosis and treatment,the long-term outcome of HCC patients after curative therapy remains very poor.Genomic instability and alterations are believed to contribute to human HCC.However,the exact etiology of HCC has not yet been fully elucidated.MiRNAs are small non-coding RNAs that function as negative regulators of protein-coding genes at the post-transcriptional level.MiRNAs can bind to complementary sequences in 3'-UTR of their target m RNAs and induce m RNA translational repression or degradation.Recently,accumulating evidence has shown that miRNAs have important roles in development and progression of cancers.MiR-709 is an abundant miRNA expressed in multiple tissues,including brain,thymus,heart,lung,liver,and kidney.Up to now,very little is known about the role of miR-709 in HCC.Hence the role and molecular mechanism of miR-709 in the development of HCC is worth in-dept investigating.Objective: To detect the role of miR-709 in HCC.Methods: The expressions of miR-709 in hepatocyte,HCC cells,HCC and paracancerous tissues were detected by q RT-PCR,and to analyze the relationship between the expression of miR-709 and the clinicopathological characteristics of HCC.After transfection of miR-709 inhibitor and miR-709 mimic,the levels of miR-709 were detected,and then CCK-8 assay,transwell assay and western blot were conducted to determine the changes of proliferation,migration and invasion of HCC cells strains.Putative miR-709 targets were predicted by bioinformatics software Luciferase reporter assay,q RT-PCR and western blotting were conducted to identify the target of miR-709.Results: ?(1)MiR-709 was elevated in HCC cell lines: Expression of miR-709 was first evaluated by q RT-PCR in HCC cell lines and one non-malignant human hepatocyte cell line,HL-7702.Epression of miR-709 was up-regulated in all HCC cell lines(Hep G2,SMMC7721 Hep3 B and Bel7402)compared to HL-7702.(2) MiR-709 was elevated in patient HCC tissues: We further quantified expression of miR-709 in 6 pairs of human HCC tissues and adjacent normal tissues by q RT-PCR.Results showed that expression level of miR-709 was generally higher in tumour tissues compared to matched non-malignant ones.Next,we validated clinical significance of miR-709 by analysing its expression in 50 pairs of HCC and normal tissues by q RT-PCR.Expression of miR-709 in HCC tissues was higher than that in adjacent non-malignant ones(P < 0.001).Of 50 HCC samples,miR-709 was up-regulated in 40 cases(40/50,80%)compared to adjacent areas.In addition,higher level expression of miR-709 was associated with p TNM stage of HCC.Tissues from lymph node metastases expressed higher levels of miR-709 compared to primary HCCs and adjacent normal tissue,indicating positive relationship between expression of miR-709 and metastatic status of HCC.?(1)Ectopic expression of miR-709 promoted HCC cell proliferation: Cells were transfected with miR-709 mimic and inhibitor,which had high transfection efficiency.CCK-8 proliferation assay showed that down-expression of miR-709 inhibited proliferation rate of HCC cells compared to control cells.Conversely,miR-709 mimic promoted proliferation of Hep G2 cells.Proliferative effect of miR-709 was further confirmed by evaluating Ki-67 expression.There was reduction in Ki-67 protein and m RNA levels of the group transfected with miR-709 mimic,compared to control or untreated groups.Conversely,miR-709 inhibitor inhibited Ki-67 expression and proliferation of HCC cells.(2)Ectopic expression of miR-709 promoted HCC cell migration and invasion: Migration assays indicated that overexpression of miR-709 promoted migration of Hep G2 cells compared to controls whereas miR-709 inhibitor prevented cell migration.Invasion assays showed that overexpression of miR-709 promoted invasion of both Hep G2 cells compared to controls,whereas miR-709 inhibitor stopped cell invasion.?GPC5 was a novel direct target of miR-709:As predicted by Pic Tar and Target Scan,there was complementarity between miR-709 and GPC5 3'-UTR.To validate whether GPC5 was a bona fide target of miR-709,a human GPC5 3'-UTR fragment containing wildtype or mutant miR-709 binding sequence was cloned downstream of the firefly luciferase reporter gene.Interestingly,relative luciferase activity of the reporter was suppressed when miR-709 mimic were cotransfected.In contrast,luciferase activity of the mutant reporter was unaffected by transfection of miR-709 mimic,indicating that miR-709 may suppress gene expression through miR-709 binding sequence at the 3'-UTR of GPC5.In addition,ectopic expression of miR-709 caused a reduction in GPC5 expression at both m RNA and protein levels.Restoration of miR-709 inhibited GPC5-inhibited HCC cell proliferation and invasion of Hep G2 cells transfected with pc DNA-GPC5 caused elevation in GPC5 protein expression,shown using western blotting.The CCK-8 proliferation assay and invasion assay showed that overexpression of GPC5 inhibited HCC cell proliferation and invasion.When miR-709 mimic and pc DNA-GPC5 were cotransfected into Hep G2 cells,miR-709 expression enhanced GPC5-inhibited HCC proliferation and invasion.Conclusions: MiR-709 was up-regulated in HCC tissue samples and that up-regulation of miR-709 expression was associated with p TNM stage.Furthermore,we demonstrated that miR-709 enhanced HCC cell invasion by regulating expression of GPC5.Suppression of miR-709 may be a potential novel strategy for inhibiting HCC metastasis.