Expression Of Mina53 In Human Hepatocellular Carcinoma And Its Effect On Proliferation,Invasion And Metastasis In Vitro

Objective We aimed to detect the expression of Mina53 in hepatocellular carcinoma(HCC)and its adjacent normal tissues and to explore its relationship to clinicopathological features and prognosis of HCC patients.Moreover,we examined the expression of Mina53 in HCC cell lines and explored its relationship with the proliferation,cell cycle,apoptosis,migration and invasion of HCC cells.The potential molecular mechanism of Mina53 underlying the cell proliferation and metastasis was also investigated.Methods The expression of Mina53 mRNA and protein in 18 pairs of fresh HCC and its adjacent normal tissues was detected by quantitative real-time fluorescence PCR(qRT-PCR)and western blot.Tissue microarray and immunohistochemistry were used to detect the expression of Mina53 in 284 cases of HCC and its adjacent normal tissues and then analyzed its relationship to clinicopathological features and prognosis of HCC.The expression of Mina53 in HCC cell lines was determined by western blot,the overexpression plasmid was transiently transfected into HCCLM3 and QGY-7703 cells,and the small interfering RNA(siRNA)was transiently transfected into Huh7 cells.The capabilities of cell proliferation and metastasis in HCC were evaluated using MTT assay,colony formation assay,transwell assay and wound healing assay.The detection of cell cycle and apoptosis were performed by flow cytometry.The morphology changes of HCC cells were observed by optical microscope.The expression of PCNA、cyclinD1、cyclinE1、pRB、ppRB、E2F1、p21、pro caspase3、caspase3、E-cadherin、N-cadherin、vimentin、MMP2、MMP9 was examined by western blot.Results The expressing level of Mina53 mRNA and protein was found significantly higher in HCC compared to that in the adjacent normal tissues assessed by q RT-PCR and western blot(t=3.100,P=0.007;t=10.616,P<0.001).Immunohistochemical analysis showed that the positive expression rate of Mina53 in HCC(88.73%,252/284)was significantly higher than that in adjacent normal tissues(13.03%,37/284,z=-18.739,P<0.001).The expression level of Mina53 in HCC tissues was significantly associated with tumor size(P=0.019),vascular invasion(P=0.003),tumor differentiation(P=0.002)and TNM stage(P<0.001).Survival analysis showed that HCC patients with high expression of Mina53 had shorter 3-year overall survival time(OS)(P=0.011)and disease-free survival time(DFS)(P<0.001)thanthose with low expression of Mina53 in HCC.High Mina53 expression of HCC patients with tumor diameter(>5cm)and vascular invasion indicated shorter 3-year DFS(P<0.001,P=0.002).High Mina53 expression of HCC patients with positive expression indicated shorter 3-year OS(P=0.002)and DFS(P < 0.001).In addition,HCC patients with high expression of Mina53 had shorter 5-year OS(P=0.048)and DFS(P < 0.001).Cox multivariate regression analysis showed that Mina53 and multiple tumors were effective independent predictors for OS and DFS of HCC patients(P<0.05).Overexpression of Mina53 in HCCLM3 and QGY-7703 cells enhanced cells proliferation,migration and invasion(P<0.05).As for cell cycle,the results revealed that the proportion of G0/G1 phase cells decreased significantly and proportion of S phase cells increased after Mina53overexpression(P<0.05).By apoptosis assay,overexpression of Mina53 inhibited cell apoptosis(P<0.05).After knocking down of Mina53 in Huh7 cells,the proliferation,migration and invasion ability were decreased(P<0.05).The results of flow cytometry found the proportion of G0/G1 phase cells was increased significantly after Mina53 knockdown and the proportion of S phase cells decreased(P<0.05),knockdown of Mina53 increased the cell apoptosis rate(P<0.05).Overexpression of Mina53 in HCC cells upregulated the expression of PCNA、cyclinD1、cyclinE1、ppRB、E2F1、MMP2 and MMP9,but a reduction was noted in p21 and caspase3 levels(P<0.05,respectively);knockdown of Mina53 in HCC cells inhibited the expression of PCNA、cyclinD1、cyclinE1、ppRB、E2F1、MMP2、MMP9and increased the expression of p21 and caspase3(P<0.05,respectively).However,there was no significant effect of Mina53 on the morphology of HCC cells and the expression of pRB、pro caspase3、E-cadherin、N-cadherin and vimentin(P>0.05).Conclusion Mina53 plays an important role in the tumorigenesis and development of HCC;Related to the survival time of HCC patients suggests that Mina53 may be used as a novel prognostic biomarker of HCC.Mina53 could affect the G1/S phase transition through the activation of ppRB/E2F1 signaling pathway,and promote the cell proliferation in HCC cells.Moreover,Mina53 may affect the migration and invasion of HCC cells by regulating the expression of MMP2 、 MMP9,but may be no obvious effect on the progression of epithelial-mesenchymal transformation(EMT).