MiR-181a Inhibits Metastasis Of Hepatocellular Carcinoma By Down-regulating HMGB2

Aims:Hepatocellular carcinoma(HCC)is one of the most common and aggressive malignancies in China,and has become a major public health problem.Prognosis remains extremely poor due to metastasis and recurrence,which are the main cause of death in HCC patients.Therefore,it is urgently needed to investigate the molecular mechanisms underlying metastatic progression and screen key targets of metastasis and recurrence in HCC to develop new diagnostic and therapeutic strategies.miRNA plays an important role in the initiation and progression of HCC,and participates in regulating metastasis of HCC.In this study,miRNA microarray revealed that miR-181a was down-regulated in Huh-7/M8 with high migratory and invasive ability.miR-181a is a multifunctional miRNA and its precise role in metastasis of HCC remains controversial.Then,identify the candidate target gene HMGB2 after searching the database.HMGB2 is a member of the high-mobility group box(HMGB)protein family,and it has been reported to be involved in the regulation of cancer metastasis,such as gastric and ovarian cancers,but the function of HMGB2 in metastasis of HCC has not yet been elucidated.Besides,the interaction of miR-181a and HMGB2 has not been studied.Therefore,the aim of this study is to investigate the role of miR-181a and HMGB2 in HCC metastasis and the underlying molecular mechanism,and to analyze the correlation between miR-181a,HMGB2 expression and clinicopathological features,prognosis of HCC patients.Methods:Firstly,we established a novel cell model,Huh-7/M8,using Transwell chamber.The migration and invasion abilities of parental Huh-7 and Huh-7/M8 cells were compared in vitro and in vivo by transwell migration and invasion assay,wound healing assay and tail vein metastatic assay.Then,miRNA microarray and qRT-PCR were used to verify the potential miRNA that regulated HCC metastasis,miR-181a.The effect of miR-181a on the metastatic ability of HCC cells was investigated by knockdown and overexpression experiments using lentiviral vectors.Secondly,the target genes of miR-181a were predicted using TargetScan,Mirdb and Mirtarbase database.qRT-PCR and Western immunoblotting confirmed that HMGB2 was downregulated in Huh-7/M8 cells overexpressing miR-181a.Then,dual luciferase reporter gene was used to study whether miR-181a directly targets the 3'UTR of HMGB2.The effect of HMGB2 on the metastatic ability of HCC cells was studied by routine knockdown and overexpression experiments.Morevoer,the effects of miR-181a and HMGB2 on HCC metastasis in vivo were investigated by tail vein metastatic assay,and regulatory relations of miR-181a and HMGB2 were futher demonstrated by rescue experiments.Furthermore,WB was used to detect the effect of knockdown or overexpression of miR-181a and HMGB2 on epithelial-mesenchymal transition(EMT)of HCC cells.Finally,qRT-PCR(n=144),in situ hybridization(n=90)and immunohistochemical staining(n=90)were used to detect miR-181a and HMGB2 expression in samples of hepatocellular carcinoma patients who underwent surgical resection.Then statistical analyses are performed to compare the correlation between miR-181a,HMGB2 expression and clinicopathological features,prognosis of HCC patients.Results:Firstly,compared with parental Huh-7 cells,the metastatic ability of Huh-7/M8 cells was significantly enhanced both in vitro and in vivo.It was demonstrated that miR-181a was significantly down-regulated in Huh-7/M8 by miRNA microarray and qRT-PCR.Overexpression of miR-181a could suppress the migration and invasion of Huh-7/M8 cells,while miR-181a inhibitor enhanced the metastatic ability of Huh-7 cells in vitro.Secondly,after searching for candidate target genes in database,we confirmed that overexpression of miR-181a could down-regulate the expression of HMGB2 mRNA and protein in Huh-7/M8 cells using qRT-PCR and WB.Moreover,dual luciferase reporter assays showed that miR-181a directly binds to the 3'-UTR region of HMGB2 mRNA.It was further found that overexpression of HMGB2 could enhance the migration and invasion of Huh-7 cells,while silencing of HMGB2 inhibited the metastatic ability of Huh-7/M8 cells.As demonstrated in rescue experiments overexpression of HMGB2 could restore the metastatic ability of Huh-7/M8 cells with miR-181a overexpression,and silencing of HMGB2 could suppress the metastatic ability of Huh-7 cells with miR-181a inhibitor.In vivo study showed that the number and size of lung metastasis nodules were significantly decreased in Huh-7/M8 with overexpression of miR-181a,but significantly increased with overexpression of HMGB2.Mechanism study showed that overexpression of miR-181a or silencing of HMGB2 could up-regulate the expression of E-cadherin and down-regulate the expression of vimentin,while miR-181a inhibitor or overexpression of HMGB2 could down-regulate the expression of E-cadherin and up-regulate the expression of vimentin.Finally,miR-181a expression was significantly down-regulated in HCC cancer tissues compared with adjacent normal tissues,and negatively correlated with vascular invasion,but positively correlated with overall survival time.HMGB2 mRNA and protein expression were significantly up-regulated in HCC cancer tissues,and HMGB2 protein expression was correlated with age,vascular invasion,differentiation,AFP,tumor grade,and negatively correlated with overall survival time.Patients with low expression of miR-181a and high expression of HMGB2 protein had the worst prognosis.Conclusions:Compared with parental Huh-7 cells,Huh-7/M8 cells had enhanced migration and invasion ability both in vitro and in vivo.miR-181a was significantly down-regulated in Huh-7/M8.miR-181a could down-regulate HMGB2 by directly targeting 3'-UTR region.miR-181a inhibits metastasis of hepatocellular carcinoma by suppressing HMGB2-mediated EMT pathway.miR-181a was significantly down-regulated in HCC cancer tissues,while HMGB2 was up-regulated.Up-regulation of miR-181 a and down-regulation of HMGB2 protein correlated with poor prognosis in patients.Patients with low expression of miR-181a and high expression of HMGB2 protein had the worst prognosis.Our study elucidates that miR-181a and HMGB2 are potential prognostic biomarkers and therapeutic targets for the prevention of HCC metastasis.