| 【Background】Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, thefifth most common cancer, the third leading cause of cancer death worldwide, and thesecond leading cause of cancer deaths in China. Its incidence and mortality is increasingyear by year. The high mortality of HCC is mainly attributed to frequent tumourmetastasis and recurrence after surgical intervention. Because of its exact mechanism isstill not clear, there is still lack of effective means of prevention and treatment. So it is themost important and urgent issues to explore the molecular mechanisms of metastasis andeffective prevention and treatment measures in the field of oncology research.MicroRNAs (miRNAs) are an abundant class of endogenous, highly conserved, noncoding20-23nt small RNAs, that have been shown to regulate gene expression andmediat complex biological processes, including cellular differentiation, apoptosis,metabolism and proliferation through directly binding to the3’-untranslated region(3’UTR) of target mRNAs in a wide range of organisms. Emerging evidence demonstratesthat dysregulation of miRNAs is linked to many disease states including cancer and acts asoncogenes or tumour suppressor genes. Recently, correlation between miRNAs expressionand tumour metastasis also has aroused widespread concern in several types of cancers.Numerous miRNAs have been shown to play important role in metastasis throughregulation of the cell adhesion, matrix degradation, EMT, angiogenesis. Different studyshowed that miR-10b serve different functions, which suggest that one miRNA may servedifferent functions in different cellular environments. For example, miR-10b can promotemetastasis in breast cancer and esophageal cancer, but promote proliferation and inhibitsapoptosis in gliomas. In addition, another study showed that miR-10b is up-regulated inneoplastic transformation of liver cancer stem cells, which suggest that up-regulatedmiR-10b may be involved in HCC carcinogenesis and progression.CADM1(Cell adhesion molecule1), a tumour suppressor, encodes animmunoglobulin superfamily molecule that is involved in cell-cell adhesion and regulationof cytoskeleton in a variety of human epithelia. Its down-regulation or inactivation isrelated to tumour metastasis and poor prognosis. CADM1protein expression has beenfound to be lost or markedly reduced in several human cancers including HCC withunknown mechanism. By using computational prediction and bioinformatics analysis, Wespeculated that CADM1might be one of the target genes of miR-10b in HCC.However，it is not clear that whether miR-10b is involved in HCC carcinogenesis andmetastasis through targeting CADM1, and whether therapeutic intervention on miR-10bexpression could effectively reverse the metastatic phenotype of HCC cells. So it is stillneed to be explored roles and mechanisms of miR-10b in HCC carcinogenesis andmetastasis.【Aims】To explore the regulatory role and the underlying mechanisms miR-10b in HCC invasion and metastasis, with the aim of better elucidating the mechanisms of HCCmetastasis and laying a foundation for formulating novel therapeutic target for treatmentof HCC.【Methods】1．A total of58HCC tissues (36metastatic HCC and22non-metastatic) and10normal control liver tissues were obtained from patients who underwent routine curativesurgery at Xijing Hospital of the Fourth Military Medical University. Expression ofmiR-10b was detected by qRT-PCR analysis, then its association with clinicopathologicfeatures was explored. The human HCC cell lines HepG2, SMMC7721, and MHCC97Has well as normal human hepatocyte HL-7702cells were maintained and cultured in ourlab. Expression of miR-10b was also detected by qRT-PCR analysis, then the relationshipbetween the miR-10b expression and cell migration and invasion was analyzed.2．Observe the effects of miR-10b on biological behavior of HCC cells in vitro:HCC cells were transfected with miR-10b mimics or inhibitors (miR-10b-AS) respectivelyto up or down-regulate miR-10b expression, then MTT assay were used for evaluating cellproliferation, flow-cytometric analysis was used for analysis of cell cycle and apoptosis,transwell migration and invasion assays were employed for investigating the metastaticproperties.3．miR-10b was up-regulated in HepG2cells with miR-10b expression lentiviralvector (mir-10b/GV254-LV), then these HepG2cells up-regulated miR-10b expressionwere injected into nude mice via tail vein to oberve the effect of miR-10b overexpressionon cell metastasis in vivo.4．Prediction and validation of miR-10b targets: By using PicTar and miRanda, wespeculated that CADM1might be one of the target genes of miR-10b in HCC. Luciferasereporter assay was used to verify miR-10b directly regulated CADM1expression.qRT-PCR and western blotting were used to examine the mRNA and protein expression ofCADM1respectively in HCC cells transfected with miR-10b mimics or inhibitors. RNAitechnique was used to down-regulate CADM1expression in HCC cells, then its effect onmigration and invasion, and cell morphology were examined by transwell migration and invasion assays, immunofluorescence and electron microscopy separately.【Results】1．Expression of miR-10b in HCC tissues: miR-10b was up-regulated both inmetastasis-free and metastatic HCC tissues, but its up-regulated degree was much moresignificant in metastatic HCC tissues(P﹤0.05).2．Relationship between the miR-10b expression and clinicopathologic features:High miR-10b expression was strongly correlated with the metastasis(P﹤0.01), AJCCstage(p=0.016). But it was not correlated with age, gender, hepatitis B, cirrhosis, tumorsize, degree of differentiation, and tumor number. Patients with high miR-10b expressionhad significantly poorer overall survival(P﹤0.01). High miR-10b expression wasindependent prognostic factors for overall survival(p=0.016).3．Expression of miR-10b in different HCC cell lines and its association with cell cellmigration and invasion ability: Compared with normal liver cells HL-7702, miR-10b wasup-regulated in HCC cell lines. There were statistically significant correlations of themiR-10b level with both cell migration and invasion potential（r=0.65，r=0.71）.4．miR-10b had no effect on HCC cell cycle，proliferation and apoptosis, butsignificantly increased potential of migration and invasion in vitro.5．In the in vivo studies, metastasis potential of up-regulated HCC cells in nudemice was significantly increased.6．Bioinformatics predicted that CADM1might be a target of miR-10b. Luciferasereporter assay showed that miR-10b over-expression remarkably repressed the expressionof luciferase from a vector also containing the wild-type miR-10b binding site(CADM1-UTR), but had no effect on mutant and negative control vector. qRT-PCR andwestern blotting analysis revealed that miR-10b over-expression did not cause degradationof CADM1mRNA but did drastically reduce its protein levels. Silencing of CADM1withsiRNA in HCC cells led to increased cell migration and invasion and induced cytoskeletonreorganization, which was similar to over-expression of miR-10b. These hinted thatmiR-10b is involved in HCC invasion and metastasis through negatively regulatingCADM1expression. 【Conclusions】1．miR-10b was up-regulated HCC tissues, especially in metastatic HCC tissues andhigh invasive and metastatic HCC cell lines, which indicated that miR-10b acts asoncogenes in HCC carcinogenesis and progression.2．miR-10b is expected to become a new prognosis reference of HCC.3．Overexpression of miR-10b increased potential of cell migration and invasion,whereas inhibition of miR-10b reduced cell migration and invasion in HCC cells, but hadno effect on cell proliferation and apoptosis. These indicated that miR-10b plays importantrole in HCC metastasis.4． miR-10b is involved in HCC invasion and metastasis through negativelyregulating CADM1expression. Both of them are likely to be new potential therapeutictarget for treatment of HCC. |
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