| AimsHepatocellular carcinoma(HCC) is one of the most highly metastatic cancer. Under normal circumstances, after detaching from the extracellular matrix, cells generallyundergo detachment-induced cell death, termed as anoikis. This is an important mechanism to maintain physiologicalhomeostasis in the body. Anoikis is believed to act as a barrier to metastasis,thus cancercells must acquire the capability to resist anoikis in the process of metastasis. EMT (Epithelial-mesenchymal transition) is a key step in metastasis of tumor cells. We found that miR-424-5p was down-regulated in anoikis-resistant HCC cells. Whether and how miR-424-5p play a role in the regulation of anoikis remained to be elucified. Our studyaimed to explore the role of abnormal expression of miR-424-5p in anchorage-deprived HCC cells and the mechanism involved in this process.Materials and Methods1Identification and verification of up-or down-regulated microRNAs in anoikis-resistant HCC cells1.1Establishment of anoikis-resistant model of HCC cellsBEL-7402cells were cultured in RPMI1640with10%FBS at37℃5%CO2conditions to reach logarithmic phase, then digested by0.25%trypsin into single cell suspension. Adjusted the concentration of cells, and cultured the cells in poly-HEMA-coated plates for24h.1.2microRNA microarray analysis to identify up-or down-regulated microRNAsMicroRNA microarray analysis was performed with above samples to identify the microRNAs that were up-or down-regulated in the anchorage-deprived HCC cells.1.3Verification of the up-or down-regulated microRNAs with real-time PCRThe selected differentially expressed microRNAs in the detached HCC cells were analyzed by quantitative real-time PCR to validate the data obtained from microRNA array analysis.2Detection of the Epithelial-mesenchymal transition (EMT) in the anoikis-resistant HCC cellsExpression of EMT markers was measured by real-time PCR and western blot at the mRNA level and protein level, respectively.3Investigating the role of miR-424-5p in anoikis-resistant HCC cellsAfter miR-424-5p mimics were transfected, we detected the malignant behavior of the anchorage-deprived HCC cells.4Analyzing the mechanism involved in detachment-induced EMT regulated by miR-424-5pTarget gene of miR-424-5p was analyzed in the miRDB database.Mir-424-5p mimics were transfected into HCC cells, expression of target proteins were measured by real-time PCR and western blot.Results1miR-424-5p was down-regulated in anoikis-resistant HCC cellsCompared with the attached HCC cells, expression of miR-424-5p was significantly down-regulated in the anoikis-resistant HCC cells.2anoikis-resistant HCC cells showed EMT behaviorEpithelia markers including E-cadherin and β-catenin were down-regulated, while mesenchymal markers including N-cadherin and vimentin were up-regulated in anoikis-resistant HCC cells.3miR-424-5p negatively regulated anoikis-resistance related malignant behavior of HCC cellsmiR-424-5p reversed anoikis-resistance of HCC cells by reversing EMTbehavior of HCC cells. It further reduced invasiveness and inhibited colony formation of anoikis-resistant HCC cells. Furthermore, miR-424-5p caused cell cycle arrest, inhibited cell proliferation, and reduced colony formation of HCC cells.4miR-424-5p played a negative regulatory role in anchorage-deprival induced EMT by targeting ICAT.4.1miR-424-5p suppressed ICAT expressionAfter transfection of miR-424-5p mimics, ICAT expression in transfected HCC cells was significantly down-regulated.4.2miR-424-5p interacted with3’UTR of ICAT and inhibited its luciferase activityThe microRNA prediction program, miRDataBase was applied and a putative miR-424-5p binding site was predicted in the3’UTR region of ICAT. Therefore,3’UTR of ICAT(WT and mutant)was cloned and fused to the downstream of the firefly luciferase gene to generate a reportor construct. Transfection of miR-424-5p mimics significantly inhibited the luciferase activity of the reportor construct with the wild type3’UTR. However, after the miR-424-5p binding site in the3’UTR of ICAT was mutated, the inhibitory effect of miR-424-5p on the luciferase activity was abrogated.4.3miR-424-5p played a negative regulatory role of anchorage-deprival induced EMT by targeting ICATCo-transfection of miR-424-5p and ICAT abrogated the effect of miR-424-5p on EMT of HCC cells, which indicated that miR-424-5p exert its function by targeting ICAT.5miR-424-5p was down-regulated in tumor tissues of HCC patientsCompared with normal liver tissues, MiR-424-5p was significantly down-regulated in tumor tissues of HCC patients, and its expression was inversely correlated with disease progression.6miR-424-5p was down-regulated in serum of HCC patients Compared with healthy control, miR-424-5p was down-regulated in serum of HCC patients, and its expression was inversely correlated with disease progression.ConclusionmiR-424-5p played a role as an anti-onco miR in HCC. By targeting ICAT, miR-424-5p inhibited EMT behavior in the anchorage-deprived HCC cells, thus suppressed the malignant behaviors of HCC.Innovation and significance In this study, it is the first time that we demonstrated as follows:1) miR-424-5p can inhibit EMT behavior of anoikis-resistant HCC cells;2) miR-424-5p directly targets ICAT by interacting with the3’UTR of ICAT;3) ICAT promotes EMT behavior of HCC cells by inhibiting the E-cadherin/β-catenin complex. |
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