| [Background]With incidence and mortality increasing annually, hepatocellular carcinoma (HCC) isone of the most serious global health burdens. It is postulated that HCC can be derivedfrom abnormal activated or malignant transformed adult hepatic stem/progenitor cells.However, the regulatory factors and molecular mechanisms underlying the process are notwell defined. As a “tumor-suppressing miRNA”, miR-200a plays an important role ininhibiting epithelial-mesenchymal transition (EMT), maintaining epithelial cell polarityand stem cell maintenance. Nevertheless, the function miR-200a exerts on adult hepaticstem/progenitor cells and HCSCs is rarely reported. Interestingly, our previous miRNAmicroarray analysis revealed a significant decrease of miR-200a level in F344rat HCCside population (SP) fraction cells versus their normal counterparts. As a result, wespeculate that miR-200a may be associated with the phenotypes and function of rat hepatic stem/progenitor cells (Hepatic oval cells, HOCs), and abnormal expression of miR-200amight induce malignant transformation of HOCs during hepatocarcinogenesis. Elucidatethis pathogenesis is expected to open new avenues for HCC prevention and therapy.[Objective]Determine miR-200a expression levels in WB-F344HOCs and rat HCC cell lines,then investigate changes of WB-F344cell phenotypes and function by miR-200aexogenous loss-of-function study, and finally explore the regulatory mechanismsunderlying this process.[Methods]1. Analyze the expression levels of miR-200a in WB-F344cells as well as in BRLnormal rat liver cells and three hepatoma cell lines (H-4-II-E, CBRH-7919, RH-35) byqRT-PCR.2. Establish lentivirus-mediated miR-200a loss-of-function WB-F344cell model(WB-anti-miR-200a) and negative control model (WB-miR-NC), and then identify thetransfection efficiency from miRNA level, mRNA level and protein level, respectively.3. Investigate HCSC-like phenotypes and function in WB-anti-miR-200a cells: theproliferation and self-renewal capacity determined by cell counting andspheroid-formation assay; the cell apoptosis levels demonstrated by FITC-Annexin V-PIstaining and Caspase3/7activity; the expression levels of EpCAM, CD133, AFP and otherHCSC-like molecules determined by qRT-PCR; the chemo-resistance ability forco-cultured with chemotherapeutic drugs illustrated by flow cytometry; in vivotumorigenicity by subcutaneous inoculation in nude mice.4. Identify EMT features in WB-anti-miR-200a cells: observing cell morphologicalchanges under light microscopy, investigating cell motility in vitro by Transwell migrationassay, and detecting E-cadherin, Vimentin, N-cadherin protein levels by western-blotmethod.5. Prediction and validation of miR-200a target genes in WB-F344cells: We selectedβ-catenin as a target gene of miR-200a by TargetScan prediction, and then employeddual-luciferase reporter gene to verify miR-200a directly regulated CTNNB1(β-catenin). Lastly, endogenous expression of β-catenin was detected in WB-anti-miR-200a cells bywestern-blot method.6. Determine Wnt/β-catenin pathway activity in WB-anti-miR-200a cells: thedistribution of β-catenin in the cytoplasm and nucleus were observed byimmunofluorescence, and TopFlah/FopFlash reporter gene assay was performed toinvestigate β-catenin/T-cell factor (TCF) transcriptional activity. Finally, the expressionlevels of Wnt/β-catenin pathway downstream molecules c-Myc and cyclin D1weredetected by western-blot method.7. RNA interference (RNAi) method was employed to downregulate β-cateninexpression in WB-anti-miR-200a cells, then the self-renewal capacity (spheroid-formationassay) and in vitro migration ability (transwell migration assay) were investigated.[Results]1. miR-200a expression was much higher in WB-F344cells than in hepatoma cells(H-4-II-E, CBRH-7919, RH-35).2. Successfully establish lentivirus-mediated miR-200a loss-of-function WB-F344cells. It was demonstrated that miR-200a expression was significantly inhibited inWB-anti-miR-200a cells by qRT-PCR analysis. Moreover, ZEB2, a well-acknowledgedtarget of miR-200a, was promoted in protein level after miR-200a silencing, although itsmRNA expression was constant.3. Several HCSC-like traits appeared in WB-anti-miR-200a cells, including enhancedspheroid-forming capacity, expressing putative hepatic CSC markers and displayingsuperior resistance to chemotherapeutic drugs in vitro. In vivo xenograft assay alsodemonstrated the acquisition of tumorigenicity of WB-F344cells after miR-200asiliencing.4. miR-200a silencing conferred a mesenchymal phenotype to WB-F344cells,including an elongated cell morphology, enhanced cell migration ability and expression ofEMT-representative markers.5. Dual luciferase reporter gene assay and western blot analysis identified β-catenin(CTNNB1) as a direct target of miR-200a, and miR-200a silencing activated Wnt/β-catenin signaling.6. Silencing of β-catenin functionally attenuated spheroid-forming capacity and cellmigration ability in vitro in WB-anti-miR-200a cells.[Conclusions]To conclude, we illustrated that downregulation of miR-200a in liver oval cellspromotes EMT and CSC-like traits, at least partially through the regulation of β-cateninsignaling. These findings underscore the importance of miR-200a in inhibiting neoplasticphenotypes in normal adult hepatic progenitor cells. Exploring miR-200a-basedprevention and therapeutics might benefit HCC clinically. |
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