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miRNA-214Inhibits Hepatocellular Carcinoma Growth by Targeting β-catenin Pathway

Author: WangXiaoJun
Tutor: JiangJiaZuo
School: Fujian Medical
Course: Internal Medicine
Keywords: miR-214 HCC CTNNB1 β-catenin lentiviral
CLC: R735.7
Type: PhD thesis
Year: 2013
Downloads: 61
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Abstract


Hepatocellular carcinoma (HCC) is one of the most common malignant tumorworldwide. Despite of great progress in the treatment of HCC, it remains the thirdleading cause of cancer-related death, and the5-year survival of HCC patients is lessthan5%. Therefore, it’s very necessary to clarify the molecular mechanisms of HCCfor development of new diagnostic, treatment and prognosis strategy. MicroRNAs(miRNAs) is a class of rich non-coding short RNA inhibiting the expression oftarget genes in post-transcriptional level by inducing mRNA degradation or inhibit itstranslation. Recently, a growing number of reports indicate that abnormal expressionof miRNAs associated with a variety of human cancers, including HCC, and miRNAas a tumor suppressor or oncogene involved in the development of human cancers.This provides a new approach to the diagnosis and treatment of HCC. MicroRNAprofile in recent studies have demonstrated that miR-124is downregulated in HCC,however, its potential function in the process of HCC and related mechanisms are notyet clear.MethodsWe detected the expression lever of mature miR-214in18paired HCC tissue andadjacent tissues,and HCC cell lines including Hep3B,SK-HEP1, SMMC-7721, Huh7,HepG2cells and normal liver cells LO2by quantitative real-time RT-PCR analysis.Lentiviral expression plasmid of miR-214(Lv-miR-214) or lentiviral control(Lv-control) and packaging systems co-transfected293T cells to produce lentivirus,then infected HepG2cells, and screened cell strains stably expressing miR-214. Cellgrowth curve and MTT assay were employed for HepG2cell proliferation assay;Colony formation assay for single tumor cell colony growth capacity assay; Flowcytometry for cell cycle analysis assay. Nude mice experiments was employed toobserve the impact of the tumorigenic ability of miR-214on HepG2cells.Bioinformatics softwares were applicated to predict the possible target genes of miR-214, and luciferase reporter experiments, quantitative real-time RT-PCR, Westernblot for target gene validation. β-catenin RNA interference and restore expressionexperiments was employed for testing the effects of β-catenin on cell proliferation.Impact of miR-214on the expression levels of β-catenin pathway downstream proteinof CyclinD1, c-Myc,TCF-1, and LEF-1was detected by Western blot.Results1. miR-214is significantly downregulated in HCC tissues and HCC cell lines.2. HepG2cell strains stably expressing miR-214were established.3. Cell growth curves showed that HepG2cell strains stably expressing miR-214growed slowly; MTT experiments showed that miR-214overexpression inhibitsHCC cell proliferation; Colony formation experiments showed that overexpression ofmiR-214reduced tumor cell colony growth ability; The flow cytometry discoveredthe proportion of cells in G1/G0phase were54.62%in the control group, while theratio in G1/G0increased to69.28%in the experimental group; the proportion of cellsin S phase was19.56%, lower than the control group, the proportion was33.48%.This suggest that miR-214caused cell cycle G1arrest.4. HepG2cell strains stably expressing miR-214were applicatied to establish nudemice HCC animal model, compared to the control group, the experimental grouptumors grow slowly; The volume and wight of the tumors in the experimental groupwere32%and37.5%of the control group25d after inoculated subcutaneously withBALB/c nude mice.5.TargetScan predicted CTNNB1may be a target genes of miR-214. Luciferasereporter experimental results show that luciferase gene activity can be suppressed byco-transfection of miR-214expression plasmid and CTNNB1-3’-UTR, but not byco-transfection of miR-214expression plasmid and CTNNB1-3’-UTR-mt. Westernblot analysis showed that miR-214can inhibit the expression of β-catenin protein;qRT-PCR results showed that miR-214didn’t influrenced β-catenin mRNA expression.6.The shβ-catenin reduceed β-catenin expression, inhibited HCC cell proliferation.Transfection of β-catenin overexpression plasmid not containing the3’-UTR ofCTNNB1restored β-catenin protein expression, reversed the inhibition of miR-214on cell proliferation.7.Western blot showed that CyclinD1, c-Myc,LEF-1, and TCF-1expression levelsdecreased in HepG2cell strains stably expressing miR-214and nude micetransplanted tumor tissue.Conclusions1.miR-214is significantly downregulated in HCC tissues and HCC cell lines.2.miR-214inhibition of the growth of HCC cells in vitro and in vivo.3. CTNNB1is a target gene of miR-214. miR-214can affect HCC cells growth by theβ-catenin pathway.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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