| Objective The incidence rate and mortality rate of hepatocellular carcinoma(HCC)rank the fifth and fourth among all cancers in China,which seriously threaten people’s life and health.Due to the lack of typical early symptoms,most HCC patients are already in the middle and late stages when first diagnosed,and the 5-year survival rate is less than 30%.The high recurrence and metastasis of HCC are the main reasons for the poor prognosis of patients.Previous studies have confirmed that angiogenesis is closely related to tumor growth and metastasis.The aim of this study was to investigate the molecular mechanism and clinical significance of miR-378a-3p in regulating HCC angiogenesis,and to search for molecular markers and potential therapeutic targets for prognosis prediction of HCC patients.Methods 1.Screening of candidate miRNAs using TCGA and GEO public databases.2.Differential expression of candidate miRNAs in hepatocellular carcinoma cells and fresh tissues by qRT-PCR and their clinical significance.3.To construct miR-378a-3p high and low expression hepatocellular carcinoma cell lines for phenotype validation,including cell models in vitro:HUVECs cells co-cultured with hepatocellular carcinoma cells were used to complete proliferation(CCK-8 and colony formation assay),migration(Wound healing and Transwell assay),and angiogenesis(tubule formation assay);and animal models in vivo:subcutaneous xenograft mouse model and matrix plug assay.4.To search for possible downstream target genes of miR-378a-3p using RNA-seq and bioinformatics prediction,and to verify the molecular interactions by luciferase reporter assay,immunofluorescence assay,immunoblotting and qRT-PCR to verify the upstream and downstream regulatory relationships,and to verify the regulatory network of miR-378a3p inhibition of angiogenesis by reversion experiments.5.Treatment of hepatocellular carcinoma cells with the demethylation reagent 5-aza-2’deoxycytidine(5-Aza-CdR)and qRT-PCR analysis of the effect of promoter methylation on miR-378a-3p expression;construction of DNA methyltransferase DNMT1,DNMT3A,DNMT3B low expression cell lines and qRT-PCR analysis of the major methyltransferases catalyzing miR-378a-3p promoter methylation.6.Correlation of p65 and DNMT1 by GEPIA and JASPAR databases,luciferase reporter assay,ChIP experiments to analyze the transcription factor regulation of DNMT1 by p65.7.In vivo subcutaneous tumor model and immunohistochemistry to verify the role of miR-378a-3p in the regulation of HCC angiogenesis and the expression of key molecules;TCGA database to verify the relevance of key molecules.Results 1.miR-378a-3p expression in hepatocellular carcinoma and its clinical significanceTCGA,GEO microarrays(GSE108724 and GSE174608)were analyzed and miR-378a-3p expression was confirmed by qRT-PCR assay to be lower in HCC tissues than in corresponding paraneoplastic tissues.In addition,miR-378a-3p expression was found to be negatively correlated with MVD in tumor tissues and positively correlated with patient prognosis.2.miR-378a-3p inhibits angiogenesis in HCCAfter interfering with the expression of miR-378a-3p in SMMC-7721 cells co-cultured with HUVECs,the angiogenesis of HUVECs were significantly enhanced,and the immunohistochemical results showed an increase in CD34 and VEGF expression.After overexpressing miR-378a3p in HCCLM3 cells co-cultured with HUVECs,the angiogenesis of HUVECs were significantly reduced,and the immunohistochemical results showed a decrease in CD34 and VEGF expression.3.miR-378a-3p inhibits angiogenesis in HCC by targeting TRAF1 to downregulate VEGFThe downstream target genes regulated by miR-378a-3p were obtained by RNA-seq using miR-378a-3p stable overexpression in SMMC-7721 cells,as well as bioinformatics prediction to find the genes that may bind to miR-378a-3p,and the two target gene sets were taken to intersect to obtain the candidate gene TRAF1.Dual luciferase reporter gene assay confirmed that miR-378a-3p could only reduce the luciferase activity of TRAF1 wild-type plasmid.It had no effect on the luciferase activity of the mutant plasmid.qRT-PCR and western blot assays showed that miR-378a-3p significantly inhibited TRAF1 mRNA and protein expression.In vitro,the rescue experiments confirmed that overexpression of TRAF1 reversed the inhibitory effects of overexpression of miR-378a3p on the proliferation,metastasis and tubule formation of HUVECs cells and VEGF expression in serum.TRAF1 siRNA reversed the inhibitory effects of interference with miR-378a-3p on the proliferation,metastasis and tubule formation of HUVECs cells and VEGF expression in serum.4.miR-378a-3p regulates NF-κB signaling pathway through TRAF1The TRAF1-related signaling pathway was predicted by STRING database,and miR-378a-3p mimic decreased NF-κB luciferase activity and decreased p65 nuclear translocation by dual luciferase reporter gene,immunofluorescence,and immunoblotting experiments,while miR-378a3p inhibitor increased NF-κB luciferase activity and increased p65 nuclear translocation.5.DNMT1 silences miR-378a-3p expression by promoting miR378a-3p promoter methylationTreatment of HCCLM3 and SMMC7721 cells with the methyltransferase inhibitor 5-Aza-CdR significantly increased miR-378a3p expression.miR-378a-3p expression was significantly up-regulated after transfection with DNMT1 siRNA in HCCLM3 and SMMC-7721 cells.After transfection of DNMT1 overexpression plasmid,miR-378a-3p expression was significantly suppressed down-regulated.qRT-PCR showed that DNMT1 expression was significantly higher in cancer tissues of HCC patients than in paraneoplastic tissues,and there was a significant negative correlation between DNMT1 and miR-378a-3p.6.Transcription factor p65 regulates miR-378a-3p through DNMT1 negative feedbackThe results of qRT-PCR assay showed that p65 could repress miR378a-3p expression;and the promoter region of gene DNMT1 had binding elements for transcription factor p65.p65 was found to elevate DNMT1 promoter activity by dual luciferase reporter gene assay,and ChIP assay further confirmed the binding of p65 to DNMT1 promoter region.7.miR-378a-3p/TRAF1/p65/DNMT1 axis regulates angiogenesis in hepatocellular carcinoma in vivoAn in vivo model of HCC in nude mice was constructed,and tumor volume,tumor weight,FISH,and immunohistochemical results showed that miR-378a-3p high expression could inhibit angiogenesis and tumor growth of HCC through TRAF1/p65/DNMT1 axis.The relationship between miR-378a-3p,TRAF1,p65,and DNMT1 was verified using fresh hepatocellular carcinoma tissues and data from the TCGA database.Conclusion The above results suggest that miR-378a-3p can inhibit angiogenesis in HCC.The mechanism is mainly that miR-378a-3p can inhibit VEGF-mediated HCC angiogenesis by targeting TRAF1 to downregulate NF-κB signaling pathway;at the same time,transcription factor p65 downregulation leads to reduced transcription of methyltransferase DNMT1,which downregulates the promoter region methylation of miR-378a-3p and activates the expression of miR-378a-3p.This further affects HCC angiogenesis and inhibits the growth and metastasis of HCC. |
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