| Background:China is a high-prevalence area of hepatocelluar carcinoma,accounting for about 40%of global hepatocelluar carcinoma,so it is very important to make clear the pathogenesis and find out effective therapeutic targets.During our search for Cristrome Data Browser chip-seq database,we found that miR-5188 is regulated by Wnt/?-catenin pathway downstream signaling molecule c-Jun.Meanwhile,bioinformatics software predicated that cancer suppressor gene FOXO1 maybe the target gene of miR-5188.As a newly discovered microRNA,the specific function of miR-5188 in hepatocelluar carcinoma and its relation with Wnt/?-catenin p.athway,FOXO1 still needs our further research.Objectives:1.To figure out the specific influence of miR-5188 on invasion,metastasis,sternness and chemo-resistance in hepatocelluar carcinoma.2.To figure out the specific mechanism of miR-5188 in hepatocelluar carcinoma.3.To verify the target gene of miR-5188 and how miR-5188 regulates this target gene.4.To analyze the expression characteristics of miR-5188 and target gene in hepatocellular carcinoma tissue and their relations.1.In vitro functional experiment was used to study the role of miR-5188 in hepatocelluar carcinoma cells(1)Hepatocelluar carcinoma cell lines Hep3B,Huh7 with stable overexpression of miR-5188 and HCCLM3,PLC/PRF/5 with miR-5188 transiently interference were constructed;(2)Scratch assay,Transwell assay and Boyden assay were used to verify the influence of miR-5188 on cell invasion and metastasis;(3)Tumorsphere formation assay,flow cytometry,the orthotopic tumor model and pulmonary metastasis mouse model were used to verify the influence of miR-5188 on tumor sternness;(4)MTT assay was used to verify the influence of miR-5188 on tumor chemosensitivity.2.Specific Mechanism of miR-5188 influencing biological function of hepatocelluar carcinoma cells(1)Western Blot was used to detect expression changes of Wnt/?-catenin pathway proteins,tumor sternness related proteins,EMT related proteins,chemo-resistance related proteins.(2)Change of cell biological behavior was observed after Wnt signaling pathway was reduced by si-?-catenin in hepatocellular carcinoma cells with miR-5188 overexpression.3.Discussion on the relation between Wnt/?-catenin downstream molecules c-Jun and miR-5188(1)Bioinformatics software was used to predict c-Jun binding sites in upstream of miR-5188 transcriptional start site;(2)Electrophoretic mobility shift assay,chromatin immunoprecipitation assay,promoter biluciferase reporter experiment were used to verify the role of c-Jun on miR-5188 promoter region;(3)The expression of miR-5188 in hepatocellular carcinoma cells was detected by Real-time PCR after interference with c-Jun.4.Verification of miR-5188 target gene(1)luciferase reporter experiment was carried out to verify FOXO1 was a target gene of miR-5188;(2)Western Blot and Real-time PCR were used to detect changes of FOXO1 protein and gene after overexpression of miR-5188;(3)Changes of cell biological behavior and Wnt/?-catenin pathway proteins were detected after transfection of FOXO1 plasmid in hepatocelluar carcinoma cell with stable overexpression of miR-5188 or si-FOXO1 in cells interfering with miR-5188.5.Discuss the relation between FOXO1 and p-catenin.Immunofluorescence and confocal microscopy were used to verify whether FOXO1 can combine with ?-catenin.6.The expression of miR-5188 and FOXO1 in hepatocellular carcinoma tissue and their clinical significance.In situ hybridization and immunohistochemistry were used to observe the expression of miR-5188 and FOXO1 in hepatocellular carcinoma tissues.Their relation with clinical parameters and prognosis,the relevance between miR-5188 and FOXO1 were analyzed.Results:1.miR-5188 can promote invasion and metastasis of hepatocellular carcinoma,enhance sternness and induce chemotherapy resistance;2.miR-5188 can upregulate Wnt/?-catenin and its downstream signal pathways;3.Wnt/?-catenin downstream signaling molecule c-Jun can promote transcription of miR-5188,miR-5188 and Wnt/?-catenin and c-Jun forms activation loop with each other;4.miR-5188 can targetly reduce the protein expression of FOXO1,FOXO1 can reverse the activation effect of miR-5188 on Wnt/?-catenin pathway;5.FOXOl can combine with ?-catenin and inhibit activation of Wnt/?-catenin pathway;6.High expression of miR-5188 is a poor prognosis factor for hepatocellular carcinoma patients.The expression of miR-5188 is negatively related to FOXO1 in hepatocelluar carcinoma tissues.Conclusions:miR-5188 plays the role of cancer-promoting gene in hepatocellular carcinorma and promotes invasion,sternness and chemo-resistance of hepatocellular carcinoma cells by targeted suppressing FOXO1 expression and upregulating Wnt/?-catenin signal pathway.Besides,Wnt/?-catenin downstream signal molecule c-Jun can also act on miR-5188,upregulate the expression of miR-5188 and form activation loop of miR-5188/Wnt/C-Jun with each other.As shown in the following 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