Screening And Function Of MicroRNA In Hepatic Fibrosis And Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a common malignant tumor in worldwide,but so far,there still have no effective measures beside surgery.And low early diagnosis and detection rate of HCC made the loss of early treatment clinically for the majority of patients were found with advanced disease.So,seeking new effective treatment and early prevention of HCC are always been the research focus and important problems.Liver fibrosis is the pathological basis of chronic liver disease,and HCC often develops on the basis of patients with chronic liver disease.Therefore,liver fibrosis is also an important pathology basis of HCC.HCC and liver cirrhosis are all progressed to an irreversible stage,but liver fibrosis is reversible.So,if we can effectively block or even reverse fibrosis,good preventive effect of the chronic liver disease,cirrhosis and HCC could be obtained.MicroRNA plays an important role in tumor development,more than 1/3 of the human protein-coding genes are subject to microRNA regulation.As research reported,microRNA can reflect the molecule nature closely related to the development of the disease,and has a great research potential role in beening applied to research the disease etiology,diagnosis,molecular typing,treatment and prognosis.Meanwhile,microRNA and mRNA crossing study can greatly improve the target gene prediction accuracy of microRNA and make finding target gene more easily.This research aiming to find new early prevention and treatment of HCC from investigating the pathogenesis of liver fibrosis and HCC,using microRNA and mRNA expression profiling screening method to analyze the differential expression of RNA bioinformatics,provides new basis for HCC early prevention and effective treatment.This study is composed of two parts.Part I:MicroRNAs and target genes screening research in intervention of the liver sinusoidal endothelial cell capillarisationObjective:To filter out the differentially expressed mRNA and microRNA in liver sinusoidal endothelial cell(LSEC)capillarisation,do bioinformational analysis,and according to the screening results,select target genes to do histological verification,providing initial basis on researching key genes in LSEC capillarisation.Methods:Cultured human hepatic sinusoidal endothelial cells(HHSEC)in vitro,investigated fenestration in scanning electron microscopy and grouped.Extracted mRNA for mRNA and microRNA microarray test,the results of the study did bioinformational and crossing analysis studies,evidently different target gene would be selected for clinical validation.Results:Fenestration could be observed gradually disappearing in in vitro cultured HHSEC.Extracted RNA for mRNA and microRNA microarray.mRNA microarray results showed that 1639 genes upregulated and 1,608 genes downregulated.532 out of these mRNA had significantly difference.MicroRNA microarray screening found 19 microRNAs had significantly relationship with HHSEC capillarisation(P<0.05),including 7 microRNAs upregulation,12 microRNAs downregulation.Bioinformational analysis methods obtained 10 109 target genes information.Further studies of mRNA and microRNA crossing analysis counted 253 target genes and chosen CALM2,one of the most obviously genes for a clinical histological verification.The results showed CALM2 expression was proportionate to liver fibrosis that confirmed the relationship of the target gene and liver fibrosis.Conclusion:Combining HHSEC mRNA and microRNA microarray screening and biological analysis results obtained target genes which confirmed with relationship of HHSEC capillarisation and liver fibrosis.This would provide a strong experimental basis for further investigation of the pathogenesis and reversal measurement of liver fibrosis.Part II:MicroRNAs regulated Glypican-3 expression in hepatocellular carcinomaObjective:To filter out Glypican-3(GPC3)expression associated microRNA in HCC,and do bioinformational analysis,then according to screening results select the most different microRNA for histological verification,giving RNA basis on GPC3 expression mechanism in HCC.Methods:GPC3 immunohistochemical staining and mRNA level in HCC samples with complete clinical pathological data were carried out and divided into GPC3 positive or negative group according to detected results.Further microRNA microarray screening was processed and bioinformatiinal analysis were done.Selected the obviously different microRNAs to validate in expanded sample.Results:MicroRNA microarray of different GPC3 groups screened out 25 different microRNAs(signal value>500),of which 10 were up-regulated and 15 were down-regulated.5 microRNAs were chosen for qPCR validation on expanded samples and successfully screened out 4 differentially expressed microRNA(miR-99a-5p,miR-125b-5p,miR-376c-3p,miR-17-5p),and obtained relevant information of GO and signaling pathways through biological information analysis.Conclusion:Using microRNA microarray and expanded sample qPCR validation method successfully screened out GPC3 associated microRNAs in HCC,and provided reliable experimental datas for subsequent experimental research by bioinformational analysis.Conclusion:A large numbers of target genes and microRNAs participate liver fibrosis and HCC formation.We screened out significantly different microRNAs or mRNAs by mRNA and microRNA expression profiling,and obtain the relevant target genes and signaling pathways information using bioinformational analysis methods,and validated on clinical expanded sample.This would provided initial basis for further research.