Screening Of Mirnas That Up-regulate RAVV-mediated Gene Expression In Human Hepatic Stellate Cell Line LX-2and Mechanism Analysis

Hepatic fibrosis is a response to chronic liver injury. The central event of hepaticfibrosis is the activation of hepatic stellate cells (HSCs), which promotes the excessproduction of extracellular matrix. It will develop into cirrhosis without treatments,even hepatocellular carcinoma. In recent years. Gene therapy become one of the newarea for hepatic fibrosis medicinal development because of its targeted drug deliveryand low dose. Developing gene drugs is a new research hotspot of anti-fibrosis.Recombinant adeno-associated virus (Recombinant AAV, rAAV) vectors is basedon Adeno-associated virus (AAV), is recognized as one of the most promising therapyvectors for gene therapy. Some clinical trials with rAAV achieved certainresults.However, the low transduction efficiency of gene expression in vivo is one ofthe restriction in the clinical application of rAAV vectors. But high doses of the drugcan cause immune response. Thus, how to improve its transduction efficiency is onehotspot for rAAV research.Based on these, the paper tries to find a way to enhance rAAV vector transductin liver, to treat liver fibrosis. RNAi is likely to be an effective tool.This thesis tries to screen out some kinds of miRNA from miRNA mimicLibrary in vitro with LX-2cell (human hepatic stellate cells). This paper analysis thetarget genes ans signaling pathways of miRNA by bioinformantics. We choosemiR-3133as a further study object because it is involved in many signaling pathways.It may effect the expression of rAAV by MAPK signaling pathway or ErbB signalingpathway. CBL is involved in ErbB signaling pathway. It is selected as a target genebecause it can effect the ubiquitin pathway which is related to the expression ofrAAV.With quantitative Realtime PCR, we confirmed the CBL gene is a target ofmiR-3133. MiR-3133can reduce CBL, that may be a mechanism which may affectmiR-3133rAAV expression. Meanwhile, miR-3133also reduced TGFBR1which isTGF-β receptor. TGF-β can promote liver fibrosis.All the results show miR-3133mayoptimize the treatment of hepatic fibrosis via inhibiting the activation of LX-2andsimultaneously increasing the expression of rAAV. In this paper, we screened out a miRNA which can enhance the expression ofrAAV from a library of miRNAs.And we analyzed the possible target genes andsignaling pathways. It is helpful to develop the research of gene therapy for liverfibrosis with rAAV-miRNA.