The Functional Role And Relevant Mechanism Of Circular RNA CircFBXW4 On Hepatic Fibrosis Mediated By Recombinant Adeno-associated Virus Vector

Hepatic fibrosis(HF)is results from persistent injury of infection,immune reaction,physical and chemical factors on liver,metabolic dysequilibrium of extracellular matrix(ECM)causes intrahepatic connective tissue dysplasia and liver architecture distortion.Clinically,there are no typical early-stage symptoms of hepatic fibrosis,high efficiency early diagnosis indexes is urgent needed currently.Reports suggest that activation of hepatic stellate cell(HSC)differentiates into myofibroblasts,which are the mainly cell type responsable for extracellular matrix secretion.Therefore,hepatic stellate cells activation is regarded as the key action in hepatic fibrosis initiation and persistence stages.The process of hepatic stellate cells activation is regulated by various noncoding RNA(nc RNA),study on molecular mechanism of hepatic fibrosis will be benefit for new treatment technology discovery.noncoding RNA play important regulatory role in hepatic fibrosis occurrence and development.A number of studies have revealed the mechanism and clinical value of micro RNA(miRNA)and long noncoding RNA(lnc RNA)in hepatic fibrosis.In recent years,as a novel member of noncoding RNA,circular RNA(circRNA)was discovered and closely related to various diseases and physiological process.Endogenous circRNAs is characterized by head-to-tail structural form,connecting by covalent bond,high stability of circRNAs causes it can tolerate Rnase R degradation,circRNAs are conserves in different species,and exhibit specific expression pattern in different developmental stage and physiological state of tissues and cells.Molecular structural characteristics of circRNAs show that it may serve as the promising novel clinical diagnostic markers,circRNAs are developed into a hot research area in biomedical sciences.The functions and mechanisms of circRNAs in various diseases have gradually attracted attention of researchers,however,the expression pattern and mechanisms of circRNAs in hepatic fibrosis remain to be clarified.In this study,the progression and reversal of mouse hepatic fibrosis model were established,primary hepatic stellate cells were isolated from mice by in situ liver perfusion technology.The expression pattern of circRNAs in primary hepatic stellate cells was analyzed by high throughput sequencing,results show that there are a large number of circRNAs differentially expressed in hepatic fibrosis.The level of circFBXW4 is reduced in the progression of hepatic fibrosis stage while restored in hepatic fibrosis reversal stage,reminding that circFBXW4 may closely relate to the pathological process of hepatic fibrosis,promoting us to investigate the functional roles of circFBXW4 in hepatic fibrosis.In this study,recombinant adeno-associated virus was used as a vector,mediating the gene target therapy of circFBXW4 to liver,results show that enhancing circFBXW4 levels inhibited liver injury in hepatic fibrosis,decreasing fibroplasia and collagen deposition in liver tissue,partly repairing the phenomenon of liver structural disorder.Moreover,stable circFBXW4 overexpression hepatic stellate cell line was constructed via lentivirus biological vehicle,overexpression of circFBXW4 inhibited the transformation of hepatic stellate cells activation,arresting its cell cycle and reducing the malignant proliferation of activated hepatic stellate cells.In addition,cationic liposome,a member of non-viral vector system,was employed in this study,we demonstrated that interfering the biological signal of circFBXW4 showed a reverse regulatory function on hepatic stellate cells.All those results suggest that circFBXW4 may be an anti-hepatic fibrosis RNA factor.Mechanistically,most of the circRNAs,which are derived from exons and located in cytoplasm,regulate target gene expression through the competitive endogenous RNA(ce RNA)mechanism,then mediating relevant biological functions.Further,micro RNAs array technology was employed to analyze the differentially expressed micro RNAs in primary hepatic stellate cells,and bioinformatics was used to predict the micro RNA targets of circFBXW4,we found that circFBXW4 binds and regulates the level of miR-18b-3p.Both micro RNAs array and experimental detection show that the expression of miR-18b-3p was upregulated in the process of hepatic fibrosis,miR-18b-3p promotes the expression of fibrous factors in hepatic stellate cells,the levels of circFBXW4 is negatively correlated with miR-18b-3p.F box and WD 40 domain containing protein 7(FBXW7),a tumor suppressor gene,is one of the target genes of miR-18b-3p,and it was found in this study that circFBXW4 regulates the FBXW7 signaling by targeting miR-18b-3p.The phenotype and mechanisms of circFBXW4 were preliminarily explored in different dimensions of animal,tissue,cell,molecule levels.This study found that circFBXW4 is an important biomolecule in regulating hepatic stellate cells activation and the development of hepatic fibrosis,circFBXW4 may serve as a potential biomarker for hepatic fibrosis progression and regression processes.