Construction Of Recombinant Adeno-associated Virus Vector Mediated CircPSD3 Delivery System And Study On Function And Mechanism Of CircPSD3 In Hepatic Fibrosis

Hepatic fibrosis（HF）,the inevitable pathological process of chronic hepatitis caused by a variety of damage factors to cirrhosis,is manifested by abnormal accumulation of extracellular matrix（ECM）in the liver,which further triggers hyperplasia of fibrous connective tissue and formation of scar septa dividing normal hepatic lobular structure,and finally leads to the formation of pseudolobules.Strictly speaking,the pathogenesis of HF is extremely hidden and the clinical symptoms are not obvious,and it is often found in the stage of liver cirrhosis.Therefore,taking the development of HF as a breakthrough point,and exploring the molecular mechanism of its pathological process is beneficial to provide ideas for developing biomarkers for early diagnosis.Studies have shown that myofibroblasts activated from hepatic stellate cells（HSCs）secrete a large amount of ECM components and thus activation and proliferation of HSCs are considered to be the central event in the occurrence of HF.Notably,an increasing number of studies have reported that non-coding RNAs（nc RNAs）are involved in the activation and proliferation of HSCs,such as micro RNAs（miRNA）and long noncoding RNA（lnc RNA）,and their clinical significance has been successively reported.In recent years,with advances in high-throughput sequencing and bioinformatics,the expression profiles and molecular functions of circular RNAs（circRNAs）in different diseases have been gradually revealed.Of note,our previous study established the expression profile of circRNAs in primary HSCs from mice with HF by high-throughput sequencing,and circ FBXW4 was found to inhibit the progression of HF by targeting the mi R-18b-3p/FBXW7 axis.But little is known about the function of circRNAs in the process of HF.In our sequencing results,circPSD3,derived from the host gene PSD3（Pleckstrin and Sec7 domain-containing 3）,was abnormally decreased in primary HSCs of mice HF compared with control mice.Noteworthily,increasing studies have revealed the key roles of conserved circRNA in liver diseases and circPSD3 was identified as a conserved circRNA that is formed in human and mouse by back-splicing of five consecutive exons of PSD3 gene on chromosome 8.However,the role of circPSD3 in HF has not been reported.In recent years,increasing studies have focused on gene therapy（GT）for diseases caused by liver gene abnormalities.GT is a method to correct abnormal gene expression levels by introducing exogenous nucleic acid into somatic cells through appropriate carrier packaging,so as to achieve therapeutic purposes.Adeno-associated virus（AAV）vector,a commonly used vector for GT,has the advantages of high safety,excellent gene transduction ability,low immunogenicity,and long-term efficacy in mediating gene expression.Notably,AAV mediated GT have been successively applied for clinical trials.Recombinant AAV（r AAV）vectors are constructed by is replace the sequence of the gene of interest with that of viral genome,and then the vectors are co-transfected with vectors determining the AAV serotype and helper vectors into AAV-293T cells to produce r AAV vector with a specific serotype.Different serotypes determine different tissue or organ tropisms of AAV in vivo.It has been reported that AAV8 has the features of prominent liver tropism,mediated gene long-term expression,low immunogenicity and promising clinical application prospect.In addition,AAV8mediated GT has shown promising results in murine HF.However,AAV8-mediated circRNA delivery system for HF has not been reported yet.Therefore,in this study,the circPSD3 delivery system mediated by r AAV vector was firstly established to provide in vivo research tools for the later study of the function of circPSD3 in HF.Then,the expression levels of circPSD3 were detected in mice liver fibrosis models and human liver fibrotic samples,and the difference of circPSD3 expression in different liver cells was further determined.Furthermore,the function and possible mechanism of circPSD3 in the progression of HF were investigated in vivo and in vitro.Methods:1.First,the full length of circPSD3 was obtained through circBase website.Then,specific primers for circPSD3 were designed to amplify the target fragment,which was introduced into the pHBAAV-CMV-ZSgreen1 vector.Enzyme digestion of the vector and full-length sequencing assays were used for determining whether the vector was successfully established.AAV-293T cells were transfected with“three plasmid system”to obtain cells containing virus particles.Then,the crude virus particles were obtained by repeatedly freezing and thawing the cells,and the supernatant was collected by centrifugation.The virus particles were further purified and tested for quality control.Finally,the purified virus particles were injected into mice through tail vein to observe whether the virus was toxic to mice and the virus distribution of the main organs in mice.2.The hepatic fibrosis models induced by different etiologies were established by long-term intraperitoneal injection of carbon tetrachloride（CCl₄）and Bile duct ligation（BDL）in mice,and the pathological indexes,serum liver injury indexes and liver fibrosis related indexes were detected to confirm whether the models were established successfully.Then,total RNAs were extracted from liver tissues and the expression levels of circPSD3 were detected by q-PCR.3.Primary hepatocytes,HSCs and macrophages of mice were isolated by in situ liver perfusion technique,and total RNAs were extracted.Then,the expression levels of circPSD3 were detected by q-PCR.4.Human liver fibrotic samples were collected.After pathological confirmation,total RNAs were extracted from liver tissues and the expression levels of circPSD3 were detected by q-PCR.5.Mice were given a certain amount of r AAV vector mediated circPSD3 or empty AAV vector by tail vein injection.One week later,hepatic fibrosis modeling induced by CCl₄ was performed for 6 weeks.The function of circPSD3 in the process of hepatic fibrosis was explored by detecting pathological indexes,serum indexes and liver fibrosis related indexes.6.The stability of circPSD3 was determined by RNase R digestion assay and Actinomycin D tolerance assay,and the subcellular localization of circPSD3 was determined by RNA fluorescence in situ hybridization（FISH）assay and nucleoplasmic RNA isolation assay.7.In vitro,LX-2,a human hepatic stellate cell lines,was stimulated with 10 ng/mL TGF-β1 to simulate the activation process of HSCs in vivo.Then,total RNAs were extracted and the expression levels of circPSD3 were detected by q-PCR.8.In LX-2 cells,the function of circPSD3 was investigated by lentiviral vector mediated circPSD3 overexpression and novel cationic liposome mediated circPSD3knockdown technique.Western blot,q-PCR,flow cytometry,CCK8 assay and Ed U click assay were used to detect the activation and proliferation condition of LX-2 cells.9.Mi RNA sequencing technology was used to detect the differentially changed miRNAs in circPSD3-overexpressing LX-2 cells.Further,the miRNA that may bind to circPSD3 was confirmed by target prediction website,q-PCR,and dual luciferase reporting assays.10.Mi RNA mimics were transfected into circPSD3-overexpressing LX-2 cells,and the effects of miRNA on circPSD3 function were detected by q-PCR,western blot,CCK8and Ed U click assays.Results:1.The results of enzyme digestion and full-length sequencing assays showed that pHBAAV-CMV-Zs Green1-circPSD3 was successfully established.After virus packaging and purification processes,circPSD3 delivery system mediated by AAV8（AAV8-circPSD3）was successfully constructed,and the sterile and mycoplasma test was passed,and the virus titer was within the ideal range.It was found that AAV8-circPSD3 was concentrated in mouse liver and had no toxicity to mouse liver.2.The expression levels of circPSD3 were abnormally reduced in CCl₄-and BDL-induced HF in mice.3.The expression levels of circPSD3 were significantly decreased in isolated primary HSCs from mice with HF.4.The expression levels of circPSD3 were significantly reduced in the collected human liver fibrosis samples compared with normal liver samples.5.Compared with the AAV8 empty vector group,mice with AAV8-circPSD3pretreatment showed the significantly reduced degree of liver fibrosis induced by CCl₄,which was manifested by significantly reduced serum ALT and AST levels,liver collagen deposition and inflammatory response.And the effect of AAV8-circPSD3 was better than circPSD3 delivery system mediated by lentivirus vector.6.circPSD3 was more resistant to RNase R digestion and Actinomycin D than linear PSD3.7.The expression levels of circPSD3 were significantly decreased in activated LX-2cells.8.Lentiviral vector-mediated overexpression of circPSD3 significantly inhibited the activation and proliferation of LX-2 cells,while knockdown of circPSD3 showed the opposite effects.9.Mi RNA sequencing results showed that overexpression of circPSD3 significantly inhibited the expression levels of mi R-92b-3p.Star Base website predicted that circPSD3 has an ideal binding site with mi R-92b-3p.Furthermore,the results of q-PCR and dual luciferase reporting assay showed that circPSD3 could bind to mi R-92b-3p.10.Overexpression of mi R-92b-3p mediated by miRNA mimics significantly weakened the inhibitory activation and proliferation effect of circPSD3 on LX-2 cells,and inhibited the promotive effect of circPSD3 on Smad7.Conclusion:Collectively,in this study,r AAV mediated circPSD3 delivery system was successfully established,and circPSD3 was found to suppress the progression of HF through targeting mi R-92b-3p/Smad7 axis.The circRNA delivery system mediated by r AAV vector provides an idea for the realization of GT targeting circPSD3,and lays a preliminary foundation for developing biomarkers for diagnosis of hepatic fibrosis.