Effect And Mechanism Of CircRNA97 On Reversing Hepatic Fibrosis Through MiRNA-146b-5p/HIPK1 Axis

Hepatic fibrosis（HF）is caused by the imbalance of extracellular matrix（ECM）metabolism in the liver under the stimulation of various external or pathological factors for a long time,which leads to the abnormal proliferation of fibroblast connective tissue and the disorder of liver structure.Although studies have shown that liver fibrosis is a reversible process,with the progress of liver fibrosis,it is very easy to cause further liver lesions,liver cirrhosis and even liver cancer.Therefore,the early diagnosis and treatment of liver fibrosis is particularly important.However,because the early symptoms of liver fibrosis are not typical clinically,early diagnosis becomes particularly difficult.The activation of hepatic stellate cell（HSC）is the central link of liver fibrosis.Activated hepatic stellate cells can differentiate into fibroblasts and are also the main source of extracellular matrix.Therefore,in-depth study of the process and molecular mechanism of hepatic stellate cell activation and search for diagnostic markers and therapeutic targets in the process of hepatic stellate cell activation are the key and difficult problems in the current clinical application research of hepatic fibrosis,and will provide a research basis for the development of new diagnostic and therapeutic methods.The activation of hepatic stellate cells and the process of liver fibrosis are regulated by noncoding RNA（ncRNA）.A large number of literatures have been reported on the research of circular RNA,long noncoding RNA and micro RNA in liver fibrosis.As a new non coding RNA rediscovered in recent years,circular RNA plays an important role in various disease processes and physiological processes.Circular RNA is a closed circular molecule with covalent bonds formed by reverse shearing.It has high stability and is highly conservative among different species,but it is differentially expressed in different tissues,cells or physiological states.Because of its above structure,function and expression characteristics,circular RNA has natural advantages as a clinical diagnostic marker.Therefore,it has become one of the hotspots in the field of medical research.However,the expression profile,differential expression,effect on the process of liver fibrosis and molecular mechanism of circular RNA in liver fibrosis are not clear,and still need to be clarified.In this study,we first constructed a rat model of liver fibrosis and obtained rat hepatic stellate cells by liver perfusion in situ;Using high-throughput sequencing technology,the differentially expressed circular RNA in activated hepatic stellate cells of rats with liver fibrosis was analyzed,and circRNA97 with significantly low expression in rats with liver fibrosis was screened;After analyzing its sequence,it was found that it was located on rat chromosome 3 and contained an open reading frame,which was highly homologous with rat smox sequence.After constructing adenovirus vector to infect rats,the level of liver fibrosis in rats overexpressing circRNA97 was significantly improved under the same CCl₄induction,suggesting that circRNA97 can effectively inhibit or reverse the process of liver fibrosis in rats;Through the online prediction of Starbase and targetscan,and the establishment of visual RNA-RNA interaction network by Cytoscape,the miR-146b-5p complementa-ry to circRNA97 sequence was screened,and the circRNA97/miR-146b-5p/HIPK1 pathway was screened;Then,the targeting of circRNA97 with miR-146b-5p and mi-146b-5p with HIPK1 was verified by double Luciferase Report Analysis;Overexpression of circRNA97 significantly reduced the expression level of miR-146b-5p,and transfection of miR-146b-5p significantly inhibited the expression of HIPK1.Subsequently,we constructed an LPS induced HSC-T6 activation model in vitro;It was found that miR-146b-5p could significantly promote the activation of HSC cells and increase the markers of liver fibrosisα-SMA,TGF-βAnd COL1A1expression level;Lentivirus overexpression of HIPK1 could effectively inhibit the activation of HSC cells induced by LPS,and the promotion of miR-146b-5p on liver fibrosis was significantly inhibited.In conclusion,we found a circular RNA that inhibits liver fibrosis-circRNA97,and analyzed the effect and mechanism of circRNA97 on the process of liver fibrosis from multiple levels and angles of molecules,cells and animals.The results showed that circRNA97 could significantly inhibit the activation of hepatic stellate cells in vitro through spongy miR-146b-5p/HIPK1 pathway,and inhibit the process of hepatic fibrosis in rats in vivo;It is a potential molecular marker and therapeutic target of liver fibrosis.