ASIC1a Regulates MiR-350/SPRY2 By M~6A Modification To Promote Hepatic Fibrosis

Hepatic fibrosis(HF)is a pathological process caused by chronic liver damage induced by various reasons,and it is a necessary stage for the development of liver cirrhosis and even liver cancer.Hepatic fibrosis is a reversible scar repair reaction,and proper intervention during its formation can block or even reverse hepatic fibrosis.It is currently believed that the activation and proliferation of hepatic stellate cells(HSC)plays a key role in the formation of hepatic fibrosis.How to regulate the activation and proliferation of HSC is the key research direction of anti-hepatic fibrosis.Acid-sensing ion channels(ASICs)are a type of cation channels activated by extracellular acidification,which belong to the ENa C / DEG family.There are seven subunit proteins encoded by four coding genes: ASIC1 a,ASIC1b,ASIC1b2,ASIC2 a,ASIC2b,ASIC3 and ASIC4.Acid-sensing ion channel 1a(ASIC1a),as a member of the ASICs family,opens when activated and mediates the influx of Na+,Ca2+ and other ions,further causing a series of pathological and physiological changes in the body.Micro RNA(mi RNA),a kind of endogenous single-stranded non-coding RNA with a length of about 21?25 nucleotides,can interfere with the transcription process of the target gene by degrading the m RNA of the target gene or combining with the 3 '-UTR base pairing of the target gene m RNA,and regulate the expression of the target gene at the protein level.mi RNA is involved in the regulation of biological activities such as HSC activation,proliferation and apoptosis,and it plays an important role in the occurrence and development of hepatic fibrosis.Through high-throughput screening of mi RNAs differentially expressed in activated HSC,we found that mi R-350 was up-regulated in activated HSC.After the specific blocking of ASIC1 a by Psalmotoxin-1(Pc Tx-1),the expression of mi R-350 was also down-regulated.N6-methyladenosine(m~6A)is the most common post-transcriptional modification,mediating over 80% of RNA methylation and participating in the processing of mi RNA,m RNA and other RNAs.It has been reported that m6 A modification can affect the synthesis and processing of mi RNA and function at multiple levels,resulting in imbalance of mi RNA levels in cells.In this study,the role of ASIC1 a in hepatic fibrosis and its regulatory mechanism for the methylation modification of m6 A and the synthesis of mi RNA were studied at the level of patients,animals,and cells.In addition,the influence of ASIC1a/m~6A/mi RNA on the activation and proliferation of HSC and its related signaling pathways was explored,which provided a basis for the study of the pathogenesis of hepatic fibrosis and provided new targets and ideas for its prevention and treatment.The study first used Western blotting,immunohistochemistry and immunofluorescence techniques to observe the expression of ASIC1 a in vivo and in vitro.The recombinant adeno-associated virus(r AAV)type 8 was injected into the tail vein of mice to silence ASIC1 a.And the specific inhibitor Pc Tx-1 and RNAi technology were used to interfere with the expression of ASIC1 a in vitro,the levels of m6 A and mi R-350,as well as the activation of HSC and the degree of liver fibrosis were detected.In vitro RNAi technology was used to further interfere with mi R-350 and its target genes to detect changes in downstream genes.The binding site of mir-350 to the target gene was detected by the dual luciferase report.Co-immunoprecipitation and RNA-IP were used to observe the mechanism of m6a-related enzymes regulating mi R-350 processing.Finally,the possible signaling pathways were explored by Western blotting.The results showed that the expression of ASIC1 a in mice and patients with hepatic fibrosis was significantly increased,and the results were the same in PDGF-BB-induced activated HSC.After down-regulating ASIC1 a expression,the level of m6 A modification was significantly reduced,the expression of mi R-350 wasalso reduced,and HSC activation was inhibited.Immunoprecipitation results showed that METTL3 regulates the level of m6 A modification of pri-mi R-350 by binding to DGCR8.The results of the dual luciferase reporting experiment showed that mi R-350 bound to the 3'UTR region of the target gene SPRY2.mi R-350/SPRY2 can participate in HSC activation through PI3K/AKT and MAPK/ERK signaling pathways.The above results indicate that m6 A and mi R-350 play an important role in HSC activation regulated by ASIC1 a.In summary,ASIC1 a regulates the processing of mi R-350 through METTL3-dependent m6 A modification,which up-regulating the expression of mi R-350 matures and inhibiting the transcription of its target gene SPRY2.The inhibition of SPRY2 activates the PI3K/AKT and MAPK/ERK pathways in HSC and promotes hepatic fibrosis.