ASIC1a Promotes Acute Lung Injury Through The CircRNA18-658/miR-127-5p/TRIM72 Axis

Acute lung injury（ALI）is a serious clinical syndrome with high morbidity and mortality.The main manifestations were decreased pulmonary stress,severe hypoxemia and progressive hypoxic respiratory failure.Acute lung injury often involves Alveolar typeⅡepithelial cells.The occurrence and development of acute lung injury is often accompanied by apoptosis of typeⅡepithelial cells.Acid-sensing ion channels（ASICs）is a voltage-independent H⁺gated cationic channel that is widely expressed in central and peripheral nerves in rodents and humans.Among its many subtypes,only ASIC1a（acid-sensitive ion channel 1a）is permeable to calcium ions,making ASIC1a an important research object.Circ RNA（circular RNA）is a novel class of RNA molecules characterized by covalently closed circular structures,which are produced by reverse splicing.Because it is closed into a ring,it is resistant to exonuclease,and can exist in cells more stably and accumulate continuously.Circrnas have a variety of roles,one of which is to specifically bind mirnas to act as molecular sponges.Circrnas also act as transcriptional regulators,molecular scaffolds,and sources of small protein/peptide translation to regulate a variety of cellular processes.Mi RNA（micro RNA）are endogenous small（about 20-25 nucleotides）non-coding Rnas that often act as negative regulators of gene expression,because they are responsible for the degradation and translation inhibition of their target m RNA.A single mi RNA can regulate the expression of multiple target genes by inhibiting or promoting the degradation of target m RNA translation.This affects many levels of developmental pathways.Therefore,it plays an extremely important role in the occurrence,biological development,epigenetics and metabolism of diseases.TRIM72（tripartite motif-containing protein 72）,a membrane repair protein,is highly expressed in bone and myocardium of mice and is a member of the trimotif family.Studies have shown that inhibition of TRIM72 can alleviate severe bacterial infection in the lung,and TRIM72 can directly or indirectly regulate entosis.Participate in alveolar epithelial cell repair by removing plasma membrane trauma.It also acts on alveolar epithelial cells to promote acute lung injury.In addition,TRIM72 has been shown to be involved in repairing cell membrane damage.It has a protective effect on hypoxia/reperfusion damage in multiple oxygen-dependent organs such as the heart,brain,lungs,kidneys and liver.The recombinant human TRIM72 also plays a unique role in I/R,sepsis,and other aspects.Funded by the provincial Jieqing Project,the preliminary research group found that acute lung injury is often accompanied by inflammatory response,and the PH value of the inflammation site will decrease.ASIC1a can sense this change,and then affect acute lung injury.However,the specific mechanism of action is still unclear.Does ASIC1a affect acute lung injury by circ RNA?Therefore,we designed an experiment to explore.In this study,the role of ASIC1a in acute lung injury and its possible mechanism were studied from the animal and cellular molecular levels,and high-throughput sequencing was conducted on ALI rat lung tissues,and the effect of circ RNA18-658/mi R-127-5p/TRIM72 axis on acute lung injury was found and verified.It provides new targets and ideas for the mechanism research of acute lung injury and the development of new drugs.The main research contents are summarized as follows:1.Expression changes of ASIC1a and apoptosis-related genes（Bcl-2,Bax,Cleaved caspase-3）in ALI ratsIn this study,LPS（5mg/kg）was injected into the trachea to construct a rat model of acute lung injury.TUNEL staining was used to observe the apoptosis of lung tissue in each group,and HE staining was used to observe the pathological changes of lung tissue in each group,and blood gas analysis results and dry-wet weight ratio of rats were observed to determine the status of lung tissue.The results showed that HE staining showed LPS-induced lung tissue structure destruction and intraluminal inflammatory cell infiltration.TUNEL staining showed that inhibition of ASIC1a alleviated the nuclear DNA fragmentation symptoms of acute lung injury.The dry/wet weight ratio also showed that the ASIC1a blocker Pc Tx-1 relieved pulmonary edema.Arterial blood gas analysis showed that Pc Tx-1 could improve arterial oxygen partial pressure（PO₂）,reduce arterial carbon dioxide partial pressure（Pa CO₂）and p H,and alleviate pulmonary dysfunction.The expression of Bcl-2,Bax,Cleaved caspase-3 and ASIC1a in lung tissues were detected by immunohistochemistry,immunofluorescence and WB.The results showed that the expression of Bax,Cleaved caspase-3,and ASIC1a was increased in ALI,decreased in Pc Tx-1,decreased in Bcl-2,and increased in Pc Tx-1.The m RNA expression levels of Bcl-2,Bax and ASIC1a in lung tissues were detected by RT-PCR.The results were consistent with those for proteins.2.Expression changes of ASIC1a and apoptosis-related genes（Bcl-2,Bax,Cleaved caspase-3）in LPS-induced acute lung injury cell modelsIn vitro cell models of acute lung injury were induced by LPS stimulation at 10μg/m L for 24h in the culture dish of alveolar epithelial cells RLE-6TN.In the ASIC1a inhibition group,Pc Tx-1（100 ng/m L）was added 24h before LPS was added to block ASIC1a.The expression of Bcl-2,Bax,Cleaved caspase-3 and ASIC1a were detected by immunofluorescence,western blot and q RT-PCR.The results showed that the expression of Bax,Cleaved caspase-3,and ASIC1a increased in ALI group,decreased in Pc Tx-1group,and the expression trend of Bcl-2 was opposite.3.High-throughput sequencing was conducted in ALI rat lung tissues,and the differential expression of circ RNA was verifiedHigh-throughput sequencing was performed on rat lung tissues,and PCR verification was performed on the top 10 circ RNA with different expression levels.It was found that the verification results of circ RNA18-658 were the same as the sequencing results,with significant expression differences.circ RNA18-658 obtained from high-throughput sequencing results database analysis was most likely to target mi RNA-127-5p for verification.The results showed that the verification results of mi RNA-127-5p were the same as the sequencing results.The most likely target gene TRIM72 targeted by mi RNA-127-5p was obtained from the database,and the expression level of TRIM72 in each group was detected by WB and q RT-PCR.The results showed that the expression trend of verification results was consistent with sequencing results.4.Effect of circ RNA18-658/mi R-127-5p/TRIM72 axis on LPS-induced apoptosis of RLE-6TN cellsWe constructed circ RNA18-658 silencing plasmid to transfect RLE-6TN cells.The expression level of circ RNA18-658 in the circ RNA18-658 silencing group（sh-circ RNA group）was significantly lower than that in the blank plasmid control group（NC group）.Compared with the NC group,the expression of Bax,TRIM72,Cleaved caspase-3 and ASIC1a decreased,and the expression of apoptosis-related protein Bcl-2 increased.Mi R-127-5p small interference si RNA was constructed to transfect RLE-6TN cells.The expression level of mi R-127-5p in the mi R-127-5p silencing group（si-mi RNA group）was significantly lower than that in the blank inhibitor control group（NC group）.Compared with the NC group,protein expressions of Bax,Cleaved caspase-3,TRIM72,and ASIC1a were increased in the si-mi RNA group,and expression of apoptosis-related protein Bcl-2 was decreased.RLE-6TN cells were transfected with TRIM72 silencing plasmid.q RT-PCR showed that the expression level of TRIM72 in the silencing group was significantly lower than that in the blank plasmid control group（NC group）.Compared with the NC group,the expression of Bax,ASIC1a,and Cleaved caspase-3decreased,and the expression of apoptosis-related protein Bcl-2 increased.These results suggest that ASIC1a may promote acute lung injury through the circ RNA18-658/mi R-127-5p/TRIM72 axis.