The Role Of CircRNA-Phf21a＿0002 Mediated Hepatocyte Pyroptosis In Hepatic Ischemia-reperfusion Injury

Objective: In liver surgery and liver transplantation,hepatic ischemia-reperfusion injury(IRI)is unavoidable,which can cause postoperative liver dysfunction and graft dysfunction.There are many mechanisms that affect IRI in liver,including oxidative stress,innate immune response,inflammatory response,apoptosis,autophagy and pyroptosis.Among them,pyroptosis is an inflammatory programmed cell death,which is closely related to inflammatory reaction.At present,it is believed that the functions of circ RNA include regulating transcription,binding proteins,and mi RNA sponge,which are closely related to the occurrence and development of various diseases such as tumors,cardiovascular,digestive system,and other diseases.However,there are few reports on the functional mechanism of circ RNA in liver IRI,and its role and function need further research and exploration.This study aims to explore the changes of circ RNA(circ RNA-Phf21a＿0002)expression and liver pyroptosis during mouse liver IRI injury,and explore the changes of circ RNA-Phf21a＿0002 on IRI treatment induced pyroptosis of hepatocytes provides a new idea for studying the mechanism of circ RNA in hepatic IRI.Methods: A mouse hepatic IRI model was established(ischemia 1h,reperfusion 2h,6h,12 h,24h).The changes of ALT and AST contents in the serum of mice were detected.HE staining and TUNEL method were used to observe the liver histopathological changes and apoptosis damage.Western blot was used to detect pyrolytic proteins(Bax,Bcl-2,Caspase-1,Cleaved-Caspase-1,IL-18 and IL-1β)in liver tissue at different reperfusion time points).The expression level of IL-1 and IL-18 in serum of each group was detected by Elisa method;IHC detected the expression of Caspase-1 and IL-1β in liver tissue.Then construct a model for circ RNA sequencing;The difference analysis and functional analysis of sequencing results were carried out,and the host genes were enriched;The mi RNA predicted by sponge adsorption was screened from the three data of mi Rnada,Target Scan and RNAHybrid;Then use the mutli Mi R and GEO database to screen the downstream m RNA and construct the competitive endogenous RNA(ce RNA)regulatory network;Analyze potential biological functions through GSEA,KEGG and GO.Through data analysis,the target circ RNA(circ RNA-Phf21a＿0002)was found.The expression of circ RNAPhf21a＿0002 in liver tissues of each group was detected by q RT-PCR;The cyclization and stability of circ RNA-Phf21a＿0002 were detected by RNase R and actinomycin D experiments,and the cellular location of circ RNA-Phf21a＿0002 was detected by RNAFish experiment to verify that it was indeed circular RNA.The model of hypoxia/reoxygenation(H/R)of AML12 cells at different reoxygenation time points(2h,6h,12 h,24h)was constructed,and the expression of circ RNA-Phf21a＿0002 was detected by q RT-PCR;The AML12 cell line was transiently transfected with circ RNAPhf21a＿0002 overexpression(OE)plasmid,and then the grouping H/R model was constructed;Western blot was used to detect the effect of the expression on pyroptosis;Hoechst/PI staining was used to detect the apoptosis and pyroptosis after OE of circ RNA-Phf21a;Through bioinformatics analysis,let-7b-5p adsorbed by circ RNAPhf21a＿0002 sponge was found and let-7b-5p mimics was constructed.After cotransfection of circ RNA-Phf21 a OE and let-7b-5p mimics into the AMl12 H/R model,will it affect the pyroptosis.Results: The contents of serum ALT and AST in the IRI group were significantly higher than those in the Sham group at different reperfusion time points(P < 0.05).After HE staining of liver tissue,the pathological changes such as edema,balloon-like degeneration,central venous congestion and focal necrosis of liver cells could be observed by microscope.TUNEL test showed that the apoptosis and pyroptosis of cells at different reperfusion time points were significantly higher than that in Sham group(P < 0.05).The pyroptosis-related protein Cleaved-Caspase-1,IL-18,and mature-IL-1β detected by western blot were also significantly changed compared with Sham group(P < 0.05).The difference analysis of circ RNA sequencing data obtained 375 different circ RNAs(P < 0.05),of which 40 were significantly different(log FC > 1.5),including39 down-regulated and 1 up-regulated.KEGG analysis of parent gene showed that it was highly correlated with Foxo signal pathway and AMPK pathway.KEGG results of the target genes of circ RNA-Phf21a＿0002 found after the ce RNA network was constructed showed to be associated with the m TOR signaling pathway,which we believe may be involved in pyroptosis in hepatic IRI.And the log FC absolute value of circ RNA-Phf21a＿0002 was the largest(-4.76)in data analysis,and the q RT-PCR results in both hepatic IRI model and AML12 cell H / R model showed that circ RNAPhf21a＿0002 showed a downregulation trend compared with the sham group(P < 0.05).RNase R and actinomycin D experiments indicated better stability of circ RNAPhf21a＿0002 compared with linear Phf21 a,which conformed to the characteristics of circ RNA.And the RNA-Fish experimental results localized its location,mainly in the nucleus.In H/R model,OE group compared with other groups,the results of Western blot showed that pyroptosis-related proteins Gsdmd,IL-18,Cleaved-Caspase-1,mature-IL-1β and so on showed a trend of up regulation expression(P < 0.05),Caspase-1 and pro-IL-1β had no obvious change.Hoechst/PI results indicated that OE of circ RNA-Phf21a＿0002 significantly increased the ratio of apoptosis and pyroptosis in H/R AML12 cells(P < 0.05);After co-transfection of let-7b-5p mimics,the aggravation of pyroptosis caused by upregulated circ RNA-Phf21a＿0002 was alleviated,and the expression of Bach1 was downregulated in the H/R model.Conclusions: Our study clarifies that circ RNA-Phf21a＿0002 aggravates the pyroptosis of hepatocytes related to IR by sponging let-7b-5p.These findings provide new molecular mechanisms and novel biomarkers for follow-up treatment.