Study On The Function And Mechanism Of PiR-16744 Regulating Cardiomyocyte Pyroptosis

Objective "Interpretation of Report on Cardiovascular Health and Diseases in China2020" pointed out that the morbidity and mortality of cardiovascular disease in China showed an increasing trend year by year,and cardiovascular disease has become one of the leading causes of death in urban and rural residents.At the same time,cardiovascular disease patients tend to be younger.Therefore,cardiovascular disease has received more and more attention.Among many types of cardiovascular diseases,myocardial infarction and ischemic injury have a high mortality rate,and ischemic/reperfusion(I/R)injury after reperfusion therapy can induce multiple programmed cell death,including apoptosis,pyroptosis,and ferroptosis.Cardiomyocyte pyroptosis plays an important role in the I/R injury of cardiomyocytes.PIWI-interacting RNA(piRNA)is a non-coding RNA about 30 nt that was discovered in recent years.It is called piRNA because it can only exert its biological function after combining with members of the PIWI protein family.piRNA has been shown to play an important role in reproductive diseases,but its regulatory mechanism in heart-related diseases remains to be explored.With the in-depth study of piRNA,researchers find role of piRNA is not only limited to germ cells,but also an increasing evidence that piRNA is expressed in the somatic cells of the heart,liver,kidney,nervous system and pancreatic islets,which indicates that piRNA may also play an important role in the physiological and pathological processes of these tissues and organs.In this study,a hypoxia/reoxygenation(H/R)model was constructed in vitro to induce the pyroptosis of primary neonatal mouse cardiomyocytes(NMCs),and found that piR-16744 was significantly downregulated in NMCs undergoing pyroptosis.Therefore,by exploring the effect of piR-16744 on H/R-induced NMCs pyroptosis and its potential mechanism of action,it provides a theoretical basis for alleviating and reversing myocardial I/R injury.Methods In this experiment,the primary neonatal mouse cardiomyocytes were used as the research object,and a model of NMCs pyroptosis induced by H/R was constructed in vitro and the time gradient of hypoxia for 6 h,12 h and 24 h reoxygenation for 6 h was set to screen out the optimal time for inducing pyroptosis of NMCs;High-throughput gene sequencing and quantitative real-time PCR(q RT-PCR)were used to screen the piRNA;Validation of the expression of piR-16744 in I/R-injured heart tissue and pyroptotic NMCs by q RT-PCR technology;piR-16744 was knocked down by antagomir in normal NMCs,and piR-16744 was overexpressed by agomir in H/R treated NMCs;The expression levels of pyroptosis-related proteins,such as Gasdermin D(GSDMD),GSDMD-C and Caspase-1 were explored through western blot,as well as inflammatory factors interleukin-1β(IL-1β)and IL-18;LDH assay and PI staining were used to detect the cell survival level;In NMCs,Caspase-1 was first knocked down by si RNA,and then piR-16744 was knocked down by antagomir to verify the signaling pathway regulated by piR-16744;In addition,the ROS levels in NMCs was detected by reactive oxygen species(ROS)detection kit;At the same time,the potential regulatory pathways of piR-16744 were analyzed using pull down experiments and bioinformatics methods.Results 1.The most obvious pyroptosis of NMCs was treated with hypoxia for 6 h and reoxygenation for 6 h;2.piR-16744 is significantly downregulated in I/R-injured cardiac tissue and in pyroptotic NMCs;3.In NMCs,knockdown piR-16744 can promote the expression of pyroptosis-related proteins,increase cell mortality and ROS expression;4.In H/R treated NMCs,over-expression of piR-16744 could inhibit the expression of pyroptosis-related proteins,reduce cell mortality and ROS expression;5.In NMCs,knockdown of Caspase-1 attenuates the expression of pyroptosis-related proteins caused by knockdown of piR-16744;6.piR-16744 may be involved in the regulation of myocardial I/R injury through pyruvate pathway,glucagon signaling pathway or type 2diabetes pathway.Conclusion In this study,H/R induced NMCs pyroptosis model was successfully constructed in vitro.And found that piR-16744 was significantly down-regulated in I/Rinjured heart tissue and pyroptotic NMCs.Knockdown piR-16744 promoted the occurrence of pyroptosis in NMCs.In H/R treated NMCs,the overexpression of piR-16744 could inhibit the occurrence of pyroptosis of NMCs.Moreover,piR-16744 is involved in the regulation of H/R-induced pyroptosis of NMCs through the Caspase-1-dependent pyroptosis canonical signaling pathway.In addition,it is predicted that piR-16744 may be involved in regulating myocardial I/R injury through pyruvate pathway,glucagon signaling pathway or type 2 diabetes pathway.In conclusion,this study provides theoretical basis for the mitigation and reversal of myocardial injury by exploring the role of piR-16744 in the process of NMCs pyroptisis and its potential molecular mechanism.