CircEYA3 Promotes Formaldehyde-induced Cardiomyocyte Pyroptosis By Targeting Smad5

Object:Formaldehyde is an air pollutant that has toxic effects on human and animal growth and development.Inhalation of or exposure to formaldehyde during pregnancy may lead to miscarriage,premature birth,and congenital heart disease(CHD)such as ventricular septal defect(VSD).At present,the mechanism of formaldehyde-induced CHD is still unclear.This study aimed to explore the regulatory effect and mechanism of circular RNA(circRNA)on formaldehyde-induced pyroptosis,so as to provide potential targets and new methods for the diagnosis and prevention of formaldehyde-induced CHD.Methods:(1)Echocardiography was performed on normal fetuses and fetuses diagnosed with CHD using an echocardiograph;(2)Real time quantitative PCR(qRT-PCR)was used to detect the hearts of adult rats and fetal rats,etc.The expression of circEYA3 in organs,rat cardiomyocytes and cardiac fibroblasts,in fetal rat heart tissue induced by formaldehyde,and in CHD patients and normal human plasma;(3)The cyclic structure of circEYA3 was verified by Sanger sequencing and actinomycin D experiments.The localization of circEYA3 in the nucleus and cytoplasm was detected by immunofluorescence in situ hybridization(FISH).Hematoxylin-eosin staining(HE)staining was used to observe the structural changes of fetal rat hearts induced by formaldehyde.Immunohistochemistry(IHC)was used to detect the expression of pyroptosis-related proteins(NLRP3,GSDMD,ASC,cleaved-caspase-1 and cleaved-IL-1β);(5)The H9C2 cells was treated with different concentrations of formaldehyde at different times,the expression of circEYA3 and NLRP3 was detected by qRT-PCR;(6)CircEYA3 small interfering RNA(siRNA)was designed and synthesized,and its knockdown efficiency in H9C2 cells was verified by qRT-PCR.SiRNA was transfected in H9C2 cells to knock down circEYA3,and after formaldehyde stimulation,the number of pyroptosis was detected by Hoechst/PI staining assay,lactate dehydrogenase(LDH)assay,Caspase-1 activity assay,and WB to detect the expression of pyroptosis-related proteins;(7)circEYA3 mimic was designed,and the expression efficiency was verified by qRT-PCR.In addition,the circEYA3 mimic was transfected in H9C2 cells,and the number of pyroptosis was detected by Hoechst/PI staining assay,LDH assay and Caspase-1 activity assay after formaldehyde stimulation,and the expression of pyroptosis-related proteins was detected by WB;(8)The direct targeting relationship between circEYA3 and Smad5 was verified by RNA pull-down assay and RNA binding protein immunoprecipitation assay(RIP).The co-localization of circEYA3 and Smad5 in H9C2 cells was detected by FISH experiment;(9)H9C2 cells were treated with different concentrations of formaldehyde at different times,and the expression of Smad5 was detected by WB experiment.After overexpression and knockdown of circEYA3 in H9C2 cells,respectively,the expression level of Smad5 was detected by WB assay.At the same time,siRNA was used to knock down circEYA3 and Smad5 in H9C2 cells,and the number of pyroptosis was detected by Hoechst/PI staining assay and LDH assay.Results:(1)We found that circEYA3 was highly expressed in neonatal rat heart,and circEYA3 was up-regulated in formaldehyde-induced neonatal rat heart tissue and plasma of CHD patients(P<0.05);(2)Sanger sequencing,actinomycin D Experiments to verify the ring structure of circEYA3.FISH experiments showed that circEYA3 was expressed in both the cytoplasm and the nucleus;(3)HE staining showed that compared with the control group,the fetal heart structure was disordered and ventricular hypertrophy was increased under formaldehyde induction.WB and IHC detected the expression of pyroptosis-related proteins in fetal rat heart tissue induced by formaldehyde(P<0.05);(4)Pyroptosis was significantly increased after formaldehyde-stimulated H9C2 cells,and the expressions of circEYA3 and NLRP3 were up-regulated in formaldehyde-stimulated H9C2 cells(P<0.05);(5)Pyroptosis was significantly increased after formaldehyde-stimulated H9C2 cells,and the expressions of circEYA3 and NLRP3 were up-regulated in formaldehydestimulated H9C2 cells(P<0.05);(6)The results of WB experiment,Hoechst/PI staining experiment,LDH experiment and Caspase-1 activity kit experiment showed that knockdown of circEYA3 can reduce the number of formaldehyde-induced pyroptosis(P<0.05);(7)WB experiment,Hoechst/PI staining experiment,LDH experiment and Caspase-1 activity kit experiment showed that overexpression of circEYA3 could increase the number of formaldehyde-induced pyroptosis(P<0.05);(8)RIP experiments screened and verified that circEYA3 can directly bind to Smad5;(9)WB experiments showed that in H9C2 cells,with the increase of formaldehyde concentration and the passage of time,the expression of Smad5 gradually decreased,it was also found that the expression of Smad5 was significantly increased after overexpression of circEYA3,whereas the expression decreased.The results of Hoechst/PI staining experiment and LDH experiment showed that knockdown of circEYA3 could inhibit the increase of pyroptosis after formaldehyde stimulation by knockdown of Smad5.Conclusion:Our results show that circEYA3 can promote the pyroptosis of cardiomyocytes after formaldehyde stimulation by directly binding to Smad5,which may lead to the occurrence of congenital heart disease.This study enriched the mechanism of formaldehyde-induced cardiomyocyte pyroptosis,and provided a potential therapeutic target for clinical diagnosis and treatment of CHD.