| Object:To explore the regulatory mechanism of fetal congenital heart disease(CHD)induced by formaldehyde exposure during pregnancy.By exploring the functional role and regulatory mechanism of circ Fam120 a in apoptosis and autophagy of cardiomyocytes induced by formaldehyde exposure,to explore whether circ Fam120 a is a key target of formaldehyde induced CHD.This may provide new strategies for the prevention,diagnosis and treatment of formaldehyde-induced CHD.Methods:(1)The fetus with a history of formaldehyde exposure was examined by echocardiography to observe the occurrence of CHD,and the normal fetal heart was used as negative control;Blood samples were collected from patients with CHD and healthy people;(2)A rat model of cardiac dysplasia induced by formaldehyde exposure was established,which was the formaldehyde exposure group;meanwhile,normal saline was injected as the negative control;the healthy group was examined by echocardiography of fetal rat hearts,and then the offspring hearts of rats in the two groups were collected respectively for full transcriptome sequencing and bioinformatics analysis;(3)The 10 circ RNAs with the most significant differential expression between the two groups were screened out from the sequencing results.Rat myocardial cell line(H9C2)was used to construct the cell model of formaldehyde exposure concentration gradient and time gradient.Real-time quantitative PCR(q RT-PCR)was used to detect the expression levels of these 10 circ RNAs in the cell model.Circ Fam120 a with the most significant trend was selected;(4)q RT-PCR was used to detect the expression of circ Fam120 a in fetal heart tissue of formaldehyde exposure group,fetal heart tissue of normal group,myocardial cells,myocardial fibroblasts,CHD patients and normal human plasma;(5)The expression level of circ Fam120 a in fetal rat heart tissue samples and its localization in H9C2 cells were determined by fluorescence in situ hybridization(FISH);(6)Hematoxylin-eosin staining(HE)was used to observe the morphologic and structural changes of fetal heart exposed to formaldehyde;(7)According to previous studies on the apoptosis of H9C2 cells induced by formaldehyde,and the known correlation between apoptosis and autophagy,Western Blot(WB)was used to detect the expressions of autophagy related genes LC3Ⅰ/Ⅱ and p62 in H9C2 cells induced by formaldehyde;(8)Autophagy of H9C2 cells induced by formaldehyde was detected by autophagy LC3 double labeled adenovirus.Detection of reactive oxygen species(ROS)in H9C2 cells stimulated by formaldehyde;(9)Small interfering RNA of circ Fam120 a was constructed and its knockdown efficiency in H9C2 cells was verified by q RT-PCR.H9C2 cells were transfected with small interfering RNA to knockdown circ Fam120 a,and WB was used to detect the expression changes of apoptosis and autophagy related proteins caspase3,cleaved caspase3,LC3Ⅰ/Ⅱ,and p62 after methylal stimulation.Td T-mediated d UTP Nick-End Labeling(TUNEL)and flow cytometry were used to measure the apoptosis of cells,autophagy was detected by LC3 double label adenovirus;(10)circ Fam120 a simulant was designed and its overexpression efficiency in H9C2 cells was verified by q RT-PCR.H9C2 cells were transfected with circ Fam120 a mimicry,and WB was used to detect the expression changes of apoptosis and autophagy related proteins caspase3,cleaved caspase3,LC3Ⅰ/Ⅱ,and p62 after being stimulated by methaldehyde.TUNEL assay and flow cytometry were used to detect apoptosis,autophagy was detected by LC3double-labeled adenovirus;(11)The downstream protein of circ Fam120 a,eukaryotic translation initiation factor 4A2(EIF4A2),was identified by biotin probe RNA pulldown and mass spectrometry.The targeted binding of circ Fam120 a with EIF4A2 was verified by RNA pulldown Assay and RNA Binding Protein Immunoprecipitation Assay(RIP).FISH assay was used to detect the colocalization of circ Fam120 a and EIF4A2 in H9C2cells;(12)H9C2 cells were stimulated by formaldehyde concentration gradient and time gradient,and the expression of EIF4A2 in H9C2 cells induced by formaldehyde was detected by WB.After circ Fam120 a was overexpressed or knockdown in H9C2 cells,the expression level of EIF4A2 was detected by WB assay under physiological conditions and formaldehyde stimulation;(13)Small interfering RNA of EIF4A2 was designed,then co-knock circ Fam120 a and EIF4A2 in H9C2 cells,and cotransfection of circ Fam120 a overexpressed plasmid with small interfering EIF4A2 was performed in H9C2 cells.The expression changes of apoptosis and autophagy related proteins caspase3,cleaved caspase3,LC3Ⅰ/Ⅱ,and p62 were detected by WB after methylal stimulation.TUNEL assay and flow cytometry were used to detect apoptosis,autophagy was detected by LC3double-labeled adenovirus;(14)The formaldehyde-induced fetal rat cardiac dysplasia model was constructed,and circ Fam120 a knockdown adeno-associated virus was constructed for treatment.Echocardiography was used to detect the cardiac function of newborn rats.HE was used to detect the cardiac morphology and structure of each group.The expression of circ Fam120 a in each group was detected by q RT-PCR,and the expression of circ Fam120 a in each group was detected by FISH assay.Expression of EIF4A2 and apoptosis,autophagy associated proteins caspase3,cleaved caspase3,LC3Ⅰ/Ⅱ,and p62 were detected by WB.Results:(1)Echocardiography revealed a ventricular septal defect in the CHD fetus;Blood samples from 15 CHD patients and 15 healthy people were collected;(2)Highthroughput sequencing found that circ Fam120 a was significantly up-regulated in the formaldehyde-exposed fetal rat heart compared with the normal group,and q RT-PCR detection showed that circ Fam120 a was up-regulated in formaldehyde-stimulated H9C2 cells,formaldehyde-induced fetal rat heart and plasma of CHD patients(P < 0.05);(3)FISH assay showed that circ Fam120 a was expressed in both nucleus and cytoplasm of H9C2;(4)The results of WB and LC3 double label adenovirus showed that the autophagy level of H9C2 cells was significantly increased by formaldehyde stimulation(P < 0.05).ROS experiment showed that formaldehyde stimulation could increase the level of reactive oxygen species in H9C2 cells;(5)The results of WB assay,TUNEL assay,flow cytometry and autophagy LC3 double labeled adenovirus showed that circ Fam120 a down-regulated could reduce the number of formaldehyde-induced apoptosis and autophagy(P< 0.05);(6)The results of WB assay,TUNEL assay,flow cytometry and autophagy LC3double-labeled adenovirus showed that circ Fam120 a overexpression could further increase the formaldehyde-induced apoptosis and autophagy levels(P < 0.05);(7)The RNA pull-down and RIP experiments found and verified the direct binding of circ Fam120 a to the downstream target protein EIF4A2;(8)WB experiment results showed that the expression level of EIF4A2 in H9C2 cells decreased gradually with the increase of formaldehyde concentration and the extension of time.It was also found that the expression of EIF4A2 increased after circ Fam120 a knockdown and decreased after circ Fam120 a overexpression;(9)The results of WB assay,TUNEL assay,flow cytometry and autophagy LC3 double-labeled adenovirus showed that circ Fam120 a could negatively regulate EIF4A2,thereby mediating apoptosis and autophagy of cardiomyocytes induced by formaldehyde;(10)In the animal model,compared with the healthy group,the formaldehyde-induced fetal heart showed decreased cardiac function and slowed heart rate,and the arrangement of myocardial cells was loose and disordered.Circ Fam120 a,LC3Ⅰ/Ⅱ and cleaved caspase3 expression levels increased,and EIF4A2 and p62 expression levels decreased in myocardial tissue(P < 0.05).After the knockdown treatment of circ Fam120 a,the cardiac function and heart rate were restored,the morphology and structure of myocardial tissue tended to be normal,and the apoptosis and autophagy levels were reduced(P < 0.05).Conclusion:Through the analysis of clinical samples and in vitro and in vivo experiments,we found that circ Fam120 a can promote the apoptosis and autophagy of cardiomyocytes induced by formaldehyde by directly targeting EIF4A2,which may lead to the occurrence of congenital heart disease.This study enriched the functional regulatory mechanism of formaldehyde-induced CHD production and proved that circ Fam120 a may be an important regulatory factor,thus providing a potential therapeutic target for clinical prevention,diagnosis and treatment of CHD. |
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