The Expression Profile Of Myocardial CircRNA In Tetralogy Of Fallot

Objective:The different expression of circRNA in the myocardial tissue of right ventricular outflow tract between TOF fetuses and healthy fetuses with non-abnormal factors,a ceRNA network was constructed to locate the genes and circRNA related to tetralogy of Fallot.Methods:(1)Sample collection and pathological anatomical observationIn this study,13 fetuses with tetralogy of Fallot(TOF,experimental group)and 13 fetuses with non-cardiac malformation’s induced abortion(control group)were selected and transferred to the pathological anatomy room for anatomical observation,and the myocardial tissue was taken for examination.An appropriate amount of myocardium was taken for HE staining section,and the pathological changes of myocardium were observed.(2)Microarray Analysis of circRNA Expression ProfileWe extractedRNA from 26 myocardial samples of both experimental and control groups,then used Nano Drop-1000 and agarose gel electrophoresis to detect the quality of each sample.The fluorescence labeled cRNA of 3 experimental groups and 3 control groups were hybridized with the Arraystar Human circRNA array(8 × 15K).Further to screen the differentially expressed circRNA by using the original data of the chip that standardized and processed with R software package.(3)Constructing circRNA-miRNA-mRNA networkmiRNA prediction software was used to analyze circRNA/miRNA binding sites.To ensure the validity of prediction results,miRNA differentially expressed in data set GSE35490(from GEO database)were screened to obtain miRNA related to TOF.We took the intersection of the above two results,and input into the Target Scan,mi RDB and mi RTar Base databases for target gene prediction.The intersection of the results is processed to obtain target mRNA,removing the unmatched miRNAs and their corresponding circRNA,and finally constructed a ceRNA network.(4)Functional GO terms and Pathway enrichment analysisEnrichr database was used to analyze the GO and Pathway enrichment function of34 key genes in ceRNA network,and CTD database was used to explain the association between 34 key genes and congenital heart disease.(5)Quantitative PCR verification and ceRNA correlation analysisWe expanded the sample size to collect the myocardial tissues of 10 experimental groups and 10 control groups.The relative expression levels of genes and circRNA in cardiac tissue were detected by RT-q PCR,so as to verify our screening results of differential expression.Combined with ceRNA theory,the correlation between the relative expression of hsa＿circ＿0007798 and HIF1 A was analyzed by querying the circ Base database.Results:(1)In this study,the diagnosis of fetal cardiovascular development and myocardial tissue structural characteristics was verified through local anatomy and HE staining of tissue sections.The myocardial tissue of fetuses with similar gestational weeks in case group and the control group were collected for microarray analysis.According to the analysis of the differential expression circRNA,compared with the control group,there were 214 up-regulated circRNA and 62 down-regulated circRNA in experimental group.(2)A total of 768 miRNA elements were obtained via miRNA prediction software.In order to ensure the validity of predicting miRNA loci,we screened the GEO/GSE35490 dataset and obtained 98 diffential expression miRNA related to tetralogy of Fallot.Taking the intersection of the above two results,25 miRNA are obtained.After miRNA target gene prediction and deletion of unmatched data,19 circRNA,8 miRNA and 34 mRNA were finally obtained to construct ceRNA network.(3)The GO and Pathway analysis of 34 key genes in ceRNA network indicated that the most statistically significant gene function was involved in cardiac development(p=0.002538).In addition,the 34 possible pathogenic genes of TOF,mentioned above all,participated in or promoted the formation of "congenital heart disease",and HIF1 A was the most related gene to CHD.(4)In order to verify the results of the study,we used RT-q PCR to detect the expression level of hsa＿circ＿0007798 in 10 TOF heart tissues and 10 healthy heart tissues.The results showed that the expression of hsa＿circ＿0007798 inexperimental group was significantly up-regulated.The query of circ Base database shows that hsa＿cic＿0007798 is located on chromosome 6 and consists of 6 exons with full 805 bp sequence.According to the ceRNA theory,the correlation analysis,between the relative expression of hsa＿circ＿0007798 and HIF1 A,showed that there was a significant positive correlation between them(pearson coefficient/r= 0.6291).Finally,we considered hsa＿circ＿0007798/ mi R-199b-5p/ HIF1 A as the key ceRNA are related to the TOF progression.Conclusions:(1)In our study,we found that the diagnosis of tetralogy of Fallot can be further verified by local anatomy and tissue section HE staining.(2)A total of 19 circRNAs,8 miRNAs and 34 mRNAs may be involved in the progression of TOF.The main function of 34 key genes is participating in cardiac development.HIF1 A is the most significant marker gene.(3)Compared with healthy controls,the expression of hsa＿circ＿0007798 in tetralogy of Fallot group was significantly up-regulated.There is a significant positive correlation between hsa＿circ＿0007798 and HIF1 A.The study successfully screened the key regulatory pathway,hsa＿circ＿0007798/mi R-199b-5p/HIF1 A,related to tetralogy of Fallot.