Prenatal Diagnosis Of Congenital Heart Disease,the Screening Of Related Biomarkers And Research On Their Mechanisms

Objectives: Congenital heart disease(CHD)is the most common congenital malformation,and the morbidity and mortality in perinatal period are relatively high.When fetal CHD is diagnosed in utero,the pregnant women can be benefit from better clinical consultation,examinations,and proper referral timely,which can reduce the perinatal mortality.At present,the prenatal detection rate of CHD is relatively low,only 30%-60% in developed countries,and even lower in developing countries.Therefore,it is of great importance to increase the prenatal detection rate of CHD for better prenatal and postnatal care.In this study,we first carried out a retrospectively study to analyze the data of fetal conotruncal malformation and aortic arch anomaly which are diagnosed in the Prenatal Diagnosis Center of our hospital.We proposed standardized protocols for three-dimensional(3D)and spatial temporal image correlation(STIC)volume data analysis to improve the efficacy of 3D modality.We aimed to explore the application value of 3D ultrasound in prenatal diagnosis of CHD and improve the accuracy of prenatal diagnosis of CHD.At the same time,we realized that ultrasound examination was mainly concentrated in the second trimester,and fetal echocardiography was difficult to popularize as a screening method due to the limitations of equipment and the technical level of examiners.Therefore,we tried to find a relatively simple and efficient method of screening fetal CHD.In order to explore more effective methods for prenatal screening of CHD,we performed transcriptomics analysis to screen noninvasive biomarkers for prenatal screening of CHD,using the maternal plasma as well as the exosomes derived from it in the pregnancies with CHD fetuses,and the heart tissue of these fetuses.The current study also aimed to explore the role and mechanism of the above genes in regulating the occurrence of CHD,and provide a theoretical basis for the optimization of prenatal diagnosis and early treatment intervention of CHD.Methods: 1.In the first part of the retrospective study,fetal 3D-STIC cardiac volumes of conotruncal malformation and aortic arch anomaly were analyzed using our newly proposed standardized protocols.First,the 3D-STIC volumes were displayed in a multiplanar mode.The origin and course of the aorta and pulmonary artery,as well as the spatial relationship of the great arteries were shown when moving the reference point step by step in the four-chamber view.This was used to determine the ventriculoarterial connections and thus make a diagnosis of conotruncal malformation.Then the fetal aortic arch in sagittal view was retrieved for 3D-STIC volumes and then displayed in multiplanar mode when rotating the y-and z-axis step by step.Tomographic ultrasound imaging(TUI)and surface rendering were used to show the fetal aortic arch and its branching patterns.This was used to make a diagnosis of fetal aortic arch anomaly.The value of the 3D modality in the diagnosis of fetal conotruncal malformation and aortic arch anomaly was analyzed when compared with traditional 2D modality.2.In the second part of this study,RNA sequencing analysis of plasma and plasma derived exosomes from pregnant women and follow-up experiments were performed to explore the diagnostic efficacy of circular RNAs(circ RNAs)and micro RNAs(mi RNAs)as biomarkers for early non-invasive prenatal diagnosis of CHD.RNA sequencing analysis of the maternal plasma,followed with bioinformatics analysis was performed to explore the differentially expressed circ RNAs.The q RT-PCR method was used to detect the maternal plasma expression levels of seven circ RNAs(hsa?circ?0001550,hsa?circ?0000992,hsa?circ?0000026,hsa?circ?0000778,hsa?circ?0005982,hsa?circ?0030883 and hsa?circ?0000825),differential expressions of which had been proved both in maternal plasma and fetal heart tissue.The receiver operator characteristic(ROC)curves were used to analyze the diagnostic ability of hsa?circ?0001550,hsa?circ?0000992,hsa?circ?0000778 as potential biomarkers for prenatal diagnosis.To clarify whether they were specifically expressed in pregnancy,the maternal plasma expression levels of the three circ RNAs mentioned above were measured by q RT-PCR before and 24 hours after delivery.Bioinformatics analysis and q RT-PCR were used to verify the differentially expressed mi RNAs in exosomes derived from maternal plasma.The ROC curves were used to analyze the ability of differentially expressed hsa-mi R-1228-5p,hsa-mi R-337-5p and hsa-mi R-1538 as potential biomarkers to screen for CHD.To clarify whether they were specifically expressed in pregnancy,the expression levels of hsa-mi R-1228-5p,hsa-mi R-337-5p and hsa-mi R-1538 in the plasma-derived exosomes of pregnant women with normal term delivery before and 24 hours after delivery were measured by q RT-PCR.The ROC curve was used to analyze the candidate markers of plasma circ RNAs and plasma-derived exosomes mi RNAs to find the optimal combination to improve the diagnostic efficiency.3.In the third part,the transcriptomics sequencing analysis of the heart tissues of CHD fetuses and normal fetuses was performed to verify the expression levels of hsa?circ?0000992,hsa-mi R-378 g,and MEIS1 in the heart tissue of CHD fetuses,their mutual regulation,and their effects of proliferation and migration in H9C2 cells.We also investigated the mechanism of the molecular regulatory network composed of the above genes in the occurrence and development of CHD.Western blot and q RT-PCR analysis were used to detect the endogenous expression levels of hsa?circ?0000992,hsa-mi R-378 g,and MEIS1 in fetal heart tissue.We cultured H9C2 cells and human embryonic kidney cells(HEK-293T).The dual luciferase reporter gene experiment was used to detect the binding effect and binding site of hsa?circ?0000992,hsa-mi R-378 g,and hsa-mi R-378 g with MEIS1.The H9C2 cell line over expressing hsa?circ?0000992 was established by transient transfection.The q RT-PCR and Western blot were used to detect the change levels of hsa?circ?0000992,hsa-mi R-378 g and MEIS1.The CCK-8 and Ed U experiment were used to detect cell proliferation ability,and the transwell experiment was used to detect cell migration ability.FISH experiment was used to detect the subcellular localization of hsa?circ?0000992 and hsa-mi R-378 g in H9C2 cells.The H9C2 cell line over expressing hsa-mi R-378 g was established by transient transfection.The q RT-PCR and Western blot methods were used to detect the change level of MEIS1.The CCK-8 and Ed U experiment were used to detect cell proliferation ability and transwell experiment was used to detect cell migration ability.The cell line with hsa?circ?0000992 and hsa-mi R-378 g overexpression was established by transient transfection.Western blot was used to detect the protein expression level of MEIS1.The CCK-8 and Ed U experiment were used to detect cell proliferation ability and transwell experiment was used to detect cell migration ability.Results: 1.The 3D modality using standardized volume-analysis protocols could improve the detection of fetal CHD.In the diagnosis of fetal transposition of the great arteries,the 3D-2 modality using standardized volume-analysis protocol,and the combined modality by which two-dimensional(2D)ultrasound(2D modality)and3D-2 modality were used in together,both showed significantly higher sensitivity than 2D modality;in diagnosis of fetal tetralogy of Fallot and persistent truncus arteriosus,the sensitivity of the combined method was significantly higher than both of the 2D method and the 3D-2 method,respectively;in the diagnosis of double-outlet right ventricle,there was no significant difference shown among the 2D,3D-2,and the combined modality.The 3D-1 modality(no specific volume analysis protocols were used)showed significantly lower sensitivity than other three modalities in detecting all conotruncal malformations.There was no significant difference in the diagnostic specificity among the four methods.The 3D modality using standardized volume-analysis protocol could clearly show the position,course and branching patterns of the aortic arch,and the spatial relationship of the great vessels.Compared with the 2D modality,the 3D method had significantly higher sensitivity and accuracy in detecting both abnormal brachiocephalic arteries and aortic arch anomalies,while there was no significant difference in determining the aortic arch position.There was no significant difference in the diagnostic specificity of the two methods.2.Maternal plasma circ RNAs and plasma-derived exosomes mi RNAs were expected to become biomarkers for prenatal screening of CHD.The seven circ RNAs(hsa?circ?0001550,hsa?circ?0000992,hsa?circ?0000026,hsa?circ?0000778,hsa?circ?0005982,hsa?circ?0030883 and hsa?circ?0000825)were differentially expressed in maternal plasma and CHD fetal heart tissue.The q RT-PCR was used in large sample verification,and the results showed that the hsa?circ?0001550,hsa?circ?0000992,hsa?circ?0000778 were significantly highly expressed in the plasma of pregnant women with CHD fetuses,which showed good diagnostic ability for fetal CHD,and the area under the curve(AUC)were 0.776,0.710 and 0.780respectively(P<0.0001).In the diagnosis using the combined 3 circ RNAs,the AUC was 0.827(95% confidence interval [CI],0.773-0.873;P<0.0001).The expression levels of the 3 circ RNAs mentioned above decreased significantly in 24 hours after delivery,which showed that they were specific in pregnancy.The hsa-mi R-1228-5p,hsa-mi R-1538 and hsa-mi R-337-5p were significantly overexpressed in exosomes in maternal-derived plasma in pregnancies with CHD fetuses.They showed good diagnostic ability for fetal CHD,and the AUC was 0.906,0.856 and 0.866(all P<0.0001),respectively.The AUC was 0.958(95% confidence interval [CI],0.887-0.990;P<0.0001)in the combined diagnosis of the three mi RNAs.The expression levels of the above three mi RNAs decreased significantly in 24 hours after delivery,which showed that they were specific in pregnancy.The hsa-mi R-1228-5p,hsa-mi R-337-5p and hsa-mi R-1538 also had good diagnostic efficacy for CHD in16-19 gestational weeks.Plasma-derived exosomes hsa-mi R-1228-5p,hsa-mi R-337-5p,hsa-mi R-1538 combined with plasma hsa?circ?0001550 could obtain better diagnostic performance,and the AUC was 0.969,(95% [CI],0.904–0.995;P<0.0001).3.In the third part of this study,the potential CHD-related genes were screened to establish the hsa?circ?0000992/hsa-mi R-378g/MEIS1 gene regulatory network.Bioinformatics analysis was used to screen possible pathogens,followed by verifications with enlarged samples.The expression levels of hsa?circ?0000992 and MEIS1 were upregulated while hsa-mi R-378 g was downregulated in the fetal heart tissue.There was a binding site between hsa-mi R-378 g and hsa?circ?0000992,which could act as a "molecular sponge".MEIS1 was a potential target gene of hsa-mi R-378 g,and the interaction between the two genes was achieved through the binding site that exists in the 3'-end untranslated region of MEIS1 m RNA.When hsa?circ?0000992 was overexpressed,the expression level of hsa-mi R-378 g decreased,and the m RNA and protein expression levels of MEIS1 increased,which inhibited the proliferation and migration of H9C2 cells.FISH experiment showed that the hsa?circ?0000992 and hsa-mi R-378 g were co-located in the cytoplasm of H9C2 cells.When hsa-mi R-378 g was overexpressed,the m RNA and protein expression levels of MEIS1 decreased,which promoted the proliferation and migration of H9C2 cells.When hsa?circ?0000992 and hsa-mi R-378 g were both overexpressed,hsa-mi R-378 g could reverse the promoting effect of hsa?circ?0000992 on the expression of MEIS1 and the inhibition of cardiomyocyte proliferation and migration.Conclusions: 1.The 3D modality using standardized volume-analysis protocol could improve the detection of fetal CHD.2.The three circ RNAs differentially expressed in maternal plasma(hsa?circ?0001550,hsa?circ?0000992,hsa?circ?0000778)had the potential to be used as biomarkers for prenatal screening of CHD.3.The three mi RNAs(hsa-mi R-1538,hsa-mi R-1228-5p and hsa-mi R-337-5p)differentially expressed in maternal plasma-derived exosomes had the potential to serve as biomarkers for prenatal screening of CHD.4.The combination of maternal plasma-derived exosomes mi RNAs and plasma circ RNAs biomarkers could improve the ability of prenatal screening for CHD.5.The hsa?circ?0000992 was highly expressed in the heart tissue of CHD fetuses.It could act as a "molecular sponge" for hsa-mi R-378 g,inhibiting the proliferation and migration of cardiomyocytes.6.The hsa-mi R-378 g was low-expressed while MEIS1 was high-expressed in CHD fetal heart tissues.The hsa-mi R-378 g promoted the proliferation and migration of cardiomyocytes by targeting the 3'-UTR of MEIS1.7.The hsa?circ?0000992/hsa-mi R-378g/MEIS1 regulatory axis played an important role in the development of the heart.