| ObjectiveThis study was performed to detect the expression of mi R-363-3p,mi R-194-5p,mi R-222-3p and let-7e-5p in infants with CHD,neonatal CHD,fetal CHD and maternal controls.To explore the feasibility and value of four plasma mi RNAs for the diagnosis of CHD in different periods,aiming to provide an effective reference for the future clinical supplementation and improvement of prenatal and postnatal diagnosis methods for conge-nital heart disease.Methods1.80 cases of CHD patients aged 1 to 3 years old who were treated and treated in pediatric cardiac surgery were used as infant and child case groups,and 89 infants and young children who underwent normal physical examination at the hospital’s physical examination center were selected as the control group.92 children with CHD who were treated and treated in the neonatology department were selected as the neonatal case group.100 newborns who underwent normal physical examination at the hospital’s physical examination center were selected as the control group.In the obstetrics and gynaecology department,the 13 fetuses(18-20 weeks)who died of congenital heart disease and their mothers were collected as fetal cases,and 9 fetuses and their mothers who had terminated pregnancy due to other reasons were selected as the fetal control group.2.Primer design and synthesis of mi R-363-3p,mi R-194-5p,mi R-222-3p,let-7e-5p and internal reference gene U6 were provided by Shanghai Gene Pharma.3.The fasting venous blood samples of the study subjects were collected,plasma was separated,total plasma RNA was extracted,and c DNA was synthesized by reverse transcription.Real-time quantitative PCR was used to detect the CT value of mi R-363-3p,mi R-194-5p,mi R-222-3p,let-7e-5p.4.Statistical analysis was performed using SPSS 24.0 software.Qualitative data were compared using four tablesX~2test.Quantitative data were compared by t-test or Mann-Whitney U rank sum test.ROC curve was used to evaluate the effectiveness of plasma mi RNAs in the diagnosis of CHD.The diagnostic parameters were evaluated by calcu-lating a series of indicators under the curve,such as area,sensitivity,specificity and correct index,and the optimal critical point for the relative expression of mi RNAs in CHD diagnosis was determined.Logistic regression was combined with multiple differentially expressed mi RNAs to establish a multi-index diagnosis model,and a combined diagnostic molecular tag was constructed to evaluate the effectiveness and value of multiple indicators in the diagnosis of CHD.Results1.The expression of mi R-363-3p,mi R-222-3p and let-7e-5p in the plasma of infants with CHD was significantly down-regulated,and the areas under the ROC curve of the three differentially expressed mi RNAs were 0.819,0.743,and 0.745,respectively.Multi-indicator logistic regression analysis showed that the combination of three plasma mi RNAs and molecular markers could improve the diagnostic efficacy of CHD in infants.The diagnostic value was 0.892.When the cutoff value was 0.583,the corresponding sensitivity and specificity were 0.844,0.811,respectively.The two-index diagnostic model was constructed.The combined diagnosis of mi R-363-3p and mi R-222-3p was 0.867,and the cut-off value was 0.585.The corresponding sensitivity and specificity were 0.781 and0.811,respectively.The combined diagnosis of mi R-363-3p and let-7e-5p AUC was 0.865.When the cutoff value was 0.542,the corresponding sensitivity and specificity were the same as those of mi R-363-3p and mi R-222-3p.The combined diagnosis of mi R-222-3p and let-7e-5p was 0.815,and the cut-off value was 0.552.The sensitivity and specificity were 0.719.2.The expression levels of mi R-194-5p,mi R-222-3p and let-7e-5p in the plasma of neonatal CHD patients were down-regulated,and the areas under the ROC curve of the three differentially expressed mi RNAs were 0.805,0.786,and 0.834,respectively.Multi-indicator logistic regression analysis showed that the area under the ROC curve of the three plasma mi RNAs could be as high as 0.900 or more,and the specific AUC value was 0.909.When the optimal critical point value was 0.486,the sensitivity and specificity of the newborn CHD diagnostic test were corresponding at 0.844.From the perspective of health economics,combining any two of mi R-194-5p,mi R-222-3p and let-7e-5p,and evaluating the ability of two mi RNAs to replace three mi RNAs in the diagnosis of neonatal CHD.The diagnostic value of the molecular tag of plasma mi R-194-5p and mi R-222-3p is0.879.When the optimal critical point is 0.420,the sensitivity is 0.875 and the specificity is 0.719.The combination of mi R-194-5p and let-7e-5p has the highest diagnostic value of0.886.When the optimal critical point is 0.497,the corresponding sensitivity and specificity are 0.813 and 0.781,respectively.The diagnostic value of mi R-222-3p and let-7e-5p was 0.858,and the optimal critical point was 0.405.The sensitivity was 0.813and the specificity was 0.719.3.The expression of mi R-194-5p and let-7e-5p was significantly down-regulated in fetal CHD patients and maternal plasma.The two mi RNAs showed a high positive correlation between fetal umbilical cord plasma and maternal plasma expression.The area under the ROC curve of maternal plasma mi R-194-5p and let-7e-5p for fetal CHD diagnosis was 0.761,0.774,respectively.After combining the two mi RNAs,the diagnostic efficiency was significantly improved,and the diagnostic value could be as high as 0.915.When the cutoff value was 0.629,the corresponding sensitivity and specificity were 0.923and 0.889,respectively.Conclusions1.Plasma mi R-363-3p,mi R-222-3p,let-7e-5p can be used for the diagnosis of CHD in infants.The combination of mi R-363-3p and mi R-222-3p or mi R-363-3p and let-7e-5p can provide higher diagnostic value than single mi RNAs,and can also achieve up to three plasma mi RNAs(mi R-363-3p,mi R-222-3p,let-7e-5p)diagnostic efficacy in combination.2.Plasma mi R-194-5p,mi R-222-3p,let-7e-5p can be used as potential markers for neonatal CHD diagnosis.The diagnostic efficacy of the combined diagnosis of mi R-194-5p and let-7e-5p was essentially the same as when the three mi RNAs were combined.3.Maternal plasma mi R-194-5p,let-7e-5p may be a promising biomarker for the diagnosis of fetal CHD,and the combination of the two molecular markers can more effectively distinguish between fetal CHD patients and control populations. |
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