| Objectives The study aimed to screen and identify differentially expressed circRNA in serum of children with autism spectrum disorder(ASD),explore the biological function and regulatory mechanism of circRNA in SH-SY5 Y cells,and search for potential biomarkers for the diagnosis of ASD.It is of great significance for further study of the pathogenesis of ASD and for diagnosis and early intervention of ASD.Methods 1 Data collection: the case-control study was conducted.A total of 184 ASD children aged 2-5 years who were diagnosed in Tangshan Maternal and Child Health Hospital and trained in Tangshan special education institutions from September 2018 to September 2021 were included in the case group.A total of 192 healthy children aged 2-5years who underwent physical examination in the hospital during the same period were included in the control group.A self-administered questionnaire was used to collect information on the general demographic characteristics and disease history of the subjects,and the Autism Behaviour Checklist(ABC)and the Childhood Autism Rating Scale(CARs)were used to assess the symptoms and severity of disease associated with ASD in children.Serum was collected from the subjects and kept refrigerated at-80°C.2 Circ RNA chip screening and verification: three ASD children with severe autism and typical symptoms and three healthy children were selected by 1:1 matching according to gender,age,ethnicity.Their serum was used for circRNA microarray screening.Bioinformatics analyzed the GO function and KEGG pathway of circRNA and the mi RNA targeted by circRNA were predicted by mi Randa database.PCR was used to verify the differentially expressed circRNAs in serum.3 Study on biological function and regulatory mechanism of hsa＿circ＿0054602: the hsa＿circ＿0054602 overexpression plasmid and si RNA and mi R-1202 inhibitor were constructed and transfected into SH-SY5 Y cells.The expression of hsa＿circ＿0054602,mi R-1202,FLT4,KI67 and TUBB3 in the cells was detected by q RTPCR and FLT4 protein levels was detected by Western Blot.The dual luciferase reporter gene assay was used to verify the binding sites of hsa＿circ＿0054602,mi R-1202 and FLT4.4 Diagnostic value of Circ RNAs: the ROC curves were plotted to analyze the area under the curve(AUC),sensitivity and specificity of selected circRNAs,and the models of logistic regression,random forest and support vector machine were constructed to evaluate the diagnostic value of the combined application of multiple circRNAs.Results 1 Twenty significantly differentially expressed circRNAs were screened by microarray in the serum of children with ASD,of which 3 were up-regulated and 17 were down-regulated.KEGG pathway analysis revealed that the ribosome,synaptic vesicle cycle,Rap1 signaling pathway,PI3K-Akt signaling pathway,circadian rhythm,et al are the major signaling pathways associated with ASD.2 After expanding the sample size,differentially expressed circRNAs were verified in 184 ASD cases and 192 healthy controls.It showed that hsa＿circ＿0054602 was overexpressed and hsa＿circ＿0004566,hsa＿circ＿0085145 and hsa＿circ＿0073642 were underexpressed in the serum of children with ASD.3 Hsa＿circ＿0054602 with up-regulated expression for ASD was selected to investigate its role in regulating the proliferation and differentiation of SH-SY5 Y cells.The results showed that overexpression of hsa＿circ＿0054602 inhibited the proliferation and promoted the differentiation of SH-SY5 Y cells.Hsa＿circ＿0054602 could regulate the expression of FLT4 through the "molecular sponge" of mi R-1202,thus affecting the proliferation and differentiation of SH-SY5 Y cells.4 ROC curve analysis of the four validated circRNAs revealed that hsa＿circ＿0054602 and hsa＿circ＿0004566 exhibited high diagnostic value for ASD with AUCs of 0.789 and 0.735,sensitivity of 65.76% and89.13%,and specificity of 81.25% and 55.20%,respectively.The AUC,sensitivity and specificity of hsa＿circ＿0085145 were 0.683,89.13% and 45.31%,respectively.Hsa＿circ＿0073642 indicated low diagnostic value.5 According to the results of multivariate Logistic regression analysis and ROC curve analysis,age,hsa＿circ＿0054602,hsa＿circ＿0004566 and hsa＿circ＿0085145 were finally determined as the input variables to construct Logistic regression,random forest and support vector machine models which were used to evaluate the diagnostic value of combined circRNAs.The results showed that the AUCs of the logistic regression,random forest and support vector machine models in the training set were 0.801,0.954 and 0.808,with sensitivities of 80.00%,98.47% and81.16%,specificity of 80.00%,92.14% and 80.00%,coincidence rates of 80.00%,95.28%and 80.58%,respectively;the AUCs of the three models in the test set were 0.808,0.902 and 0.840,with sensitivities of 79.54%,92.68% and 82.22%,and specificities of 79.54%,86.36% and 84.09%,coincidence rates of 79.54%,90.18% and 83.21%,respectively.The chi-square test indicated that three fitted models were stable(P>0.05).These rusluts indicated that the random forest model had the highest diagnostic efficiency.It was found that Logistic regression model and support vector machine model had the lowest diagnostic value for the two-year-old group,with AUC of 0.757 and 0.797 respectively,while random forest model had the highest diagnostic value for the two-year-old group,with AUC of0.986 and the sensitivity,specificity and coincidence rate were all 98%.So the random forest model showed higher diagnostic value and early diagnostic effect for ASD.Conclusions 1 There are differentially expressed circRNAs in the serum of children with ASD which may be involved in the development of ASD disease.2 Hsa＿circ＿0054602regulates the expression of FLT4 through the "molecular sponge" of mi R-1202,thereby inhibiting the proliferation and promoting the differentiation of SH-SY5 Y cells.3 The random forest model constructed from the combination of age,hsa＿circ＿0054602,hsa＿circ＿0004566 and hsa＿circ＿0085145 has high diagnostic value and early diagnostic effect for ASD.Figure 37;Table 44;Reference 215... |
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