Differential Expression Profile And Gene Function Analysis Of Maternal Fetal-derived CircRNA For Down's Syndrome

Down syndrome,also known as Trisomy 21 Syndrome,is caused by a copy of chromosome 21,is the most common birth defect disease caused by abnormal number of chromosomes.DS patients generally have more common clinical manifestations,including mild-to-moderate learning disabilities,craniofacial abnormalities and physical development retardation,and often accompanied by low immunity,congenital heart disease,leukemia and earlier Alzheimer's diseases and so on.DS brings a heavy financial burden to families and society.At present,the only medical means to avoid the birth of children with DS is the comprehensive prenatal screening and prenatal diagnosis of pregnant woman.However,traditional prenatal screening has the disadvantages of low detection rate,high leakage rate and high rate of false positives,fetal chromosome prenatal diagnosis related methods are due to invasive sampling means prone to bleeding,abortion and suffer from other complications,and the existing noninvasive prenatal diagnosis method has the steps cumbersome,high cost and other issues.Therefore,based on the defects of traditional prenatal screening and prenatal diagnosis,as well as the lack of noninvasive prenatal diagnosis methods,it is very important to explore the new noninvasive Down syndrome fetal prenatal diagnostic markers.Aims:Taking circRNA as the entry point,this research intended to use the gene chip technology to screen out the differentially expressed circRNA from the cord blood of pregnant women with Down's syndrome fetuses and the ones with normal fetuses,and then the selected fetal differentially expressed circRNA were identified by RT-PCR technology in peripheral blood to find potential markers.At the same time,the mRNA was screened out by the above same method.Moreover,the correlation between circRNA and mRNA was analyzed.Finally,the function analysis on differential genes by bioinformatics analysis were made,and to provide a basis for establishing a new noninvasive DS fetal prenatal screening diagnosis.Methods:12 umbilical cord blood samples were collected from pregnant females?including 6 cases by G-banding karyotype analysis diagnosed with Down syndrome fetus,and 6 cases of healthy fetal umbilical cord blood?;total RNA was extracted using Trizol method;using NanoDrop ND-1000 to detect whether RNA degradation and the concentration of RNA;the expression profile of the circRNA and mRNA in the sample was analyzed by Agilent microarray technology;RT-PCR was used to verify the expression of differentially expressed circRNA and mRNA in peripheral blood from children with Down syndrome and healthy children;and the miRNA binding sites on the circRNA was predicted by Arraystar's home-made miRNA target prediction software;finally,we analyzed the expression profiles of differentially expressed circRNA and mRNA and conducted a correlation analysis between circRNAs and mRNAs to explore the function of circRNA in DS and its mechanism.Results:?1?A total of 4568 differentially expressed circRNAs were detected in the two groups of umbilical cord blood samples,among which 735 were differentially expressed,including 414 up-regulated circRNAs and 321 down-regulated circRNAs.The results of RT-PCR in peripheral blood showed that the expression difference of hsa_circRNA₁03135,hsa_circRNA₁03137,hsa_circRNA₁01116 do not have statistically significant?P>0.05?,however there have statistical significance in the expression difference of hsa_circRNA₁03127,hsa_circRNA₁03112,hsa_circRNA₁04907?P<0.05?.?2?A total of 17567 differentially expressed mRNAs were detected,of which 6619 were significantly differentially expressed,including 3411 up-regulated mRNAs and 3208 down-regulated mRNAs.The results of RT-PCR in peripheral blood showed that the expression difference of DYRK1A and STRBP do not have statistically significant?P>0.05?,however there have statistical significance in the expression difference of USP25?P<0.05?.?3?Through the GO analysis of differentially expressed genes,it was found that there were 805 genes involved in the biological process,of which 315 genes were up-regulated and 490 genes were down-regulated,the five most enriched biological processes are:cellular processes,primary metabolic processes,cellular metabolic processes,biological regulation,and nitrogen compound metabolic processes.There ware 189 genes involved in cell composition,of which 66 genes were up-regulated and 123 genes were down-regulated,the five most enriched cell compositions were:intracellular,intracellular part,organelle,intracellular organelle and membrane-bound organelle.There were 155 genes involved in molecular function,of which 77 genes were up-regulated and78 genes were down-regulated,the five most enriched molecular functions are:binding,protein binding,RNA binding,ligase activity and catalytic activity.Through the pathway analysis of differentially expressed genes,it was found that the differential gene was significantly enriched 73 pathways,of which,23 genes were up-regulated and 50 genes were down-regulated.Differentially expressed genes were mainly involved in adhesive spots,ECM-receptor interaction,gap junction,protein digestion and absorption,vitamin digestion and absorption,TNF signaling pathway and so on,also involved in primary immunodeficiency,systemic lupus erythematosus,rheumatoid arthritis,type I diabetes mellitus,arrhythmogenic right ventricular cardiomyopathy?ARVC?,homologous recombination and other processes.?4?By associating the 2-fold difference of the circRNA with the corresponding differential gene expression profile information,we found that 254 differentially expressed circRNAs could regulate the expression of target mRNA by adsorbing specific miRNAs.Conclusion:?1?circRNAs are differentially expressed in DS samples of this experiment.?2?Differential expression of the circRNA has its corresponding miRNA binding site,may act as miRNA sponges to bind miRNAs so as to regulate the expression of the target gene.?3?The differentially expressed genes are mainly involved in cell division and development process,also involved in primary immunodeficiency,rheumatoid arthritis,type I diabetes mellitus,as well as homologous recombination processes.The gene expression disorder may caused by abnormal copies of chromosome 21,may be associated with DS fetal immune system developmental defects,and the emergence of low immunity,mental development abnormalities,physical retardation and other clinical symptoms.?4?Hsa_circRNA₁03112 not only showed significant difference in the cord blood of DS fetus,but also showed significant difference in the peripheral blood,and its corresponding gene USP25 is likely to be involved in the pathogenesis of DS.The results suggest that overexpression of Hsa_circRNA₁03112 may lead to the expression imbalance of diploid gene through the interaction between circRNA-miRNA-mRNA,and thus participated in the pathogenesis of DS.Hsa_circRNA₁03112 in peripheral blood of pregnant woman may be one of the potentially important markers of fetal DS noninvasive prenatal screening.