The Expression Analysis Of Plasma Exosomes MiRNA In Patients With Non-syndromic Cleft Lip And Palate

Objective: In this study,we extracte the plasma exosome from the non-syndromic cleft lip and palate patients and normal people,and the concentration of exosomes was measured to analyze the relationship between plasma exosome concentration and cleft lip and palate.Then we extracte the miRNA from exosome and find them which abnormally expressed in plasma exosomes of children with non-syndromic cleft lip and palate.Finally,we predict the biological processes and signaling pathways by using the bioinformatics analysis.Methods: We collect 10 ml of peripheral blood of 8 patients and 8 healthy children.After separation of plasma,we using the QIAGEN exoEasy Maxi Kit kit to Extract the exosomes.Observing the morphology of exosomes under transmission electron microscopy,measuring their size,and counting.Then we use the BCA method to Determine the exosome protein concentration and statistical analysis.We use the Trizol method to extract the miRNA and the miRNA library was constructed and analyzed by Agilent 2100 miRNA microarray.The differentially expressed miRNAs were screened,the corresponding target genes were predicted,and GO enrichment analysis and KEGG pathway enrichment analysis were performed.Results:Part Ⅰ: We successfully isolate the exosomes from 16 plasma samples.Under transmission electron microscopy,vesicle-like structures with different sizes of 30-100 nm were observed,which conformed to the electron microscopic manifestations of exosomes.The total number of exosomes was 47,the minimum diameter was 40 nm,the maximum diameter was 99 nm,and the diameter was concentrated in the 60-80 nm range.Statistical analysis of plasma exosome concentration showed that there was no significant difference among the groups.Part Ⅱ: In this study,the concentration of microRNAs met the requirement of Library construction,and the results of quality control showed that they met the requirement of sequencing.The results of sequencing total reads of microRNAs showed that the clean reads of microRNAs in plasma exosomes of patients with non-syndromic cleft lip and palate were higher than those of normal people.The results showed that there was no significant difference in the number of microRNAs in plasma exosomes of patients with non-syndromic cleft lip and palate between the two groups,and there was no significant difference in the number of microRNAs in plasma exosomes of normal people.However,the number of microRNAs in plasma exosomes of patients with non-syndromic cleft lip and palate is slightly higher than the normal people,which indicates that abnormal expression of microRNAs may affect the occurrence of non-syndromic cleft lip and palate.T-test was used to analyze the significant difference between the experimental group(non-syndromic cleft lip and palate group)and the control group(normal children group).After screening 18 differentially expressed microRNAs between the experimental group and the control group,the P values were < 0.05,with statistical significance.There were 14 up-regulated and 4 down-regulated expressions in the experimental group compared with the control group.GO and KEGG pathway enrichment analysis was carried out on predictive targets of differentially expressed microRNAs,and6747 possible target genes were produced.The enriched GO terminology shows that the target gene function is enriched in 30 biological processes,such as multicellular biological development,transcriptional regulation,signal transduction,protein binding and exosome.Abnormally expressed target genes of microRNAs are enriched and analyzed by KEGG database.The target gene signaling pathways are enriched in 304 signaling pathways,such as lysine degradation,Wnt signaling,MAPK signaling,TGF-beta signaling and calcium signaling.Conclusion:1.Exosomes are present in peripheral blood plasma of patients with non-syndromic cleft lip and palate.,and the shape of plasma exosome is the same as the normals;2.Exosomes have no significant changes in peripheral blood of patients with non-syndromic cleft lip and palate than normal people;3.There are 4 kinds of miRNAs with differential expression down-regulation in children with cleft lip and palate,and 14 kinds of miRNAs with up-regulated miRNA.4.Differentially expressed miRNAs predict that target genes are enriched in multiple signaling pathways such as Wnt 、 TGF-β,and related signaling pathways affect the occurrence of non-syndromic cleft lip and palate.