Transcriptome Profiling Of Peripheral Plasma In Congenital Talipes Equinovarus Patients Based On RNA Sequencing

Objective:To sequence the whole transcriptomics of congenital talipes equinovarus(CTEV)through RNA sequencing(RNA-Seq),the differential expressions of mRNA,lncRNA,circRNA and miRNA between normal children and those with CTEV were compared.In order to identify the differentially expressed genes(DEGs)and differentially expressed transcripts(DETs),the possible molecular mechanisms in the development of CTEV were revealed,which provided the molecular biological bases for follow-up studies Methods:1.RNA-SeqThe total RNA was extracted from plasma samples of 3 normal children and another 3 with CTEV for construction of RNA-Seq libraries.The sequencing libraries of different samples into the sequencing processes were mixed.Transcriptomes of 3 blood samples were analyzed through RNA sequencing,and DEGs and DETs of normal children and those with CTEV were screened out.Gene ontology(GO)function significance enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG)pathway significance enrichment analysis were carried out for genes with differential expressions2.qRT-PCRIn order to verify the sequencing results,qRT-PCR was used to detect expressions of target differential genes of 20 cases in experimental group and 13 cases in control group3 Statistical analysisPrism 8.2.0 statistical software was used in statistical analyses,and t test was used to analyze relative expression of the target geneResults:1.RNA-Seq resultsThere were 181 DGEs in the experimental group and the control group,including 58 up-regulated genes and 123 down-regulated genes.A total of 23 lncRNA,12 up-regulated and 11 down-regulated,were differentially expressed in this study.There were 23 miRNA differentially expressed,including 11 up-regulated and 12 down-regulated miRNA.No differential expression of circRNA was found.The expression of lncRNA growth arrest specific 5(GAS5)(fold change,FC=0.0184979910605766)was significantly different2.Bioinformatical analysis GO analysis showed that 181 DEGs participated in 717 BP,130 CC and 119 MF Significant DEGs were mainly involved in cellular metabolic,hydrogen peroxide metabolic,oxygen transport and other processes.Analysis of KEGG showed that 181 DEGs were mainly enriched in 43 signaling pathways.DEGs were mainly involved in PI3K-Akt and HIF-1 signaling pathway as well as other metabolic pathways.23 differentially expressed miRNAs were involved in 1945 BP,270 CC and 349 MF Significant differentially expressed miRNAs were mainly involved in BP such as cell-cell signaling and regulation of cellular processes.KEGG analysis showed that 23 DEGs were mainly enriched in 83 signal pathways.Differentially expressed miRNAs are mainly involved in Hippo and PI3K-Akt signaling pathway as well as other metabolic pathways 3.qRT-PCRThrough quantitative detection of lncRNA GAS5,expressions of GAS5 in plasma samples of experimental group were lower than those of control group,which was consistent with the sequencing results,and the differences were statistically significant(P=0.0011284781)Conclusion:Through RNA-Seq,we found that there were large numbers of DEGs in plasma of normal children and those with CTEV.In plasma samples of children with CTEV,expressions of lncRNA GAS5 were significantly down regulated,which might be related to pathogeneses of CTEV.