Mechanism Research Of Vitamin A Deficiency Mediated CeRNA Dysregulation In The Somitogenesis Of Congenital Scoliosis

Part I:Identification of Competing Endogenous RNA Regulatory Networks in Vitamin A Deficiency-Induced Congenital Scoliosis by Transcriptome Sequencing AnalysisBackground:Congenital scoliosis（CS）is a result of anomalous development of the vertebrae including failure of formation or segmentation and frequently associate with somitogenesis malformation.The pathogenesis of CS has not yet been fully identified,but vitamin A deficiency（VAD）during early pregnancy could induce CS.Objective:This part of the study is based on the rat model of vitamin A deficiency in early pregnancy induced congenital scoliosis.The rat embryos of the somite development stage will be used for high-throughput sequencing of the whole transcriptome and detected the pathogenetic related ncRNAs and mRNAs.The ceRNA regulatory network was constructed by multiple bioinformatics analysis,and the specific molecular functions and mechanisms involved in the specific ceRNA network were studied to elucidate the pathogenesis of VAD-CS.Methods:Rat embryonic tissues on gestation day（GD）9 were taken,including 9 cases of vitamin A deficiency group（VAD）and 4 cases of control group（CON）for total RNA extraction by real-time quantitative PCR（qRT-PCR）.Identification of key enzymes of vitamin A metabolism and nuclear receptor expression,and perform high-throughput sequencing of whole transcriptome.After sequencing and quality control,the sequencing results of the selected mRNA（messenger RNA）,long non-coding RNA（lncRNA）,circular RNA（circRNA）and microRNA（miRNA）were compared and verified.The ceRNA regulatory network was constructed by verified the interaction between differentially expressed mRNAs and ncRNAs and analyzing the molecular function through GO and KEGG.Results:qRT-PCR results showed that the expression levels of retinal dehydrogenase（RALDH）and retinoic acid receptor（RAR）in VAD group were significantly lower than CON group.A total of 749 mRNAs,56 miRNAs,685 lncRNAs,and 70 circRNAs were identified to have significantly different expression levels between the two groups.The up-and down-regulated mRNAs（Cyp4f18,Foxo4）,miRNA（rno-miR-466c-3p,rno-miR-187-5p）s IncRNA（NOINRATG024332.1,NONRATG027649.1）and circRNA circRNA（chr15₂3792823₂3793342₊,chr5₅0556456₅1183813_-）were selected to test the sequencing results.The sequencing results of 8 genes were found to be consistent with the expression results of qRT-PCR,indicating that the sequencing data with good quality and the reliability.Wnt,PI3K-ATK,FoxO,EGFR,and mTOR were found to be the most significant pathways involved in VAD-CS pathogenesis.The circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA networks of CS were built and visualized.Conclusion:We comprehensively identified ceRNA regulatory networks of embryonic somite development in VAD-CS as well as revealed the contribution of different ncRNA expression profiles.Our data demonstrate the association between mRNAs and ncRNAs in the pathogenic mechanism of CS.Part Ⅱ:Molecular mechanism of IncRNA SULT1C2A-miR-466c-5p-Foxo4 axis in rat embryonic somitogenesisBackground:Although noncoding RNAs（ncRNAs）have been recently determined to be involved in the pathogenesis of CS,the specific competing endogenous RNA（ceRNA）regulatory network and molecular mechanism in CS remain largely unknown.Objective:The purpose of this part study was to explore the potential molecular mechanism of IncRNA SULT1C2A,mir-466c-5p,Foxo4 and signaling pathway involved in abnormal somitogenesis in the rat model of vitamin A deficiency（VAD）-induced CS.Methods:The ceRNA mechanism was validated by bioinformatics analysis,a dual luciferase reporter gene and siRNA assay.The expression of SULT1C2A,rno-miR-466c-5p,and Foxo4 in embryos was detected by quantitative real-time PCR（qRT-PCR）and Northern blotting.Western blot was performed to examine the expression of phophorylated AKT（p-AKT）and PI3K.Results:Bioinformatics analysis and qRT-PCR indicated that SULT1C2A expression was down-regulated in VAD group,accompanied by increased expression of rno-miR-466c-5p but decreased expression of Foxo4 and somitogenesis-related genes such as Paxl,Nkx3-2 and Sox9 on gestational day（GD）9.Luciferase reporter and siRNA assays showed that SULT1C2A funnctioned as a ceRNA to inhibit rno-miR-466c-5p expression by direct binding,and rno-miR-466c-5p inhibited Foxo4 expression by binding to its 3’untranslated region.The spatiotemporal expression of SULT1C2A,rno-miR-466c-5p,and Foxo4 axis were dynamically altered on GDs 3,8,11,15,and 21 as detected by qRT-PCR and Northern blot analyses,with parallel changes in AKT phosphorylation and PI3K expression on GD 11.Conclusion:The IncRNA SULT1C2A-miR-466c-5p-Foxxo4axis participates in the regulation of early-mid stage somitogenesis.SULT1C2A enhanced Foxo4 expression by negatively modulating rno-miR-466c-5p expression via the PI3K-ATK signaling pathway in the VAD-CS rat model.