Primary Study Of The Osteogenic-related MiRNAs In The Thoracic Ossification Of Ligamentum Flavum

Objective: Thoracic ossification of ligamentum flavum(TOLF)is a genetic disease which characterized by pathological heterotopic ossification of ligamentum flavum.microRNAs(miRNAs)are a class of small(~22 nucleotides long),single-stranded endogenous non-coding RNAs that participate in regulating numerous biological processes including cell differentiation,proliferation,and apoptosis.miRNAs degrade their targert mRNA or repress mRNA translation by binding the complementary regions.Many studies have shown that miRNAs play critical roles in the bone development,osteoblast differentiation and osteoporosis pathophysiology.However,less is konown about the roles of miRNAs in the pathological process of TOLF.In this study,we use high throughput sequencing to identify the differentially expressed miRNAs in TOLF cells compared to normal ligament flavum fibroblast.To explore the target genes of differentially expressed miRNAs and explore their role in the development of TOLF by GO and KEGG analysis.The results revealed the mechanisms of miRNAs in TOLF,and provided theoretical guidance and experimental evidence for future TOLF research.Methods: There are four TOLF specimens and four normal ligamentum flavum specimens collected during surgery.All the specimens were put to primary cell culture inmmediately.Then we identified the differentially expressed miRNAs with high throughput sequencing from TOLF cells and LF cells.There are seven differentially expressed miRNAs were found through sequencing and confirmed by qRT-PCR.Furthermore,the bioinformatics analysis of TOLF differentially expressed miRNAs were performed using gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Results: There are seven differentially expressed miRNAs were identified in TOLF,including 4 upregulated and 3 downregulated.Through the bioinformatics analysis,172 target genes were picked out and these genes are associated cell cycle,cell differentiation and gene expression.Furthennore,the miRNAs and target genes predominating in the miRNAs-gene regulatory networks were established successfully.Conclusions: 1.We identified seven differentially expressed miRNAs in TOLF cells.2.This study investigated 5 miRNAs target genes,which involve in cell cycle,cell differentiation and gene expression.3.Multiple osteoblastic signaling pathways(Notch signaling pathway,Wnt signaling pathway,TGF-? signaling pathway,PI3K/Akt signaling pathway,etc.)may be involved in TOLF osteogenesis.