Primary Study Of Ossification Related MiRNA In The Ossification Ligamentum Flavum In DISH

Objective; miRNA (microRNA) are very short length of non-coding RNA, by reducing the stability of target genes or inhibit its translation level to influence cell differentiation, proliferation, and apoptosis.MiRNAs have been shown to offer great potential in the ossification process. The global miRNA expression profile of DISH with OLF has not been reported.We seek to identify the differentially expressed miRNAs in the DISH (Diffuse idiopathic skeletal hyperostosis) ossification ligament flavum chondrocytes(OLFD) and ligament flavum fibroblast(LF).To explore the target genes of differentially expressed miRNAs and their mutual cross talks with corresponding miRNAs in DISH and explore its role in the development of DISH.The result is proved the mechanisms regulating miRNAs of DISH, and the prognosis for future research to provide theoretical guidance and experimental evidence.Methods:4 cases ossification of ligamentum flavum specimens and 4 normal li gamentum flavum specimens as normal control were collected, cell cultures were performed and were identified by immunofluorescence.We performed a microRNA microarray analysis to compare the microRNA expression levels between OLFD and their normal counterpart—LF.The differential expression of six microRNAs was confirmed by quantitative RT-PCR. Bioinformatics analysis was also performed. According to the bioinformatic techniques, target genes of deregulated miRNAs in DISH in comparison to normal counterpart were picked out based on the miRNA bank. In addition, these target genes were narrowed according to the Unigene data base which specific to ossification. Then the target genes of deregulation miRNAs were subjected to gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis. The miRNAs and target genes predominating in the miRNAs gene regulatory net works were further investigated.Transcription factor binding sites were predicted base on the procedures patser.Results:A total of 15 dysregulated miRNAs were identified in OLFD, including 12 upregulated miRNAs and 3 downregulated miRNAs, many of these miRNAs have been reported to be dysregulated in other experiment and have a potential role in ossification. Through the bioinformatic bank, several target genes were found specific to seperatly differentially expressed miRNAs. According to the gene ontology and KEGG analysis, categorys of high enrichment GOs and specific pathways targeted by deregulation miRNAs were found involving ossification.Furthermore, miRNAs-genes net work of overlapping target genes selected in GOs and pathways analysis and their differentially miRNAs were performed successfully.Preliminary analyses showed these targets can be directly involved in DISH with ossified ligamentum flavum. Functional analysis suggested that the 67 miRNA targets constitute molecular pathways previously associated with ossification. These pathways appeared to be regulated both transcriptionally as well as by miRNAs at posttranscriptional level. These data are a rich and novel resource for DISH and miRNA research and suggest that the 4 miRNAs and their cognate mRNA target sequences constitute pathways that promote DISH.Conclusions:1. Ossification of ligamentum flavum cells were osteoblastic cells, normal ligamentum flavum cell was fibroblasts.2. We identified microRNAs that are dysregulated in OLFD and bioinformatics analysis suggests that these microRNAs may be involved in DISH with ossification of ligamentum flavum and have the potential to be biomarkers.3. Real-time quantitative PCR, verified 6 differentially expressed miRNAs, the chip proved accurate and reliable.4. This study found five miRNAs target gene, they target a very wide, involving cell cycle regulation, cell differentiation, transcription regulation, etc, some target genes play a role in promoting ossification of the role of direct or indirect effect, which 5 miRNAs genes in the pathogenesis of DISH may play in promoting or inhibiting the potential role of bone formation.5. Multiple pathways (MAPK pathway, Wnt pathway, TGF-βpathway, Focal adhesion) may be involved in DISH osteogenesis.