The diffuse idiopathic skeletal hyperostosis related miRNAs analysis

Abstract

Objective To obtain and anlysis the diffuse idiopathic skeletal hyperostosis(DISH)related miRNAs under 3-D adhesion for cell culture. Methods From January 2012 to January 2014, 4 ossific ligamenta flava tissues were obtained from DISH patients and 4 normal ligamenta flava tissues were obtained from trauma patients surgically. Fibroblasts were separated by using collagenase technique and then cultured on human acellular amniotic membrane(HAAM). Each sample was identified by immunofluorescence before harvested. Total RNA was extracted and then quantified by microfluidics analysis. The small RNAs(< 300 nt)were isolated by using a YM-100 Microcon centrifugal filter. μParaflo™ MiRNA microarray assay was performed using a service provider to identify miRNAs whose expression was significantly different between the two groups. Part of differential expression miRNAs were verified by qRT-PCR. Targets of miRNAs were obtained using PicTar 2005, miRanda v5, TargetScan 5.1, their function were analyzed by using Gene Ontology. Functional pathway analysis of miRNAs was performed using KEGG Path-way Analysis. TRANSFAC 7.0 public and Patser were used to get the distribution of transcription factor binding sites. Results When grown on HAAM, fibroblasts kept their morphology, distributed in the way of cluster, lived in multi-level of HAAM, and established linkage. Collagen I and III were tested positive in normal group cells. Collagen I, II, III and Osteocalcin were tested positive in DISH group cells by immunefluorescence. In total 15 miRNAs showed differential expression, 12 were up-regulated and 3 were down-regulated. The result of qRT-PCR was consistent with MiRNA microarray assay. Totally 67 target genes were predicted which participated in cell differentiation, cell adhesion, mineralization et al, and had function in regulating MAPK, Wnt, TGF-β, Focal adhesion signal pathway et al. In total 10 transcription factors were predicted in differentially expressed miRNAs. Conclusion HAAM can provide fibroblasts with 3D adhesion growth, Some differentially expressed miRNAs may participate in the pathogenesis of DISH. Key words: MicroRNAs; Ligamentum flavum; Ossification, heterotopic; Microarray analysis