The DISH Related MicroRNA Analysis Under3D Adhesion

Objectives:To obtain and discuss the diffuse idiopathic skeletal hyperostosis (DISH) related microRNA (miRNA) under3D adhesion by using naturally occurring matrix (human acellular amniotic membrane, HAAM) for cell culture.Methods:The HAAM was created by the patented technologies of the amniotic cells from non-acid-base system.4ossific ligamenta flava tissues were obtained from DISH patients who were combined of ossification of ligament flava,4normal ligamenta flava tissues were obtained from fracture of cervical/thoracic spine patients. These patients were divided to DISH group and NORMAI group. The ligamenta flava tissue fibroblast were separated by using collagenase technique and then cultured on HAAM. The cells were collected when approximately80%covered. Total RNA was extracted and then quantified by microfluidics analysis. The small RNAs (<300nt) were isolated by using a YM-100Microcon centrifugal filter. μ ParafloTM MicroRNA microarray Assay was performed using a service provider (LC Sciences) to to identify miRNAs whose expression was significantly different between the two groups. Predicted targets for miRNAs were analyzed using PicTar2005, miRanda v5, TargetScan5.1. Functional pathway analysis of miRNAs was performed using KEGG Pathway Analysis.Results:The cells were fibroblast in both DISH group and NORMAI group, and mean cultured14days to80%covered. The cells distributed in the way of cluster, lived in multi-level of HAAM, and cells established linkage between each other. In total we identified6miRNAs that showed differential expression between DISH and normal group,2miRNAs (let-7c, miR-21) were down-regulated and4up-regulated (let-7b, miR-214, miR-4298, miR-1975). The targets were participate in cell differentiation, cell adhesion, proliferation et al, and had function in regulating MAPK signal pathway, Jak-STAT signal pathway, NOTCH signal pathway et al.Concluti ons:Using HAAM for fibroblast obtained from ligment flavum to live can achieve3D adhesion, which can get rid off the effect of3D adhesion on miRNA expression. By doing this research, we obtain DISH related miRNAs and find some breakthrough points for further study. The results are helpful to gene diagnosis and therapy of DISH in the near future. This research either provides scientific data to figure out whether NOM has impact on gene expression.