一个新克隆MiR-X对成骨分化影响的研究

bstract Objective: To investigate the role of miR-X in osteoblast differentiations of mouse stromal cells ST2 Method: The primers designed according to pre-miRNA-X sequence were annealed and linked to the linearized pSilencer 4.1 CMV puro vector to construct miR-X expression vector termed "pre-miR-X". In order to make gain or loss of function analysis, pre-miR-X vectors or 2′-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-X) were transfected into BMP-2 induced ST2 cells (300ng/ml) respectively. 48 hours after transfection, alkaline phosphatase (ALP) activity was evaluated by spectrophotometric measurement of P-nitrophenol release and osteocalcin (OC) secretion was detected by radioimmunoassay. Runt related transcription factor 2 (Runx2) proteins and mRNA expression were determined by western blot and real-time quantitative PCR respectively. Results: (1) miR-X overexpression increased levels of ALP activity and OC secretion and also promoted Runx2 protein and mRNA expression in osteoblast differentiation of BMP-2 induced ST2 cells. (2)The inhibition of miR-X attenuated the increase of ALP activity and OC secretion and also partly reduced the increase of Runx2 protein and mRNA levels. Conclusion: miR-X increased Runx2 expression further promoted osteoblast differentiation.