The Research For MiR-762targeted The IFITM5Gene Inhibits Mineralization In Saos-2Cells

Interferon-induced transmembrane protein5(IFITM5) is one of theinterferon-induced transmembrane protein family members, with the promotion of therole of osteoblast mineralization. Its expression increases with osteoblast differentiation,peaking with matrix production and mineralization. At present, two independent studiesfound that a mutation in the5’UTR of the IFITM5gene is the cause of osteogenesisimperfecta (OI) type V. OI type V is characterized by an autosomal-dominant inheritancepattern, propensity to hyperplastic callus formation, and calcification of the fore-arminterosseous membrane.microRNA (miRNA) is an abundant class of endogenous, small (21-23nucleotide),single strand, and noncoding RNA molecules that can bind to the3’untranslated region(3’UTR) of target mRNA and regulate the stability and translation of mRNA, resulting ineither inhibition of translation or degradation of the target mRNA. miRNA has confirmedthe broad participation of a variety of physiological and pathological processes, such ascell proliferation, differentiation, apoptosis. Studies have shown that miRNA can regulateosteoblast differentiation by targeting the key genes in the regulation of osteoblastdifferentiation. So far biological evidences found there are no miRNA targeted IFITM5,thereby regulating osteoblast mineralization.Objective: This study aimed to discovery the microRNA genes targeted IFITM5,verify the effect of osteoblasts mineralization, differentiation by microRNA.Methods: We predicted micoRNA via the TargetScan, miRanda, Microcosm andDIANA-microT. microRNA must be predicted by at least two sofewares in order to determine whether IFITM5and microRNA had the target relationships. SyntheticmicroRNA mimics, construct the wild-type and mutant pmirGLO-IFITM5-3’UTRluciferase reporter vectors and then detect firefly luciferase activity relative to filter outmicroRNA that integrated with IFITM53’UTR, further screen protein microRNA viaWestern blotting in protein levels. After microRNA mimics transfected Saos-2cells andinduction of mineralization, we detected mineralized nodules by Alizarin Red staining,and applied real-time PCR method to detect differentiation marker proteins ALP, Runx2,OCN genes expression.Results:(1) Bioinformatics predicted28related microRNA, where15microRNAwere found in the three or more databases.(2) The wild-type dual luciferase reportervectors were co-transfected with microRNA mimics into Saos-2cells, fluorescence ofexperimental group decrease30%than relative to the control group as the principle:miR-1296, miR-2861, miR-4316, miR-661, miR-762; then these five mutant dualluciferase report were co-transfected with microRNA mimics into Saos-2cells, filter outmiR-2861, miR-4316, miR-762.(3) The above three microRNA transfected into Saos-2cells, Western blotting showed that miR-762and miR-4316could significantly reduceexpression level of IFITM5protein.(4) Compaired with the control group, miR-762group can significantly reduce the formation of mineralized nodules via Alizarin redstaining after induction of mineralization after72hours; compaired with the controlgroup, The expression of Runx2, ALP and OCN also decreased after72hours.Conclusions: miR-762and miR-4316could negative regulation the expression ofIFITM5and miR-762inhibited osteoblast mineralization.