The IFITM5 Mutation C.-14C>T Effects On The Founction In Saos-2 Cells

Background: Interferon-induced transmembrane protein 5(IFITM5) is one of the interferon-induced transmembrane protein family members,is an osteoblast-specific membrane protein.Its expression increases with osteoblast differentiation, peaking with matrix production and mineralization, with the promotion of the role of osteoblast mineralization. At present, two independent studies found that a mutation c.-14C>T in the 5’UTR of the IFITM5 gene is the cause of osteogenesis imperfecta(OI) type V. OI type V is characterized by an autosomal-dominant inheritance pattern, propensity to hyperplastic callus formation, and calcification of the fore-arm interosseous membrane. In addition, IFITM5 was also involved in tumor suppressor pathways with antiproliferative action and may have the modulation before the tumor formation.Therefore,abnormal expression of IFITM5 in cancer cell lines may be associated with the occurrence or progression of the tumor.Objective: This study aimed to detect the effect of IFITM5 mutation c.-14C>T in Saos-2 cell on the biological founction.Methods: Real-time PCR and Western blotting were performed to detect the gene and protein expression levels of IFITM5 in 15 different cancer cell lines.After pc DNA4-IFITM5-E12-W and pc DNA4-IFITM5-E12-MU plasmids which had been constructed transfected Saos-2 cells,MTT,scratches, apoptosis and transwell methods were used to determinate the cell proliferation,migration,apoptosis, and invasion Afterinduction 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: There was remarkable difference in the expression of IFITM5 in this cancer cell lines.The lowest expression level of m RNA and protein were detected in H7402,RD and HEP2 cell lines.The highest IFITM5 expression was observed in SAOS2 cells,followed by 3AO.After pc DNA4-IFITM5-E12-W and pc DNA4-IFITM5-E12-MU plasmids which had been constructed transfected Saos-2 cells,MTT,scratches, apoptosis and transwell methods were used to determinate the cell proliferation,migration,apoptosis and invasion.The results showed that the cell proliferation,migration,apoptosis and invasion were not obvious.Compaired with the control group, pc DNA4-IFITM5-E12-W and pc DNA4-IFITM5-E12-MU group can significantly increase the formation of mineralized nodules via Alizarin red staining after induction of mineralization after 72 hours; compaired with the control group, The expression of Runx2, ALP and OCN also increased after 72 hours,the gene levels of IFITM5 in pc DNA4-IFITM5-E12-MU was higher than pc DNA4-IFITM5-E12-W.Real-time PCR and Western blotting showed that the gene and protein levels of IFITM5 have significantly increased expression, the gene and protein levels of IFITM5 in pc DNA4-IFITM5-E12-MU were higher than pc DNA4-IFITM5-E12-W.Conclusions:The study showed that there was significant differential expression of IFITM5 in cancer cell lines. pc DNA4-IFITM5-E12-W and pc DNA4-IFITM5-E12-MU plasmids can promote the gene and protein expression levels of IFITM5,but have no obvious effect on the cell proliferation,migration,apoptosis and invasion. The over-expression of IFITM5 can promote osteoblast mineralization.