The Research For MiR-4316 Targeted IFITM5 Gene To Inhibit Mineralization Of Saos-2 Cell

MicroRNAs(miRNAs)are highly conserved endogenous single-stranded noncoding small RNAs that play an important role in the regulation of gene expression.Through sequence specific binding to the target gene,the gene post transcriptional regulation,leaded to mRNA degradation or inhibit target gene expression and corresponding protein,thereby participating in cell the proliferation,differentiation and apoptosis process.Osteoblasts are the main functional cells of bone formation.In the process of differentiation,osteoblasts involve the involvement of multiple signaling pathways and transcription factors.MicroRNA can play a positive or negative role in osteoblast differentiation by targeting cytokines involved in osteoblast differentiation.Interferon induced transmembrane protein 5(IFITM5),also known as the bone-restricted IFITM-like protein(Bril),has been recognized as a bone-specific regulator,and its expression level increases with the differentiation of osteoblasts and reached a peak in the early mineralization and maturation of osteoblasts.In the pre-laboratory study,IFITM5 was identified as a target gene for miR-4316 through bioinformatics software prediction and dual luciferase reporter vectors.But,so far,there is no biological evidence that mir-4316 targeted regulation of IFITM5,thus affecting the mineralization of osteoblasts.Objective: To investigate the effect of miR-4316 on the mineralization and differentiation of Saos-2 osteoblasts by targeting IFITM5.Methods: Saos-2 cells were induced with an induction medium containing 50?g/mL ascorbic acid and 10mmol/L beta glycerophosphate.Alizarin red staining and quantification,Von Kossa staining,ALP staining,and activity assays were used to evaluate mineralization after 3,7,and 10 days of induction effect.RT-qPCR and Western blot were used to analyze the mRNA expressions of Runx2,OCN,ALP,COL1A1,Osterix,OPG and IFITM5 and the expression of Runx2,OCN and IFITM5 proteins.RiboFECTTM CP transfection reagent was used to transfect miR-4316 mimic,miR-NC and inhibitor at 100 nM concentration.Three days after transfection of Saos-2 cells,the formation of mineralized nodules was detected by alizarin red staining and quantification and Von Kossa staining.ALP staining was used to detect the activity of ALP.RT-qPCR and Western blot were used to detect the expression of osteogenic genes and proteins of Runx2,OCN,ALP,COL1A1 and IFITM5.Results:(1)Alizarin red staining showed that Saos-2 cells started to show a small amount of mineralized nodules at 3 days of induction,and the number of nodules gradually increased with the induction time.Von Kossa staining showed that black nodules were visible in the induction group after addition of silver nitrate.After the neutral red was dyed,the pink cell structure around the cells were observed under light microscopy.ALP staining and activity identification showed that the expression and activity of ALP in the induction group was significantly higher than that in the uninduced group,and the expression was highest at the 7th day of induction.The results of RT-PCR showed that the expression of osteogenic genes COL1A1 and OPG was highest in Saos-2 cells at 3rd days of induction.The expression of osteogenic genes Runx2 and ALP was highest at 7 days after induction,and the expression of osteogenic gene OCN and Osterix was highest at 10 days after induction.Western blot results showed that protein expression of Runx2 and OCN was highest at 7 and 10 days of induction.The results of RT-PCR and Western blot showed that the expression of IFITM5 mRNA and protein increased at first and then decreased with the prolongation of induction time,and the expression level was the highest at 7th days.(2)Alizarin red and Von Kossa staining showed that miRNA-4316 could inhibit the formation of mineralized nodules in Saos2 cells compared with the control group.ALP staining and activity identification show that miRNA-4316 can inhibit the activity of ALP during mineralization.RT-qPCR and Western blot results showed that miRNA-4316 could inhibit the mRNA expression of the osteogenic genes Runx2,OCN,ALP and COL1A1 and the target gene IFITM5,and the protein expression of Runx2 and IFITM5.Conclusions: The expression of IFITM5 increased with the differentiation of Saos-2 osteoblasts and was highest in the early and mature stages of osteoblast mineralization.This study was the first to confirm that miR-4316 inhibited the mineralization of Saos-2 osteoblasts,and this function It may be achieved by inhibiting its target gene IFITM5.