The Regulation Of Puerarin On MiRNAs In Osteoblasts And The Relationship Between MiRNA-204 And Runx2

Objective:To study multiplication capacity and alkaline phosphatase vitality of cell and the target relationship between microRNA and Runx2 after effect of puerarin on osteoblasts MC3T3-E1.Methods:1. The multiplication capacity of cell was detected by MTT after effect of puerarin on osteoblasts MC3T3-E1. Do with a final concentration of 0.01 mg/ml of puerarin treatment,and set up a blank control group, add such as volume of alpha MEM cultures,each group of six complex hole respectively continue to develop after 24 h,48 h,72 h then detect the OD value. 2.The vitality of osteoblasts was detected by activity of alkaline phosphatase after effect of puerarin on it.Culture of osteoblasts and cells to climb, for cells to climb after the success, abandon to culture,washed with PBS for three times by alkaline phosphatase staining kit instruction method, for cell staining.3.The expression level of mRNA and protein of Runx2 was detected by real-time quantitative PCR and western blot after Puerarin on osteoblast of r the extraction of RNA and protein, respectively.4. miRNA which been targeted to Runx2 was predicted by target predicted software of Target Scan, PicTar and so on. Compaired the miRNA which been targeted possiblly to Runx2 been detected by miRNA expression spectrum after effect of puerarin on osteoblasts with the miRNA which been targeted to Runx2 been predicted by target predicted software of Target Scan, PicTar and so on.5.The expression level of miRNAs been possiblly targeted to Runx2 was detected by real-time quantitative PCR.6.The RhoE 3’UTR vector and RhoE mut 3’UTR vector were constructed.miRNA-204mimics and miRNA-204NC were synthetised.The target gene were verified by dual luciferase report gene assay.7.After transfecting the cell of MC3T3-E1 with miRNA-204mimics and miRNA-204 inhibitor, the expression level of mRNAof Runx2 was detected by real-time quantitative PCR and and protein of Runx2 was detected by western blot.Results:Compaired with blank after effect of puerarin, multiplication capacity and activityof cell were both rose, the expression level of mRNA and protein of Runx2 were both rose, the expression level of miRNA-204 and miRNA-344f-5p were declined, the expression level of miRNA-2861 was rose, the expression level of miRNA-23a-5p, miRNA-770-5p and miRNA-871-5p were changed unobviously.After transfection of the miRNA-204 mimics. According to the results of dual luciferase reporter gene method after cell transfection of 48 h,only set of 3 ’UTR Runx2+mimics the miRNA-204 of fluorescein protein expression level decreased significantly,showing only the miRNA-204 inhibits Runx2 3’UTR report gene expression. The expression protein level of Runx2 was rose, it was declined after transfection of the miRNA-204 inhibitor.Conclusion:On osteoblasts, puerarin promoted cell proliferation and differentiation and regulated the miRNAs which been possiblly targeted to Runx2. It could provide reliable basis of research for further study promoted effect of puerarin on osteoblasts.