The Effect Of MiR-23a-3p On Osteogenic Differentiation Factors ALP And RUNX2 Of Periodontal Stem Cells In Vitro

Objective:In order to determine the expression of miR-23a-3p during the induction of osteogenesis,HPDLSCs were induced to transform into osteoblasts in vitro.Lentivirus-mediated inhibition and overexpression of miR-23a-3p gene in HPDLSCs were followed by detection of the expression of osteogenic factors ALP and RUNX2,in order to investigate whether miR-23a-3p is involved in periodontal bone remodeling and explore its possible mechanism of action.Methods:1.Primary HPDLSCs were isolated by the method of explant culture and purified by using limited dilution in vitro for passage.The cells were identified in combination with the three experiments of cyto-immunofluorescence staining technique,flow cytometry and multidirectional differentiation induction in vitro to provide support for subsequent studies.2.The second or third generation of HPDLSCs were inoculated in a six-well plate to be induced into osteoblasts.After 0,3,5,7,14 and 21 day’s of osteogenic induction,q RT-PCR and WB were used to detect the expression level of ALP,RUNX2 m RNA and protein in HPDLSCs at each timepoint,respectively.Meanwhile,the relative expression of miR-23a-3p was detected.3.Synthetic lentivirus miR-23a-3p mimics,LV-hsa-miR-23a-3p(9746-1),and the mimic negative controls(CON220)were transfected into HPDLSCs,and four groups of MOI=50,60,80,100 were set up.The fluorescence intensity of GFP in the control group,the CON220 group and the miR-23a-3p mimics group were observed under inverted fluorescence microscope to determine the optimal MOI.HPDLSCs were then infected with the optimal MOI of LV-hsa-miR-23a-3p and CON220 virus,and the Cell Counting kit-8 method was used to detect the influence of Lv-hsa-miR-23a-3p and CON220 virus on the proliferation capacity of HPDLSCs.4.After transfection synthetic miR-23a-3p mimics,miR-23a-3p inhibitor,and also mimic and inhibitor negative controls into HPDLSCs for 48 h,purinomycin was added to the cells.QRT-PCR detection was used to test the inhibition and overexpression efficiency of the virus on miR-23a-3p after the transfection of HPDLSCs.5.After inhibiting and overexpressing of miR-23a-3p,HPDLSCs were induced with osteogenic induction solution.QRT-PCR and WB were used to detect the relative m RNA and protein expression in ALP and RUNX2 on the 7th day,respectively.The experiment was randomly divided into 5 groups: Negative Control group(NC group),miR-23a-3p mimics group(miR-23 a group),miR-23a-3p mimics negative control group(miR-23 a NC group),miR-23a-3p inhibitor group(miR-23 a I group)and miR-23a-3p inhibitor negative control group(miR-23 a NCI).Results:1.The cultured cells were spindle-shaped and fibroblasts-like.The sources of cells were identified as mesenchymal by immunohistochemical staining.The cells,surfaces were strongly positive for STRO-1,CD90 and CD146,which were all the specific surface markers of stem cells.while the marker called CD45 was showed very low level of expression.HPDLSCs can differentiate into osteoblasts and adipocytes by induction in vitro using corresponding differentiation medium.In conclusion,it can be identified as periodontal membrane stem cells.2.The expression of ALP,RUNX2 mRNA and protein was increased during the osteogenic differentiation of HPDLSCs compared with non-induced group.The HPDLSCs were successfully differentiated into osteoblasts.The expression of miR-23a-3p in the groups which were added the osteoblast inducing conditional media was significantly lower than the uninduced group(P < 0.01).And the general trend of miR-23a-3p expression is declining.3.After 48 h of lentivirus transfection,the form of cells were similar to the normal and had a good cellular growth behavior.The optimal MOI value for virus infection was 80.CCK-8 detection showed that the proliferation ability of HPDLSCs after infected had no statistical difference compared with the nontransfected group.4.After virus transfection,the relative expression of miR-23a-3p in miR-23 a NC group and miR-23 a NCI group was not significantly different from that in the NC group.The expression level of miR-23a-3p in the miR-23 a group was significantly higher than the NC group and the miR-23 a NC group(P<0.01).The virus were effectively transfected into cells.The expression of miR-23a-3p in the miR-23 a I group was significantly lower than the NC group(P=0.025),but there was no statistical difference between the miR-23 a I group and the miR-23 a NCI group.5.After HPDLSCs were induced by Mesenchymal Stem Cell Osteogenic Differentiation Medium in vitro for 7 days,no significant difference were found in the m RNA and protein expression of ALP,RUNX2 between miR-23 a NC group and the NC group.Moreover,the expression level of ALP、RUNX2 mRNA and protein in the group where miR-23a-3p had been overexpressed was significantly lower than the NC group and the mimics negative control group.After inhibiting the expression of miR-23a-3p,the m RNA expression of ALP and RUNX2 decreased significantly compared with the NC group(P<0.05).Meanwhile,the ALP and RUNX2 m RNA of the negative control group were also lower than those of the NC group.However,there was no statistical difference in protein expression between miR-23 a I group,miR-23 a NCI group and NC group.Conclusion: The overall expression of miR-23a-3p showed a declining trend in osteogenic induced HPDLSCs in vitro.Overexpression of miR-23a-3p inhibited the expression of ALP,RUNX2 m RNA and proteins in HPDLSCs.It is suggested that miR-23a-3p may have a negative regulatory effect on the osteogenic differentiation of HPDLSCs.