Mechanisms Of Osteogenic Differentiation Of RBMSCs Regulated By Quercetin Through MiR-214-3p

Objective:In this experiment,we observed the changes of miR-214-3p and Wnt/β-catenin signaling during the osteo-differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)promoted by quercetin(QUE),and the effects of overexpression of miR-214-3p on the osteogenic differentiation and Wnt/β-catenin signaling of rBMSCs promoted by QUE,in order to explore the miR-214-3p regulated the mechanism of QUE pro-osteogenic differentiation of rBMSCs.Method:1.ERα silencing model of rBMSCs was constructed,and the expression of ERα was verified by Western-blot and q PCR,followed by the expression of osteogenic factors such as COLΙ,ALP,BMP2,RUNX2 and miR-214-3p after ERα silencing.2.3,7,10 and 14 d of osteogenesis induction,q PCR was performed to detect the expression of osteogenesis-related factors COLΙ,ALP,BMP2,RUNX2 and miR-214-3p.3.CCK8 kit to detect the effect of QUE on cell viability of rBMSCs;ALP kit to detect the effect of QUE on synthesis and secretion of ALP by rBMSCs;alizarin red staining to detect the effect of QUE on the formation of mineralized nodules by rBMSCs.4.q PCR to detect the effects of QUE on osteogenesis-related factors COLΙ,ALP,BMP2,RUNX2 and miR-214-3p in rBMSCs,Western-blot to detect the effects of QUE on osteogenesis-related factors COLΙ,ALP,BMP2,RUNX2 and Wnt3 a,β-catenin in rBMSCs.5.miR-214-3p overexpression model of rBMSCs was constructed.q PCR and Western-blot were performed to detect the expression of miR-214-3p,COLΙ,ALP,BMP2,RUNX2 and Wnt3 a,β-catenin in miR-214-3p overexpression model in the presence or absence of QUE intervention.Result:1.After ERα silencing,the m RNA and protein expression of ERα was significantly lower in the Lv-sh ERα group compared to the CON group(*P<0.05),and the m RNA and protein expression of the osteogenesis-related factors COLΙ,ALP,BMP2,and RUNX2 was lower(*P<0.05),while the expression of miR-214-3p was significantly higher(*P<0.05)..2.Gene expression of osteogenesis-related factors COLΙ,ALP,BMP2,and RUNX2 was significantly increased(*P<0.05)and expression of miR-214-3p was significantly decreased(*P<0.05)in osteogenesis-induced 3,7,10,and 14 d rBMSCs compared with the CON group.3.Compared with the CON group,different concentrations of QUE(0.1,0.5,1 and5 μM)significantly promoted the cell proliferation of rBMSCs at 48 h and 72 h of intervention(*P<0.05);significantly promoted the synthesis and secretion of ALP by rBMSCs at 7 d and 14 d of intervention(*P<0.05);after 28 d of intervention,all groups increased The number of mineralized nodules of rBMSCs was increased in all groups after 28 d of intervention;the greatest increase was observed at 1 μM concentration.4.QUE significantly promoted gene and protein expression of the osteogenesisrelated factors COLΙ,ALP,BMP2 and RUNX2(*P<0.05)and significantly decreased the expression of miR-214-3p(*P<0.05)compared to the CON group.5.Overexpression of miR-214-3p significantly decreased the gene and protein expression of osteogenesis-related factors COLΙ,ALP,BMP2,RUNX2(*P<0.05),and also significantly decreased the expression of Wnt3 a,β-catenin protein(*P<0.05)compared to the CON group.Compared to the QUE+NC group,QUE and miR-214-3p together significantly reduced the expression of genes and proteins of osteogenic factors COLΙ,ALP,BMP2,RUNX2(*P<0.05),and also Wnt3 a,β-catenin(*P<0.05).Conclusion:1.Silencing ERα significantly inhibited osteogenic differentiation of BMSCs and promoted the expression of miR-214-3p.2.After osteogenic differentiation of rBMSCs,osteogenic index expression increased and miR-214-3p expression decreased.3.QUE promoted proliferation and osteogenic differentiation of rBMSCs,increased Wnt3 a,β-catenin expression and decreased miR-214-3p expression.4.Overexpression of miR-214-3p inhibited osteogenic differentiation and decreased the expression of Wnt3 a and β-catenin in rBMSCs;overexpression of miR-214-3p inhibited the promotion of osteogenic-related factors and Wnt3 a and β-catenin by QUE in rBMSCs.