The Study Of Estrogen Regulation Of The Expression Of Osteoprotegerin Mechanism

Objective: To observe the celluar vitality of estrogen on human osteosarcoma cell lines(MG-63)in vitro,as well as protein expression of osteoprotegerin(OPG)and changes in OPGmRNA transcription level,and to further explore:(1)which promoter sequence that transcription factors acting on the OPG gene;(2)the changes of miR-145 expression,and investigate whether estrogen targeting regulate the expression of OPG by miR-145 after the effect of estrogen on MG-63 cells.Methods:(1)MG-63 cells vitality were measued using MTT assay cells at various concentrations of 17?-estradiol(E2)(0nM,1nM,10 nM,100nM)on 1d,2d,3d,4 d,5 d,6 d,7 d at different point changes,and to detect the expression change of OPG protein after 48 h on the different concentrations of estrogen effect by Western Blot analysis and quantitative ELISA analysis and then analyze changes of OPG mRNA using Realtime-PCR.(2)Amplify different lengths OPG promoter sequences(upstream of the structural gene sequence of OPG: 986 bp / 769 bp / 527 bp / 255bp)with human genomic DNA as template,respectively inserted into the promoter region of the plasmid pGL3-basic,and then pGL3-basic-986/769/527/255-OPG were transfected into MG-63 cells,using luciferase luciferase reporter gene to detected the role of different lengths OPG promoter sequence affecting the sequence of luciferase expression at different concentrations(0nM,1nM,10 nM,100nM)E2,to explore which optimum sequence range of OPG gene that promoter transcription factors combined.(3)To observe the changes of miR-145 at different concentrations(0nM,1nM,10 nM,100nM)estrogen E2 acting the MG-63 cells,and then miR-145 analogues mimics-145 were introduced into cells,or import antisense oligonucleotide fragments ASO-145,with increasement or decreasement of miR-145,to observe the expression of OPG protein and changes in OPGmRNA.(4)Utilizing the dual fluorescence luciferase gene technology to verify whether miR-145 targeting regulate the OPG gene expression.Results:(1)It is detected that the vitality of MG-63 cell were obviously elevated by MTT assay after estrogen.With the extension of an increase in estrogen concentrations and duration of action,the MG-63 cell proliferation was significantly enhanced,but there was no significant difference between 10 nM and 100 nM concentrations groups increased the rate of cell proliferation activity(P>0.05).(2)After estrogen E2 and cells co-cultured with 48 h,it showed that the ability of cells expressing OPG protein were significantly enhanced by WesternBlot protein analysis and ELISA assay,especially in 10 nM group.By Realtime-PCR analysis,the level of OPGmRNA was significantly increased after 48 h,especially when the concentration come to 10 nM,the highest level of transcription of OPGmRNA was found.The results were similar to Western Blot and ELISA analysis of changes in OPG protein.(3)Use of dual luciferase reporter gene to observe cells pGL3-basic-986 plasmid expression of osteoprotegerin promoter at different concentrations of estrogen E2,and finally find at the maximum fluorescence intensity expressed at a concentration of 10 nM.(4)Thus selectiing 10 nM estrogen act MG-63 cells containing plasmid vector pGL-basci-986/769/527/255-OPG,and the results showed that the plasmid pGL-basci-986 and the plasmid pGL-basci-769 the fluorescence intensity,estrogen stimulation group were significantly higher than the control(P<0.05);the group pGL-basci-527 plasmid and pGL-basci-255 plasmid group,there were no statistically significant differences between the two(P>0.05),pGL-basci-986 plasmid and pGL-basci-769 plasmid fluorescence intensity was significantly higher than pGL-basci-527 plasmid and pGL-basci-255 plasmid group(P<0.05),but there was no statistically significant difference between pGL-basci-986 between the plasmid and the plasmid pGL-basci-769 group(P> 0.05),although the fluorescence intensity pGL-basci-255 plasmid group was slightly lower than pGL-basci-527 plasmid group,there was no significant different(P>0.05).(5)After estrogen E2 acted on cell,miR-145 expression downregulated.(6)When import mimics-145 into MG-63 cells,miR-145 expression increased,and OPG protein and OPGmRNA expression decreased,on the contrary,import antisense oligonucleotide fragments ASO-145,miR-145 expression downregulated,and OPG protein and OPGmRNA expression rised.(7)Dual-Luciferase technology prompted that OPG gene is mi R-145 target genes.Conclusion:(1)Estrogen could enhance the vitality of cells and promote the transcription process osteoprotegerin gene,increased expression of OPG protein.(2)After the estrogen acted on the MG-63 cells,transcription factors work the OPG gene promoter sequence in its upstream 527-769 range,specific binding sequence needs further research.(3)Estrogen E2 could targeted regulate the expression of OPGmRNA by miR-145 in the MG-63 cells,thereby affecting the expression of OPG protein.