The Effects Of Expression Of OPG And RANKL In Human Osteoblastic MG63 Cells After Estrogen Receptor Beta Being Silenced By RNAi Mediated By Retroviral Vector

Objective:It is commonly think that estrogens were believed to act via estrogen receptors. After estrogen receptor beta(ERP) subtype was found, new understanding of function mode of estrogen was produced. And the finding of ERβdeveloped new ways for studying the mechanism whose estrogen regulars bone metabolism. Otherwise, bone formation and bone resorption which conducted by osteoblast and osteoclast pass through bone metabolism process.OPG/RANKL system which express in the osteoblast have important meaningful in revealing mechanism of bone metabolism, especially the alteration of OPG/RANKL ratio may influence bone formation and bone resorption, which affect bone metabolism.The main object of our study include①To construct retro viral-mediated short hairpin RNA (shRNA) expression vector of estrogen receptor (3 pRNA-H1.4/Retro-shRNA and to be packaged as antivirus.②To explore the specific inhibition effect of estrogen receptorβin human osteoblast-like cell MG63 after MG63 was transfected by the anti virus.③To establish ERβgene knockdown human osteoblast-like cells models and to observe the effects of down-regulation of ERβon the expression of OPG、RANKL mRNA and protein in human osteoblast-like cell MG63.To provide the experimental evidence to further explore how ERβregular bone metabolism.Methods:①Genome sequences of ERβwas retrieved from Genebank, siRNA(small interfering RNA)was designed and was converted into cDNA coding expression of shRNA(small hairpin RNAs) for ERβgene.The cDNA was synthesized and inserted into plasmid pRNAT-H1.4/Retro. The recombinant shRNA expression vector pRNAT-H1.4/Retro-ERβ-shRNAwere constructed and identified by restriction enzyme digestion and the sequence analysis.The recombinant vector was transfected into 293 cells by Lipofectamine 2000,which was packaged as retro virus.②The cultured human-like cell MG63 was observed at morphology and Trypan blue-Wright’s staining.The retrovirus was transiently transfected into human osteoblast-like cell MG63, while untreated and transfected scramble shRNAnc served as the blank control and nonspecific shRNA control separately. Transfecting efficiency was detected by flow cytometry.Total RNA and protein of the cells were extracted after 48 h of transfection. The expression of ERβin mRNA and protein levels were assessed by semiquantitive RT-PCR and Western blot respectively in order to ascertain the most effective shRNA sequences.③Then retrovirus with the most effective shRNA sequence and scramble shRNAnc was used to stablely transfect human osteoblast-like cell MG63, while untreated and transfected scramble shRNA served as the blank control and nonspecific shRNA control separately. Stable transfective clone was selected by hygromyc and were amplified and cultured.The stable inhibition rate of ERβin mRNA and protein levels were assessed by semiquantitive RT-PCR and Western blot respectively. Cell proliferation was tested by MTT assay. Total RNA and protein of the three group cells, that is, MG63、MG63 transfected with ERβshRNA retrovirus、MG63 transfected with shRNAnc retrovirus, were extracted after 48 h of administration of 10-8mol/L E2. The mRNA and protein levels expression of OPG and RANKL in MG63 were assessed by semiquantitive RT-PCR and Western blot respectively.Using SPSS 13.0 statistical software to analyze the results of the measurement data to express X±S, One way ANOVA is conducting a number of the samples were found between the number of significant test, test standard a=0.05.P value<0.05 indicated significant difference.Results:①Three retroviral vector-ERβ-shRNA(pRNAT-H 1.4/ Retro-ERβ-shRNA1,pRNAT-H1.4/Retro-ERβ-shRNA2, pRNAT-H1.4/ Retro-ERβ-shRNA3) were constructed.Recombinant plasmid by enzyme digestion and sequence analysis confirmed that the purpose of identification of sequence has been inserted into the site which is expected to meet the design requirements.The recombinant vector was transfected into 293 cells by Lipofectamine 2000,which was packaged as antivirus.②The results of transfection showed that retrovirus can transfect MG63 cells efficiently and stablely.The transfecting rate was about 70%.The results of instantaneous transfection show that pRNAT-H1.4/Retro-ERβ-shRNA 1, pRNAT-H 1.4/Retro-ERβ-shRNA 2, pRNAT -H1.4/Retro-ER(3-shRNA3 all could decrease the ERβmRNA expression level and inhibition rate were 44.77±5.41%.61.21±4.47%、80.11±1.72% respectively, while the protein inhibition rate was 44.84±0.96%,62.52±1.02%,81.18±0.73% respectively (P<0.05);pRNA-H1.4/Retro-ERβ-shRNA3 could most effectively decrease the ERβmRNA and protein expression level to 80.11±1.72% and 81.18±0.73% respectively (P<0.05).③The stable inhibition rate of ERp in MG63 was 88.17±1.17%(P<0.05) in mRNA level and 89.01±1.22%(P<0.05)in protein level after stablely transfecting into MG63 by retrovirus with ERβ-shRNA3.MTT method showed that MG63 cells proliferation was not affected after transfected ERβ-shRNA..According to the results of semiquantitive RT-PCR and Western blot, the expression of OPG mRNA were up-regulated by 15.51±1.72% and protein were up-regulated by 20.35±1.15%(P<0.05) and RANKL mRNA were down-regulated by 22.17±0.94% and protein were down-regulated by 27.22±1.48%(P<0.05),and OPG/RANKL ratio were up-regulate (P<0.05) in MG63 whose ERp was stablely inhibited after administration of E2 for 48h when compared with the blank control and nonspecific shRNA control (P<0.05)Conclusion:①Three kinds of retro viral vector pRNAT-H1.4/Retro-ERβ-shRNA were constructed successfully by using pRNAT-H1.4/Retro vector.pRNAT-H1.4/Retro-ERβ-shRNA antivirus were packaged.②Three kinds of ERβ-shRNA retrovirus can transfect MG63 cells efficiently and stablely and inhibit the expression of ERβremarkablely, whose ERβ-shRNA3 was most efficient shRNA sequences③ERβgene knockdown human osteoblast-like cells models was constructed successfully.Inhibition of ERβexpression with administration of estrogen can up-regulates the mRNA and protein expression of OPG and down-regulates mRNA and protein expression RANKL in human osteoblast-like cell MG63,and up-regulates OPG/RANKL ratio.ERβmay play an important role in bone metabolism through regulating OPG/RANKL ratio.