| Objective:1.Bisulfite sequencing PCR(BSP)sequencing technology was used to detect the methylation of ER? in the blood of postmenopausal osteoporosis patients,and to explore the effect of gene methylation on the proliferation and differentiation of osteoblasts and its molecular mechanism;2.The effect of ER?-AMPK-Wnt/?-catenin signaling pathway on the proliferation and differentiation of osteoblasts was verified;3.The effect of Inokosterone on ER ?-AMPK-Wnt/?-catenin signaling pathway and its mechanism of action on osteoblasts was verified.Method:1.The effect of ER? gene methylation on osteoporosisFrom June 2017 to December 2018,43 blood samples were collected from the Third Affiliated Hospital of Guangzhou University of traditional Chinese medicine,including 14 blood samples of healthy people,14 blood samples of postmenopausal osteoporosis people and 15 blood samples of postmenopausal non osteoporosis people.The methylation frequency of ESR1 was detected by BSP sequencing technology,and the expression level of ESR1 mRNA was verified by RT-qPCR technology.Serum estradiol and ESR1 were measured by ELISA Assay.2.In vitro verification of the effect of DNA methylation of ER? on osteogenic differentiationMouse osteoblasts type MC3T3E1-colon 24(MC3T3E1-24)were cultured in vitro;Specific DNMT1 siRNA was designed and synthesized to interfere with DNMT1 expression and DNA methylation in MC3T3E1-24 cells,RT-qPCR and Western blot were used to detect ER?,OPG and RANKL expression,and to verify the effect of ESR1 gene methylation on osteoblast differentiation;Alizarin red staining and ALP staining assays were used to further test the effect of ESR1 gene methylation on osteoblast maturation;MTT assay technology was used to detect the activity of osteoblasts and verify the effect of ESR1 gene methylation on osteoblasts proliferation.3.ER? regulates AMPK a and Wnt/?-catenin signaling pathway and affects osteoblast activityIn order to verify the regulatory effect of ER? on AMPK? expression,online websites PROMO and GPMiner were used to predict the binding sites of ER? in Promoter region of AMPK? gene.To further verify the regulatory effect and molecular mechanism of ER? on the activity of osteoblasts,ESR1 shRNA and over expression lentivirus vector were constructed and then transfected into the MC3T3E1-24 cell;Cells without treatment and transfected with negative lentivirus vector were as control group;RT-qPCR and Western blot technologies were used to detect the expression of important molecules such as ER?,AMPK?,p-AMPK?,frizzled,GSK3 ?,LRP5,?-Catenin,phspho-?-Catenin,OPG,and ALP;ALP activity assay further confirmed the effect of ESR1 gene expression on osteoblast maturation;The activity of osteoblasts was detected by MTT assay.4.AMPK? mediates Wnt/?-catenin signaling pathway to affect osteoblast activityIn the last step,it was verified that ER? regulates the expression and activation of AMPK? and affects the activity of osteoblasts.In order to further confirm the regulatory effect and mechanism of AMPK ? on osteoblasts,AMPK agonist AICAR was used to activate AMPK signaling pathway in MC3T3E1-24 cells;RT-qPCR and Western blot were used to detect the expression of AMPK?,p-AMPK?,frizzled,GSK3 ?,LRP5,?-Catenin,phspo-?-Catenin,and the expression of OPG and ALP were also detected;Alizarin red staining and ALP activity assay were used to further confirm the effect of ESR1 gene expression on osteoblast maturation;The activity of osteoblasts was detected by MTT assay.5.Inokosterone mediates ER ?-AMPK-Wnt/?-catenin signaling pathway and affects osteoblast activityIn order to explore the effect of genistein on ER-AMPK-Wnt/?-catenin signaling pathway,the expression of ER?,AMPK?,frizzled,GSK3?,LRP5,?-catenin,in MC3T3E1-24 cells was detected by RT-qPCR after intervention of Inokosterone(10 ng/?L);In order to verify the effect of Inokosterone on the differentiation of osteoblasts,the expression of OPG and ALP were detected by RT-qPCR,and the calcification of osteoblasts was detected by alizarin red staining and ALP activity assay kit;The activity of osteoblasts was detected by MTT assay,and the apoptosis of osteoblasts was detected by TUNEL assay.Results:1.Osteoporosis induced by DNA methylationThe BSP sequencing results of clinical blood samples showed that the frequency of ER? methylation in postmenopausal osteoporosis patients was higher than that in healthy people(P<0.01)and postmenopausal non osteoporosis people(P<0.01).In addition,RT-qPCR detection results showed that the ER? transcription level in blood samples of postmenopausal osteoporosis patients was significantly higher than that of healthy people(P<0.05)and postmenopausal non osteoporosis people(P<0.01).At the meantime,there was significant low expression of estradiol(E2)and ER?protein in serum samples detected by ELISA assay(P<0.05).Postmenopausal osteoporosis is related to the DNA hypermethylation level of ER?.2.Inhibition of DNA methylation of osteoblasts in vitro can promote osteogenic differentiationMC3T3E1-24 cells were cultured in vitro,and DNA methylation was interfered by DNMT1 siRNA.The results showed that inhibition of DNA methylation promoted the mRNA and protein expression of OPG and ALP,and inhibited the expression of RANKL.The results of alizarin red staining and ALP assay showed that the cell calcification level of DNMT1 siRNA intervention group was significantly higher than that of control group(P<0.05).MTT assay results showed that compared with the blank control group and negative siRNA intervention group,DNMT1 siRNA intervention group had stronger cell activity(P<0.05).3.ER? phosphorylation mediates the activation of AMPK and Wnt/?-catenin signaling pathway and promotes the differentiation and proliferation of osteoblasts1)Prediction of ER? at AMPK transcription binding siteER?,as a transcription factor,can regulate the level of transcription activation and expression of target genes.According to the prediction results of PROMO and GPMiner online websites,ER? had binding sites in the promoter region of the PRKAA2 gene encoding AMPK?.Effects of ER a phosphorylation on AMPK-Wnt/?-catenin signaling pathway and osteoblastsER? shRNA lentiviral vector inhibited ER? transcription and protein expression in MC3T3E1-24 cells.In addition,compared with blank control group and negative lentivirus vector group,the results of RT-qPCR and WB showed that expression of AMPK? 1/2 increased;In the Wnt/?-catenin signaling pathway,the mRNA expression of frizzled,GSK3 ?,LRP5,?-Catenin prominently decreased;At the same time,the expression of OPG and ALP decreased.The results of ALP activity assay showed that compared with the control group,the calcification level of osteoblasts in the ER? low expression group decreased,the difference was statistically significant;And the activity of osteoblasts was also significantly reduced in the MTT results.2)ER? overexpression lentiviral vector promotes ER? protein express ion and phosphorylation in MC3T3E1-24 cells.The results of RT-qPCR and WB showed that the levels of AMPK? and p-AMPK? were significantly higher than those of blank control group and ne gative lentiviral vector group;The mRNA expression of frizzled,GSK3 ?,LRP5,?-Catenin increased significantly,which indicated Wnt/?-catenin signaling pathway was activated.The results showed that the expression of OPG and ALP increased significantly compared with the control group;T he results of ALP protein assay and MTT assay showed that the calcificati on and proliferation of osteoblasts in the ER? high expression group wer e significantly increased.4)The effect of AMPK on Wnt/?-catenin signaling pathway and osteobla sts.After activating AMPK signaling,the expression and activation level of AMPK in MC3T3E1 cells increased.The results of RT-qPCR showed that th e expression of frizzled,GSK3?,LRP5,?-catenin in Wnt/?-catenin sign al pathway increased significantly,phspo-?-catenin protein expression s ignificantly decreased,the expression of OPG and ALP increased;The resu Its of ALP protein assay and MTT assay showed that compared with the cont rol group,the level of calcification and proliferation of osteocytes inc r eased in AMPK overexpression group.4.Inokosterone promotes the differentiation and proliferation of ost eoblasts by promoting ER? phosphorylation,activating AMPK and Wnt/?-ca tenin signaling pathway.After incubation with Inokosterone for 48 h,RT-qPCR technology was u sed to detect the expression of ER ?-AMPK-Wnt/?-catenin signaling pathwa y related genes in MC3T3E1-24 cells.The results showed that the expressi ons of ER?,AMPK?,frizzled,GSK3 ?,LRP5,?-catenin in the treatment group were significantly higher than that in the control group,the diffe rences were statistically significant,which indicated that ER ?-AMPK-Wnt/?-catenin signaling pathway was activated;The mRNA expression of OPG a nd ALP increased significantly,indicated the differentiation ability of osteoblasts increased;The results of alizarin red staining and ALP activ ity showed that genistein promoted the calcification of bone cells;TUNEL staining was used to detect apoptosis and MTT assay was used to detect c ell proliferation,which showed that there was no significant difference in cell apoptosis and proliferation rate between the control group and In okosterone treatment group.Conclusion:1.Hypermethylation of ER? gene promotes the occurrence of osteoporo sis,which is mainly related to the phosphorylation level of ER?;2.The low expression of ER? and low phosphorylation level inhibit t he expression of OPG and ALP,promote the expression of RANKL,inhibit th e differentiation of osteoblasts,and decrease the activity of osteoblast s;3.ER? phosphorylation promotes AMPK? activation,up regulates Wnt/?-catenin signaling pathway related gene expression,to promote osteobla st differentiation and proliferation;4.Inokosterone promotes osteoblast differentiation by activating ER?-AMPK and Wnt/?-catenin signaling pathway. |
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