OBJECTIVE:To explore whether electroacupuncture(EA could alleviate osteoarthritis(OA)through affecting the DNA methylation regulated transcription of miR-146a and mi R-140-5p.METHODS:Sixty male eight-week-old Sprague-...OBJECTIVE:To explore whether electroacupuncture(EA could alleviate osteoarthritis(OA)through affecting the DNA methylation regulated transcription of miR-146a and mi R-140-5p.METHODS:Sixty male eight-week-old Sprague-Dawley rats were divided into three groups:normal group(normal healthy rats;no treatment),model group(OA rats;no treatment)and EA group(OA rats treated with EA).Safranin O staining and modified Mankin’s score were performed to evaluate the histopathological alterations and degeneration of cartilage 8 weeks after 8 consecutive weeks of treatment.Quantitative real time polymerase chain reaction(qR T-PCR)assay was employed to evaluate the expression of miR-146a in the cartilage tissue and miR-140-5p in the synovium tissue,respectively.The bisulfite sequencing analysis and quantitative methylation specific PCR(qMSP)were used to analyze the status of methylation in the regulatory regions of miR-146a and miR-140-5p.Chromatin immunoprecipitation(ChIP)assay were performed to assess the binding of nuclear factor-kappa B(NF-κB)and signal transducer and activator of transcription 3(SMAD-3)in the regulatory regions of miR-146a and miR-140-5p.Western blot analysis was performed to detect the expressions of DNA Methyltransferase 1(DMNT1),DNA Methyltransferase 3A(DMNT3A),and DNA Methyltransferase 3A(DMNT3b),NF-κB,SMAD3 levels.RESULTS:Our results showed that EA treatment significantly upregulated miR-146a and miR-140-5p expressions.qMSP analysis showed that EA significantly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium.Bisulfite DNA sequencing(BDS)and ChI P analysis showed that EA significantly increased binding affinity of SMAD3 and NF-kB on the hypermethylated miR-140 regulatory region and miR-146a promoter,respectively.Western Blot analysis demonstrated that EA also significantly decreased expressions of methylation related proteins-DMNT1,DMNT3a,and DMNT3b as well as NF-κB and SMAD3.CONCLUSIONS:Electroacupuncture stimulating Neixiyan(EX-LE5)and Dubi(ST35)may alleviate OA via affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.展开更多
文摘OBJECTIVE:To explore whether electroacupuncture(EA could alleviate osteoarthritis(OA)through affecting the DNA methylation regulated transcription of miR-146a and mi R-140-5p.METHODS:Sixty male eight-week-old Sprague-Dawley rats were divided into three groups:normal group(normal healthy rats;no treatment),model group(OA rats;no treatment)and EA group(OA rats treated with EA).Safranin O staining and modified Mankin’s score were performed to evaluate the histopathological alterations and degeneration of cartilage 8 weeks after 8 consecutive weeks of treatment.Quantitative real time polymerase chain reaction(qR T-PCR)assay was employed to evaluate the expression of miR-146a in the cartilage tissue and miR-140-5p in the synovium tissue,respectively.The bisulfite sequencing analysis and quantitative methylation specific PCR(qMSP)were used to analyze the status of methylation in the regulatory regions of miR-146a and miR-140-5p.Chromatin immunoprecipitation(ChIP)assay were performed to assess the binding of nuclear factor-kappa B(NF-κB)and signal transducer and activator of transcription 3(SMAD-3)in the regulatory regions of miR-146a and miR-140-5p.Western blot analysis was performed to detect the expressions of DNA Methyltransferase 1(DMNT1),DNA Methyltransferase 3A(DMNT3A),and DNA Methyltransferase 3A(DMNT3b),NF-κB,SMAD3 levels.RESULTS:Our results showed that EA treatment significantly upregulated miR-146a and miR-140-5p expressions.qMSP analysis showed that EA significantly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium.Bisulfite DNA sequencing(BDS)and ChI P analysis showed that EA significantly increased binding affinity of SMAD3 and NF-kB on the hypermethylated miR-140 regulatory region and miR-146a promoter,respectively.Western Blot analysis demonstrated that EA also significantly decreased expressions of methylation related proteins-DMNT1,DMNT3a,and DMNT3b as well as NF-κB and SMAD3.CONCLUSIONS:Electroacupuncture stimulating Neixiyan(EX-LE5)and Dubi(ST35)may alleviate OA via affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.
