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Fig. 5 | Arthritis Research & Therapy

Fig. 5

From: Long non-coding XIST raises methylation of TIMP-3 promoter to regulate collagen degradation in osteoarthritic chondrocytes after tibial plateau fracture

Fig. 5

LncRNA XIST inhibits the expression of TIMP-3 by recruiting DNA methyltransferases into the TIMP-3 promoter region. a The mRNA expression of TIMP-3 after silencing and over-expression of lncRNA XIST determined by RT-qPCR. b, c The protein expression of TIMP-3 after silencing and over-expression of lncRNA XIST determined by Western blot analysis. d Prediction of subcellular localization of lncRNA XIST available on lncATLAS website. e Detection of subcellular localization of lncRNA XIST in OA chondrocytes by FISH assay (× 400). f Blast alignment between lncRNA XIST sequence and TIMP-3 promoter sequence. g Prediction of CpG island enrichment in TIMP-3 promoter region by MethPrimer website. h Determination of methylation status of TIMP-3 promoter in each group using different primers by MS-PCR assay. i Prediction of combining ability between lncRNA XIST sequence and DNA methyltransferase DNMT1, DNMT3A, and DNMT3B protein sequences, and the combining ability is reflected by when random forest (RF) > 0.5 and support vector machine (SVM) > 0.5. j The binding of lncRNA XIST to DNMT1, DNMT3A, and DNMT3B measured by RNA pull-down assay. k RIP assay used to detect the enrichment of lncRNA XIST by DNMT1, DNMT3A, and DNMT3B, and the amount of enriched lncRNA XIST enriched detected by RT-qPCR. l, m The enrichment of DNMT1, DNMT3A, and DNMT3B in TIMP-3 promoter detected by ChIP, and TIMP-3 promoter fragment is measured by RT-qPCR; * p < 0.05 vs. the sh-NC group (cells transfected with pGPU6/Neo); #, p < 0.05 vs. the oe-NC group (cells transfected with pCMV6-AC-GFP); the measurement data were expressed as the mean ± standard deviation, which were analyzed by the independent sample t test or one-way analysis of variance; the experiment was repeated three times to obtain the mean value

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