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Fig. 6 | BMC Biology

Fig. 6

From: A pair of long intergenic non-coding RNA LINC00887 variants act antagonistically to control Carbonic Anhydrase IX transcription upon hypoxia in tongue squamous carcinoma progression

Fig. 6

887S inhibits CA9 expression through regulating DNMT1-mediated DNA methylation upon hypoxia. A DNMT1 was identified as 887S-associated protein in a hypoxia-dependent manner, by RNA pull down and Western blot. IRE: a control RNA provided by Pierce RNA 3′ End Desthiobiotinylation kit. Related to Figure S11. B DNMT1 RIP assay showed 887S, instead of 887L RNA was immunoprecipitated under hypoxia (n=3). C A schematic view of the process to detect methylation status of the CpG dinucleotides. M1 and M2: two primer sets to amplify the converted DNA sequence of CpG site on CA9 promoter. D Representative image of methylation status of the CpG site on CA9 promoter with or without 5-Aza treatment by the indicated PCR primer sets. PC: a primer set to detect the C to T conversion which is provided by the MSP kit. E Relative expression levels of CA9 in TSCC15 cells in the presence of carrier (DMSO) or 5-Aza (n=3). F, G Effects of the indicated DNMT1 siRNAs on DNMT1 (F) and CA9 (G) mRNA level (n=3). H, I Relative expression levels of DNMT1 (H) or CA9 (I) under the indicated treatments (n=3). J, K Transwell assay showed that knockdown of CA9 significantly inhibited the effect of DNMT1 knockdown in TSCC15 cells (n=3). L DNMT1 ChIP assay on the indicated CpG site and control region of CA9 upon normoxia and hypoxia (n=3). M Overexpression efficiency of 887S (n=3). N Representative image of methylation status of the CpG site on CA9 promoter detected by M1 in the control or 887S-overexpressed cells. O DNMT1 ChIP assay on the CpG site of CA9 promoter in 887S knockout cells under hypoxia (n=3). P Capture of 887S RNA by RNA pull-down assays in 293T cells, that do not express 887S, and TSCC15 cells. NC: non-probe control; even and odd: two separated pools of 887S probes containing either even or odd numbered probes based on their positions along the 887S sequence. 887S-P1 and 887S-P2: primers for 887S detection; GAPDH, primers for GAPDH detection. (n=2). Q ChIRP assay showed the association of the 887S RNA with the CA9’s CpG site under normoxia and hypoxia (n=2). R, S Relative expression level of DNMT1 (R) or CA9 (S) in control and 887S overexpressed TSCC15 in the presence of siDNMT1 (n=3). T Relative expression levels of CA9 in Ctrl and 887S overexpressed TSCC15 cells in the presence of carrier (DMSO) or 5-Aza (n=3). Data are shown as means ± SEMs. P values are calculated using Student’s t test

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