Fig. 3
HOXD-AS1 inversely regulates the expression of neighbouring gene HOXD3. a Expression of HOXD3 in CRC cells were detected by real-time PCR (upper) and Western blot (down) when HOXD-AS1-overexpression or -depletion, respectively. b Expression levels of HOXD-AS1 in 35 paired CRC and adjacent non-cancerous tissues. c The expression correlation between HOXD-AS1 and HOXD3 was detected by real-time PCR in 35 paired CRC and adjacent non-cancerous tissues. d FISH analysis of the subcellular location of HOXD-AS1 in CRC cells. HOXD-AS1 mainly enriches in nucleus. e To identify the proteins associated with HOXD-AS1 by RNA pull-down assays. Biotinylated HOXD-AS1 and antisense RNA were incubated with cell extracts, and the associated proteins were resolved by SDS-PAGE. The HOXD-AS1-sense-special bands (arrows) in SW620 nuclear lysates group were excised and analyzed by mass spectrometry. f Western blot was used to detect SUZ12 and EZH2 in pull-down products. g RIP assays were performed in SW620 cells using anti-SUZ12, anti-EZH2 or nonspecific IgG antibodies respectively. Real-time PCR was performed to determine amount of RNA associated with SUZ12, EZH2 or IgG compared with the input control. h ChIP assays were performed in HOXD-AS1 overexpressed(SW620-HOXD-AS1)and control cells using anti-EZH2-, anti-SUZ12-, and anti-H3K27me3-antibodies or IgG antibody respectively. To determine the specific binding site of PRC2 complex and the promoter region of HOXD3, we divided HOXD3 promoter region into 7 segments. The enrichments of 7 segments of HOXD3 gene promoter DNA associated with antibodies were examined by real-time PCR. i-j The expression of HOXD3 were detected by i real-time PCR and j Western blot in SUZ12 or EZH2-depleted SW620 and control cells, respectively. k The quantitative data analysis of j to show the expression pattern between EZH2 or SUZ12 and HOXD3, respectively. For a, b, c, g, h, i and k, data were presented as means ± SD in three independent experiments. *P < 0.05, **P < 0.01