Fig. 6

5-Aza-dC induced demethylation and re-expression of silenced HOXD10. a Higher HOXD10 methylation in tumor tissues compare to paired normal tissues. ‘Pos’ represented the positive controls for the methylated (M) and unmethylated (U) allele. The normal peripheral lymphocytes DNA was used as negative control. b HOXD10 was confirmed to be hypermethylated in colon adenocarcinoma cell lines SW480 and LoVo compared with normal colon cell line CCD-18Co cell line. The change of DNA methylation level was minimal in LS180 (COAD cell line) compared with CCD-18Co cells. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the CCD-18Co cell lines. c Minimum effective dose of 5-Aza-dC was determined by MTT. 1 μM showed difference. * P < 0.05, ** P < 0.01, compared with the 0 μM group. d The methylation level of HOXD10 decreased after the treatment with demethylation agent 5-Aza-dC in SW480, LoVo, LS180, HT29 and HCT-116 cell lines. e Relative DNMT activity was decreased after treatment of the cells with 5-Aza-dC (1 μM) for 72 h. Compared with the control group, ** P < 0.01. f 5-Aza-dC (1 μM) treatment decreased HOXD10 methylation in SW480, LoVo, LS180, HT29 and HCT-116 cell lines. Compared with the control group, *** P < 0.001. g The mRNA expression of HOXD10 in SW480, LoVo, LS180, HT29 and HCT-116 cell lines was higher after treatment with 5-Aza-dC (1 μM) for 72 h or overexpression of HOXD10. *** indicated P < 0.001 compared with the control group; ## indicated P < 0.01 compared with the vector congtrol group. h Cell proliferation was suppressed in SW480 and LoVo cells after treatment with 5-Aza-dC (1 μM) for 72 h or overexpression of HOXD10, determined by MTT assay. Compared with the control group, *** P < 0.001. Compared with the vector control group, ###P < 0.001