Fig. 4

HOTAIR modulates DNA methylation within MTHFR promoter region. shRNA was delivered into TE-1 cells to specifically blunt HOTAIR (sh-lncRNA HOTAIR group). Expression vectors containing the lncRNA HOTAIR were introduced into TE-1 cells (oe-lncRNA HOTAIR group). Scramble shRNA-treated cells and empty vectors-treated cells were served as negative controls (sh-NC group and oe-NC group). Moreover, TE-1 cells treated with restored HOTAIR were added with either 5-Aza-CdR or DMSO. (a) MTHFR mRNA level in EC tissues (n = 70) and adjacent normal tissues (n = 70) determined by RT-qPCR, normalized to GAPDH. (b) Distribution of CpG islands within MTHFR promoter region. (c) MSP was adopted to detect methylation level of MTHFR promoter region in EC tissues (n = 70) and adjacent normal tissues (n = 70). (d) The binding sites between the lncRNA HOTAIR and MTHFR promoter regions predicted using Blast comparison website. (e) Target relationship verified by dual luciferase reporter gene assay. (f) RIP was utilized to detect the enrichment of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) affected by HOTAIR in TE-1 cells. (g) CHIP was employed to detect the enrichment of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) within MTHFR promoter region in TE-1 cells. (h), MSP was used to detect methylation level of TE-1 cells. (i) MTHFR mRNA level in TE-1 cells after restoration or depletion of HOTAIR determined by RT-qPCR, normalized to GAPDH. MTHFR (75kD), GAPDH (36kD) (j) MTHFR protein level in TE-1 cells after restoration or depletion of HOTAIR determined by Western blot analysis, normalized to GAPDH. (k) Protein band patterns of MTHFR in TE-1 cells after restoration or depletion of HOTAIR determined by Western blot analysis, normalized to GAPDH. Values obtained from three independent experiments in triplicate are expressed as mean ± SD and analyzed by paired t test between EC tissues and adjacent normal tissues, by unpaired t test between EC cells and HEEC cells, and by ANOVA followed by Tukey’s post hoc test among three or more groups. *p < 0.05 compared with adjacent normal tissues, or TE-1 cells treated with IgG, oe-lncRNA HOTAIR + DMSO, sh-NC, or oe-NC group