Fig. 4

HOTAIR and Dnmt3b downregulates HOXA5 through promoting its methylation. a The subcellular localization of HOTAIR predicted by lncATLAS website and verified by FISH (×200). b Luciferase assay to confirm the binding of HOTAIR to HOXA5 promoter. c Distribution of CpG islands within the HOXA5 promoter region by MethPrimer and methylation level of HOXA5 promoter region detected by MSP. d The binding of Dnmt3b to HOTAIR after alteration of HOTAIR or Dnmt3b expression detected by RIP. e RNA pull down biotin-labeled HOTAIR and the expression of Dnmt3b in immunoprecipitation examined by western blot analysis. f The binding of Dnmt3b with HOXA5 promoter region after alteration of HOTAIR or Dnmt3b expression detected by CHIP-PCR. g Expression change of HOXA5 after alteration of HOTAIR or Dnmt3b expression examined by western blot analysis. *p < 0.05 vs. the oe-NC group; ^#p < 0.05 vs. the oe-HOTAIR + sh-NC group. RT-qPCR reverse transcription quantitative polymerase chain reaction, NC negative control, FISH fluorescence in situ hybridization, RIP RNA immunoprecipitation, CHIP chromatin immunoprecipitation, HOTAIR Hox transcript antisense intergenic RNA, HOXA5 homeobox A5, Dnmt3b DNA methyltransferase 3b. The results were measurement data. Comparisons between two groups were conducted by means of independent sample t-test, and comparisons among multiple groups were assessed by one-way analysis of variance. The experiment was independently repeated three times