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. 2019 Nov 20;28(2):613–630. doi: 10.1016/j.ymthe.2019.11.015

Figure 4.

Figure 4

The Molecular Mechanism of ZFAT-AS1 Inhibits CDX2 Transcription and Expression by Binding PRC2 to Induce H3K27me3 Modification in the CDX2 Promoter Region

(A and B) An RNA immunoprecipitation (RIP) assay confirmed that EZH2 bound to ZFAT-AS1. Relative enrichment was measured using quantitative real-time PCR. (A) Relative enrichment of U1 snRNA in SNRNP70 relative to normal IgG immunoprecipitates. (B) Relative enrichment of ZFAT-AS1 in anti-EZH2 relative to normal IgG immunoprecipitates. Normal mouse IgG was used as a negative control, SNRNP70 was used as a positive control, and GAPDH was used as a negative RNA control. Data are presented as the mean ± SD (n = 3, each group). **p < 0.01 versus anti-normal IgG group; ##p < 0.01 versus GAPDH group by Student’s t test. (C) RNA pull-down confirmed that ZFAT-AS1 bound to EZH2. EZH2 was detected using western blot. NC was representative of biotin-labeled negative RNA control (poly(A)25 RNA). Bio-ZFAT Trspts and Bio-ZFAT-AS1 are representative of 3′ end desthiobiotinylated ZFAT transcripts and ZFAT-AS1, respectively. (D) CDX2 mRNA in ZFAT-AS1 overexpression or knockdown cells. NC, group treated with negative control empty vector plasmids with non-targeting sequence. Data are presented as the mean ± SD (n = 3, each group). **p < 0.01 versus ZFAT-AS1(+)NC group; ##p < 0.01 versus ZFAT-AS1(−)NC group by one-way ANOVA. (E) H3K27me3 reaction stripe was detected in the CDX2 promoter region 500 bp (PCR1) upstream of the transcription start site (TSS) in U87 and U251 cells of control, ZFAT-AS1(+)NC, and ZFAT-AS1(+) groups. Schematic representation of the human CDX2 promoter region 2,500 bp upstream of TSS, which was designated as +1, is shown. PCR was conducted with the resulting precipitated DNA. (F) Percentage of PCR1 relative to input was analyzed by quantitative real-time PCR. Data are presented as the mean ± SD (n = 3, each group). **p < 0.01 versus ZFAT-AS1(+)NC group by one-way ANOVA. (G) Effect of ZFAT-AS1 and CDX2 on the proliferation of U87 and U251 cells. (H) Cell apoptosis of U87 and U251 cells. (I) Effect of ZFAT-AS1 and CDX2 on the migration and invasion of U87 and U251 cells. Scale bars, 50 μm. Data are presented as the mean ± SD (n = 3, each group). *p < 0.05 versus ZFAT-AS1(+)NC + CDX2(+)NC group; **p < 0.01 versus ZFAT-AS1(+)NC + CDX2(+)NC group; ##p < 0.01 versus ZFAT-AS1(+) + CDX2(+)NC group by one-way ANOVA.