Fig. 7

FTO-induced epigenetic up-regulation of LINC00022 is YTHDF2-dependent. (A) Western blot was used to examine the protein levels of YTHDF2 in 14 pairs of ESCC tissues. N represents normal and T represents tumor. (B) Person’s Coefficient analysis revealed the negative correlation between YTHDF2 protein and LINC00022 transcription in 14 cases of ESCC tumors. (C) The protein levels of YTHDF2 in Het-1A, KYSE70 and KYSE150 was examined by Western blot. (D) The direct binding affinity of LINC00022 transcript with YTHDF2 protein was determined by RIP-qRT-PCR (upper panel) and agarose gel electrophoresis (nether panel). (E) The knockdown or over-expression efficiencies of YTHDF2 mediated by recombinant lentivirus were verified by Western blot in KYSE70 and KYSE150 cells. (F) Knockdown or over-expression of YTHDF2 resulted in a significant increase or decrease in LINC00022 expression in ESCC cells revealed by qRT-PCR, **p < 0.01; ***p < 0.001. (G-H) The effect of knockdown or over-expression of YTHDF2 on the stability of LINC00022 transcript in ESCC cells in the presence of ActD (5 μg/mL) was tested by qRT-PCR, *p < 0.05. (I) Knockdown of YTHDF2 (si-Y2) significantly abolished FTO ablation-induced down-regulation of LINC00022 in KYSE150 cells, *p < 0.05. (J) The up-regulation of LINC00022 caused by FTO over-expression was relieved by increase of YTHDF2 (OE-Y2) in KYSE70 cells, *p < 0.05; ***p < 0.001