Fig. 7
HIFα, in turn, upregulates ALKBH5 to demethylate LINC00115 m6A and enhance LINC00115 stability. A WB of the proteins from antisense LINC00115 and LINC00115 pull-down assays. LINC00115 combined with ALKBH5 and YTHDF2. B A schematic model of LINC00115 stability regulated by ALKBH5 and YTHDF2. The stability of LINC00115 may be regulated through such a mechanism, which ALKBH5 demethylates of LIN00115 m6A and decreases YTHDF2-mediated m6A LIN00115 degradation. C Schematic representation of the position of m6A motifs within LINC00115 predicted by SRAMP. D m6A RNA immunoprecipitation (MeRIP)-qPCR was used to quantify relative LINC00115 m6A levels in ALKBH5 KD (shALKBH5-1 and shALKBH5-2) MDA-MB-231 and BT549 BCSCs. E QRT-PCR of LINC00115 levels in MDA-MB-231/shALKBH5and BT549/shALKBH5 BCSCs. F Interaction between YTHDF2 and LINC00115 was determined by RIP assay in MDA-MB-231 BCSCs with transfection of indicated DNAs. G QRT-PCR analysis of LINC00115 stability. MDA-MB-231 BCSCs and MDA-MB-231 YTHDF2 KD (shYTHDF2-1 and shYTHDF2-2) BCSCs were treated with Actinomycin D. H HIF1α knockdown impaired ALKBH5 expression in MDA-MB-231 and BT549 BCSCs. I Scatter plot of median standard-score-normalized (z-score) expression levels of HIF1α and ALKBH5 across human TNBC metastases after chemotherapy in the TCGA database (n = 160). J Effects of ectopic expression of HIF1α on ALKBH5 expression in LINC00115 KD MDA-MB-231 and BT549 BCSCs. K Effects of ALKBH5 knockdown on SETDB1 association with PLK3. L Effects of ectopic expression of Flag-SETDB1 WT or the H1224K mutant on PLK3 methylation and HIF1α and ALKBH5 expression in MDA-MB-231 BCSCs/tetO-shSETDB1 and BT549 BCSCs/tetO-shSETDB1 cells. MDA-MB-231 and BT549 BCSCs with a tetO-shSETDB1 were pretreated with or without Dox. EV, an empty vector control. M, N Effects of ectopic expression of SETDB1 WT or H1224K mutant on LINC00115 m6A (M) and LINC00115 (N) levels. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. **P < 0.01, by paired two-way Student’s t-test