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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: Long non-coding RNA HUMT hypomethylation promotes lymphangiogenesis and metastasis via activating FOXK1 transcription in triple-negative breast cancer

Fig. 4

HUMT interacted with YBX1 to regulate FOXK1 expression and cell proliferation. a Venn diagram showed that HUMT interacted with YBX1 using PAR-CLIP in two independent biological replicates (GSE133620). b RNA immunoprecipitation (RIP) using the YBX1 antibody followed by qRT-PCR for HUMT. c Validation of the interaction between HUMT and YBX1 by RNA pulldown assay. HUMT antisense biotinylated probes were used as the negative control. d CHIP-seq identified YBX1 binding peaks on the promoter region of FOXK1 in three independent cells (ENCODE). e Schematic illustration of the PCR-amplified region of FOXK1 promoter. f Chromatin immunoprecipitation (CHIP) was performed using the YBX1 antibody to identify the YBX1-binding regions on the FOXK1 promoter. IgG was used as a negative control. g Schematic summary of the dCas9-CHIP. A 3xFLAG-dCas9-HA-2xNLS fusion protein (FLAG-dCas9) consisting of an N-terminal triple FLAG (3xFLAG) epitope tag and catalytically inactive Cas9 endonuclease (dCas9) was expressed with guide RNAs (gRNAs) in appropriate cell context to validate HUMT interaction with FOXK1 promoter. h Enrichment of HUMT on the FOXK1 promoter region. IgG and scrambled gRNA were used as negative controls. i Correlation between YBX1, HUMT, and FOXK1 RNA expression in GSE76124. j FOXK1 expression in YBX1-KD and HUMT-KO cells was detected by qRT-PCR. k FOXK1 expression in YBX1 and HUMT modulated cells by western blot. l Cell proliferation in YBX1-KD and HUMT-KO cells. m Comprehensive analysis of basal-like breast cancer in GEO datasets using bc-GenExMiner-identified FOXK1 to be a prognostic factor for metastatic relapse-free survival. Data were shown as mean ± SD; *P < 0.05; **P < 0.01

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