Fig 1.
The level of HOTAIR expression in CSC-MCF7 and CSC-MB231 populations.
(A) Enrichment of CSC subpopulations from MCF7 and MB231 cells were incubated and photographed on day 5 and day 17. (B) Colony formation by CSCs and their original cells on soft agar when seeded at 103 cells per well or 104 cells per well. (C) Colonies in each well were counted after staining with 0.05% crystal violet. (D) Tumor formation ability of CSCs or their original cells at different amounts was measured in mice, and the tumor volumes were measured from day 10 to day 32. (E) RT-qPCR measurement of the expression levels of MALAT-1, HOTAIR, HN-1 and HIF1A-AS in CSC-MCF-7 or CSC-MB231. *P<0.05.
Fig 2.
Expression of HOTAIR in CSC-MCF7 regulates proliferation, colony formation, migration and self-renewal capacity.
(A) Detection of eGFP confirmed the successful infection of LV-HOTAIR or LV-HOTAIRKD. (B) A CCK-8 assay was performed to detect proliferation in MCF7 cells transfected with LV-HOTAIR, LV-scrambled or LV-HOTAIRKD. (C) A colony formation assay was performed in MCF7 cells transfected with LV-HOTAIR, LV-scrambled or LV-HOTAIRKD (Left panel); the colonies in each well were counted after nitro blue tetrazolium (NBT) staining (Right panel). (D) A migration assay was performed in uncoated transwell chambers (Left panel); the cells that migrated through the membrane were stained with 0.05% crystal violet and counted (Right panel). (E) Self-renewal capacity was detected in serum-free medium; spheres (>40 μm in diameter) were counted per well at different passages. *P<0.05.
Fig 3.
Expression of HOTAIR transcriptionally inhibits expression of miR-34a.
(A) The levels of miR-16, miR-29b, miR-34a, miR-34c, miR-375 and miR-101 expression were detected in CSC-MCF7 or CSC-MB231 by RT-qPCR. (B) After transfection of LV-scrambled, LV-HOTAIRKD, or LV-HOTAIR into CSC-MCF7 cells, the miRNAs mentioned above were detected by RT-qPCR. (C) HOTAIR truncations were cloned. (D) After the transfection of the truncations, the levels of miR-34a and miR-34c in transfected CSC-MCF7 cells were detected by RT-qPCR. (E) For comparison of the effect of HOTAIR and miR-34a on proliferation, CCK-8 assays were performed in these transfected cells. (F) After co-transfection of LV-HOTAIR with miR-34a mimics or scrambled mimics, CCK-8 assays were performed. *P<0.05, **P<0.01.
Fig 4.
HOTAIR regulates the Sox2 expression via miR-34a and affects CSC self-renewal capacity.
(A) The levels of Bmi1, β-catenin, c-Myc, Nanog, Sox2 and Oct4 expression in CSC-MCF7 cells transfected with LV-HOTAIRKD, LV-scrambled or LV-HOTAIR were detected by RT-qPCR. (B) The mRNA levels (Left panel) and protein levels (right panel) of Sox2 in CSCs were analyzed by RT-qPCR and semi-quantitative Western blotting. (C) To detect the effects of HOTAIR or miR-34a on CSC self-renewal capacity, a sphere formation assay was performed. (D) To detect the effects of HOTAIR or miR-34a on colony formation, a soft agar assay was performed in which the colonies per well were counted. *P<0.05.
Fig 5.
HOTAIR modifies the p53/p21 pathway and thus regulates CSC proliferation and colony formation.
(A) RT-qPCR and semi-quantitative western blotting were applied to detect the mRNA and protein levels of p53 and p21 in original or CSC-MCF7 and CSC-MB231 cells. (B) After introduction of HOTAIR or shRNA targeting HOTAIR into CSCs, expression of p53 and p21 was detected. (C and D) A ChIP assay was performed to detect the binding of native p53 in MCF7 cells (C) or ectopic expressed Flag-P53 in CSCs (D) to the p21 promoter region. (E) A CCK-8 assay to detect CSC-MCF7 cells transfected with Flag-p53R248Q or Flag-p53. (F and G) CCK-8 (F) and colony formation (G) assays to detect the proliferation rate of CSC-MCF7-LV-HOTAIR with p53 reintroduction and co-culture with 30 μM PFTα. *P<0.05.