Fig. 1.

tcons1221 expression is induced upon hypoxia. (A) hierarchical clustering analysis of the top 60 lncRNAs (30 upregulated and 30 downregulated) that were differentially expressed between hypoxia-treated (1% O₂, 24 h) and normoxia-treated (21% O₂) GC cells (MKN45 and MKN28 cell lines). (B) The expression profiling of seven cancer-associated lncRNAs in GC cells under hypoxia (1% O₂, 24 h). (C) MKN45 cells were transfected with siRNA-NC, siRNA-HIF-1α, siRNA-HIF-2α, or siRNA-p53. Twenty-four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 h. The expression levels of target genes were determined by western blot analysis. The levels of CBSLR expression were determined by qRT-PCR. (D) MKN45 cells were transfected with pcDNA3.1-HIF-1α. Twenty-four hours after transfection, the expression level of HIF-1α was determined by western blot analysis. The levels of CBSLR expression were examined by qRT-PCR. (E) Schematic illustration of HIF-1α responsive element (HRE) in CBSLR locus. MKN45 and MKN28 cells were cultured under normoxia or hypoxic conditions (1% O₂) for 24 h, then, a CHIP assay was employed to examine the binding of HIF-1α to each HRE. (F) (Upper) MKN45 cells were cotransfected with Renilla luciferase plasmid and the indicated reporter constructs. (Lower) MKN45 cells expressing shRNA-NC or shRNA-HIF-1a were cotransfected with Renilla luciferase plasmid and reporter constructs containing the sequence around the second HRE site. Twenty-four hours after transfection, cells were cultured under normoxic or hypoxic conditions (1% O₂) for 24 h. Luciferase activity was then determined and normalized to Renilla luciferase activity. Results were expressed as mean ± S.D. (n = 3).