CD24 knockdown in TMK-1 cells dramatically decreased cell invasion activities in both normal and hypoxia conditions
CD24 knockdown in TMK-1 cells dramatically decreased cell invasion activities in both normal and hypoxia conditions. cell surface protein that is expressed in putative stem cells and is overexpressed in various human malignancies, yet the significant functions of CD24 in gastric cancer development are still elusive. We investigated the involvement of CD24 in gastric cancer aggressiveness, which is usually attributed to its heterogeneity. Cultured gastric cancer cells showed diverse expression patterns in CD24, whereas other defined cell surface markers, such as CD44 and CD133, were homogenous. Purely sorted CD24-unfavorable gastric cancer cells showed strong alteration into the CD24-positive cell type in an autochthonous manner, and reached to constant TPCA-1 expression levels. Our clinicopathological study revealed that CD24 positivity was an independent prognostic factor in both intestinal and diffuse types of gastric cancer. CD24 expression was correlated with the advanced TPCA-1 stages, invasiveness, and lymph node metastasis of gastric cancer. Silencing of CD24 in cultured cells significantly decreased cell migration and invasion. Hypoxic treatment TPCA-1 upregulated CD24 expression, and simultaneously induced cell motility and invasion of gastric cancer cells. Hypoxic treatment-induced CD24 expression was significantly attenuated by knockdown of hypoxia-inducible transcription factors. These data suggest that CD24-unfavorable cells are capable of gaining cell motility and invasiveness through the induction of CD24, which is usually mediated by hypoxia. CD24 would be a stylish marker to define not only the heterogeneity but also the aggressiveness of gastric cancer cells. The mechanisms by which hypoxia induces CD24 expression would also be a potential therapeutic target for gastric cancer. 0.05; **0.01. CD24 expression was induced by hypoxia in gastric cancer cells We attempted to explore TPCA-1 the mechanisms of how CD24 expression is regulated in GCa. In the 5-flanking region of up to ?3.4 kb upstream from the transcription start site (National Center for Biotechnology Information; accession “type”:”entrez-nucleotide”,”attrs”:”text”:”Y14692″,”term_id”:”2765419″,”term_text”:”Y14692″Y14692), there are some consensus sequences that might be bound by several transcriptional factors, all of which might be potential molecules to induce cancer aggressiveness (Fig. ?(Fig.4a,4a, left panel). In these upstream promoter elements, we focused on the hypoxic responsive element (HRE) since low oxygen concentrations can directly influence stem cell renewal and differentiation(36) and is essential for the maintenance of those stemness.(37) Open in a separate window Fig 4 Induced CD24 expression in TMK-1 cells by hypoxia. (a) Localization TPCA-1 of the putative binding sites of several transcriptional factors in the region of the Rabbit Polyclonal to CCBP2 CD24 promoter (left panel). Western blot analyses of HIF-1 and HIF-2 in TMK-1 cells under hypoxia (1% O2) for up to 72 h (right panel). (b) FACS analyses for the positive rates of CD24, CD44, and CD133 expressions in TMK-1 cells under hypoxia (1% O2, left panel). Western blot analyses of HIF-1 and HIF-2 in TMK-1 cells cultured in hypoxia (1% O2) for 48 h (right panel). (c) FACS analyses of CD24 positive populace in TMK-1 cells transfected with siControl, or siHIF-1 and/or siHIF-2 and cultured in normoxia or hypoxia for 48 h (left panel). Western blot analyses of HIF-1 and HIF-2 in each corresponding cell (right panel). Paired cells were used for FACS to determine the percentage of cells in CD24-positive groups. *0.05; **0.01. To examine our hypothesis that low oxygen tension would recess CD24 expression in GCa, hypoxic culture was performed on GCa. When TMK-1 was exposed to hypoxia for up to 72 h, HIF-1 was firstly stabilized within 24 h in hypoxia, and then HIF-2 was upregulated subsequently at 24 h onwards (Fig. ?(Fig.4a,4a, right panel). Concomitantly with the increased HIF-2, CD24 expression rather increased gradually from 63% to 82% (48 h; = 0.0007) and to 87% (72 h; = 0.0002), whereas the expression level of other cell surface markers such as CD44 and CD133 were not influenced by hypoxia (Fig. ?(Fig.4b,4b, left panel). Hypoxic treatment within 72 h didn’t influence the viability of TMK-1 cells (data not shown). Cellular responses to low oxygen tension were also monitored by immunoblotting to measure the stabilization of HIF-1 and HIF-2 in the nuclear fraction of TMK-1 cells at the time point of 48 h in hypoxia (Fig. ?(Fig.4b,4b,.