247 2 040 ± 0 360 2 531 ± 0 524 * P > 0 05, compared with EC9706/

247 2.040 ± 0.360 2.531 ± 0.524 * P > 0.05, compared with EC9706/pcDNA3.1 ECRG4 overexpression blocked cell cycle click here progression The stable-transfected EC9706/pcDNA3.1-ECRG4 cells exhibited detectable ECRG4 protein expression compared with EC9706/pcDNA3.1 cells, as shown in Figure 1B. The percentages of cells in the G1, S and G2/M phase of cell cycle demonstrated that overexpression of ECRG4 in EC9706 cells resulted in an accumulation of cells in G1 phase and a decrease in S and G2/M phase compared with EC9706/pcDNA3.1 control cells (P < 0.05) (Table 2). Flow cytometric analysis suggested that ECRG4 overexpression

could arrest EC9706 cells at the G1/S checkpoint and delay cell cycle into S phase. Consequently, ECRG4 overexpression slowed down cell cycle

progression and caused cell cycle G1 phase block. Table 2 ECRG4 overexpression caused cell cycle G1 phase block Group G1 S G2/M EC9706/pcDNA3.1-ECRG4* 73.7 ± 1.86 Small molecule library purchase 14.8 ± 1.13 11.5 ± 0.92 EC9706/pcDNA3.1 59.8 ± 2.06 25.0 ± 1.39 15.2 ± 1.64 * P < 0.05, compared with EC9706/pcDNA3.1 ECRG4 may be involved in p53 pathway In exploring the molecular mechanism of cell cycle G1 phase block caused by ECRG4 overexpression in EC9706 cells, we found that p53 and p21 protein expression levels were increased in EC9706/pcDNA3.1-ECRG4 cells compared with in EC9706/pcDNA3.1 cells (Figure 4). It indicated that ECRG4 may be involved in p53 pathway in ESCC. ECRG4 might induce p21 upregulation through p53 pathway to block cell cycle progression in ESCC. Figure 4 ECRG4 may be involved selleck screening library in p53 pathway. Representative photos and statistic plots of relative protein expression levels in EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1. Analysis of cell’s total proteins by Western blot showed that p53 and p53 target gene p21 expressions were increased in EC9706/pcDNA3.1-ECRG4 cells

compared with in EC9706/pcDNA3.1 cells (P < 0.05). Lane 1: EC9706/pcDNA3.1-ECRG4; Lane 2: EC9706/pcDNA3.1. *, P < 0.05, compared with EC9706/pcDNA3.1. Discussion ESCC is a highly invasive and clinically challenging cancer in China, and its molecular basis GNA12 remains poorly understood. ECRG4 is a novel gene identified and cloned in our laboratory [5, 6]. ECRG4 gene is highly conserved among various species, suggesting an important role for ECRG4 in eukaryotic cells [10]. However, its exactly biological function in carcinogenesis is still unclear. Our previous study demonstrated that ECRG4 gene promoter hypermethylation accounted for decreased expression in ESCC, and the low expression of ECRG4 protein in patients with ESCC was associated with poor prognosis [7, 8]. These findings were also supported by similar studies of other research groups [11, 12]. Furthermore, restoration of ECRG4 expression in ESCC cells inhibited tumor cells growth in vitro and in vivo [7, 8].

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