摘要目的 探讨未成熟同源蛋白增强子(ERH)基因敲除对人膀胱癌T24细胞增殖和凋亡的影响.方法 通过克隆有干扰ERH基因序列的慢病毒感染T24细胞,建立稳定的ERH基因抑制的T24细胞克隆,采用实时荧光定量聚合酶链反应(qPCR)检测ERH mRNA的表达.通过四甲基偶氮唑盐(MTT)法、克隆形成法和流式细胞术检测ERH基因敲除对细胞增殖和凋亡的影响.通过裸鼠皮下成瘤实验验证体内ERH基因敲除对T24细胞成瘤效果的影响.结果 转染慢病毒后,经qPCR检测,ERH基因敲除效果满意[敲除组ERH mRNA相对表达量为0.079±0.007,未经处理的T24细胞(ERH正常组)为1.006±0.126;t=12.72,P=0.0002].MTT实验结果提示,敲除ERH基因的T24细胞增殖能力减弱[ERH敲除组490 nm波长处吸光度(A490)值为0.13±0.00,ERH正常组为0.66±0.01;t=104.61,P<0.0001].克隆形成实验结果提示其克隆能力减弱[ERH敲除组克隆数为(10.5±1.2)个,ERH正常组为(196.4±4.0)个;t=73.63,P<0.0001].流式细胞术检测结果示细胞凋亡率增加[ERH敲除组凋亡率为(11.0±0.5)％,ERH正常组为(4.2±0.5)％;t=16.06,P<0.0001].裸鼠皮下成瘤实验活体成像结果显示,ERH敲除组肿瘤区域总荧光强度为(4.67±0.59)×1010μW/cm2,ERH正常组相应部位为(9.54±4.20)×1010μW/cm2,差异有统计学意义(t=3.64,P=0.0051);ERH敲除组肿瘤重量为(0.80±0.62)g,ERH正常组为(1.79±0.71)g,差异有统计学意义(t=3.33,P=0.0037).结论ERH基因敲除可以抑制人膀胱癌T24细胞的增殖,促进其凋亡.

abstractsObjective To investigate the effect of ERH gene knockdown on the proliferation and apoptosis of human bladder cancer T24 cells. Methods T24 cells infected by lentivirus with interference on ERH gene sequence were cloned to establish stable T24 cells clone in ERH gene suppression. The expression of ERH mRNA gene in bladder cancer was detected by using quantitative real time polymerase chain reaction&nbsp;(qPCR). The effects of ERH knockout on the cell proliferation and apoptosis were examined by using methylthiazolyl tetrazolium (MTT) assay, colony formation assay and flow cytometry. The effect of ERH knockout on the tumorigenic effect of T24 cells in vivo was verified by subcutaneous tumor formation in nude mice. Results After lentiviral transfection, qPCR results showed that the knockdown effect of ERH mRNA in ERH normal group (untreated T24 cells) was better than that in ERH gene knockdown group, and the difference was statistically significant [(1.006±0.126) vs. (0.079±0.007); t=12.72, P=0.0002]. After knocking out ERH gene, MTT assay showed that the proliferation ability of T24 cells in ERH gene knockdown group was weakened compared with ERH normal group, and the difference was statistically significant [A490 value: (0.13±0.00) vs. (0.66±0.01);t=104.61, P<0.0001]. Colony formation assay indicated that the ability of clone in ERH normal group was weakened compared with ERH gene knockdown group [(10.5 ±1.2) vs. (196.4 ±4.0); t= 73.63, P< 0.0001]. Flow cytometry showed that the cell apoptosis rate in ERH gene knockdown group was higher than that in ERH normal group [(11.0 ±0.5) ％ vs. (4.2 ±0.5) ％; t= 16.06, P<0.0001]. Imaging results of subcutaneous tumor formation in nude mice showed that the total fluorescence intensity of the tumor area in ERH gene knockdown group was (4.67 ±0.59) × 1010 μW/cm2, and the corresponding part in ERH normal group was (9.54±4.20) × 1010μW/cm2 (t=3.64, P=0.0051);tumor weight in ERH gene knockdown group was (0.80±0.62) g, and in ERH normal group was (1.79±0.71) g (t=3.33, P=0.0037). Conclusion ERH gene knockout can inhibit the proliferation of human bladder cancer T24 cells, and promote the cell apoptosis.