摘要
目的探讨沉默YWHAE表达对胃癌细胞增殖的影响。方法实验分为三组,未转染组,构建YWHAE短发夹RNA(sh RNA)表达载体及相应空载载体,慢病毒包装后分别感染胃癌MGC803细胞(干扰组,对照组),应用Western blot、RT-PCR方法检测YWHAE沉默效率;MTT技术观察YWHAE表达沉默前后细胞增殖变化;流式细胞术、细胞克隆实验检测细胞周期、凋亡及克隆形成变化情况。结果慢病毒包装YWHAE-sh RNA后成功感染胃癌MGC803细胞;RT-PCR、Western blot检测结果显示,与对照组比较,干扰组MGC803细胞YWHAE基因表达明显降低,抑制效率为72.7%。MTT结果显示,干扰组细胞增长速率明显低于对照组细胞,72 h增长倍数分别为1.56、2.43,差异有显著统计学意义(P<0.01);细胞克隆实验结果显示,干扰组及对照组克隆形成率分别为6.24%、31.00%,差异有显著统计学意义(P<0.01)。流式细胞术检测显示,YWHAE基因表达沉默后,MGC803细胞凋亡率增加,由未消减前的3.23%增加到9.83%,差异有显著统计学意义(P<0.01);相比对照组细胞在G1期的比例(57.88%),干扰组G1期细胞比例明显增加(72.42%),差异有显著统计学意义(P<0.01)。结论沉默YWHAE表达能抑制胃癌MGC803细胞增殖,可能与诱导细胞周期阻滞,促进细胞凋亡有关。
Objective To investigate the effect of YWHAE expression silencing on the proliferation of gastric cancer cells. Methods The expression vector of short hairpin RNA（sh RNA） sequences for YWHAE（interference group） and the control vector（control group） were constructed, which were used to infect the gastric cancer cells MGC803 after they were produced and packaged by lentiviruses. The expression levels of YWHAE were assayed by Western blot and RT-PCR. Proliferation was evaluated by MTT（3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide） colorimetric assay. Colony formation analysis was used to determine growth properties of transduced cells.Cell cycle and apoptosis were determined by flow cytometry. Results Lentivirus-mediated sh RNA-YWHAE was successfully transfected into gastric cancer cells. Compared with the control group, the expression of YWHAE was significantly decreased by 72.7% in the interference group by Western blot, RT-PCR analysis. The results of MTT showed that the proliferation ability of the interference group cells was significantly slower than that of the control group, and the cell growth times were 1.56 and 2.43 for 72 h, respectively（P〈0.01）. Cell cloning experiment results showed that the clone formation rate of the interference group was 6.24%, which was significantly lower than 31.00% of the control group（P〈0.01）. Flow cytometry showed that reduced YWHAE expression in gastric cancer cells lead to the increasing of cell apoptosis rate（from 3.23% to 9.83%）, and the difference was statistically significant（P〈0.01）. Compared with the control group（57.88%）, the proportion of the interference group cells（72.42%） in G1 phase increased significantly（P〈0.01）.Conclusion The expression silencing of YWHAE inhibits the proliferation ability of gastric cancer MGC803 cells,which may be associated with cell apoptosis promotion and cell cycle G1 arrest.
作者
黄幼生
解娜
张艺馨
孙芳娇
罗志飞
薛逢贵
HUANG You-sheng XIE Na ZHANG Yi-xin SUN Fang-jiao LUO Zhi-fei XUE Feng-gui(Department of Pathology, the First Affiliated Hospital of Hainan Medical University, Haikou 571101, Hainan, CHINA)
出处
《海南医学》
CAS
2017年第16期2581-2585,共5页
Hainan Medical Journal
基金
国家自然科学基金(编号:81260321)
关键词
YWHAE
胃癌
RNA干扰
细胞增殖
凋亡
YWHAE
Gastric cancer
RNA interference（RNAi）
Proliferation
Apoptosis
