转染ELF5mRNA对子宫内膜癌细胞凋亡及周期的影响

Effect of Transfection of ELF5 mRNA on Apoptosis and Cycle of Endometrial Carcinoma Cells

目的:检测人子宫内膜癌Ishikawa细胞转染pc DNA3.1-ELF5+EGFP后ELF5基因的表达,并观察其对Ishikawa细胞凋亡情况及周期变化的影响。方法:通过脂质体介导的方法将pc DNA3.1-ELF5+EGFP真核细胞表达载体体外转染至子宫内膜癌Ishikawa细胞,设立实验组(转染pc DNA3.1-ELF5+EGFP真核载体)、空质粒组(转染pc DNA3.1-EGFP空载体)、空白对照组(未经转染的Ishikawa细胞)。体外实验:用qRT-PCR法检测子宫内膜癌Ishikawa细胞中ELF5mRNA的表达情况,MTT法检测ELF5mRNA对Ishikawa细胞增殖的影响,流式细胞仪检测三组细胞周期及凋亡情况,Western blot法检测三组肿瘤组织的细胞周期调控蛋白P21及凋亡调控因子Caspase-3表达情况的变化。体内实验:将三组细胞分别接种于NOD/SCID雌性裸鼠,观察肿瘤生长情况及测定成瘤能力,应用TUNEL法检测三组肿瘤组织细胞凋亡的情况,采用蛋白印迹法检测三组肿瘤组织的细胞周期调控蛋白P21及凋亡调控因子Caspase-3表达情况的变化。结果:重组质粒pc DNA3.1-ELF5+EGFP成功转染至子宫内膜癌Ishikawa细胞;重组质粒组细胞ELF5mRNA的表达明显高于空质粒组及空白对照组,差异均有统计学意义(P<0.05);重组质粒组各时间点的细胞增殖能力较较其他两组明显下降,差异均有统计学意义(P<0.05);重组质粒组G_0/G_1期的细胞比例均高于空质粒组及空白对照组,差异均有统计学意义(P<0.05);重组质粒组细胞凋亡率分别与空质粒组及空白对照组比较,差异均有统计学意义(P<0.05);重组质粒组细胞中P21及Caspase-3的表达明显高于空质粒组及空白对照组,差异均有统计学意义(P<0.05)。重组质粒组裸鼠体内的成瘤能力(瘤组织最大直径、重量)明显低于空质粒组及空白对照组,差异均有统计学意义(P<0.05);重组质粒组裸鼠体内瘤组织细胞凋亡率分别与空质粒组及空白对照组比较,差异均有统计学意义(P<0.05);(9)重组质粒组裸鼠体内瘤组织P21及Caspase-3的表达明显高于空质粒组及空白对照组,差异均有统计学意义(P<0.05)。结论:ELF5基因可抑制子宫内膜癌Ishikawa细胞的增殖,将细胞周期阻滞于G_0/G_1期,并诱导其凋亡,其机制可能与P21及Caspase-3有关。

Objective：To study the effects of ELF5 gene on cycles and apoptosis of human endometria cancer cells and its probable mechanism.Methods： The eukaryotic expression vectedical or pcDNA3.1-ELF5＋EGFP was transfected into human endometria cancer cells in vitro （recombinant plasmid group）, and positive cell clones were selected by G418 and amplified.Transfected pcDNA3.1-EGFP cells （empty plasmid group） and untransfected cells （blank control group） were served as control groups.In vitro, the cell prolifetraion ability of three groups was analyzed by MTT;the cycle and apoptosis was analyzed by flow cytometry;the expression of protein P21 and Caspase-3 was detected by Western blot.In vivo, three groups of cells were transplanted into NOD/SCID nude mice, and observed the growth ability of tumors;the apoptosis of tumors was determined by TUNEL;the expression of protein P21 and Caspase-3 in tumors was detected by Western blot.Results： The recombinant plasmid pcDNA3.1-ELF5＋EGFP was successfully transfected into human endometria cancer cells;The expression of ELF5 mRNA in the recombinant plasmid group was higher than that in the empty plasmid group and blank control group （P〈0.05）;At each time point, the cell proliferation ability of the recombinant plasmid group was significantly decreased than that of the empty plasmid group and blank control group, the differences were statistically significant （P〈0.05）;The number of cells arrested in G0/G1 phase of the recombinant plasmid group was higher than that of the empty plasmid group and blank control group, the differences were statistically significant （P〈0.05）;The apoptosis rate of the recombinant plasmid group was compared with the empty plasmid group and the blank control group, the differences were statistically significant （P〈0.05）;The expression of protein P21 and Caspase-3 in the recombinant plasmid group was higher than that in the empty plasmid group and blank control group （P〈0.05）.In vivo, the growth ability of tumors （maximum diameter, weight） in recombinant plasmid group was lower than that in the empty plasmid group and blank control group, the differences were statistically significant （P〈0.05）;In vivo, the apoptosis rate of the recombinant plasmid group was compared with the empty plasmid group and the blank control group, the differences were statistically significant （P〈0.05）;In vivo, the expression of protein P21 and Caspase-3 in the recombinant plasmid group was higher than that in the empty plasmid group and blank control group （P〈0.05）.Conclusions： ELF5 gene has obvious effects on cycles and apoptosis of human endometria cancer cells and its mechanism connected with the protein P21 and Caspase-3.