STAT3真核表达质粒的构建及对胃癌细胞增殖的影响

Construction of STAT3 eukaryotic over-expression vector and its effects on the proliferation of gastric cancer cells

目的构建人信号转导和转录激活因子3(STAT3)真核表达载体,研究STAT3基因对人胃癌细胞株MGC803的影响。方法构建pcDNA3-STAT3真核表达质粒,经脂质体介导转染胃癌细胞株MGC803。RT-PCR和Western blotting检测转染后STAT3、P-STAT3(Y705)、P-STAT3(S727)、Survivin和PCNA在MGC803细胞中mRNA和蛋白水平的表达,CCK-8法检测pcDNA3-STAT3对细胞MGC803的影响。结果测序及酶切鉴定证实,真核表达质粒pcDNA3-STAT3构建成功。Western blotting和RT-PCR实验结果证实,与转染空载体组相比,转染重组质粒组STAT3、P-STAT3(Y705)、P-STAT3(S727)、Survivin和PCNA在蛋白水平和mRNA水平的表达均明显升高,差异有统计学意义(P<0.05)。CCK-8实验结果显示,与转染空载体组相比,转染重组质粒组人胃癌细胞株MGC803增殖能力明显升高,差异有统计学意义(P<0.05)。结论真核表达质粒pcDNA3-STAT3可以提高STAT3基因在人胃癌细胞株MGC803中的表达,并且增强胃癌细胞的增殖能力。

Objective To investigate the effects of overexpression human signal transducer and activator of transcription 3 （STAT3） on human gastric cancer cell line MGC803 by the construction of a STAT3 gene eukaryotic expression vec- tor. Methods The eukaryotic expression vector pcDNA3-STAT3 was constructed and transfected into the gastric canc- er cell line （ MGC803 ） by the liposome. The mRNA and protein expression levels of STAT3, P-STAT3 （ Y705 ）, P-STAT3 （$727）, Survivin and PCNA in MGC803 were detected by RT-PCR and Western blotting. The effects of STAT3 on MGC803 cell proliferation were detected through the CCK-8 assay. Results The DNA sequencing and re- striction endonuclease identification results confirmed that the eukaryotic expression plasmid pcDNA3-STAT3 was suc- cessfully constructed. Western blotting and RT-PCR results confirmed that STAT3, P-STAT3 （Y705）, P-STAT3 （ $727 ）, Survivin and PCNA protein and mRNA expression levels were significantly increased in the recombinant plas- mid group compared with the empty vector group. CCK-8 results proved that, compared with the empty vector group, the recombinant plasmid group showed higher proliferative capacity, and the difference was statistically significant （ P 〈 0.05 ）. Conclusion eukaryotic expression plasmids of pcDNA3-STAT3 can increase the expression of STAT3 gene in human gastric cancer cell line MGC803, and enhance the proliferation of gastric cancer cells.