FUT3-miRNA重组质粒对人胃癌KATO-III细胞增殖的影响

目的:研究利用miRNA干扰技术抑制FUT3基因表达对人胃癌KATO-Ⅲ细胞增殖的影响.方法:将前期实验构建成功的2对针对FUT3基因的特异性miRNA表达载体,用脂质体转染入人胃癌KATO-Ⅲ细胞,RT-PCR检测FUT3基因表达水平的变化;免疫细胞化学法、流式细胞术检测其合成抗原sLeA表达变化;MTT法、克隆形成实验检测FUT3基因的表达抑制对KATO-Ⅲ细胞增殖的影响.结果:转染FUT3-miRNA的2个干扰组FUT3基因mRNA相对表达量分别为0.41±0.01,0.36±0.02,明显低于对照组(0.71±0.05)和空载体组(0.65±0.03,P<0.05);细胞表面合成抗原sLeA的表达水平,FUT3-miRNA1组为35.51%±0.36%,FUT3-miRNA2组为26.05%±1.14%,明显低于对照组(52.79%±2.62%)与空载体组(49.75%±1.29%,P<0.05);与对照组(5.60%±0.63%)和空载体组(8.90%±0.91%)相比较,FUT3-miRNA1组(38.10%±1.96%)和FUT3-miRNA2组(49.04%±2.37%)能明显抑制细胞的增殖(P<0.05);细胞的克隆形成能力FUT3-miRNA1组(14.10%±1.70%)和FUT3-miRNA2组(12.50%±1.96%),显著低于对照组(29.79%±3.05%)和空载体组(28.92%±2.10%,P<0.05).结论:FUT3靶向miRNA真核表达载体可有效抑制胃癌细胞的增殖能力.

Transfection of miRNAs targeting the FUT3 gene inhibits cell proliferation in human gastric cancer cell line KATO-III

AIM:To investigate the effect of inhibition of FUT3 gene expression with miRNAs on the proliferation of gastric cancer cells(KATO-Ⅲ).METHODS:Vectors carrying two miRNAs targeting the FUT3 gene were constructed and transiently transfected into KATO-III cells using lipidosome-mediated method.RT-PCR was performed to detect the expression of FUT3 mRNA,and immunocytochemistry and flow cytometry analysis were carried out to test expression variation of sLeA antigen.MTT assay and colony-forming assay were used to analyze cell proliferation and to detect the effect of decreased FUT3 expression on cell growth.RESULTS:Compared to non-transfected cells and cells transfected with empty vector,the relative expression levels of FUT3 mRNA were significantly decreased(0.41 ± 0.01 vs 0.71 ± 0.05,0.65 ± 0.03,both P < 0.05;0.36 ± 0.02 vs 0.71 ± 0.05,0.65 ± 0.03,both P < 0.05);the sLeA antigen expression levels were also significantly reduced(35.51% ± 0.36% vs 52.79% ± 2.62%,49.75% ± 1.29%,both P < 0.05;26.05% ± 1.14% vs 52.79% ± 2.62%,49.75% ± 1.29%,both P < 0.05);cell growth was significantly inhibited(38.10% ± 1.96% vs 5.6% ± 0.63%,8.9% ± 0.91%,both P < 0.05;49.04% ± 2.37% vs 5.6% ± 0.63%,8.9% ± 0.91%,both P < 0.05);and colony-forming ability was significantly reduced(14.10% ± 1.70% vs 29.79% ± 3.05%,28.92% ± 2.10%,both P < 0.05;12.50% ± 1.96% vs 29.79% ± 3.05%,28.92% ± 2.10%,both P < 0.05) in FUT3-miRNA and FUT3-miRNA2 transfeced cells.CONCLUSION:Transfection of miRNAs targeting the FUT3 gene can effectively inhibit the proliferation of KATO-Ⅲ cells.