摘要
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.
AIM： To investigate the effect of inhibition of FUT3 gene expression with miRNAs on the pro- liferation of gastric cancer cells （KATO-III）. METHODS： Vectors carrying two miRNAs targeting the FUT3 gene were constructed andtransiently transfected into KATO-III cells us- ing lipidosome-mediated method. RT-PCR was performed to detect the expression of FUT3 mRNA, and immunocytochemistry and flow cy- tometry analysis were carried out to test expres- sion 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 an- tigen 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 abil- ity 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 target- ing the FUT3 gene can effectively inhibit the proliferation of KATO-III cells.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第25期2341-2346,共6页
World Chinese Journal of Digestology
基金
山东大学科技局科技计划基金资助项目
No.200705086-2
山东大学卫生局科技发展计划基金资助项目
No.2007-17~~
