靶向细胞外信号调节激酶2短发夹式RNA对人食管癌Eca109细胞增殖的影响

目的 观察靶向细胞外信号调节激酶2(ERK2)短发夹式RNA(shRNA-ERK2)对人食管癌细胞株Eca109细胞增殖的影响.方法 构建重组质粒pGeneClipTM-shRNA-ERK2脂质体介导重组质粒转染人食管癌Eca109细胞,荧光倒置显微镜观察转染效率;噻唑蓝(MTT)比色法检测转染食管癌Eca109细胞后细胞的增殖能力;Western blot检测转染后食管癌Eca109细胞ERK2基因的表达和凋亡抑制基因Survivin的表达.结果 测序证实载体构建成功,荧光倒置显微镜观察转染后72 h转染效率50%～70%;转染重组质粒96 h后食管癌Eca109细胞生长抑制率最高为10.45%,与阴性对照组(非特异性序列对照)和空白对照组比较抑制效果明显(P＜0.05);转染重组质粒72 h和96 h后食管癌Eca109细胞株中ERK2和Survivin的表达与U0126对照组和阴性对照组比较均下降(P＜0.05).结论 重组质粒pGeneClipTM-shRNA-ERK2在食管癌Eca109细胞株中能发挥靶基因沉默作用,影响Survivin基因的表达,抑制食管癌细胞增殖.

Effection of short hairpin RNA targeting extracellular signal-regulated kinase 2 on proliferation of Eca109 cells

Objective To study the effect of short hairpin RNA (shRNA) targeting extracellular signal-regulated kinase 2 (ERK2) on proliferation of human esophageal cancer cell line Eca109. Methods The recombinant plasmid (pGeneClipTM-shRNA-ERK2) was constructed and transfected into the human esophageal cancer cell line Eca109 by liposome, and the transfection efficiency was observed by reverse fluorescence microscope. The proliferation ability of esophageal cancer Eca109 cells after transfection was analyzed by methyl thiazolyl tetrazolium (MTF). The expression of ERK2 and inhibitor apoptosis gene Survivin following transfection was detected by Western blotting. Results Sequencing proved that recombinant plasmid was successfully constructed, and the transfection efficiency was about 50%-70% after 72 h following transfection. The Eca109 cell growth at 96 h after transfection was significantly inhibited by 10.45% as compared with negative group and blank group,P ＜0. 05. The protein expression of ERK2 and Survivin was decreased in transfection group as compared with the U0126 group and negative group at 72 h and 96 h ( P ＜ 0. 05 ). Conclusion The recombinant plasmid ( pGeneClipTM-shRNA-ERK2) has been proved not only to be effective for down-regulation of ERK2, but also can affect the expression of Survivin and significantly inhibit the Eca109 cells proliferation.