Aim : To observe the effects of single-strand ( shRNAg) on the expression of p70S6K in EC9706 cells. Methods; According to the sequence of p70S6K mRNA,shRNAs were synthesized. Sense and antisense shRNA were annealed. The annealed products and the linear pSIREN-RetroQ-DsRed-Express were ligated by T4 ligase to create a recombi-nant plasmid pshRNA-p70S6K, and then the plasmid was transfected to EC9706 cells by Lipofectamine?2000. The expression levels of mRNA and p-p70S6K were analyzed, respectively, using RT-PCR and Western blot. EC9706 cells without tansfection was used as control. Results: The expression levels of p70S6K mRNA and p-p70S6JC in the 6 groups had significant differences (F = 106. 343,721. 632,P <0. 001). The expression of p70S6K mRNA decreased at 24,48 and 72 h after transfection(P < 0.05 ) , which come to the lowest at 48 h. The expression of p-p70S6K decreased at 24,48,72 and 96 h after transfection(P <0.05 ) .which come to the lowest at 48 h. Conclusion:p70S6K specific shRNA can downregulate the expressions of p70S6K mRNA and p-p70S6K in EC9706 cells.%目的:观察核糖体40 s小亚基S6蛋白激酶(p70S6K)特异性短发夹RNA(shRNA)对EC9706细胞p70S6K表达的影响.方法:根据GenBank p70S6K的mRNA序列,设计并合成p70S6K特异性的shRNA序列,退火形成双链后插入pSIREN-RetroQ-DsRed-Express载体,构建表达载体pshRNA-p70S6K,转染EC9706细胞.采用RT-PCR和Western blot方法分别检测转染24、48、72、96及120 h后细胞中p70S6K mRNA和磷酸化p70S6K的表达,设未转染的EC9706细胞为对照.结果:各组细胞p70S6K mRNA和磷酸化p70S6K蛋白的表达差异有统计学意义(F=106.343,721.632,P均＜0.001).转染细胞p70S6K mRNA的表达在转染24、48和72 h后均较对照组下降(P＜0.05),在第48 h表达最低;磷酸化p70S6K蛋白的表达在转染24、48、72和96 h后均较对照组下降(P＜0.05),第48 h表达最低.结论:p70S6K特异性shRNA能够下调EC9706细胞中p70S6K mRNA和蛋白的表达.
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