RNAi对结肠癌HCT-8细胞桩蛋白表达的沉默作用观察

目的观察RNA干扰（RNAi）对结肠癌HCT-8细胞桩蛋白（Paxillin）表达的沉默作用。方法构建针对Paxillin基因序列的短发卡RNA（shRNA）表达载体,并转染至结肠癌HCT-8细胞,72 h后分别用实时定量PCR和Western blot法测定转染细胞中Paxillin mRNA及蛋白。结果根据针对Paxillin基因设计了小干扰RNA（siRNA）序列,构建了shRNA表达载体,并成功转染至HCT-8细胞。以空白对照组作均一化,转染Paxillin-shRNA_1、Paxillin-shRNA_2、Paxillin-shRNA_3、Paxillin-shRNA_NC的细胞及空白对照组细胞Paxillin mRNA相对表达量分别为0.66±0.28、0.31±0.11、1.00±0.22、1.05±0.32、1.00±0.29,Paxillin蛋白相对表达量分别为0.62±0.11、0.39±0.13、0.74±0.21、0.94±0.09、1.00±0.16。结论 RNAi可沉默结肠癌HCT-8细胞Paxillin的表达。

Silencing expression of Paxillin gene in colon cancer HCT-8 cells by RNA interference

Objective To observe the interference effect of RNA interference（RNAi） for silencing the the expression of Paxillin gene in colon cancer HCT-8 cells.Methods According to Paxillin＇s gene sequence of Genebank to construct paxillin gene targeted short harpin RNA（shRNA）,and transfect to human colon cancer HCT-8 cells.The Paxillin gene in transfected cells at mRNA and protein was detected after transfected for 72 h by RT-PCR and Western blot respectively.Results shRNA sequence was designed according to small interfering RNA（siRNA） oligonucleotides and its expression vector were constructed and transfected into HCT-8 cells successfully.The cell strain were divided into Paxillin-shRNA_1,Paxillin-shRNA_2,Paxillin-shRNA_3,Paxillin-shRNA_NC group and un-treated group.The expression of Paxillin mRNA after transfection were 0.66 ± 0.28,0.31 ± 0.11,1.00 ± 0.22,1.05 ± 0.32,1.00 ± 0.29 respectively,while Paxillin protein expression levels were 0.62 ± 0.11,0.39 ± 0.13,0.74 ± 0.21,0.94 ± 0.09 and 1.00 ± 0.16.Conclusion RNAi can decrease the expression of Paxillin in colon cancer HCT-8 cells.