Lu Shi, Huang Qi, Wang Zehua, Song Yinfeng, Wang Lijun
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2009 Oct;29(5):620-4. doi: 10.1007/s11596-009-0517-2. Epub 2009 Oct 11.
In order to investigate the effects of vector-based hairpin small interference RNA (shRNA) on the reversal of multi-drug resistance (mdr) of A2780/Taxol cells, a novel vector pEGFP-H1/mdr1 containing mdr1-shRNA targeting at position 2943-2963 of mdr1 was designed and synthesized. Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/mdr1, and the expression of mdr1 mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration (IC(50)) of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdr1 mRNA was decreased to (52.1+/-1.0)% and (0.01+/-1.7)%, and that of P-gp decreased to (88.3+/-2.1)% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdr1, and then administrated Taxol. The tumor volume in pEGFP-H1/mdr1-transfected group was significantly reduced as compared with that in blank control group or pEGFP-H1-transfected group (807.20+/-103.16 vs 1563.78+/-210.54 or 1480.78+/-241.24 mm(3), both P<0.01). These results suggested that transfection of pEGFP-H1/mdr1 could efficiently down-regulate the expression of mdr1 mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol cells to Taxol both in vitro and in vivo.
为研究基于载体的发夹状小干扰RNA（shRNA）对A2780/Taxol细胞多药耐药（mdr）逆转的影响，设计并合成了一种新型载体pEGFP-H1/mdr1，其含有靶向mdr1基因2943 - 2963位的mdr1-shRNA。随后，用pEGFP-H1/mdr1转染A2780/Taxol细胞，分别采用RT-PCR和Western blot检测mdr1 mRNA和P-糖蛋白（P-gp）的表达。用MTT法测定紫杉醇对A2780/Taxol细胞的半数抑制浓度（IC50）。结果显示，转染后第24小时和48小时，mdr1 mRNA表达分别降至（52.1±1.0）%和（0.01±1.7）%，P-gp表达分别降至（88.3±2.1）%和0%。转染后第48小时，A2780/Taxol细胞对紫杉醇的相对逆转率为69.54%。在体内，对裸鼠移植瘤注射pEGFP-H1/mdr1，然后给予紫杉醇。与空白对照组或pEGFP-H1转染组相比，pEGFP-H1/mdr1转染组的肿瘤体积显著减小（807.20±103.16 vs 1563.78±210.54或1480.78±241.24 mm3，均P<0.01）。这些结果表明，转染pEGFP-H1/mdr1可有效下调A2780/Taxol细胞中mdr1 mRNA和P-gp的表达，并在体外和体内有效恢复A2780/Taxol细胞对紫杉醇的敏感性。
