沉默多药耐药基因逆转大肠癌耐药细胞株LoVo/5-氟尿嘧啶细胞耐药性研究

Effect of silencing multi-drug resistance gene 1 on reversing the drug resistance of colorectal cancer drug-resistant cell line LoVo/5-fluorouracil

目的探讨沉默多药耐药基因(multi-drug resistance 1,MDR1)对大肠癌耐药细胞株LoVo/5-氟尿嘧啶(fluorouracil,Fu)细胞耐药性的逆转作用。方法将MDR1逆转的大肠癌耐药细胞株LoVo/5-Fu细胞分为EASYshRNA-MDR1(实验组)、shRNA-control(对照质粒组)和normal(未转染组),3组采用MTT法检测细胞对氟尿嘧啶的敏感性,采用流式细胞仪检测细胞凋亡情况,采用RT-PCR法检测细胞MDR1mRNA表达水平,采用ELISA法检测细胞P-糖蛋白水平,比较3组半数抑制浓度(half maximal inhibitory concentration,IC50)、耐药指数(resistance index,RI)、MDR1mRNA和P糖蛋白表达水平、对氟尿嘧啶的敏感性相对逆转率和细胞凋亡率。结果实验组IC50[(2.197±0.245)g/L]、RI(4.980)、MDR1 mRNA表达水平[(7.380±1.501)×107拷贝/μg]、P-糖蛋白表达水平[(46.569±4.200)%]低于未转染组[(7.283±0.461)g/L、15.221、(8.891±1.362)×108拷贝/μg、(97.810±1.921)%]和对照质粒组[(7.012±0.027)g/L、14.928、(7.346±1.201)×108拷贝/μg、(95.301±2.334)%](P<0.05),对5-Fu的敏感性相对逆转率(72.2%)、细胞凋亡率[(20.904±0.493)%]高于未转染组[0、(6.215±0.721)%]和对照质粒组[3.2%、(6.580±0.188)%](P<0.05);未转染组与对照质粒组各指标比较差异均无统计学意义(P>0.05)。结论 RNA干扰可明显抑制P-糖蛋白表达,降低LoVo/5-Fu细胞对药物的耐药性,通过促进细胞凋亡和在mRNA环节中表达而降低耐药性。

Objective To explore the effect of silencing multi-drug resistance gene 1 （MDR1） on reversing the drug resistance of colorectal cancer drug-resistant cell line LoVo/5-fluorouracil（Fu）. Methods MDR1 reversed colorectal cancer drug-resistance cell line LoVo/5-Fu cells were divided into EASY-shRNA-MDR1 group, shRNA control group and no-transfection group. The LoVo/5-Fu sensitivity was detected by MTT, apoptosis was detected by flow cytometer, and MDR1 mRNA level was detected by ELISA. The half maximal inhibitory concentration （IC50）, resistance index （RI）, the expression of MDR1 mRNA p-glycoprotein （P-gp）, the sensitivity relative reverse transcription rate to 5-Fu and apoptosis rate were detected and compared among three groups. Results IC50 （（2. 197 ± 0. 245） g/L）, RI （4. 980）, MDR1 mRNA （（7. 380±1. 501）× 10^7 copies/μg） and P-gp （（46. 569 ± 4. 200）%） were significantly lower in EASY- shRNA-MDR1 group than those in no-transfection group （（7. 283 ±0. 461） g/L, 15. 221, （8. 891±1. 362）× 10^8 copies/μg, （97. 810±1. 921）%） and shRNA control group （（7. 012 ±0. 027） g/L, 14. 928, （7. 346±1. 201） × 10^8 copies/μg, （95. 301±2. 334）%） （P〈 0.05）. The sensitivity relative reverse transpcription rate to LoVo/5-Fu （72.2%） and the apoptosis rate （（20. 904 ± 0. 493）%） were significantly higher in EASY-shRNA-MDRI group than those in no-transfection group （0, （6. 215±0. 721）%） and shRNA control group （3.2%, （6.580±0. 188）%） （P〈 0.05）, and there were no significant differences between shRNA-control group and no-transfection group （P〉0.05）. Conclusion RNA interference can significantly inhibit the expression of P-gp and reduce drug resistance of LoVo/5-Fu by promoting the apoptosis and mRNA expression.