αv整合素沉默对结肠癌多细胞球药物敏感性的影响

Effect of silencing αv integrin gene on drug sensitivity of colon carcinoma multicellular spheroids

目的应用反转录病毒介导的shRNA沉默αv整合素,探讨后者在结肠癌多细胞球(MCSs)对奥沙利铂耐药中的作用。方法构建针对αv整合素的反转录病毒质粒,脂质体转染质粒至结肠癌细胞株HT29。采用改良liquid overlay技术培养MCSs;Western blotting检测MCSs中αv整合素蛋白的表达;Cell Counting Kit-8检测梯度浓度(0、5、50、500、5 000μg/L)的奥沙利铂处理后MCSs的存活率。结果测序证实质粒构建成功。Western blotting结果显示反转录病毒质粒介导的shRNA能有效、特异、稳定地沉默αv整合素基因。沉默αv整合素基因后,MCSs对奥沙利铂的药物敏感性显著增加。奥沙利铂50、500μg/L处理48h后,MCSs的存活率为0.86%±0.05%和0.43%±0.04%,转染反转录病毒的MCSs则下降为0.77%±0.06%和0.30%±0.04%(P<0.05)。结论反转录病毒介导的shRNA能有效沉默αv整合素基因。沉默αv整合素能有效提高结肠癌MCSs对化疗药物奥沙利铂的敏感性。

Objective To investigate the effect of αv integrin in drug resistance of multicellular spheroids of colon carcinoma to oxaliplatin by using retroviral-induced RNA silence technology. Methods Retroviral plamids expressing αv integrin-specific shRNA were constructed and transfected by liposome into colon carcinoma cell line HT29. Multicellular spheroids （MCSs） were obtained with improved liquid overlay technique. The expression of αv integrin in MCSs was detected by Western blotting, and Cell Counting Kit-8 kit was employed to assess the cell survival of HT29 MCSs after being treated with oxaliplatin at gradient concentrations of 0, 5, 50, 500 and 5000μg/L. Results It was demonstrated by sequencing that the retroviral plasmids expressing αv integrin specific small hairpin RNA had been successfully constructed, and could stably express the green fluorescent protein in HT29. Western blotting revealed that the retroviral plasmids-mediated small hairpin RNA could effectively, specifically and stably silence the av integrin gene. Drug sensitivity of MCSs to oxaliplatin was elevated obviously after αv integrin gene silencing： after treated with oxaliplatin for 48 hours at concentrations of 50 and 500 μg/ L, the cell survival rate of HT29 MCSs was 0. 86%±0. 05% and 0. 43%±0. 04%, respectively, while of those MCSs transfected with retroviral plasmids decreased to 0. 77%±0. 06% and 0. 30%±0. 04%, respectively （P〈0. 05）. Conclusions Retroviral-mediated shRNA can effectively silence the αv integrin in colon carcinoma cell line HT29. Down-regulation of αv integrin by gene silence may elevate the drug sensitivity of colon carcinoma MCSs to oxaliplatin.