RNAi沉默Nrf2基因增强煤焦沥青烟提取物对BEAS-2B细胞的毒性作用
The role of RNAi-mediated Nrf2 gene silencing on malignant transformation of BEAS-2B cells induced by coal tar pitch smoke extract

目的:探讨RNA干扰(RNAi)技术沉默Nrf2基因对煤焦沥青烟提取物(CTPE)致人支气管上皮细胞(BEAS-2B)发生恶性转化过程中的作用,为肺癌的发生机制提供线索。方法:应用RNAi技术建立Nrf2基因沉默的BEAS-2B细胞稳定表达株; 设立二甲基亚砜(DMSO)阴性对照组、苯并(a)芘[B(a)P]阳性对照组、CTPE组、RNAi组; 采用软琼脂克隆形成实验分析细胞恶变情况; 采用RT-PCR法检测Nrf2和NQO1 mRNA的表达; Western blot法检测Nrf2和NQO1蛋白的表达。结果:20代以后,B(a)P组、CTPE组和RNAi组的克隆形成率高于DMSO组(P<0.001); 而RNAi组的克隆形成率高于B(a)P组和CTPE组(P<0.001)。B(a)P组和CTPE组Nrf2 mRNA和蛋白表达水平均较DMSO组增加(P<0.001),B(a)P组、CTPE组和RNAi组NQO1 mRNA和蛋白水平均较DMSO组上调,RNAi组的表达量低于B(a)P组和CTPE组(P<0.001); 诱导后(CTPE组)第10代、20代、30代细胞中Nrf2与NQO1的蛋白表达呈正相关(r=0.913,P<0.001)。结论:Nrf2可能通过上调 NQO1的表达对抗CTPE对BEAS-2B细胞的毒性作用从而抑制细胞恶化; Nrf2和NQO1在BEAS-2B细胞癌前病变阶段已呈现较明显表达,这可能有助于接触CTPE高危人群罹患肺癌的预警及筛查。
Aim: To investigate the role of RNAi-mediated Nrf2 gene silencing on malignant transformation of BEAS-2B cells induced by coal tar pitch smoke extract(CTPE)and to find out the potential mechanism of lung cancer under the condition of CTPE exposure.Methods: The stably expressed cell line of human bronchial epithelial cells(BEAS-2B)with Nrf2 gene silencing was established through RNA interfering(RNAi)method in vitro. Through adding dimethyl sulfoxide(DMSO)and Benzo(a)pyrene [B(a)P] to the culture media to get the negative and positive controls respectively; the BEAS-2B cells treated with CTPE exposure or RNAi technique were the observational groups. The malignancy of cells was estimated by soft agar colony formation assay. The genetic and protein expressions of Nrf2 and NQO1 in BEAS-2B cells were detected by RT-PCR and Western blot, respectively.Results: After the 20th generation, the quantities of colony formation in CTPE group, B(a)P group, and RNAi group were significantly higher than that in DMSO group(P<0.001), and that in RNAi group was significantly higher than CTPE group and B(a)P group(P<0.001). The expression levels of mRNA and protein for Nrf2 were significantly higher in CTPE and B(a)P groups than that in negative control group(P<0.001). The expressions levels NQO1 of mRNA and protein were all up-regulated in B(a)P, CTPE, and RNAi groups(P<0.001). The Nrf2 expression was positively associated with the expression of NQO1 at protein level in the 10th,20th, and 30th generations cell in CTPE group(r=0.913,P<0.001).Conclusion: Nrf2 might inhibit the malignant transformation process in BEAS-2B cells exposed to CTPE through up-regulation the expression of NQO1. The high expressions of Nrf2 and NQO1 could be detected in pre-malignant cells, which might help to screen lung cancer or provide pre-warning in the high risk population under constant CTPE exposure