E盒结合锌指蛋白2对非小细胞肺癌预后的意义及其介导的多药耐药机制

Prognostic value of Zinc finger E-box-binding homeobox 2 in non-small cell lung cancer and its role in multidrug resistance

摘要:

目的探讨E 盒结合锌指蛋白2（ZEB2）对非小细胞肺癌（NSCLC）预后的意义及介导的多药耐药机制。方法选取72例NSCLC患者癌组织及癌旁组织，免疫组织化学染色检测组织ZEB2的表达，分析ZEB2表达与NSCLC 患者临床资料及生存预后的关系。按照转染类型将细胞分为3 组：ZEB2-siRNA 组、ZEB2-NC组和空白对照组（Mock）。采用小干扰RNA（siRNA）技术降低A549 细胞ZEB2 表达，分别采用不同浓度顺铂、紫杉醇处理A549细胞，CCK-8 法检测细胞对化疗药物的敏感性，流式细胞术检测细胞周期和凋亡率，Western blot检测细胞耐药蛋白P-糖蛋白（P-gp）、肺耐药相关蛋白（LRP）表达。结果ZEB2蛋白在癌组织阳性表达率为77.8%（56/72），癌旁组织阳性表达率为23.6%（17/72），ZEB2蛋白在癌组织阳性表达率高于癌旁组织（ P<0.01）。肿瘤直径≥5 cm 组ZEB2 阳性表达率高于肿瘤直径<5 cm 组，Ⅲ、Ⅳ期组ZEB2 阳性表达率高于Ⅰ、Ⅱ组，淋巴结转移组ZEB2 阳性表达率高于无淋巴结转移组，中低分化组ZEB2 阳性表达率高于高分化组，各组间比较差异有统计学意义（P <0.05）。ZEB2 阳性组总生存率和无病生存期低于阴性组（P <0.05）。Mock 组、ZEB2-NC组和ZEB2-siRNA组细胞存活率均随化疗药物浓度增加而降低，其中ZEB2-siRNA组细胞存活率低于ZEB2-NC组（P =0.000）。经Cisplatin、Paclitaxel处理后，与Mock组、ZEB2-NC组比较，ZEB2-siRNA组细胞凋亡率增 加，G0/G1期所占百分率降低，S期所占百分率升高，组间比较差异有统计学意义（P =0.000）。ZEB2-siRNA组P-gp、LRP蛋白表达水平低于Mock组和ZEB2-NC 组（P =0.000），ZEB2-NC、Mock 组P-gp、LRP蛋白表达比较差异无统计学意义（P >0.05）。结论NSCLC 组织ZEB2 表达上调，抑制ZEB2 可以降低耐药蛋白P-gp、LRP 表达，并逆转肺癌的多药耐药特性。

Abstract:

Objective To investigate the prognostic value of Zinc finger E -box -binding homeobox 2(ZEB2) in non-small cell lung cancer (NSCLC) and its role in multidrug resistance. Methods In study, a total of 72 patients diagnosed with NSCLC were included in this study, and tumor tissue and adjacent normal tissue samples of them were collected. Expression of ZEB2 was measured by Immunohistochemical staining. Potential relationship between ZEB2 and clinic survival data was analyzed. In study, genetic knockdown models of ZEB2 in A549 cells line were established by siRNA. Cells were divided into 3 groups: ZEB siRNA group, sequence control group, and negative control (Mock) group. Drug cytotoxicity was measured by CCK-8 assays after co-incubation with Cisplatin or Paclitaxel. Cell cycle and apoptosis rate was measured by flow cytometry. Expression levels of p-glycoprotein (P-gp) and LRP was determined by Western blot. Results ZEB2 positive cells in cancer tissue was significantly increased when compared with normal tissue (77.8% vs 23.6%,P = 0.000). ZEB2 positive cells in group with tumor diameter larger than 5 cm, Ⅲ～Ⅳphase, lymphatic metastasis, and poor and middle differentiation were dramatically increased when compared with those in group with tumor diameter <5 cm, Ⅰ～Ⅱphase, metastasis-free, and well differentiation (P < 0.05), respectively. Overall survival and cancer-free survival in ZEB2 positive group was significantly lower than those in ZEB2 negative group (P < 0.05). Cellular survival of A549 cell line in Mock group, ZEB2-NC group and ZEB2-siRNA group was significantly decreased with treatment of Cisplatin or Paclitaxel in a dose-dependent manner. Cellular survival of A549 cell line in ZEB2-siRNA group was significantly lower than that of Mock group and ZEB2-NC group (P = 0.000). Flow Cytometry data suggested that, after treatment with Cisplatin and Paclitaxel, cell apoptosis and S phase ratio increased while G0/G1 phase ratio decreased significantly compared with those in Mock group and ZEB2-NC group (P = 0.000). Expression levels of P-gp, LRP in ZEB2-siRNA group were significantly downregulated compared with Mock group and ZEB2-NC group (P = 0.000) while no significant difference of P-gp, LRP protein between Mock group and ZEB2-NC group (P > 0.05). Conclusion ZEB2 are up-regulated in NSCLC tissue, and inhibition of ZEB2 reverses the P-gp- and LRP-dependent multidrug resistance of NSCLC.