摘要
目的探讨靶向沉默水通道蛋白4(AQP4)基因对非小细胞肺癌细胞化疗敏感性影响,并研究其分子作用机制。方法实时荧光定量PCR( qRT-PCR)和 Westernblot分别检测非小细胞肺癌A549细胞以及顺铂耐药菌株A549/DDP中AQP4mRNA和蛋白的表达。设计靶向AQP4的siRNA-1和siRNA-2,采用siRNA抑制A549/DDP细胞中AQP4的表达,筛选出最优siRNA,将其转染A549/DDP细胞,设立siRNA-NC组和空白对照组。CCK-8法检测细胞增殖能力并计算各组细胞的半数抑制浓度(IC50);流式细胞术检测细胞凋亡。Westernblot检测P53和Bcl-2蛋白的表达。结果 AQP4mRNA和蛋白在A549/DDP细胞中的表达水平显著高于A549细胞,AQP4表达沉默后A549/DDP细胞增殖抑制,细胞凋亡增加,对顺铂的敏感性增加,凋亡相关蛋白P53表达增加,而Bcl-2蛋白表达降低。结论通过靶向调控AQP4的表达可以增加非小细胞肺癌细胞对化疗药物的敏感性。
Objective To investigate the effect of targeting silenced aquaporin 4 (AQP4) gene on the chemotherapy sensitivity of non-small cell lung cancer (NSDCLC) and its mechanism. Methods The qRT-PCR method and Western bolt assay were used to detect the expression of AQP4 in A549 and A549/DDP cell lines (human cisplatin-resistant NSDCLC cell line). The siRNA-1 and siRNA-2 targeting AQP4 were designed. AQP4 expression was blocked by the siRNA in A549/DDP cells. The optimal siRNA was screened and transfected into A549/DDP cells. siRNA-NC and blank cells were set up as controls. The proliferation and half inhibitory concentration (IC50) of A549/DDP cells were detected by CCK8 method. The apoptosis of U251 cells was detected by AnnexinV- FITC/PI double marker flow cytometry. The expressions of P53 and Bcl- 2 were detected by Western blot assay. Results The expressions of AQP4 mRNA and protein were significantly increased in A549 /DDP cells compared with A549 cells. After silencing AQP4 expression, the proliferation was inhibited and the apoptosis rate was significantly increased in A549 / DDP cells. Silencing AQP4 expression significantly increased the sensitivity to cisplatin in lung cancer cells. AQP4 knockdown could also up-regulate the expression of P53, and down- regulate the expression of Bcl-2. Conclusion The selective targeting of the AQP4 expression can increase the sensitivity of NSDCLC cells to chemotherapeutic drugs.
作者
郭守俊
谢传华
黄萍
邱伊连
王硕
班振英
张威
GUO Shou-jun;XIE Chuan-hua;HUANG Ping;QIU Yi-lian;WANG Shuo;BAN Zhen-ying;ZHANG Wei(Department of Medical Oncology, Ganzhou Cancer Hospital, Ganzhou 341000, China;Department of Pathology, the Third Affiliated Hospital of Zhengzhou University)
出处
《天津医药》
CAS
北大核心
2019年第5期468-472,共5页
Tianjin Medical Journal
基金
河南省高等学校重点科研项目(17A310027)
