下调miR-205-5p增强鼻咽癌细胞HNE1/DDP对顺铂诱导凋亡的敏感性  

Down-regulation of miR-205-5p sensitizes HNE1/DDP to cisplatin induced apoptosis in vitro

作　　者：赵伟曼 董智勇 时宗芬 鲁星月 刘浩 张配 ZHAO Wei-man;DONG Zhi-yong;SHI Zong-fen;LU Xing-yue;LIU Hao;ZHANG Pei(School of Pharmacy,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院药学院

出　　处：《药学学报》2019年第7期1200-1206,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81603155);蚌埠医学院自然科学基金资助项目(BYKY1763)

摘　　要：探讨下调miR-205-5p增强鼻咽癌耐药细胞HNE1/DDP对顺铂诱导凋亡的敏感性及其可能机制。qRT-PCR检测HNE1、HNE1/DDP细胞中miR-205-5p的表达差异及HNE1/DDP细胞转染miR-205-5p抑制剂后miR-205-5p的变化;MTT法检测顺铂(DDP)和miR-205-5p抑制剂对HNE1/DDP或HNE1细胞的增殖抑制作用;PI单染流式技术检测细胞凋亡情况;DAPI染色观察细胞核的形态;Western blot检测Bax、Bak、Mcl-1、Bcl-2蛋白的表达水平。结果显示,相比于HNE1细胞,HNE1/DDP高表达miR-205-5p,转染miR-205-5p抑制剂后其表达明显下降。下调miR-205-5p可显著提高HNE1/DDP细胞对顺铂的敏感性(P<0.05)。PI单染结果显示,转染miR-205-5p抑制剂可增强HNE1/DDP细胞对顺铂的诱导凋亡作用。miR-205-5p抑制剂联合DDP (8μmol·L^-1)作用HNE1/DDP细胞24 h的凋亡率为(28.93±2.50)%,相比单用miR-205-5p抑制剂(9.83±1.31)%或DDP的凋亡率(10.83±1.70)%显著升高(P<0.05)。联合作用组细胞表现为细胞核明显皱缩浓集呈碎片状。miR-205-5p抑制剂与DDP合用能上调Bax的表达和下调Bcl-2的表达。提示下调miR-205-5p可增强HNE1/DDP细胞对DDP的敏感性,其作用机制可能是促凋亡蛋白Bax的升高和抗凋亡蛋白Bcl-2的下降。This study aims to investigate the effect of down-regulation of miR-205-5 p by transfection of miR-205-5 p inhibitor on the sensitivity of HNE1/DDP cells to cisplatin(DDP) induced apoptosis and explore the underlying mechanism.qRT-PCR was used to detect the expression of miR-205-5 p in HNE1 or HNE1/DDP cells.The expression level of miR-205-5 p was analyzed after transfecting HNE1/DDP cells with miR-205-5 p inhibitor.MTT assay was used to evaluate the inhibitory effect of DDP alone or in combination with miR-205-5 p inhibitor on the proliferation of HNE1/DDP or HNE1 cells.Apoptosis of cells treated with miR-205-5 p inhibitor alone or in combination with DDP(8 μmol·L^-1) was assessed using flow cytometry with PI staining,with the nucleus was counterstained with DAPI staining.The expression of Bax,Bak,Mcl-1,or Bcl-2 was analyzed by Western blot.HNE1/DDP cells showed a high level of expression of miR-205-5 p,and the expression of miR-205-5 p was significantly decreased by transfection of miR-205-5 p inhibitor.Down-regulation of miR-205-5 p significantly increased the sensitivity of HNE1/DDP cells to DDP(P<0.05).Transfection of miR-205-5 p inhibitor enhanced the sensitivity of HNE1/DDP cells to DDP induced apoptosis.Treatment of HNE1/DDP cells with miR-205-5 p inhibitor combined with DDP(8 μmol·L^-1) for 24 h resulted in an apoptotic rate of 28.93%± 2.50%,significantly higher than that treated with miR-205-5 p inhibitor(9.83%± 1.31%) or DDP alone(10.83%± 1.70%)(P<0.05).DAPI staining showed that HNE1/DDP cell nucleus became significantly condensed and fragmented in miR-205-5 p inhibitor combined with DDP group.The combined group up-regulated the expression of Bax and down-regulated the expression of Bcl-2 in HNE1/DDP cells.Therefore,down-regulation of miR-205-5 p can enhance the sensitivity of HNE1/DDP cells to cisplatin induced apoptosis,and the mechanism may involve up-regulation of Bax and down-regulation of Bcl-2 expression.

关 键 词：鼻咽癌细胞 顺铂 miR-205-5p 耐药性 凋亡 

分 类 号：R966[医药卫生—药理学]