细胞缝隙连接对miR-124抗肿瘤作用的影响和机制研究  

Effect of gap junctions on anti-proliferation effect of miR-124 and its mechanism

作　　者：张素枝 陶亮 张晓坚[1] 郭三星[3] ZHANG Su-zhi;TAO Liang;ZHANG Xiao-jian;GUO San-xing(Dept of Pharmacy, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China;Dept of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China;Dept of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China)

机构地区：[1]郑州大学第一附属医院药学部,河南郑州450052 [2]中山大学中山医学院药理学教研室,广东广州510080 [3]郑州大学第一附属医院肿瘤科,河南郑州450052

出　　处：《中国药理学通报》2019年第11期1528-1534,共7页Chinese Pharmacological Bulletin

基　　金：国家重点研发计划(No 2017YFC0909900);郑州大学第一附属医院院内青年创新基金(No YNQN2017137)

摘　　要：目的 探讨不同连接蛋白(connexin,Cx)组成的缝隙连接(gap junction,GJ)对miR-124抗肿瘤作用的影响及机制。方法 在稳定转染表达Cx26或Cx32的Hela细胞(Hela26、Hela32),以及稳定转染shRNA-Cx43的人胶质瘤U87细胞(U87 shRNA-Cx43 )中,应用Western blot和荧光示踪实验分别测定Cx表达和GJ功能;集落形成实验检测miR-124对细胞增殖影响;膜片钳检测Cy3荧光标记的miR-124在细胞间的传递。结果 多西环素可诱导Hela26和Hela32细胞上Cx表达和GJ形成;miR-124抑制Hela细胞增殖,但在有GJ(Dox诱导)和无GJ形成(无Dox诱导)条件下统计学上无明显差异。相较于U87 shRNA-NC ,U87 shRNA-Cx43 细胞中Cx43表达、GJ功能及miR-124的增殖抑制作用均明显降低;显微镜下可见导入到“供体细胞”中的荧光miRNA逐渐布满整个细胞并传递到相邻非“导入”细胞内。结论 GJ可影响miR-124的抗肿瘤作用,但具有Cx异质性。相较Cx26或Cx32,Cx43组成的GJ可调控miR-124的抗肿瘤作用,这可能与该GJ通道对miR-124的传递作用较强有关。Aim To investigate the role of gap junction(GJ) composed of Cx26, Cx32 and Cx43 respectively on the anti-tumor effect of miR-124 and the underlying mechanisms. Methods Hela cells, stably transfected by PBI plasmid coding for Cx32 or Cx26(called Hela32 or Hela26 cells), and U87 cells, transfected with shRNA-Cx43(called U87shRNA-Cx43) were cultured in vitro. Connexin expression was examined by Western blot, and GJ function was estimated by parachute assay. The impact of miR-124 on cell proliferation was detected by standard colony forming assay, and the transfer of cy3-labelled miR-124 through GJ channel was observed by patch clamp. Results Cx32 or Cx26 expression and GJ function were induced by doxycycline( Dox,the promotor for PBI plasmid) in transfected Hela cells. MiR-124 reduced the proliferation of Hela cells,dox incubation alone did not affect Hela cell growth,and also had no effect on anti-tumor effect of miR-124 when combined with miR-124 transfection.Compare with U87 shRNA-NC,the Cx43 expression and GJ function significantly decreased in U87 shRNA-Cx43. Similar to the effect on Hela cells,MiR-124 also reduced U87 cell growth. Reducing Cx43 expression did not influence U87 cell proliferation, but attenuated the growth-inhibition effect of miR-124 when combined with miR-124 transfection. Under the microscope,the transfer of fluorescence-labelled miR-124 from"donor"cell to adjacent "non-injection " cell was observed.Conclusions The role of GJ on anti-tumor effect of miR-124 possesses connexin heterogeneity. Compare with Cx26 or Cx32,GJ composed of Cx43 has more obvious effect,which may be related to the maximum permeability of junction channel to miR-124.

关 键 词：细胞缝隙连接 microRNA-124 缝隙连接蛋白26 缝隙连接蛋白32 缝隙连接蛋白43 肿瘤 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]