摘要
目的探讨毒蕈碱型胆碱能受体M3(muscarinic cholinergic receptors 3,ChRM3)活化对人肝癌细胞侵袭、转移能力的影响及其分子机制。方法 Western blot检测两种人肝癌细胞株Hep G2和SMMC-7721中ChRM3受体的表达;采用氯贝胆碱(Beth)及达菲那新(UK88525)处理肝癌Hep G2和SMMC-7721细胞,分为空白对照组、Beth组、Beth+UK88525组、UK88525组,经处理48 h后,Transwell实验检测两种肝癌细胞经不同分组处理后侵袭、迁移能力的变化,Western blot检测各组细胞中上皮-间质转化(epithelial-to-mesenchymal transition,EMT)标志蛋白的表达及下游信号通路PI3K/Akt的变化;采用shRNA干扰质粒经Lipofectamine 2000TM转染肝癌细胞Hep G2以沉默ChRM3,分为空白对照组、Beth组、Beth+sh ChRM3组、sh ChRM3组,分别检测细胞的侵袭、转移能力及EMT、下游PI3K/Akt的改变。结果 Hep G2和SMMC-7721两种肝癌细胞中均有ChRM3受体的表达;Transwell实验显示:Beth单独处理组对肝癌细胞的迁移和侵袭有明显的促进作用,而UK88525能够抑制Beth对肝癌细胞迁移侵袭能力的促进作用(P<0.05);Western blot结果显示:Beth单独处理组能够上调EMT标志蛋白N-Cadherin和Vimentin,下调E-Cadherin的表达,UK88525处理后能够明显抑制Beth促进Hep G2细胞EMT的过程(解除Beth对Hep G2细胞EMT的促进过程),同时也能够抑制PI3K/Akt的活化(P<0.05);干扰质粒沉默ChRM3受体同样能够抑制Beth促肝癌细胞迁移、侵袭的作用,并且抑制Beth诱导的EMT和下游PI3K/Akt的活化(P<0.05)。结论 ChRM3活化能够通过PI3K/Akt信号通路促进肝癌细胞的侵袭和转移。
Objective To explore the effect of activated muscarinic cholinergic receptor 3（ ChRM3）on the invasion and migration of hepatocellular carcinoma cells and its preliminary mechanism. Methods Western blotting was used to detect the expression of ChRM3 in hepatocellular carcinoma cell lines Hep G2 and SMMC-7721. The cells were treated with bethanechol chloride（ Beth） or darifenacin（ UK88525）,and then 4groups were designed： blank control group,Beth group,Beth ＋ UK88525 group and UK88525 group. After the cells were treated for 48 h,Transwell chamber assay was used to detect the cell invasion and migration,and Western blotting was employed to detect the epithelial-mesenchymal transition（ EMT） marker proteins（ E-cadherin,N-cadherin and Vimentin） and analyze the change of PI3K/Akt signaling pathway. In addition,ChRM3 was silenced by ChRM3 interference plasmid that was constructed and transfected by Lipofectamine2000 TMin Hep G2 cells. Then the cells were divided into blank control group,Beth group,Beth ＋ sh ChRM3 group and sh ChRM3 group. The cell invasion and migration,EMT markers and PI3K/Akt pathway were tested in the above 4 groups. Results ChRM3 was expressed in both Hep G2 cells and SMMC-7721 cells. The invasion and migration were improved and the EMT process was promoted in HCC cells after Beth stimulation.The stimulation effects of Beth on HCC cells were inhibited after ChRM3 was blocked by UK88525 or silenced by shRNA（ P〈0. 05）. Compared with the Beth group,the expression of E-cadherin was increased,but that of N-cadherin and Vimentin were decreased in the Beth ＋ UK88525 and Beth ＋ sh ChRM3 groups（ P〈0. 05）.Meanwhile,the blockade of ChRM3 also inhibited the activation of PI3K/Akt signaling pathway. Conclusion The activation of ChRM3 can promote the invasion and migration of the HCC cells via PI3K/Akt signaling pathway.
作者
张亮
宦宏波
温旭东
杨大鹏
吴黎雳
夏锋
Zhang Liang Huan Hongbo Wen Xudong Yang Dapeng Wu Lili Xia Feng(Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, 400038, Chin)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2017年第6期509-514,共6页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81272224)~~
关键词
肝细胞癌
毒蕈碱型胆碱能受体M3
侵袭
转移
上皮-间质转化
hepatocellular carcinoma
muscarinic cholinergic receptor 3
invasiveness
metastasis
epithelial-mesenchymal transition
