毒蕈碱型胆碱能受体M3调控肝癌细胞侵袭转移的研究

发布时间：2019-01-06 07:00

【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(P0.05);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(P0.05);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(P0.05)。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。
[Abstract]:Objective to investigate the effect of the activation of muscarinic cholinergic receptor M3 (muscarinic cholinergic receptors 3 (ChRM3) on the invasion and metastasis of human hepatoma cells and its molecular mechanism. Methods Western blot was used to detect the expression of ChRM3 receptor in Hep G2 and SMMC-7721 cells. The hepatoma Hep G2 and SMMC-7721 cells were treated with chloroblcholine (Beth) and Tamiflu (UK88525). The cells were divided into three groups: blank control group, Beth UK88525 group and UK88525 group. After 48 hours of treatment, Transwell assay was used to detect the invasion of the two kinds of hepatoma cells treated by different groups. The expression of epithelial-mesenchymal transformation (epithelial-to-mesenchymal transition,EMT) marker protein and the change of downstream signal pathway (PI3K/Akt) were detected by, Western blot. Hep G2 cells were transfected with shRNA interference plasmid (Lipofectamine 2000TM) to silence ChRM3, into control group, Beth sh ChRM3 group and sh ChRM3 group. The invasion and metastasis ability of the cells and the changes of PI3K/Akt downstream of EMT, were detected. Results ChRM3 receptors were expressed in Hep G2 and SMMC-7721 cells. Transwell assay showed that Beth alone could significantly promote the migration and invasion of HCC cells, while UK88525 could inhibit the effect of Beth on the migration and invasion of HCC cells (P0.05). Western blot results showed that Beth alone could up-regulate the expression of E-Cadherin by EMT marker protein N-Cadherin and Vimentin, and UK88525 treatment could significantly inhibit the process of Beth promoting EMT in Hep G2 cells (remove the process of Beth promoting EMT of Hep G2 cells). The activation of PI3K/Akt was also inhibited (P0.05). Interference plasmid silencing ChRM3 receptor also inhibited the migration and invasion of hepatoma cells induced by Beth, and inhibited the activation of EMT and downstream PI3K/Akt induced by Beth (P0.05). Conclusion ChRM3 activation can promote the invasion and metastasis of hepatoma cells through PI3K/Akt signaling pathway.
【作者单位】：第三军医大学西南医院全军肝胆外科研究所;
【基金】：国家自然科学基金面上项目(81272224)~~
【分类号】：R735.7

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