副交感神经M1受体促进前列腺癌转移并抵抗肿瘤细胞凋亡的研究

Muscarinic Acetylcholine Receptor M1 Promotes Cell Metastasis and Resists Cell Apoptosis in Prostate Cancer

该文旨在探讨副交感神经M1受体(muscarinic acetylcholine M1 receptor,CHRM1)通过调节PI3K/AKT信号通路对前列腺癌增殖、转移以及肿瘤细胞凋亡影响的研究。选用Western blot、免疫荧光等方法检测CHRM1在前列腺癌细胞中表达情况。体外培养人前列腺癌细胞系PC-3、LNCaP、DU145,然后用CHRM1激动剂卡巴胆碱(CAR)及特异性抑制剂哌仑西平(PIN)处理细胞。用CHRM1 RNAi慢病毒感染细胞,构建前列腺癌CHRM1敲低的稳转株。选用CCK8细胞增殖实验、平板克隆实验、细胞迁移侵袭实验、流式细胞术检测及透射电镜观察等方法探究CHRM1在前列腺癌中增殖、转移和凋亡水平。最后,用Western blot方法检测敲低的CHRM1前列腺癌细胞中上皮间质标志物及PI3K/AKT信号表达水平。结果显示,CHRM1大量表达在前列腺癌各细胞系细胞中。CAR处理后细胞增殖能力、克隆形成水平、转移能力及抗凋亡能力提高,而PIN处理后其增殖能力、克隆形成能力、转移能力及抗凋亡能力降低。敲低CHRM1后,细胞的转移能力及抗凋亡能力降低,且电镜下出现凋亡小体。在细胞迁移与侵袭实验中发现其转移能力与肿瘤的上皮–间充质转化(EMT)有关。同时,CHRM1通过PI3K/AKT信号通路调控肿瘤细胞进程。该研究结果提示,CHRM1在前列腺癌细胞中调节PI3K/AKT信号通路促进,前列腺癌增殖、转移并抵抗肿瘤细胞凋亡。

The aim of this study was to investigate the effects of CHRM1 (muscarinic acetylcholine receptor M1) on the proliferation,metastasis and anti-apoptosis of prostate cancer cells by regulating PI3K/AKT signaling pathway.Western blot,immunofluorescence and the other methods were used to detect the expression of CHRM1 in prostate cancer cells.Human prostate cancer cells PC-3,LNCaP,DU145 were cultured in vitro,and the cells were treated by CHRM1 agonist CAR (carbachol) and CHRM1 specific inhibitor PIN (pirenzepine).PC-3 cells were infected with CHRM1 RNAi lentivirus and a stably transfected strain of CHRM1 knockdown was constructed.CCK8 cell proliferation assay,plate cloning assay,cell migration and invasion experiments,flow cytometry detection and observation under transmission electron microscope were used to explore the proliferation,metastasis and apoptosis levels of CHRM1 in prostate cancer.Western blot was used to detect the expression of epithelial-mesenchymal markers and PI3K/AKT signals in prostate cancer cells knocking down CHRM1.The results showed that CHRM1 expressed abundantly in prostate cancer cells.In our assays,the group treated with CAR promoted the proliferation,colony formation,migration,invasion and anti-apoptosis abilities of prostate cancer,while the group PIN inhibited the proliferation,colony formation,migration,invasion and anti-apoptosis abilities.What’s more,CHRM1 knockdown also inhibited the migration,invasion and anti-apoptosis abilities,and apoptotic bodies appeared under electron microscope.The ability of metastasis was related to EMT (epithelial mesenchymal transformation) in the experiment of cell migration and invasion.Moreover,CHRM1 regulates tumor cell progression through PI3K/AKT signaling pathway.The results of this study suggested that CHRM1 could regulate PI3K/AKT signaling pathway to promote proliferation,metastasis and resist apoptosis in prostate cancer cells.