P2X7受体通过激活AKT信号通路促进小鼠Lewis肺癌细胞的迁移和侵袭

P2X7R promotes migration and invasion of Lewis lung cancer cells by activating the AKT signaling pathway

目的 探究P2X7受体(P2X7R)对小鼠Lewis肺癌(LLC)细胞迁移和侵袭能力的影响及其作用机制,同时探究体内P2X7R对荷LLC小鼠成瘤能力的影响。方法 体外实验:RT-PCR检测LLC细胞P2X7R表达。将LLC细胞分别给予P2X7R激动剂三磷酸腺苷(ATP)、2’-3’-O-(4-苯甲酰-苯甲酰)ATP(BzATP)干预或预先给予P2X7R拮抗剂oxATP、A438079然后再给予激动剂干预,共分为7组。使用细胞划痕实验和Transwell实验检测LLC细胞的迁移和侵袭能力;Western blot检测蛋白激酶B(AKT)信号通路关键蛋白的活化水平。将LLC细胞给予BzATP、A438079、磷酸肌醇3-激酶(PI3K)/AKT信号通路抑制剂LY294002干预,共分为5组。利用Transwell实验检测LLC细胞的迁移和侵袭能力。体内实验:构建荷LLC细胞的皮下移植瘤的野生(WT)型和P2X7R基因敲除(P2X7)型小鼠模型,比较两组肿瘤的体积及质量。结果 LLC细胞中表达P2X7R。划痕实验和Transwell实验结果显示:与对照组相比,激动剂组迁移和侵袭能力增强;与激动剂组相比,拮抗剂可以抑制激动剂促进LLC细胞的迁移及侵袭(P<0.001)。Western blot显示:BzATP组p-AKT蛋白表达水平相比于对照组增加;与BzATP组相比,拮抗剂组p-AKT蛋白表达水平降低(P<0.01)。Transwell实验结果显示:与BzATP组相比,拮抗剂组均可抑制BzATP促进的LLC细胞的迁移及侵袭(P<0.001)。小鼠荷瘤显示:与WT小鼠相比,P2X7小鼠瘤体体积和瘤体重量均降低(P<0.05)。结论 P2X7R通过激活AKT信号通路促进LLC细胞的迁移及侵袭,P2X7小鼠降低LLC细胞的体内成瘤能力。

Objective To explore the role of P2X7 receptor(P2X7R) in migration and invasion of mouse Lewis lung cancer(LLC)cells and examine the tumorigenic ability of LLC cells in P2X7R-knockout mice. Methods RT-PCR was used to examine P2X7R mRNA expression in LLC cells. LLC cells were treated with ATP(as a P2X7R agonist) or 2’-3’-O-(4-benzoyl-benzoyl)-ATP(BzATP)(a P2X7R agonist) with or without pretreatment with P2X7R antagonist oxATP or A438079. The changes in migration and invasive abilities of the cells were evaluated using wound healing assay and Transwell assay;Western blotting was performed to determine the activation level of the key proteins in the AKT signaling pathway. The effects of BzATP, A438079,and LY294002(a inhibitor of the PI3K/AKT pathway) on migration and invasion of LLC cells were also examined. In wild-type(WT) and P2X7R knockout(P2X7) C57BL/6 mice, the growth of subcutaneous LLC cell xenografts were observed by measuring tumor volume and weight. Results P2X7R expression was detected in LLC cells. Treatment with P2X7R agonist significantly enhanced migration and invasion abilities of LLC cells, and this effect was inhibited by application of P2X7R antagonists(P<0.001). Western blotting showed that BzATP treatment of LLC cells significantly increased the expression level of p-AKT protein,which was obviously lowered by treatment with P2X7R antagonist(P<0.01). P2X7R antagonist strongly inhibited BzATP-induced enhancement of LLC cell migration and invasion(P<0.001). In the tumor-bearing mice, the tumor volume and weight were significantly lower in P2X7mice than in WT mice(P<0.05). Conclusion P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7mice.