NHE1通过PI3K/AKT/mTOR信号通路调节肺癌免疫抑制及对癌细胞的作用

NHE1 Regulates Lung Cancer Immunosuppression and Its Effect on Cancer Cells Through PI3K/AKT/mTOR Signaling Pathway

目的探究钠氢交换蛋白1(Na[+]/H[+]hydrogen exchanger 1,NHE1)通过PI3K/AKT/mTOR信号通路调节肺癌免疫抑制及对癌细胞的作用。方法将肺癌细胞在梯度浓度Cariporide下孵育,并计算得到Cariporide的IC50值为34 mmol/L。将细胞分为对照组(0 mmol/L)、低剂量Cariporide组(17 mmol/L)、中剂量Cariporide组(34 mmol/L)和高剂量Cariporide组(68 mmol/L)。培养48 h后分别通过克隆形成、划痕愈合的实验和Transwell测定细胞增殖、迁移和侵袭。并通过皮下注射手段对裸鼠模型构建30只,分为对照组、CariporideⅠ组和CariporideⅡ组(N=10),通过腹腔注射Cariporide(3 mg/kg,6 mg/kg)干预。建模28 d后处死小鼠检测肿瘤生长情况,通过Western blot检测增殖和转移相关蛋白。通过流式细胞术检测CD_(4)^(+)、CD_(8)^(+)细胞水平。结果4组细胞的增殖、迁移、侵袭以及PI3K/AKT/mTOR通路中蛋白表达水平比较,有统计学差异(P<0.05)。对照组、低剂量Cariporide组、中剂量Cariporide组和高剂量Cariporide组的克隆形成数目、划痕前缘迁移距离和侵袭细胞数目均依次逐渐降低(P<0.05)。3组小鼠的肿瘤体积、质量、增殖和转移相关蛋白水平以及CD_(4)^(+)和CD_(4)^(+)/CD_(8)^(+)水平比较,差异显著(P<0.05)。CariporideⅠ组和CariporideⅡ组的肿瘤体积和质量均显著低于对照组(P<0.05),CyclinD1、Ki67、MMP2、MMP9的水平显著低于对照组(P<0.05),CD_(4)^(+)和CD_(4)^(+)/CD_(8)^(+)的水平显著高于对照组(P<0.05);CariporideⅡ组的肿瘤体积、质量、CyclinD1、Ki67、MMP2、MMP9的水平显著低于CariporideⅠ组,而CD_(4)^(+)和CD_(4)^(+)/CD_(8)^(+)显著高于CariporideⅠ组(P<0.05)。结论使用Cariporide抑制NHE1可通过抑制PI3K/AKT/mTOR通路抑制肺癌细胞的增殖和转移,并解除免疫抑制,从而发挥抗癌作用。

Objective To explore the mechanism of Na[+]/H[+]hydrogen exchanger 1(NHE1)through PI3K/AKT/mTOR signaling pathway to regulate lung cancer immunosuppression and its effect on cancer cells.Methods Lung cancer cells were incubated at a gradient concentration of Cariporide,and the IC50 value of Cariporide was calculated to be 34 mmol/L.The cells were divided into the control group(0 mmol/L),low-dose Cariporide group(17 mmol/L),medium-dose Cariporide group(34 mmol/L)and high-dose Cariporide group(68 mmol/L).After 48 hours of culture,cell proliferation,migration and invasion were detected by colony formation,scratch healing test and Transwell.Thirty nude mouse models were constructed by subcutaneous injection,and they were divided into the control group,CariporideⅠgroup and CariporideⅡgroup(N=10).Intervention by intraperitoneal injection of Cariporide(3 mg/kg,6 mg/kg).Mice were sacrificed 28 days after modeling to detect tumor growth.Proliferation and metastasis related proteins were detected by Western blot.The CD_(4)^(+)and CD_(8)^(+)cell levels were detected by flow cytometry.Results The proliferation,migration,invasion of the 4 groups of cells and the protein expression levels in the PI3K/AKT/mTOR pathway were obvious different among the 4 groups(P<0.05).Compared with the control group,the number of clone formation,the migration distance of the leading edge of the scratch and the number of invasive cells in the low-dose Cariporide group,the medium-dose Cariporide group and the high-dose Cariporide group were gradually decreased in tur-n(P<0.05).There were significant differences in tumor volume,weight,proliferation and metastasis related protein levels,CD_(4)^(+)and CD_(4)^(+)/CD_(8)^(+)levels among the 3 groups(P<0.05).The tumor volume and mass of the CariporideⅠand CariporideⅡgroups were obvious lower than control group(P<0.05),the levels of CyclinD1,Ki67,MMP2,and MMP9 were obvious lower than the control group(P<0.05),CD_(4)^(+)and CD_(4)^(+)/CD_(8)^(+)The level was obvious higher than the control group(P<0.05).The tumor volume,mass,CyclinD1,Ki67,MMP2,and MMP9 levels in the CariporideⅡgroup were obvious lower than CariporideⅠgroup),while CD_(4)^(+)And CD_(4)^(+)/CD_(8)^(+)were obvious higher than CariporideⅠgroup(P<0.05).Conclusion Using Cariporide to inhibit NHE1 can inhibit the proliferation and metastasis of lung cancer cells by inhibiting the PI3K/AKT/mTOR pathway,and relieve immunosuppression,thereby exerting anti-cancer effects.