Pim1介导非小细胞肺癌进展的作用及机制研究

Study on role and mechanism of Pim1 mediating NSCLC progression

目的探讨原癌基因Pim1对小鼠Lewis肺癌细胞(LLC)凋亡、增殖、侵袭和迁移功能的作用及可能存在的机制。方法收集2021年8-10月于湖北医药学院附属太和医院行肺叶切除患者的正常肺组织和非小细胞肺癌(NSCLC)组织标本,采用免疫组织化学法及Western blot检测两种组织中Pim1蛋白表达情况。CCK-8实验验证Pim1特异性抑制剂SMI-4a的最适浓度。LCC细胞分为实验组和对照组,实验组孵育50μmol/L SMI-4a,对照组孵育等体积二甲基亚砜(DMSO),Western blot检测Bcl-2、Bax、含半胱氨酸的天冬氨酸蛋白水解酶(caspase)3、cleaved-caspase3、基质金属蛋白酶(MMP)2、MMP9、丝氨酸/苏氨酸激酶(Akt)和磷酸化(p)-Akt表达情况,DAPI染色检测细胞凋亡,Edu试剂盒检测细胞增殖,Transwell和细胞划痕实验检测细胞侵袭和迁移能力。3只C57 BL/6小鼠腹股沟注射LLC,收获肿瘤后等体积接种到12只小鼠腹股沟构建小鼠瘤荷模型,并分为实验模型和对照模型,实验模型瘤内注射SMI-4a 80 mg·kg^(-1)·d^(-1),对照模型注射等体积DMSO,15 d后处死并称取肿瘤组织质量,苏木素-伊红(HE)染色观察肿瘤组织凋亡核心变化,免疫组织化学法及Western blot检测肿瘤组织Pim1表达情况。结果与正常肺组织比较,NSCLC组织中Pim1蛋白的棕黄色染色增多,且NSCLC组织中Pim1蛋白表达水平高于正常肺组织(P<0.05)。与对照组比较,实验组Bax蛋白、cleaved-caspase3、caspase3水平升高,Bcl-2蛋白水平降低,凋亡细胞数量增多,p-Akt蛋白水平降低,DNA合成和细胞增殖受到抑制,MMP2、MMP9表达水平降低,侵袭能力和细胞垂直迁移能力降低(P<0.05),细胞水平迁移能力减弱。与对照模型比较,实验模型肿瘤进展受到明显抑制,肿瘤质量更小(P<0.05),此外,肿瘤组织嗜伊红染色减少,肿瘤内部凋亡核心面积增加,凋亡核心可见较多碎裂的细胞核,凋亡核心Pim1棕黄色染色减少,且肿瘤组织Pim1表达水平降低(P<0.05)。结论抑制Pim1可能通过减少Akt的磷酸化蛋白水平抑制小鼠LLC肿瘤的进展。

Objective To investigate the effects of proto-oncogene Pim1 on the apoptosis,proliferation,invasion and migration functions of mouse Lewis lung cancer cells(LLC)and the possible mechanisms.Methods The normal lung tissue and non-small cell lung cancer(NSCLC)tissue samples from the patients with lobectomy in the Affiliated Taihe Hospital of Hubei University of Medicine from August to October 2021 were collected.The Pim1 protein expression in these two kinds of tissues was detected by the immunohistochemistry and Western blot.The CCK-8 assay confirmed the optimal concentration of SMI-4a,a Pim1-specific inhibitor.The LCC cells were divided into the experiment group and the control group.The experiment group was incubated with 50μmol/L SMI-4a,while the control group was incubated with the same volume of dimethyyl sulfoxide(DMSO).Western blot was used to detect the expression of Bcl-2,Bax,caspase3,cleaved caspase3,matrix metalloproteinase(MMP)2,MMP9,serine/threonine kinase(Akt)and phosphorylated(p)-Akt.The cell apoptosis was detected by the DAPI staining.The Edu kit was used to detect the cell proliferation,and the Transwell and cell scratch assay were used to detect the cell invasion and migration ability.Three C57 BL/6 mice were injected by LLC in the groin,after the tumor was harvested,the same volume was inoculated into the groin in 12 mice to construct the mouse tumor charge model,which were divided into the experiment model and the control model.The intratumor injection of SMI-4a 80 mg·kg^(-1)·d^(-1) was performed in the experiment model and the same volume of DMSO was injected in the control model.The mouse was executed and the mass of tumor tissue was measured.The changes of apoptosis core in the tumor tissues were observed by hematoxylin-eosin(HE)staining,and the Pim1 expression in tumor tissues was detected by immunohistochemistry and Western blot.Results Compared with the normal lung tissues,the brown-yellow staining of Pim1 protein in the NSCLC tissues was increased,moreover the expression level of Pim1 protein in the NSCLC tissues was higher than that in the normal lung tissues(P<0.05).Compared with the control group,the protein levels of Bax,cleaved-caspase3 and caspase3 in the experiment group were increased,the Bcl-2 protein level was decreased,and the number of apoptotic cells was increased,the p-Akt protein level was decreased,the DNA synthesis and the cell proliferation was inhibited,the MMP2 and MMP9 expression levels were decreased,the invasion ability and cellular vertical migration ability were decreased(P<0.05).The cellular horizontal migration ability was significantly weakened.Compared with the control model,the tumor progress in the experiment model was significantly inhibited,and the tumor quality was lower(P<0.05).I n addition,the eosinophilic staining in the tumor tissues was decreased,the intra-tumoral apoptotic core area was increased,the apoptotic core showed more fragmented nuclei,apoptotic core Pim1 brown and yellow staining was decreased,moreover the Pim1 expression level in the tumor tissues was decreased(P<0.05).Conclusion Inhibiting Pim1 may inhibit the progression of LLC tumor in mice by reducing the phosphorylated protein level of Akt.