S100A8/A9通过磷脂酰肌醇3-激酶/蛋白激酶B信号通路调控非小细胞肺癌

S100A8/A9 regulates non-small cell lung cancer through phosphati-dylinositol 3-kinase/protein kinase B signaling pathway

目的探讨S100A8/A9-磷脂酰肌醇3-激酶/蛋白激酶信号轴调控非小细胞肺癌进展的分子机制。方法通过GTEx、CCLE和GEPIA2数据库分析S100A8/A9在肺癌中的表达及临床意义;通过慢病毒转染,建立A549S100A8/A9-和A549S100A8/A9+模型;通过CCK8法、细胞迁移法、Annexin V/PI双染法检测A549S100A8/A9-和A549S100A8/A9+的增值、侵袭、转移和凋亡,分析其细胞功能的改变;应用DNA芯片技术检测A549S100A8/A9-和A549S100A8/A9+细胞中上调和下调基因,KEGG富集相关通路;根据富集通路结果,通过蛋白免疫印记法(Western blot)检测磷脂酰肌醇3-激酶/蛋白激酶(PI3K/AKT)通路蛋白的表达。结果S100A8/A9在包括肺癌的多种肿瘤组织中异常高表达,而且低表达S100A8/A9的肺癌患者生存期更长。筛选得到A549S100A8/A9-模型。与A549S100A8/A9+相比,A549S100A8/A9的细胞活力在24 h(0.735±0.182和1.414±0.128)、48 h(1.209±0.156和2.436±0.264)、72 h(1.645±0.162和3.153±0.273)均受到抑制、侵袭(545.33±18.145和994.33±43.03)和迁移(980.43±96.52和1653.45±136.27)能力降低,且早期凋亡比例增加(18.567±2.680和6.417±1.166,P<0.05差异具有统计学意义)。由DNA芯片结果可知,在A549S100A8/A9-细胞有2513个基因表达下调,1836个基因表达上调,KEGG通路富集结果提示细胞凋亡和PI3K/AKT相关通路均发生明显差异。Western blot结果显示,A549S100A8/A9-细胞的PI3K、Bcl-2、MMP-9、P-AKT、Bcl-2蛋白水平降低,Caspase-3、Bad蛋白表达增多。结论S100A8/A9可通过调节PI3K/AKT信号通路来促进A549细胞的增殖,侵袭和迁移。

Objective To explore the molecular mechanism of S100A8/A9-phosphatidylinositol 3-kinase/protein ki⁃nase signal axis regulating the progression of non-small cell lung cancer.Methods The expression and clinical signifi⁃cance of S100A8/A9 in lung cancer was analyzed through GTEx database,CCLE database and GEPIA2 database;es⁃tablish A549S100A8/A9-and A549 S100A8/A9+models were established through lentiviral transfection,CCK8 method,cell mi⁃gration method,Annexin V/PI double staining method were used to detect the proliferation,invasion,metastasis and apoptosis of A549 S100A8/A9-and A549 S100A8/A9+,and the changes in cell function were analyzed;DNA chip technology was used to detect A549 S100A8/A9-and A549 S100A8/A9+cells up-regulation and down-regulation of genes,related pathways were enriched by KEGG;according to the enrichment pathway results,the expression of phosphatidylinositol 3-kinase/protein kinase(PI3K/AKT)pathway protein was detected by Western blot.Results S100A8/A9 expressed abnormally highly in a variety of tumor tissues including lung cancer,and lung cancer patients with low expression of S100A8/A9 had a longer survival time.A549S100A8/A9-model was obtained by screening.Compared with A549S100A8/A9+,the cell viabili⁃ty of A549S100A8/A9-in 24 h(0.735±0.182 and 1.414±0.128),48 h(1.209±0.156 and 2.436±0.264),72 h(1.645±0.162 and 3.153±0.273)were inhibited,invaded(545.33±18.145 and 994.33±43.03)and migration(980.43±96.52 and 1653.45±136.27)were reduced,and the proportion of early apoptosis increased(18.567±2.680 and 6.417±1.166,P<0.05,the difference was statistically significant).According to the results of DNA chip,there were 2513 genes down regulated and 1836 genes up-regulated in A549S100A8/A9-cells.The enrichment results of KEGG pathway suggested that apoptosis and PI3K/AKT related pathways were significantly different.Western blot results showed that the protein lev⁃els of PI3K,Bcl-2,MMP-9,P-AKT,and Bcl-2 in A549S100A8/A9-cells decreased,while the expression of Caspase-3 and bad proteins increased.Conclusion S100A8/A9 can promote the proliferation,invasion and migration of A549 cells by regulating the PI3K/AKT signaling pathway.