肺癌细胞中PIK3AP1基因表达变化及其过表达对细胞增殖、侵袭、迁移的影响

Expression changes of PIK3AP1 gene in lung cancer and effects of PIK3AP1 gene overexpression on cell proliferation,invasion,and migration

目的观察肺癌细胞中PIK3AP1基因的表达情况,分析其表达与患者预后的关系,以及PIK3AP1基因过表达对肺癌细胞增殖、侵袭、迁移能力的影响。方法体外培养正常支气管上皮细胞系HBEC,非小细胞肺癌细胞系A549、HCC827、H358,小细胞肺癌细胞系H69、H526、H82。采用RT-PCR法检测上述细胞中PIK3AP1 mRNA的表达。利用Kaplan-Meier plotter在线生存分析数据库,对肺癌组织中PIK3AP1 mRNA表达与患者预后的相关性进行分析。取不表达PIK3AP1的肺癌细胞,将其分为观察组和对照组,以慢病毒转染技术分别转染PIK3AP1过表达载体和空载体,采用BrdU法观察细胞增殖能力,Transwell实验观察细胞侵袭能力,细胞划痕实验观察细胞迁移能力,Western blotting法检测AKT、p-AKT蛋白表达。结果PIK3AP1基因在小细胞肺癌细胞系H69、H526、H82中高表达,在非小细胞肺癌细胞系A549、HCC827、H358中低表达或不表达,在正常支气管上皮细胞中不表达。PIK3AP1基因高表达和低表达肺癌患者的中位生存期分别为60.73、92.63个月,PIK3AP1基因高表达者预后较差(P<0.05)。取不表达PIK3AP1的A549细胞进行转染,与对照组比较,观察组细胞增殖能力增高,侵袭能力增强(P均<0.01),迁移能力无明显变化(P>0.05);p-AKT蛋白表达增高(P<0.01),AKT蛋白表达无明显变化(P>0.05)。结论PIK3AP1在具有高转移能力的小细胞肺癌细胞中高表达,其高表达提示预后不良;PIK3AP1可能通过激活PI3K/AKT信号通路促进肺癌细胞的增殖和侵袭。

Objective To observe the expression of PIK3AP1 gene in lung cancer cells,and to analyze the relationship between its expression and the prognosis of patients,and the effects of PIK3AP1 gene overexpression on proliferation,invasion,and migration of lung cancer cells.Methods PIK3AP1 mRNA expression was detected by RT-PCR in the normal bronchial epithelial cell lines HBEC,non-small-cell lung cancer cell lines A549,HCC827,H358 and small cell lung cancer cell lines H69,H526,H82.The prognostic roles of PIK3AP1 in lung cancer was analyzed by using Kaplan-Meier Plotter database.We tested the functional consequences of PIK3AP1 expression in the lung cancer,and lentiviral constructs were introduced into A549 cells.We selected the lung cancer cells without PIK3AP1 expression and divided them into the observation group and control group,which were seperately transfected with PIK3AP1 overexpression vector and empty vector by lentiviral transfection technology.BrdU cell proliferation assay in vitro experiment,transwell cell invasion and the cell scratch test were used to detect cell proliferation,cell invasion and cell migration abilities.Western blotting was used to detect the expression levels of AKT and p-AKT proteins.Results PIK3AP1 gene was highly expressed in small cell lung cancer cell lines,low or not expressed in non-small-cell lung cancer cell lines,and not expressed in the normal bronchial epithelial cells.The median survival time of lung cancer patients with high expression and low expression of PIK3AP1 was 60.73 and 92.63 months,respectively;the prognosis of lung cancer patients with low expression of PIK3AP1 was better than that of patients with high expression of PIK3AP1(all P<0.05).Compared with the control group,the rate of cell proliferation and the number of invasive cells increased in the observation group,but the migration did not change(all P<0.01).The expression level of p-AKT was significantly up-regulated in the observation group(P<0.01),and there was no significant change in the expression of total AKT protein(P>0.05).Conclusion PIK3AP1 is highly expressed in small cell lung cancer with high metastatic capacity,and the high expression of PIK3AP1 gene in lung cancer shows poor prognosis;PIK3AP1 promotes the proliferation and invasion of small cell lung cancer cells by activating the PI3K-AKT signaling pathway.