POU4F3表达对118例肺腺癌患者预后评估及对肺腺癌细胞株迁移的影响  

Prognostic value of POU4F3 in 118 patients with lung adenocarcinoma and its effect on migration of lung adenocarcinoma cells

作　　者：柴小雪 叶辉[1] 吕欣然 丁续超 甄秋来 杜娟[3] 曹莉莉[1,2] CHAI Xiaoxue;YE Hui;LYU Xinran;DING Xuchao;ZHEN Qiulai;DU Juan;CAO Lili(Oncology Department,Shandong Qianfoshan Hospital,Cheeloo College of Medicine,Shandong University,Jinan 250014,Shandong,China;Oncology Department,The First Affiliated Hospital of Shandong First Medical University,Jinan 250014,Shandong,China;Department of Immunology,Guilin Medical College,Guilin 541004,Guangxi,China)

机构地区：[1]山东大学附属山东省千佛山医院肿瘤科,山东济南250014 [2]山东第一医科大学第一附属医院肿瘤科,山东济南250014 [3]桂林医学院免疫学教研室,广西桂林541004

出　　处：《山东大学学报（医学版）》2021年第11期8-18,28,共12页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(30901712);山东省重点研发计划(2019GSF108180);济南市科技发展计划(201907119);山东第一医科大学第一附属医院培育基金(QYPY2019NSFC1015);山东省自然科学基金面上项目(ZR2020MH203);广西科技基地人才计划项目(桂科AD20297024)。

摘　　要：目的揭示POU结构域第4类转录因子3(POU4F3)在肺腺癌组织的表达及其与肺腺癌患者预后的关系,并探索POU4F3对肺腺癌细胞迁移的作用。方法免疫组织化学染色检测人肺腺癌及其癌旁组织(样本量均为118)中POU4F3的表达水平。利用Log-rank(Mantel-Cox)检验POU4F3表达水平与总生存期的关联性。以肺腺癌株为实验对象,构建稳定过表达(Lv-POU4F3)及其对照(Lv-control)或敲低POU4F3(shPOU4F3)及其对照(control shRNA)的慢病毒载体并分别转染至SPCA1和A549细胞系,Western blotting验证POU4F3过表达或敲低转染效率。运用划痕实验、Transwell迁移实验和鬼笔环肽荧光实验检测细胞迁移能力。结果免疫组化分析显示,118例肺腺癌组织中POU4F3的表达评分低于118例癌旁组织评分(1.5 vs 4.0),差异有统计学意义(P<0.001)。与POU4F3低表达肺腺癌患者相比,POU4F3高表达患者的总生存期延长(HR=2.04,95%CI:1.158~3.607,P=0.028)。细胞实验结果显示,有效构建了过表达或敲低POU4F3的稳定转染肺腺癌细胞株。过表达POU4F3后,划痕实验表明,Lv-POU4F3组细胞划痕愈合率低于Lv-control组,差异有统计学意义(F_(SPCA1处理)=69.86,P_(SPCA1)=0.001;F_(A549处理)=492.10,P_(A549)<0.001);Transwell迁移实验表明,SPCA1-Lv-POU4F3组穿膜细胞数少于SPCA1-Lv-control组(599.0±11.36 vs 806.3±18.72,t=16.40,P<0.001),A549-Lv-POU4F3组穿膜细胞数少于A549-Lv-control组(181.0±18.68 vs 314.7±23.46,t=7.72,P=0.002);鬼笔环肽荧光实验表明,过表达POU4F3后,SPCA1细胞微丝变细、伪足较少,Lv-POU4F3组的平均荧光强度低于Lv-control组(23.30±1.34 vs 33.45±1.85),差异有统计学意义(t=7.67,P=0.002)。敲低POU4F3后,划痕实验表明,shPOU4F3组细胞划痕愈合率高于control shRNA组,差异有统计学意义(F_(SPCA1处理)=114.60,P_(SPCA1)<0.001;F_(A549处理)=1 710.00,P_(A549)<0.001)。Transwell迁移实验表明,SPCA1-shPOU4F3组穿膜细胞数多于SPCA1-control shRNA组(970.00±14.53 vs 585.00±25.53,Objective To reveal the expression of POU domain class 4 transcription factor 3(POU4 F3) in lung adenocarcinoma(LUAD) tissues and its association with the prognosis of lung adenocarcinoma(LUAD) patients,and to explore the effect of POU4 F3 on the migration of LUAD cells.Methods Immunohistochemical staining(IHC) was used to detect the expression of POU4 F3 in human LUAD and its adjacent tissues,both of the sample sizes being 118.Log-rank(Mantel-Cox) was used to examine the correlation between POU4 F3 expression and the overall survival of LUAD patients.Taking the LUAD cells as the experimental subject,stable overexpression(Lv-POU4 F3) and its control(Lv-control) or knockdown of POU4 F3(shPOU4 F3) and its control(control shRNA) lentivirus were constructed and transfected into SPCA1 and A549 cells,respectively.Western blotting was conducted to verify the efficiency of POU4 F3 overexpression or knockdown transfection.Cell migration was detected by wound healing,Transwell migration assay,and phalloidine fluorescence experiment.Results IHC analysis showed that the expression of POU4 F3 in LUAD tissues was significantly lower than that in adjacent tissues(1.5 vs 4.0),and the difference was statistically significant(P<0.001).Compared with LUAD patients with low POU4 F3 expression,patients with high POU4 F3 expression had significantly longer overall survival(HR=2.04,95%CI:1.158-3.607,P=0.028).Cell experiment results showed that stably transfected LUAD cell lines overexpressing or knocking down POU4 F3 were constructed effectively.After overexpression of POU4 F3,the wound healing expreriment showed that the wound healing rate of cells in the Lv-POU4 F3 group was lower than that in the Lv-control group,the difference was statistically significant(F_(SPCA1 treatment)=69.86,P_(SPCA1)=0.001;F_(A549 treatment)=492.10,P_(A549)<0.001).Transwell migration assay showed that the number of transmembrane cells in the SPCA1-Lv-POU4 F3 group was less than that in the SPCA1-Lv-control group(599.0±11.36 vs 806.3±18.72,t=16.40,P<0.001),

关 键 词：肺腺癌 POU结构域第4类转录因子3 总生存期 迁移 

分 类 号：R734.2[医药卫生—肿瘤]