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

doi:10.6040/j.issn.1671-7554.0.2021.0193

摘要/Abstract

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,t=22.70,P<0.001),A549-shPOU4F3组穿膜细胞数多于A549-control shRNA组(528.00±29.14 vs 185.30±9.50,t=19.37,P<0.001);鬼笔环肽荧光实验表明,敲低POU4F3后,SPCA1细胞微丝变粗、伪足增多,shPOU4F3组的平均荧光强度高于control shRNA组(48.53±3.30 vs 31.07±2.32),差异有统计学意义(t=7.50,P=0.002)。结论 POU4F3在人肺腺癌患者癌组织中表达低于癌旁组织,POU4F3高表达者预后较佳,且POU4F3能够抑制肺腺癌细胞迁移。POU4F3可能是LUAD的抑癌基因并有望成为诊断和治疗LUAD的新靶标。

关键词: 肺腺癌, POU结构域第4类转录因子3, 总生存期, 迁移

Abstract: Objective To reveal the expression of POU domain class 4 transcription factor 3(POU4F3)in lung adenocarcinoma(LUAD)tissues and its association with the prognosis of lung adenocarcinoma(LUAD)patients, and to explore the effect of POU4F3 on the migration of LUAD cells. Methods Immunohistochemical staining(IHC)was used to detect the expression of POU4F3 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 POU4F3 expression and the overall survival of LUAD patients. Taking the LUAD cells as the experimental subject, stable overexpression(Lv-POU4F3)and its control(Lv-control)or knockdown of POU4F3(shPOU4F3)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 POU4F3 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 POU4F3 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 POU4F3 expression, patients with high POU4F3 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 POU4F3 were constructed effectively. After overexpression of POU4F3, the wound healing expreriment showed that the wound healing rate of cells in the Lv-POU4F3 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-POU4F3 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), and the number of transmembrane cells in the A549-Lv-POU4F3 group was less than that in the A549-Lv-control group(181.0±18.68 vs 314.7±23.46, t=7.72, P=0.002). The phalloidine fluorescence experiment showed that SPCA1 cells with overexpression of POU4F3 had thinner microfillets and fewer pseudopodia. The mean fluorescence intensity of Lv-POU4F3 group was lower than that of Lv-control group(23.30±1.34 vs 33.45±1.85), and the difference was statistically significant(t=7.67, P=0.002). After POU4F3 was knocked down, wound healing experiment showed that the cell wound healing rate of shPOU4F3 group was higher than that of control shRNA group, the difference was statistically significant(F_{SPCA1 treatment}=114.60, P_SPCA1<0.001; F_{A549 treatment}=1 710.00, P_A549<0.001). Transwell migration assay showed that the number of transmembrane cells in SPCA1-shPOU4F3 group was higher than that in the SPCA1-control shRNA group(970.00±14.53 vs 585.00±25.53, t=22.70, P<0.001), and the number of transmembrane cells in A549-shPOU4F3 group was higher than that in the A549-control shRNA group(528.00±29.14 vs 185.30±9.50, t=19.37, P<0.001). The phalloidine fluorescence experiment showed that after knockdown of POU4F3, SPCA1 cell microfilules became thicker and pseudopodia increased. The mean fluorescence intensity of shPOU4F3 group was higher than that of control shRNA group(48.53±3.30 vs 31.07±2.32), and the difference was statistically significant(t=7.50, P=0.002). Conclusion The expression of POU4F3 in human LUAD tissues is significantly lower than that in para-cancerous tissues. LUAD patients with high expression of POU4F3 have a better prognosis. POU4F3 can inhibit the migration of LUAD cells. POU4F3 may be a novel tumor suppressor gene of LUAD and a new target for the diagnosis and treatment of LUAD.

Key words: Lung adenocarcinoma, POU domain class 4 transcription factor 3, Overall survival, Migration

中图分类号:

R734.2

柴小雪,叶辉,吕欣然,丁续超,甄秋来,杜娟,曹莉莉. 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[J]. 山东大学学报 (医学版), 2021, 59(11): 8-18.

CHAI Xiaoxue, YE Hui, LYU Xinran, DING Xuchao, ZHEN Qiulai, DU Juan, CAO Lili. Prognostic value of POU4F3 in 118 patients with lung adenocarcinoma and its effect on migration of lung adenocarcinoma cells[J]. Journal of Shandong University (Health Sciences), 2021, 59(11): 8-18.

参考文献

[1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020[J]. CA Cancer J Clin, 2020, 70(1): 7-30.
[2] Chen W, Zheng R, Baade PD, et al. Cancer statistics in china, 2015 [J]. CA Cancer J Clin, 2016, 66(2): 115-132.
[3] Singh D, Attri BK, Gill RK, et al. Review on EGFR inhibitors: critical updates [J]. Mini Rev Med Chem, 2016, 16(14): 1134-1166.
[4] Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer-molecular and clinical predictors of outcome [J]. N Engl J Med, 2005, 353(2): 133-144.
[5] Barreca A, Lasorsa E, Riera L, et al. Anaplastic lymphoma kinase in human cancer [J]. J Mol Endocrinol, 2011, 47(1): R11-R23.
[6] Bhatnagar V, Gormley NJ, Luo L, et al. Fda approval summary: daratumumab for treatment of multiple myeloma after one prior therapy [J]. Oncologist, 2017, 22(11): 1347-1353.
[7] Kasamon YL, de Claro RA, Wang Y, et al. FDA approval summary: nivolumab for the treatment of relapsed or progressive classical Hodgkin lymphoma [J]. Oncologist, 2017, 22(5): 585-591.
[8] Fashoyin-Aje L, Donoghue M, Chen H, et al. FDA approval summary: pembrolizumab for recurrent locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma expressing PD-L1 [J]. Oncologist, 2019, 24(1): 103-109.
[9] Kazandjian D, Suzman DL, Blumenthal G, et al. FDA approval summary: nivolumab for the treatment of metastatic non-small cell lung cancer with progression on or after platinum-based chemotherapy [J]. Oncologist, 2016, 21(5): 634-642.
[10] Herr W, Sturm RA, Clerc RG, et al. The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products [J]. Genes Dev, 1988, 2(12A): 1513-1516.
[11] Vahava O, Morell R, Lynch ED, et al. Mutation in transcription factor POU4F3 associated with inherited progressive hearing loss in humans [J]. Science, 1998, 279(5358): 1950-1954.
[12] Weiss S, Gottfried I, Mayrose I, et al. The DFNA15 deafness mutation affects POU4F3 protein stability, localization, and transcriptional activity [J]. Mol Cell Biol, 2003, 23(22): 7957-7964.
[13] Masuda M, Li Y, Pak K, et al. The promoter and multiple enhancers of the POU4F3 gene regulate expression in inner ear hair cells [J]. Mol Neurobiol, 2017, 54(7): 5414-5426.
[14] He L, Pang X, Chen P, et al. Mutation in the hair cell specific gene POU4F3 is a common cause for autosomal dominant nonsyndromic hearing loss in Chinese Hans [J]. Neural Plast, 2016, 2016: 9890827. doi: 10.1155/2016/9890827.
[15] Freitas ÉL, Oiticica J, Silva AG, et al. Deletion of the entire POU4F3 gene in a familial case of autosomal dominant non-syndromic hearing loss [J]. Eur J Med Genet, 2014, 57(4): 125-128.
[16] Sayyad Z, Sirohi K, Radha V, et al. 661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively [J]. Sci Rep, 2017, 7(1): 16855.
[17] Leonard JH, Cook AL, Van Gele M, et al. Proneural and proneuroendocrine transcription factor expression in cutaneous mechanoreceptor(Merkel)cells and Merkel cell carcinoma [J]. Int J Cancer, 2002, 101(2): 103-110.
[18] Chen YC, Huang RL, Huang YK, et al. Methylomics analysis identifies epigenetically silenced genes and implies an activation of β-catenin signaling in cervical cancer [J]. Int J Cancer, 2014, 135(1): 117-127.
[19] Kocsis A, Takács T, Jeney C, et al. Performance of a new HPV and biomarker assay in the management of hrHPV positive women: subanalysis of the ongoing multicenter TRACE clinical trial(n>6,000)to evaluate POU4F3 methylation as a potential biomarker of cervical precancer and cancer [J]. Int J Cancer, 2017, 140(5): 1119-1133.
[20] Wu X, Rauch TA, Zhong X, et al. CpG island hypermethylation in human astrocytomas [J]. Cancer Res, 2010, 70(7): 2718-2727.
[21] 周春燕,药立波.生物化学与分子生物学[M].9版.北京:人民卫生出版社,2018: 405.
[22] Morris LG, Chan TA. Therapeutic targeting of tumor suppressor genes [J]. Cancer, 2015, 121(9): 1357-1368.
[23] Klein CA. Parallel progression of primary tumours and metastases [J]. Nat Rev Cancer, 2009, 9(4): 302-312.
[24] 丁兰,柳志军,令利军,等.虎刺醛对人肝癌细胞HepG2生长抑制、细胞迁移抑制及其机制的研究[J].中国细胞生物学学报, 2013, 35(4): 442-449. DING Lan, LIU Zhijun, LING Lijun, et al. Study of damnacanthal on anti-proliferation, cell migration inhibition and its mechanism in human hepatoma cell HepG2 [J]. Chinese Journal of Cell Biology, 2013, 35(4): 442-449.
[25] 黄蕾,滕春莹,雷鹏,等.伪足、有氧糖酵解与肿瘤细胞迁移的研究进展[J].癌变·畸变·突变, 2020, 32(1): 72-75.
[26] Chazotte B. Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging [J]. Cold Spring Harb Protoc, 2010, 2010(5): pdb.prot4947. doi: 10.1101/pdb.prot4947.

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