中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (12): 1185-1193.doi: 10.19401/j.cnki.1007-3639.2021.12.006

• 论著 • 上一篇    下一篇

PLK4基因在人食管鳞癌组织中的表达及对癌细胞增殖、侵袭迁移影响的研究

张进忠 1 ,李悦淇 2 ,石 科 1 ,杨 亮 1 ,郭 丹 1   

  1. 1. 河南医学高等专科学校检验系,河南 郑州 451191 ;
    2. 郑州大学医学院临床医学系,河南 郑州 450000
  • 出版日期:2021-12-30 发布日期:2022-01-07
  • 通信作者: 郭 丹 E-mail: danguohmc@163.com
  • 基金资助:
    河南省科技攻关计划项目(162102310047、192102310110);河南省医学科技攻关计划联合共建项目(2018020529、LHGJ20190716);河南省高等职业学校青年骨干教师项目(2019GZGG086)。

Expression and clinical significance of PLK4 gene in esophageal squamous cell carcinoma and its effect on cell proliferation, invasion and migration

ZHANG Jinzhong 1 , LI Yueqi 2 , SHI Ke 1 , YANG Liang 1 , GUO Dan 1   

  1. 1. Department of Laboratory Medicine, Henan Medical College, Zhengzhou 451191, Henan Province, China; 2. Department of Clinical Medicine of Medical College, Zhengzhou University, Zhengzhou 450000, Henan Province, China
  • Published:2021-12-30 Online:2022-01-07
  • Contact: GUO Dan E-mail: danguohmc@163.com

摘要: 背景与目的:作为调节细胞周期的蛋白激酶,polo样激酶4(polo-like kinase 4,PLK4)参与有丝分裂启动、中心体成熟、胞质分裂及DNA损伤检测等,在多种肿瘤中呈高表达,但PLK4是否参与食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)的增殖和侵袭迁移尚不清楚。研究PLK4在ESCC细胞系和临床组织标本中的表达以及对癌细胞增殖、侵袭迁移的影响。方法:采用TRIzol提取细胞总RNA,反转录试剂盒合成cDNA,采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测PLK4基因在正常食管上皮细胞Het-1A和ESCC细胞系TE-1、TE-8和TE-13中的mRNA表达水平。离心收集细胞后用RIPA裂解液提取细胞总蛋白,采用蛋白质印迹法(Western blot)检测正常食管上皮细胞Het-1A和ESCC细胞系TE-1、TE-8和TE-13中PLK4蛋白水平。选取2017年1月—2019年12月河南医学高等专科学校附属医院93例经病理组织学检查确诊为ESCC的患者癌组织及其配对的癌旁组织(距原发灶边缘5 cm以上),临床组织用液氮速冻后加入RIPA裂解液研磨提取组织总蛋白,采用Western blot检测PLK4蛋白水平。绘制受试者工作特征曲线,分析PLK4表达与临床病理学参数之间的关系。利用siRNA干扰技术抑制TE-13细胞中PLK4的表达,设计并合成PLK4的siRNA干扰片段,采用Lipofectamine TM 2000转染细胞抑制PLK4在TE-13细胞中的表达,RTFQ-PCR实验和Western blot检测siRNA干扰片段对PLK表达的影响。PLK4在TE-13细胞中表达下调后,用细胞计数试剂盒-8(cell counting kit-8,CCK-8)实验和克隆形成实验检测细胞的增殖能力,transwell小室实验检测细胞的侵袭能力,划痕愈合实验检测细胞的迁移能力。Western blot实验检测PLK4下调后对mTOR/p70S6K信号转导通路中关键蛋白mTOR、p70S6K、p-mTOR Ser2448 和p-p70S6K Thr421/Ser424 表达的影响。结果:RTFQ-PCR和Western blot检测结果显示,PLK4基因在ESCC细胞系中的mRNA和蛋白表达水平显著高于正常食管上皮细胞(P<0.05)。与癌旁正常组织相比,ESCC组织标本中PLK4的蛋白表达水平异常增高(P<0.05),绘制的受试者工作特征曲线的曲线下面积为0.841,95%CI为0.786~0.895,灵敏度为74.2%(69/93),特异度为89.2%(83/93)(P<0.000 1)。PLK4蛋白在ESCC组织中的表达水平与性别、年龄和肿瘤大小均无关(均P>0.05),但与分化程度、临床分期和淋巴结是否转移有关(均P<0.05)。ESCC分化程度越低,PLK4高表达率越高,低分化程度的ESCC组织中PLK4高表达率为86.7%,Ⅲ~Ⅳ期患者ESCC组织中PLK4蛋白高表达率为92.3%。PLK4高表达与临床分期呈正相关(P<0.05)。CCK-8和克隆形成实验结果显示,下调PLK4的表达可以显著抑制TE-13细胞的增殖(P<0.05),transwell小室实验和划痕实验结果显示,下调PLK4的表达可以显著抑制TE-13细胞的侵袭迁移能力(P<0.05)。抑制PLK4的表达使TE-13细胞中mTOR和p70S6K蛋白的表达下降(P<0.05),且p-mTOR Ser2448 和p-p70S6K Thr421/Ser424 的表达下降(P<0.05)。结论:PLK4在ESCC细胞和组织中均呈高表达,抑制PLK4的表达可以抑制ESCC细胞的增殖和侵袭迁移能力,PLK4可能通过mTOR/p70S6K信号转导通路促进ESCC细胞恶性进程。

关键词:  polo样激酶4, 食管鳞癌, 临床病理学特征, 增殖, 侵袭迁移

Abstract: Background and purpose: As a protein kinase that regulates the cell cycle, polo-like kinase 4 (PLK4) is involved in mitosis initiation, centrosome maturation, cytokinesis, DNA damage detection, etc., which is highly expressed in a variety of tumors. Whether it is involved in the proliferation, invasion and migration of esophageal squamous cell carcinoma (ESCC), and the specific molecular mechanism still remain unclear. This study examined the expression of PLK4 in ESCC cell lines and clinical tissue specimens, and its effect on cancer cell proliferation, invasion and migration. Methods: Total cell RNA was extracted with TRIzol, and cDNA was synthesized with reverse transcription kit for real-time fluorescence quantitative polymerase chain reaction (RTFQ- PCR) to detect the mRNA expression level of PLK4 gene in normal esophageal epithelial cells Het-1A and ESCC cell lines TE-1, TE-8 and TE-13. After the cells were collected by centrifugation, total cell protein was extracted with RIPA lysate. Western blot was used to detect the expression level of PLK4 protein in normal esophageal epithelial cells Het-1A and ESCC cell lines TE-1, TE-8 and TE-13. A total of 93 cases of ESCC tissues and paired adjacent tissues (more than 5 cm from the edge of the primary tumor) confirmed by histopathology were collected at the Affiliated Hospital of Henan Medical College from January 2017 to December 2019. The clinical tissues were quick-frozen by liquid nitrogen followed by total tissue protein extraction by RIPA lysate. Western blot was used to detect the expression level of PLK4 protein in ESCC tissues. We constructed a receiver operating characteristic curve and analyzed the relationship between PLK4 expression and clinicopathological parameters. The expression of PLK4 in TE-13 cells was inhibited by siRNA interference technology. The siRNA interference fragments of PLK4 were designed and synthesized, followed by transfection with Lipofectamine TM 2000 to inhibit the expression of PLK4 in TE-13 cells. The effect of siRNA interference fragments on PLK expression was detected by RTFQ- PCR experiments and Western blot. After the expression of PLK4 was down-regulated in TE-13 cells, cell counting kit-8 (CCK-8) experiment and clone formation experiment were used to detect cell proliferation ability, and transwell chamber experiment and scratch healing experiment were conducted to detect cell invasion and migration abilities. The effects of down-regulation of PLK4 on the expressions of key proteins mTOR, p70S6K, p-mTOR Ser2448 and p-p70S6K Thr421/Ser424 in the mTOR/p70S6K signaling pathway were detected by Western blot experiments. Results: The results of RTFQ-PCR and Western blot experiments showed that the mRNA and protein expression levels of PLK4 gene in ESCC cell lines were significantly higher than those in normal esophageal epithelial cells (P<0.05). Compared with normal tissues adjacent to cancer, the protein expression level of PLK4 in ESCC tissue specimens was abnormally increased (P<0.05). The receiver operating characteristic curve exhibited an area under curve (AUC) of 0.841, a 95% CI of 0.786-0.895 with 74.2% (69/93) sensitivity and 89.2% (83/93) specificity (P<0.0001). There was no relationship between expression level of PLK4 protein and gender, age and tumor size (all P > 0.05) in ESCC tissues. However, expression level of PLK4 protein was related to the degree of differentiation, clinical stage and lymph node metastasis (all P<0.05). The lower degree of ESCC differentiation had higher expression rate of PLK4. The high expression rate of PLK4 in ESCC tissues of poorly differentiated degree was 86.7%, and the high expression rate of PLK4 protein in ESCC tissues of patients with stage Ⅲ-Ⅳ was 92.3%. The high expression of PLK4 was positively correlated with clinical stage (P<0.05). The results of CCK- 8 and clone formation experiments showed that down-regulating the expression of PLK4 significantly inhibited the proliferation of TE-13 cells (P<0.05). The results of the transwell chamber experiment and the scratch experiment showed that down-regulating the expression of PLK4 significantly inhibited the invasion and migration abilities of TE-13 cells (P<0.05). Inhibiting the expression of PLK4 decreased the protein expressions of mTOR and p70S6K in TE-13 cells (P<0.05), and the expressions of p-mTOR Ser2448 and p-p70S6K Thr421/Ser424 decreased (P<0.05). Conclusion: PLK4 is highly expressed in ESCC cells and tissues. Inhibition of PLK4 expression inhibited the proliferation, invasion and migration of ESCC cells. PLK4 may promote the malignant process of ESCC cells through the mTOR/p70S6K signaling pathway.

Key words: Polo-like kinase 4, Esophageal squamous cell carcinoma, Clinicopathological characteristics, Proliferation, Invasion and migration