Cdc42在舌鳞状细胞癌中的表达及其对细胞增殖、迁移、侵袭的影响

doi:10.19438/j.cjoms.2023.03.001

中国口腔颌面外科杂志 ›› 2023, Vol. 21 ›› Issue (3): 209-217.doi: 10.19438/j.cjoms.2023.03.001

Cdc42在舌鳞状细胞癌中的表达及其对细胞增殖、迁移、侵袭的影响

慕家宁^1,2, 袁文清³, 陆洲⁴, 赵阳¹, 王奇民², 童磊², 陈正岗²

1.大连医科大学口腔医学院,辽宁大连 116044;
2.康复大学青岛医院（青岛市市立医院）口腔医学中心,山东青岛 266071;
3.康复大学青岛医院（青岛市市立医院）基层医疗管理部,山东青岛 266071;
4.潍坊医学院口腔医学院,山东潍坊 261021

收稿日期:2022-11-11 修回日期:2023-01-17 出版日期:2023-05-20 发布日期:2023-08-16
通讯作者: 陈正岗,E-mail: chenzhg1973@163.com
作者简介:慕家宁（1997-）,男,硕士,E-mail: 2810272428@qq.com
基金资助:
国家自然科学基金（81372908）

Expression of Cdc42 in tongue squamous cell carcinoma and its influence on cell proliferation, migration and invasion

MU Jia-ning^1,2, YUAN Wen-qing³, LU Zhou⁴, ZHAO Yang¹, WANG Qi-min², TONG Lei², CHEN Zheng-gang²

1. School of Stomatology, Dalian Medical University. Dalian 116044, Liaoning Province;
2. Stomatology Center, 3. Grassroots Medical Management Department, Qingdao Hospital, University of Health and Rehabilitation Sciences(Qingdao Municipal Hospital), Qingdao University. Qingdao 266071, Shandong Provinc;
4. College of Stomatology, Weifang Medical University. Weifang 261021, Shandong Province, China

Received:2022-11-11 Revised:2023-01-17 Online:2023-05-20 Published:2023-08-16

摘要/Abstract

摘要： 目的：观察细胞分裂周期蛋白42（Cdc42）在舌鳞癌中的表达及与临床病理特征和预后的关系,探讨体外沉默Cdc42基因对舌鳞癌细胞增殖、迁移、侵袭及上皮-间质转化（EMT）的影响及作用机制。方法：通过免疫组织化学法检测Cdc42基因在舌鳞癌及癌旁组织中的表达,分析Cdc42表达与舌鳞癌临床病理学参数的相关性及对预后的影响。将人舌鳞状细胞癌CAL27和SCC-4细胞随机分为3组,分别为Cdc42-siRNA组、阴性对照组和空白对照组。设计并构建针对人Cdc42基因序列的3条小干扰RNA（siRNA）,应用脂质体介导转染方法分别将Cdc42-siRNA和NC-siRNA转染至Cdc42-siRNA组和阴性对照组,空白对照组仅加入转染试剂。通过实时荧光定量反转录PCR（qRT-PCR）和蛋白免疫印迹法（Western blot）检测各组细胞Cdc42的mRNA及蛋白表达,将沉默效率最高的Cdc42-siRNA转染组作为后续实验组。Western blot检测EMT及MAPK JNK/p38通路相关蛋白的表达,CCK-8、细胞划痕实验和Transwell侵袭实验分别检测各组细胞的体外增殖、迁移和侵袭能力。采用SPSS 22.0软件包对数据进行统计学分析。结果：Cdc42在44例舌鳞癌患者中的阳性表达率为70.5%(31/44),在癌旁组织中的阳性率为38.6%(17/44）,差异具有统计学意义（P<0.05）。Cdc42表达与不同颈淋巴结转移、临床分期、浸润深度相关（P<0.05）。Kaplan-Meier生存分析显示,Cdc42阳性表达的舌鳞癌患者预后与阴性表达患者无显著差异（P>0.05）。体外实验中,Cdc42-siRNA组细胞Cdc42 mRNA和蛋白表达显著下降（P<0.05）,EMT相关蛋白间叶标志物N-cadherin、Vimentin表达显著降低（P<0.05）,MMP-9表达显著降低（P<0.05）,而上皮标记物E-cadherin相对表达量显著增加（P<0.05）;MAPK JNK/p38通路相关蛋白p38-MAPK和JNK蛋白相对表达量差异无统计学意义（P>0.05）,而p-p38-MAPK、p-JNK蛋白表达水平显著下降（P<0.05）,Cdc42-siRNA组细胞增殖、侵袭和迁移能力显著下降（P<0.05）。结论：舌鳞癌患者中Cdc42表达增加,并且其表达与颈淋巴结转移、临床分期及浸润深度相关。沉默Cdc42可能通过阻断MAPK JNK/p38通路抑制舌鳞癌细胞EMT过程,进而抑制侵袭迁移,同时可负向调节舌鳞癌细胞增殖。

关键词: Cdc42, 舌鳞癌, 增殖, 侵袭, 迁移, 上皮-间质转化

Abstract: PURPOSE: The study aimed to observe the expression of cell division cycle protein 42(Cdc42) in tongue squamous cell carcinoma (TSCC) and its relationship with clinicopathological features and prognosis, and explore the effect of silencing Cdc42 gene in vitro on proliferation, migration, invasion and epithelial-mesenchymal transformation (EMT) of TSCC and its mechanism. METHODS: The expression of Cdc42 gene in TSCC and adjacent tissues was detected by immunohistochemistry, the correlation between the expression of Cdc42 and clinicopathological parameters of TSCC and its influence on prognosis were analyzed. Human TSCC CAL27 and SCC-4 cells were randomly divided into 3 groups, namely Cdc42-siRNA group, negative control group and blank control group. Three small interfering RNA(siRNA) targeting human Cdc42 gene sequence were designed and constructed, Cdc42-siRNA and NC-siRNA was transfected into Cdc42-siRNA group and negative control group by liposome-mediated transfection, while the blank control group only added transfection reagent. Expression of Cdc42 mRNA and protein was detected by qRT-PCR and Western blot, and the Cdc42-siRNA transfection group with the best silencing effect was used as the follow-up experimental group. The expression of EMT and MAPK JNK/p38 pathway-related proteins was examined by Western blot. The proliferation, migration and invasion ability of cells in vitro were tested by CCK-8, wound healing test and Transwell invasion test. SPSS 22.0 software package was used for data analysis. RESULTS: The positive expression rate of Cdc42 was 70.5%(31/44) in 44 patients with TSCC, and 38.6%(17/44) in adjacent tissues, the difference was statistically significant(P<0.05). The expression of Cdc42 was related to cervical lymph node metastasis, clinical stage and depth of invasion(P<0.05). Kaplan-Meier survival analysis showed that the prognosis of TSCC patients with positive expression of Cdc42 was not significantly different from those with negative expression(P>0.05). In vitro experiments, in Cdc42-siRNA group, the expression of Cdc42 mRNA and protein decreased significantly(P<0.05), the relative expression of EMT-related protein mesenchymal markers N-cadherin and Vimentin decreased significantly(P<0.05), and the relative expression of MMP-9 decreased significantly(P<0.05); however, the relative expression of E-cadherin was increased significantly(P<0.05). The relative expression levels of p38-MAPK and JNK proteins related to MAPK JNK/p38 pathway had no significant difference(P>0.05), but the expression levels of p-p38-MAPK and p-JNK proteins decreased significantly (P<0.05). In addition, the ability of proliferation, invasion and migration of cells in Cdc42-siRNA group decreased significantly(P<0.05). CONCLUSIONS: The expression of Cdc42 increased in TSCC patients, and the expression was related to cervical lymph node metastasis, clinical stage and depth of invasion. The silencing of Cdc42 may inhibit the transformation of EMT process of TSCC cells by blocking MAPK JNK/p38 pathway, thus inhibiting its invasion and migration, and negatively regulating TSCC cell proliferation.

Key words: Cdc42, Tongue squamous carcinoma cell, Proliferation, Invasion, Migration, Epithelial-mesenchymal transformation

中图分类号:

R739.8

慕家宁, 袁文清, 陆洲, 赵阳, 王奇民, 童磊, 陈正岗. Cdc42在舌鳞状细胞癌中的表达及其对细胞增殖、迁移、侵袭的影响[J]. 中国口腔颌面外科杂志, 2023, 21(3): 209-217.

MU Jia-ning, YUAN Wen-qing, LU Zhou, ZHAO Yang, WANG Qi-min, TONG Lei, CHEN Zheng-gang. Expression of Cdc42 in tongue squamous cell carcinoma and its influence on cell proliferation, migration and invasion[J]. China J Oral Maxillofac Surg, 2023, 21(3): 209-217.

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Cdc42在舌鳞状细胞癌中的表达及其对细胞增殖、迁移、侵袭的影响

Expression of Cdc42 in tongue squamous cell carcinoma and its influence on cell proliferation, migration and invasion

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