体外沉默异戊二烯基半胱氨酸羧基甲基转移酶基因对舌鳞状细胞癌迁移和侵袭的影响

doi:10.7518/hxkq.2021.03.013

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (3): 328-335.doi: 10.7518/hxkq.2021.03.013

体外沉默异戊二烯基半胱氨酸羧基甲基转移酶基因对舌鳞状细胞癌迁移和侵袭的影响

周男¹(), 迟增鹏¹, 李文健², 赵开³, 王少如², 王奇民⁴, 童磊⁴, 何宗轩⁵, 韩红钰⁴, 王莹⁶, 陈正岗⁴()

^1.潍坊医学院口腔医学院，潍坊 261021
^2.大连医科大学口腔医学院，大连 116044
^3.青岛大学口腔医学院，青岛 266003
^4.青岛大学附属青岛市市立医院口腔医学中心，青岛 266071
^5.青岛大学附属医院口腔颌面外科，青岛 266005
^6.济南市第四人民医院口腔科，济南 250031

收稿日期:2020-06-18 修回日期:2021-02-05 出版日期:2021-06-01 发布日期:2021-05-26
通讯作者: 陈正岗 E-mail:1123862614@qq.com;chenzhg1973@163.com
作者简介:周男，硕士，E-mail：1123862614@qq.com
基金资助:
国家自然科学基金面上项目(81372908)

Effects of isoprenylcysteine carboxylmethyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma

Zhou Nan¹(), Chi Zengpeng¹, Li Wenjian², Zhao Kai³, Wang Shaoru², Wang Qimin⁴, Tong Lei⁴, He Zongxuan⁵, Han Hongyu⁴, Wang Ying⁶, Chen Zhenggang⁴()

^1.College of Stomatology, Weifang Medical University, Weifang 261021, China
^2.School of Stomatology, Dalian Medical University, Dalian 116044, China
^3.School of Stomatology, Qingdao University, Qingdao 266003, China
^4.Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
^5.Dept. of Oral and Maxillafacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266005, China
^6.Dept. of Stomatology, Fourth People's Hospital of Jinan, Jinan 250031, China

Received:2020-06-18 Revised:2021-02-05 Online:2021-06-01 Published:2021-05-26
Contact: Chen Zhenggang E-mail:1123862614@qq.com;chenzhg1973@163.com
Supported by:
The National Natural Science Foundation of China(81372908)

摘要/Abstract

摘要： 目的

通过构建异戊二烯基半胱氨酸羧基甲基转移酶（ICMT）小分子干扰RNA（siRNA）沉默ICMT，研究沉默ICMT基因对舌鳞状细胞癌（TSCC）迁移和侵袭的影响。

方法

通过脂质体转染的方法将siRNA转染至人TSCC CAL-27和SCC-4细胞（ICMT-siRNA组），并设阴性对照组（转染NC-siRNA）和空白对照组（仅加转染试剂，不转染siRNA）。应用实时荧光定量聚合酶链反应（qRT-PCR）检测转染后各组细胞中ICMT、RhoA的mRNA表达并明确沉默效率。蛋白质免疫印迹法检测各组ICMT、总RhoA、膜RhoA、Rho关联含卷曲螺旋结合蛋白激酶1（ROCK1）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）蛋白表达。划痕实验测定细胞迁移能力，Transwell侵袭实验测定细胞侵袭能力。

结果

CAL-27和SCC-4细胞转染ICMT-siRNA后，实验组相较于阴性对照组和空白对照组ICMT基因和蛋白表达明显降低（P<0.05），RhoA基因和总蛋白表达比较的差异无统计学意义（P>0.05），RhoA膜蛋白、ROCK1、MMP-2、MMP-9表达降低（P<0.05）。迁移和侵袭能力明显降低（P<0.05）。

结论

ICMT-siRNA可显著抑制人TSCC CAL-27和SCC-4细胞迁移及侵袭能力，其机制可能与RhoA-ROCK信号通路有关。

关键词: 异戊二烯基半胱氨酸羧基甲基转移酶, RhoA, 舌鳞状细胞癌, 迁移, 侵袭

Abstract: Objective

The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.

Methods

Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.

Results

After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (P<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (P>0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (P<0.05). The migration and invasion abilities were inhibited (P<0.05).

Conclusion

The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.

Key words: isoprenylcysteine carboxylmethyltransferase, RhoA, tongue squamous cell carcinoma, migration, invasion

中图分类号:

R 782

周男, 迟增鹏, 李文健, 赵开, 王少如, 王奇民, 童磊, 何宗轩, 韩红钰, 王莹, 陈正岗. 体外沉默异戊二烯基半胱氨酸羧基甲基转移酶基因对舌鳞状细胞癌迁移和侵袭的影响[J]. 华西口腔医学杂志, 2021, 39(3): 328-335.

Zhou Nan, Chi Zengpeng, Li Wenjian, Zhao Kai, Wang Shaoru, Wang Qimin, Tong Lei, He Zongxuan, Han Hongyu, Wang Ying, Chen Zhenggang. Effects of isoprenylcysteine carboxylmethyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma[J]. West China Journal of Stomatology, 2021, 39(3): 328-335.

图/表 6

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ICMT-siRNA	产品编号	产品名称	靶序列
ICMT-siRNA-1	StB0010313A	genOFFTMst-h-ICMT_001	GAAGAAGAAAT-CTCACTAA
ICMT-siRNA-2	StB0010313B	genOFFTMst-h-ICMT_002	GTTAGAGTTCACACTTGAA
ICMT-siRNA-3	StB0010313C	genOFFTMst-h-ICMT_003	CTTCCGCGATCGAACAGAA

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体外沉默异戊二烯基半胱氨酸羧基甲基转移酶基因对舌鳞状细胞癌迁移和侵袭的影响

Effects of isoprenylcysteine carboxylmethyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma

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