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
作者：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
Author：Zhou Nan, Chi Zengpeng, Li Wenjian, Zhao Kai, Wang Shaoru, Wang Qimin, Tong Lei, He Zongxuan, Han Hongyu, Wang Ying, Chen Zhenggang
收稿日期：2020-06-18 年卷（期）页码：2021,39(3):328-328-335
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：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,
Key words：isoprenylcysteine carboxylmethyltransferase,RhoA,tongue squamous cell carcinoma,migration,invasion,
基金项目：国家自然科学基金面上项目(81372908)
中文摘要
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。
英文摘要
ObjectiveThe 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.
MethodsThrough 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.
ResultsAfter 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).
ConclusionThe 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.
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