College of Stomatology, Weifang Medical University, Weifang 261021, China.
School of Stomatology, Dalian Medical University, Dalian 116044, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2021 Jun 1;39(3):328-335. doi: 10.7518/hxkq.2021.03.013.
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.
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.
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 (<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (<0.05). The migration and invasion abilities were inhibited (<0.05).
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.
构建异戊烯基半胱氨酸羧甲基转移酶（ICMT）的小干扰 RNA（siRNA），研究其沉默对舌鳞癌细胞迁移和侵袭的影响。
通过脂质体转染，将 siRNA 转染入人舌鳞癌细胞 CAL-27 和 SCC-4 细胞（ICMT-siRNA 组），同时设阴性对照组（转染 NC-siRNA）和空白对照组（转染试剂但不转染 siRNA）。转染后，采用实时定量聚合酶链反应分析各组细胞中 ICMT 和 RhoA 的 mRNA 表达，测量其沉默效率。Western blot 检测各组细胞中 ICMT、总 RhoA、膜 RhoA、ROCK1、基质金属蛋白酶（MMP）-2 和 MMP-9 蛋白的表达水平。通过划痕愈合和 Transwell 迁移实验评估迁移和侵袭能力。
CAL-27 和 SCC-4 细胞转染 ICMT-siRNA 后，实验组细胞中 ICMT 基因和蛋白的表达水平明显低于阴性对照组和空白对照组（<0.05）。两组 RhoA 的 mRNA 和总蛋白表达水平无明显差异（0.05）。RhoA 膜蛋白、ROCK1、MMP-2 和 MMP-9 的表达水平降低（<0.05）。迁移和侵袭能力受到抑制（<0.05）。
转染 ICMT-siRNA 后，CAL-27 和 SCC-4 细胞的迁移和侵袭能力明显降低，其涉及的机制可能与 RhoA-ROCK 信号通路有关。
