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. 2022 Jul 25;40(4):394-402.
doi: 10.7518/hxkq.2022.04.004.

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells

[Article in English, Chinese]
Wenjian Li  1   2 , Lei Tong  1 , Qimin Wang  1 , Hongyu Han  1 , Zhenggang Chen  1
Affiliations

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells

[Article in English, Chinese]
Wenjian Li et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

Objectives: This study aimed to investigate the effects of farnesyltransferase (FTase) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism.

Methods: Three small interfering RNA (siRNA) sequences were designed and constructed based on the human FTase gene sequence. The SACC-LM and SACC-83 cells in the logarithmic growth period were used, and the expression of FTase was suppressed by liposomal transient transfection. The tested cells were categorized as the FTase-siRNA-1, FTase-siRNA-2, and FTase-siRNA-3 groups. Both negative control group (NC-siRNA) and blank control group (only transfection reagent was added) were set. The mRNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction, and the silencing efficiency was determined. The expression levels of FTase, HRAS, protein kinase B (AKT), phospho-AKT, p65, phospho-p65 (Ser563), E-cadherin, vimentin, matrix metalloproteinase (MMP)-9 protein, and HRAS membrane protein were detected by Western blot. Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells.

Results: The relative expression of FTase mRNA and protein in the FTase-siRNA-1 group decreased compared with those in the control group (P<0.05). HRAS mRNA and total protein expression had no significant difference (P>0.05), and the relative expression of HRAS membrane protein decreased (P<0.05). The relative expression of E-cadherin increased (P<0.05), vimentin decreased (P<0.05), and MMP-9 decreased (P<0.05). There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65 (P>0.05), but the relative expression levels of phospho-AKT and phospho-p65 decreased. The invasion and migration ability of the FTase-siRNA-1 group significantly decreased compared with that in the control group (P<0.05).

Conclusions: Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.

目的: 通过体外实验探讨法尼基转移酶（FTase）对涎腺腺样囊性癌SACC-LM和SACC-83细胞迁移侵袭及上皮间充质转化（EMT）的作用及其机制。方法: 针对人FTase基因序列设计构建3条小干扰RNA（si-RNA），采用处于对数生长期的SACC-LM及SACC-83细胞，经脂质体瞬时转染技术抑制细胞FTase表达，分别命名为FTase-siRNA-1组、FTase-siRNA-2组、FTase-siRNA-3组，同时设置阴性对照组（NC-siRNA），空白对照组（仅加入转染试剂）。采用实时荧光定量逆转录聚合酶链反应检测FTase、HRAS的mRNA表达，选择沉默效率最高的FTase-siRNA进行后续实验；蛋白质免疫印迹法检测FTase、HRAS、蛋白激酶B（AKT）、磷酸化AKT、p65、磷酸化p65（Ser563）、上皮钙依赖黏附蛋白、波形蛋白、基质金属蛋白酶9（MMP-9）的蛋白表达以及HRAS膜蛋白表达；Transwell小室及细胞划痕实验检测细胞的侵袭和迁移能力。结果: 与空白对照组及阴性对照组相比，FTase-siRNA-1组mRNA及蛋白相对表达量均降低（P<0.05），HRAS mRNA和总蛋白表达的差异无统计学意义（P>0.05），HRAS膜蛋白相对表达量降低（P<0.05），上皮钙依赖黏附蛋白相对表达量升高（P<0.05），波形蛋白相对表达量降低（P<0.05），MMP-9蛋白相对表达量降低（P<0.05），RAS/PI3K/AKT/核因子-κB信号通路相关蛋白AKT、p65相对表达量的差异无统计学意义（P>0.05），但磷酸化AKT、磷酸化p65蛋白相对表达量降低；与空白及阴性对照组相比，FTase-siRNA-1组细胞侵袭及迁移能力显著下降（P<0.05）。结论: 体外沉默FTase可有效抑制人涎腺腺样囊性癌细胞SACC-LM和SACC-83的侵袭、迁移能力，其作用可能是通过干扰HRAS膜蛋白的定位，调控RAS/PI3K/AKT/核因子-κB信号通路，介导EMT而实现。.

Keywords: HRAS; farnesyltransferase; invasion; migration; salivary adenoid cystic carcinoma.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. 转染后FTase、HRAS mRNA的表达
Fig 1 Expression of FTase and HRAS mRNA after FTase-siRNA transfection A、B：SACC-LM细胞；C、D：SACC-83细胞。a、b、c、d、e分别为空白对照组、阴性对照组、FTase-siRNA-1组、FTase-siRNA-2组、FTase-siRNA-3组。与对照组相比，***P<0.001，****P<0.000 1。
图 2
图 2. 转染后FTase、HRAS相关蛋白的表达
Fig 2 Expression of FTase and HRAS protein in each group 上、下分别为SACC-LM、SACC-83细胞；a、b、c分别代表空白对照组、阴性对照组、FTase -siRNA-1组；与对照组相比，*P<0.05，**P<0.01，***P<0.001，ns为差异无统计学意义。
图 3
图 3. 转染后EMT和侵袭迁移相关蛋白的表达
Fig 3 Expression of invasion and migration and EMT related proteins 上、下分别为SACC-LM、SACC-83细胞；a、b、c分别代表空白对照组、阴性对照组、FTase -siRNA-1组；与对照组相比，**P<0.01，***P<0.001，ns为差异无统计学意义。
图 4
图 4. 转染后RAS/PI3K/AKT/核因子-κB信号通路的相关蛋白表达变化
Fig 4 Expression of RAS/PI3K/AKT/nuclear factor-κB signaling pathway proteins 上、下分别为SACC-LM、SACC-83细胞；a、b、c分别代表空白对照组、阴性对照组、FTase -siRNA-1组；与对照组相比，*P<0.05，**P<0.01，***P<0.001，ns为差异无统计学意义。
图 5
图 5. Transwell侵袭实验结果 倒置相差显微镜 × 100
Fig 5 The results of Transwell assay inverted phase contrast microscope × 100 上、下分别为侵袭细胞结晶紫染色、侵袭细胞计数结果；a、b、c分别代表空白对照组、阴性对照组、FTase-siRNA-1组；与对照组相比，*P<0.05，**P<0.01，***P<0.001，ns为差异无统计学意义。
图 6
图 6. 划痕实验结果 倒置相差显微镜 × 40
Fig 6 The results of wound healing assay inverted phase contrast microscope × 40 上为SACC-LM细胞，下为SACC-83细胞；a、b、c分别代表空白对照组、阴性对照组、FTase-siRNA-1组；与对照组相比，**P<0.01，***P<0.001，ns为差异无统计学意义。
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