沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用

doi:10.7518/hxkq.2022.04.004

华西口腔医学杂志 ›› 2022, Vol. 40 ›› Issue (4): 394-402.doi: 10.7518/hxkq.2022.04.004

沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用

李文健¹^,²(), 童磊¹, 王奇民¹, 韩红钰¹, 陈正岗¹()

^1.青岛大学附属青岛市市立医院口腔医学中心，青岛 266071
^2.大连医科大学口腔医学院，大连 116044

收稿日期:2022-01-19 修回日期:2022-05-04 出版日期:2022-07-25 发布日期:2022-07-27
通讯作者: 陈正岗 E-mail:17862890529@163.com;chenzhg1973@163.com
作者简介:李文健，医师，硕士，E-mail：17862890529@163.com
基金资助:
国家自然科学基金(81372908)

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

Li Wenjian¹^,²(), Tong Lei¹, Wang Qimin¹, Han Hongyu¹, Chen Zhenggang¹()

^1.Stomatology Center, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
^2.School of Stomatology, Dalian Medical University, Dalian 116044, China

Received:2022-01-19 Revised:2022-05-04 Online:2022-07-25 Published:2022-07-27
Contact: Chen Zhenggang E-mail:17862890529@163.com;chenzhg1973@163.com
Supported by:
The National Natural Science Foundation of China(81372908);Correspondence: Chen Zhenggang, E-mail: chenzhg1973@163.com

摘要/Abstract

摘要：

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。

关键词: 法尼基转移酶, HRAS, 涎腺腺样囊性癌, 迁移, 侵袭

Abstract:

Objective 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). Conclusion 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.

Key words: farnesyltransferase, HRAS, salivary adenoid cystic carcinoma, migration, invasion

中图分类号:

R 739.8

李文健, 童磊, 王奇民, 韩红钰, 陈正岗. 沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用[J]. 华西口腔医学杂志, 2022, 40(4): 394-402.

Li Wenjian, Tong Lei, Wang Qimin, Han Hongyu, Chen Zhenggang. Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells[J]. West China Journal of Stomatology, 2022, 40(4): 394-402.

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沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用

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

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siRNA	靶序列
FTase-siRNA-1	GTCCATAGAACAGGCAAAAGTAG
FTase-siRNA-2	TCCATAGAACAGGCAAAAGTAGA
FTase-siRNA-3	GGCAAAAGTAGAAGAAAAGATCC