体外沉默二牛龙牛儿基转移酶Ⅰ对舌癌细胞迁移和侵袭的影响

doi:10.7518/hxkq.2017.06.003

华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (6): 576-582.doi: 10.7518/hxkq.2017.06.003

体外沉默二牛龙牛儿基转移酶Ⅰ对舌癌细胞迁移和侵袭的影响

陈正岗^1,²(), 王树人³, 李敬华⁴, 韩金宏⁵, 王奇民¹, 童磊¹, 刘文君⁶, 杨芳¹, 郭庆圆¹, 郭大伟¹, 王莹^7,⁸()

1.青岛市市立医院口腔科,青岛 266071
2.上海交通大学医学院附属第九人民医院口腔颌面外科,上海 200011
3.胶州市人民医院口腔科,胶州 266300
4.青岛市市立医院中心实验室,青岛 266071
5.烟台市口腔医院,烟台 264008
6.青岛市市立医院耳鼻喉科,青岛 266071
7.济南市第四人民医院口腔科,济南 250031
8.潍坊医学院口腔医学院,潍坊 261021

收稿日期:2016-12-17 修回日期:2017-09-05 出版日期:2017-12-20 发布日期:2017-12-01
作者简介:
陈正岗,副主任医师,博士,E-mail：chenzhg1973@163.com
基金资助:
国家自然科学基金面上项目（81372908）;青岛市卫计委计划项目（2014-WJZD009,2013-WSZD011）

Effects of geranylgeranyltransferase Ⅰ gene silencing by RNA interference on the migration and invasion of tongue carcinoma

Zhenggang Chen^1,²(), Shuren Wang³, Jinghua Li⁴, Jinhong Han⁵, Qimin Wang¹, Lei Tong¹, Wenjun Liu⁶, Fang Yang¹, Qingyuan Guo¹, Dawei Guo¹, Ying Wang^7,⁸()

1. Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao 266071, China
2. Dept. of Oral and Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
3. Dept. of Stomatology, Jiaozhou People’s Hospital, Jiaozhou 266300, China
4. Central Laboratory, Qingdao Municipal Hospital, Qingdao 266071, China
5. Yantai Stomatological Hospital, Yantai 264008, China
6. Dept. of Ear-nose-throat, Qingdao Municipal Hospital, Qingdao 266071, China
7. Dept. of Stomatology, Fourth People’s Hospital of Jinan, Jinan 250031, China
8. College of Stomatology, Weifang Medical University, Weifang 261021, China

Received:2016-12-17 Revised:2017-09-05 Online:2017-12-20 Published:2017-12-01
Supported by:
Supported by: National Natural Science Foundation of China (81372908);Qingdao Municipal Commission of Health and Family Planning (2014-WJZD009, 2013-WSZD011).

摘要/Abstract

摘要：

目的应用RNA干扰技术,体外沉默二牛龙牛儿基转移酶Ⅰ（GGTase-Ⅰ）,研究其在舌癌迁移、侵袭中的作用。方法登录Genebank确定人GGTase-Ⅰ基因序列,针对GGTase-Ⅰ的基因序列设计并构建3条siRNA,分别将RNA干扰组（GGTase-Ⅰ siRNA1、GGTase-ⅠsiRNA2、GGTase-Ⅰ siRNA3）、阴性对照组（NC-siRNA）和空白对照组转染至舌癌细胞Cal-27,实时定量聚合酶链反应（qRT-PCR）和蛋白质印迹法（Western blot）检测各组细胞转染后GGTase-Ⅰ、RhoA基因的mRNA、蛋白表达;选取沉默效率最高的一组siRNA作为实验组,蛋白质印迹法检测各组细胞基质金属蛋白酶（MMP）-2、MMP-9的表达,GST-pull down实验检测GTP-RhoA蛋白的表达,划痕实验检测细胞迁移能力变化,Transwell小室检测细胞侵袭能力变化。结果干扰后细胞的GGTase-Ⅰ mRNA、蛋白表达明显下降（P<0.05）,RhoA mRNA和蛋白表达无明显改变,MMP-2、MMP-9、GTP-RhoA的表达下降,迁移、侵袭能力明显下降（P<0.05）。结论抑制GGTase-Ⅰ表达,可降低舌癌细胞的迁移、侵袭能力,对GGTase-Ⅰ的深入研究可能为舌癌的治疗提供新的有效分子靶点。

关键词: 二牛龙牛儿基转移酶Ⅰ, RhoA, 舌癌, 迁移, 侵袭, 基质金属蛋白酶-2, 基质金属蛋白酶-9

Abstract:

Objective RNA interference was used to silence geranylgeranyltransferase Ⅰ(GGTase-Ⅰ) in vitro and to study the effect of GGTase-Ⅰ on the migration and invasion of tongue squamous cancer cells. Methods Three small interfering RNAs (siRNA) were designed according to the GGTase-Ⅰ sequence by Genebank and were transfected into tongue squamous cancer cells Cal-27 to knock down GGTase-Ⅰ expression. The tested cells were divided into three groups, as follows: the RNA-interfered groups (GGTase-Ⅰ siRNA1, GGTase-Ⅰ siRNA 2, GGTase-Ⅰ siRNA 3), a negative control group (disrupted by random sequence NC-siRNA), and a blank control group. GGTase-Ⅰ and RhoA gene expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The optimum interference group was screened by qRT-PCR and Western blot and was assigned as the experimental group. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were examined by Western blot. GTP-RhoA expression of protein was examined by GST-pull down. The migration and invasion abilities were analyzed by wound healing assay and Transwell motility assay. Results GGTase-Ⅰ mRNA and protein expression in Cal-27 decreased significantly after transfection of GGTase-I siRNA (P<0.05). No significant difference of RhoA gene expression was detected. MMP-2, MMP-9, and GTP-RhoA protein expressions decreased significantly (P<0.05). The migration and invasion abilities were inhibited (P<0.05). Conclusion To inhibit GGTase-Ⅰ expression, the migration and invasion abilities of tongue squa-mous cancer cells should also be inhibited. Further studies on GGTase-Ⅰ may provide novel effective molecular targets for tongue squamous cancer cells.

Key words: geranylgeranyltransferase Ⅰ, RhoA, tongue squamous cancer, migration, invasion, matrix metallo-proteinase-2, matrix metalloproteinase-9

中图分类号:

陈正岗, 王树人, 李敬华, 韩金宏, 王奇民, 童磊, 刘文君, 杨芳, 郭庆圆, 郭大伟, 王莹. 体外沉默二牛龙牛儿基转移酶Ⅰ对舌癌细胞迁移和侵袭的影响[J]. 华西口腔医学杂志, 2017, 35(6): 576-582.

Zhenggang Chen, Shuren Wang, Jinghua Li, Jinhong Han, Qimin Wang, Lei Tong, Wenjun Liu, Fang Yang, Qingyuan Guo, Dawei Guo, Ying Wang. Effects of geranylgeranyltransferase Ⅰ gene silencing by RNA interference on the migration and invasion of tongue carcinoma[J]. West China Journal of Stomatology, 2017, 35(6): 576-582.

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名称	产品编号	产品名称	靶序列
GGTase-Ⅰ siRNA1	siG000005229A	si-h-PGGT1B_001	GGAACAGCTCATCCTTATG
GGTase-Ⅰ siRNA2	siG000005229B	si-h-PGGT1B_002	CGACTTAAGCCGAGTAAAT
GGTase-Ⅰ siRNA3	siG000005229C	si-h-PGGT1B_003	GACTTCTGAACGCCTTCTA

体外沉默二牛龙牛儿基转移酶Ⅰ对舌癌细胞迁移和侵袭的影响

Effects of geranylgeranyltransferase Ⅰ gene silencing by RNA interference on the migration and invasion of tongue carcinoma

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