体外沉默Rce1对舌鳞状细胞癌细胞侵袭和迁移能力的影响

doi:10.7518/hxkq.2019.02.004

华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (2): 143-148.doi: 10.7518/hxkq.2019.02.004

体外沉默Rce1对舌鳞状细胞癌细胞侵袭和迁移能力的影响

孙君君¹,陶匀雅²,周元²,何宗轩³,盛善桂²,王奇民²,童磊²,赵开²,王少如⁴,陈正岗²()

1.青岛市城阳区人民医院口腔科,青岛 266109
2.青岛大学附属青岛市市立医院口腔医学中心,青岛 266071
3.青岛大学附属医院口腔颌面外科,青岛 266005
4.大连医科大学口腔医学院,大连 116044

收稿日期:2018-06-11 修回日期:2019-01-09 出版日期:2019-04-01 发布日期:2019-04-28
通讯作者: 陈正岗 E-mail:chenzhg1973@163.com
作者简介:孙君君,住院医师,硕士,E-mail： 852764479@qq.com
基金资助:
国家自然科学基金(81372908)

Effects of silencing Rce1 in vitro on the invasion and migration of tongue carcinoma

Junjun Sun¹,Yunya Tao²,Yuan Zhou²,Zongxuan He³,Shangui Sheng²,Qimin Wang²,Lei Tong²,Kai Zhao²,Shaoru Wang⁴,Zhenggang Chen²()

1. Dept. of Stomatology, Qingdao Chengyang District Hospital, Qingdao 266109, China
2.Medical Center of Stomatology, Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
3.Dept. of Oral and Maxillafacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266005, China
4.Stomatology College, Dalian Medical University, Dalian 116044, China

Received:2018-06-11 Revised:2019-01-09 Online:2019-04-01 Published:2019-04-28
Contact: Zhenggang Chen E-mail:chenzhg1973@163.com
Supported by:
The National Natural Science Foundation of China(81372908)

摘要/Abstract

摘要：

目的通过RNA干扰技术沉默Rce1基因,探讨Rce1对舌癌细胞侵袭和迁移能力的影响。方法体外培养舌鳞状细胞癌Cal-27和SCC-4细胞,设计合成Rce1基因的小干扰RNA（siRNA）,采用脂质体载体瞬时转染沉默Rce1基因。根据转染的siRNA不同,将实验组分为Rce1-siRNA-1组、Rce1-siRNA-2组、Rce1-siRNA-3组,脂质体载体分别转染相对应序列的siRNA。脂质体转染siCON作为阴性对照组,不转染siRNA作为空白对照组。采用实时定量聚合酶链反应检测各组Rce1、RhoA以及K-Ras基因的表达,蛋白质印迹法（Western blot）检测Rce1、RhoA、K-Ras、金属基质蛋白酶（MMP）-2及MMP-9的表达,采用体外侵袭实验检测Cal-27和SCC-4的侵袭能力,细胞划痕实验检测细胞迁移能力。结果实时定量聚合酶链反应及Western blot检测结果显示,与阴性对照组和空白对照组相比,实验组的Rce1基因及蛋白表达均下调（P<0.05）,RhoA、K-Ras基因及蛋白表达无统计学差异（P>0.05）,MMP-2、MMP-9表达下降（P<0.05）。体外侵袭实验表明,与对照组相比,实验组在聚碳酯膜上的细胞总数明显减少（P<0.05）。细胞划痕实验表明,实验组划痕处细胞闭合时间明显长于对照组（P<0.05）。结论体外沉默Rce1可以有效下调其在舌癌细胞Cal-27和SCC-4中的表达水平,降低迁移和侵袭能力。

关键词: Rce1, 鳞状细胞癌, 侵袭, 迁移

Abstract:

Objective This study aimed to explore the influence of Rce1 on invasion and migration of tongue squamous cell carcinoma cells by silencing the Rce1 gene with RNA interference. Methods The tongue squamous cell carcinoma Cal-27 and SCC-4 cells were cultured in vitro. The small interfering RNA (siRNA) of the Rce1 gene was designed, and the Rcel gene expression was silenced vialiposome transfection. According to the siRNA transfected by liposome, the experimental group was divided into three groups, namely, Rce1-siRNA-1, Rce1-siRNA-2, and Rce1-siRNA-3 groups. Negative control group was transfected by siCON, and the blank control group was untransfected by siRNA. The Rce1, RhoA, and K-Ras gene expression levels in each group were analyzed by real-time quantitative polymerase chain reaction. The Rce1, RhoA, K-Ras, MMP-2, and MMP-9 protein expression levels were analyzed by Western blot. The invasiveness of tongue cancer cell Cal-27 and SCC-4 were determined by Transwell invasion assay, and cell migration assay was performed by cell scratch assay. Results Real-time quantitative polymerase chain reaction and Western blot results showed that compared with the negative and blank control groups, the Rce1 gene and protein expression levels in three experimental groups decreased (P<0.05). The RhoA, K-Ras gene and protein expression levels were insignificantly different among groups (P>0.05). Meanwhile, the MMP-2 and MMP-9 expression levels decreased (P<0.05). Transwell invasion assay results showed that the total number of cells in the PET film of the experimental groups was significantly decreased compared with the control group (P<0.05). The cell scratch test showed that the cell closure time of the scratch in the interference group was significantly longer than those in the control and blank groups (P<0.05). Conclusion Silencing Rce1 in vitro can effectively downregulate its expression in tongue squamous cell carcinoma cells Cal-27 and SCC-4 and reduce the migration and invasion abilities of these cells.

Key words: Rce1, squamous cell carcinoma, invasion, migration

中图分类号:

R739.8

孙君君,陶匀雅,周元,何宗轩,盛善桂,王奇民,童磊,赵开,王少如,陈正岗. 体外沉默Rce1对舌鳞状细胞癌细胞侵袭和迁移能力的影响[J]. 华西口腔医学杂志, 2019, 37(2): 143-148.

Junjun Sun,Yunya Tao,Yuan Zhou,Zongxuan He,Shangui Sheng,Qimin Wang,Lei Tong,Kai Zhao,Shaoru Wang,Zhenggang Chen. Effects of silencing Rce1 in vitro on the invasion and migration of tongue carcinoma[J]. West China Journal of Stomatology, 2019, 37(2): 143-148.

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参考文献 20

[1]	Ferrari D, Codecà C, Fiore J , et al. Biomolecular markers in cancer of the tongue[J]. J Oncol, 2009,2009:412908.
[2]	Sano D, Myers JN . Metastasis of squamous cell carcinoma of the oral tongue[J]. Cancer Metastasis Rev, 2007,26(3/4):645-662. doi: 10.1007/s10555-007-9082-y URL
[3]	Kruaysawat W, Aekplakorn W, Chapman RS . Survival time and prognostic factors of oral cancer in Ubon Ratcha-thani Cancer Center[J]. J Med Assoc Thai, 2010,93(3):278-284.
[4]	Wang YH, Wu MW, Yang AK , et al. COX-2 Gene increases tongue cancer cell proliferation and invasion through VEGF-C pathway[J]. Med Oncol, 2011,28(S1):360-366. doi: 10.1007/s12032-010-9737-3 URL
[5]	Jaworski J, Govender U , McFarlane C , et al. A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17[J]. Biochem J, 2014,457(2):289-300. doi: 10.1042/BJ20131213 URL
[6]	Manolaridis I, Kulkarni K, Dodd RB , et al. Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1[J]. Nature, 2013,504(7479):301-305. doi: 10.1038/nature12754 URL
[7]	Wahlstrom AM, Cutts BA, Karlsson C , et al. Rce1 deficiency accelerates the development of K-Ras-induced myeloproli-ferative disease[J]. Blood, 2007,109(2):763-768. doi: 10.1182/blood-2006-05-024752 URL
[8]	Wang HB, Liu XP, Liang J , et al. Expression of RhoA and RhoC in colorectal carcinoma and its relations with clinico-pathological parameters[J]. Clin Chem Lab Med, 2009,47(7):811-817.
[9]	Kasuya K, Nagakawa Y, Hosokawa Y , et al. RhoA activity increases due to hypermethylation of ARHGAP28 in a highly liver-metastatic colon cancer cell line[J]. Biomed Rep, 2016,4(3):335-339. doi: 10.3892/br.2016.582 URL
[10]	Cui J, Cai YH, Hu Y , et al. Epigenetic silencing of TPM2 contributes to colorectal cancer progression upon RhoA activation[J]. Tumor Biol, 2016,37(9):12477-12483. doi: 10.1007/s13277-016-5103-1 URL
[11]	Fujimura K, Choi S, Wyse M , et al. Eukaryotic translation initiation factor 5A (EIF5A) regulates pancreatic cancer metastasis by modulating RhoA and rho-associated kinase (ROCK) protein expression levels[J]. J Biol Chem, 2015,290(50):29907-29919. doi: 10.1074/jbc.M115.687418 URL
[12]	Yan GX, Zou RH, Chen ZG , et al. Silencing RhoA inhibits migration and invasion through Wnt/β-catenin pathway and growth through cell cycle regulation in human tongue cancer[J]. Acta Biochim Biophys Sin, 2014,46(8):682-690. doi: 10.1093/abbs/gmu051 URL
[13]	Young A, Lyons J, Miller AL , et al. Ras signaling and the-rapies[J]. Adv Cancer Res, 2009,102:1-17. doi: 10.1016/S0065-230X(09)02001-6 URL
[14]	Hoa M, Davis SL, Ames SJ , et al. Amplification of wild-type K-Ras promotes growth of head and neck squamous cell carcinoma[J]. Cancer Res, 2002,62(24):7154-7156.
[15]	严国鑫, 樊兵, 邹荣海 , 等. 沉默RohA基因对舌鳞状细胞癌细胞增殖的影响[J]. 华西口腔医学杂志, 2016,34(6):620-625. doi: 10.7518/hxkq.2016.06.014
	Yan GX, Fan B, Zou RH , et al. Effects of RhoA silencing on proliferation of tongue squamous cancer cells[J]. West Chin J Stomatol, 2016,34(6):620-625. doi: 10.7518/hxkq.2016.06.014
[16]	Huang LL, Li MC, Wang DL , et al. Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer[J]. Human Pathol, 2016,47(1):109-114. doi: 10.1016/j.humpath.2015.08.021 URL
[17]	Mohammed I, Hampton SE, Ashall L , et al. 8-Hydroxyqui-noline-based inhibitors of the Rce1 protease disrupt Ras mem-brane localization in human cells[J]. Bioorg Med Chem, 2016,24(2):160-178. doi: 10.1016/j.bmc.2015.11.043 URL
[18]	Bergo MO, Ambroziak P, Gregory C , et al. Absence of the CAAX endoprotease Rce1: effects on cell growth and trans-formation[J]. Mol Cell Biol, 2002,22(1):171-181. doi: 10.1128/MCB.22.1.171-181.2002 URL
[19]	Grzelczyk WL, Szemraj J, Józefowicz-Korczyńska M . The matrix metalloproteinase in larynx cancer[J]. Postepy Hig Med Dosw, 2016,70:1190-1197.
[20]	邓莅霏, 齐淑轶, 陈福进 , 等. 舌癌组织中MMP-2、9表达的预后意义[J]. 现代肿瘤医学, 2008,16(8):1303-1306.
	Deng LF, Qi SY, Chen FJ , et al. Prognostic significance of MMP-2 and 9 expression in tongue cancer[J]. J Modern Oncol, 2008,16(8):1303-1306.

实验组	产品编号	产品名称	靶序列
Rce1-siRNA-1	siG1471135732	si-h-RCE1_001	CCATGTTAGCACCGTGCAT
Rce1-siRNA-2	siG1471135741	si-h-RCE1_002	CCATGTTAGCACCGTGCAT
Rce1-siRNA-3	siG1471135750	si-h-RCE1_003	GCCATTCCTTCTGCAATTA

体外沉默Rce1对舌鳞状细胞癌细胞侵袭和迁移能力的影响

Effects of silencing Rce1 in vitro on the invasion and migration of tongue carcinoma

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