体外沉默异戊二烯基半胱氨酸羧基甲基转移酶对人舌鳞状细胞癌细胞增殖和凋亡的影响

doi:10.7518/hxkq.2021.01.010

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (1): 64-73.doi: 10.7518/hxkq.2021.01.010

体外沉默异戊二烯基半胱氨酸羧基甲基转移酶对人舌鳞状细胞癌细胞增殖和凋亡的影响

王少如¹^,²(), 孙伟¹, 周男³, 赵开⁴, 李文健², 迟增鹏³, 王莹⁵, 王奇民¹, 童磊¹, 何宗轩⁶, 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
^3.潍坊医学院口腔医学院，潍坊 261021
^4.青岛大学口腔医学院，青岛 266003
^5.济南市第四人民医院口腔科，济南 250031
^6.青岛大学附属医院口腔颌面外科，青岛 266005

收稿日期:2020-03-18 修回日期:2020-10-27 出版日期:2021-02-01 发布日期:2021-03-02
通讯作者: 陈正岗 E-mail:wangxiaorukw@163.com;chenzhg1973@163.com
作者简介:王少如，硕士，E-mail：wangxiaorukw@163.com
基金资助:
国家自然科学基金面上项目(81372908)

Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma

Wang Shaoru¹^,²(), Sun Wei¹, Zhou Nan³, Zhao Kai⁴, Li Wenjian², Chi Zengpeng³, Wang Ying⁵, Wang Qimin¹, Tong Lei¹, He Zongxuan⁶, Han Hongyu¹, Chen Zhenggang¹()

^1.Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
^2.School of Stomatology, Dalian Medical University, Dalian 116044, China
^3.College of Stomatology, Weifang Medical University, Weifang 261021, China
^4.School of Stomatology, Qingdao University, Qingdao 266003, China
^5.Dept. of Stomatology, Fourth People, s Hospital of Jinan, Jinan 250031, China
^6.Dept. of Oral and Maxillafacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266005, China

Received:2020-03-18 Revised:2020-10-27 Online:2021-02-01 Published:2021-03-02
Contact: Chen Zhenggang E-mail:wangxiaorukw@163.com;chenzhg1973@163.com
Supported by:
The National Natural Science Foundation of China(81372908)

摘要/Abstract

摘要： 目的

通过小干扰RNA（siRNA）干扰技术体外沉默异戊二烯基半胱氨酸羧基甲基转移酶（Icmt）基因，探讨体外沉默Icmt对舌鳞状细胞癌（TSCC）细胞增殖和凋亡能力的影响。

方法

针对人Icmt基因序列设计并构建3条siRNA，经脂质体瞬时转染技术转染抑制TSCC细胞系CAL-27和SCC-4细胞Icmt表达，同时设置空白对照组（只加入转染试剂，不加入siRNA）和阴性对照组（转染NC-siRNA）。应用荧光组（荧光基团Cy3标记siRNA）检测siRNA转染效率，实时荧光定量聚合酶链反应（qRT-PCR）检测沉默Icmt后各组细胞的Icmt、RhoA mRNA表达，选取沉默效率最高组作为实验组进行后续实验。采用蛋白质免疫印迹法（Western blot）检测转染后各组细胞Icmt、RhoA的蛋白表达及RhoA膜蛋白的表达、细胞周期蛋白1（Cyclin D1）、p21蛋白表达及细胞外调节蛋白激酶（ERK）、磷酸化的细胞外调节蛋白激酶（p-ERK）蛋白的表达；细胞增殖活性检测试剂盒和流式细胞术检测细胞的增殖能力、细胞周期变化；Annexin V-APC/碘化丙啶（PI）荧光双染法检测各组细胞的凋亡能力。采用GraphPad Prism 8.2.1软件对实验数据进行统计学分析。

结果

与阴性对照组和空白对照组相比，实验组细胞Icmt mRNA和蛋白表达下降（P<0.05），RhoA mRNA和总蛋白表达无明显改变（P>0.05），但RhoA膜蛋白表达显著降低（P<0.05）；细胞周期相关蛋白Cyclin D1表达下降，p21表达升高；同时检测到实验组ERK蛋白相对表达量较对照组相比无明显差异（P>0.05），但ERK的磷酸化水平明显下降（P<0.05）。CAL-27及SCC-4细胞周期发生改变，细胞比例在G1期增加，S期降低，G2期无明显改变，细胞周期被阻滞在G1/S期（P<0.05）；细胞增殖活性下降、凋亡能力增加。

结论

体外沉默TSCC细胞Icmt基因，可降低RhoA膜靶向定位，抑制细胞增殖、诱导凋亡，提示Icmt在TSCC增殖和凋亡中可能发挥重要作用，可能作为TSCC基因治疗的分子靶点。

关键词: 异戊二烯基半胱氨酸羧基甲基转移酶, RhoA, 舌鳞状细胞癌, 细胞增殖, 细胞周期, 细胞凋亡

Abstract: Objective

This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC).

Methods

Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.

Results

The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05).

Conclusion

Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.

Key words: isoprenylcysteine carboxyl methyltransferase, RhoA, tongue squamous cell carcinoma, cell proli-feration, cell cycle, apoptosis

中图分类号:

R 739.8

王少如, 孙伟, 周男, 赵开, 李文健, 迟增鹏, 王莹, 王奇民, 童磊, 何宗轩, 韩红钰, 陈正岗. 体外沉默异戊二烯基半胱氨酸羧基甲基转移酶对人舌鳞状细胞癌细胞增殖和凋亡的影响[J]. 华西口腔医学杂志, 2021, 39(1): 64-73.

Wang Shaoru, Sun Wei, Zhou Nan, Zhao Kai, Li Wenjian, Chi Zengpeng, Wang Ying, Wang Qimin, Tong Lei, He Zongxuan, Han Hongyu, Chen Zhenggang. Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma[J]. West China Journal of Stomatology, 2021, 39(1): 64-73.

图/表 9

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参考文献 33

1	Alves AM, Diel LF, Lamers ML. Macrophages and prognosis of oral squamous cell carcinoma: a systematic review[J]. J Oral Pathol Med, 2018, 47(5): 460-467.
2	Almangush A, Heikkinen I, Makitie AA, et al. Prognostic biomarkers for oral tongue squamous cell carcinoma: a systematic review and meta-analysis[J]. Br J Cancer, 2017, 117(6): 856-866.
3	Feng Z, Xu QS, Qin LZ, et al. Predicting radiotherapy necessity in tongue cancer using lymph node yield[J]. J Oral Maxillofac Surg, 2017, 75(5): 1062-1070.
4	Sharma D, Singh G. Squamous cell carcinoma of the oral cavity and oropharynx in young adults[J]. Indian J Cancer, 2016, 53(3): 399-401.
5	Mohideen K, Krithika C, Jeddy N, et al. Meta-analysis on risk factors of squamous cell carcinoma of the tongue in young adults[J]. J Oral Maxillofac Pathol, 2019, 23(3): 450-457.
6	Haga RB, Ridley AJ. Rho GTPases: regulation and roles in cancer cell biology[J]. Small GTPases, 2016, 7(4): 207-221.
7	Glomset JA, Farnsworth CC. Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes[J]. Annu Rev Cell Biol, 1994, 10: 181-205.
8	Dai Q, Choy E, Chiu V, et al. Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum[J]. J Biol Chem, 1998, 273(24): 15030-15034.
9	Chang KW, Kao SY, Wu YH, et al. Passenger strand miRNA miR-31* regulates the phenotypes of oral cancer cells by targeting RhoA[J]. Oral Oncol, 2013, 49(1): 27-33.
10	Liao YC, Ruan JW, Lua I, et al. Overexpressed hPTTG1 promotes breast cancer cell invasion and metastasis by regulating GEF-H1/RhoA signalling[J]. Oncogene, 2012, 31(25): 3086-3097.
11	Yang X, Zheng F, Zhang S, et al. Loss of RhoA expression prevents proliferation and metastasis of SPCA1 lung cancer cells in vitro[J]. Biomed Pharmacother, 2015, 69: 361-366.
12	Yao J, Gao P, Xu Y, et al. α-TEA inhibits the growth and motility of human colon cancer cells via targeting RhoA/ROCK signaling[J]. Mol Med Rep, 2016, 14(3): 2534-2540.
13	Xu XT, Song QB, Yao Y, et al. Inhibition of RhoA/ROCK signaling pathway promotes the apoptosis of gastric cancer cells[J]. Hepatogastroenterology, 2012, 59(120): 2523-2526.
14	Li XR, Wu W, Qian LY, et al. Underexpression of dele-ted in liver cancer 2 (DLC2) is associated with overexpression of RhoA and poor prognosis in hepatocellular carcinoma[J]. BMC Cancer, 2008, 8: 205.
15	周峻, 何勇, 董绍忠, 等. RhoA小干扰RNA对舌癌细胞Tca8113增殖、侵袭影响的体外实验[J]. 中华口腔医学杂志, 2010, 45(9): 520-524.
	Zhou J, He Y, Dong SZ, et al. Effects of RhoA siRNA on the proliferation, adhesion, migration and invasion of tongue squamous cell carcinoma Tca8113 cells in vitro[J]. Chin J Stomatol, 2010, 45(9): 520-524.
16	Yan G, Zou R, Chen Z, 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.
17	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.
	Yan GX, Fan B, Zou RH, et al. Effects of RhoA silen-cing on proliferation of tongue squamous cancer cells[J]. West China J Stomatol, 2016, 34(6): 620-625.
18	Winter-vann AM, Casey PJ. Post-prenylation-processing enzymes as new targets in oncogenesis[J]. Nat Rev Cancer, 2005, 5(5): 405-412.
19	Cushman I, Casey PJ. Role of isoprenylcysteine carboxylmethyltransferase-catalyzed methylation in Rho function and migration[J]. J Biol Chem, 2009, 284(41): 27964-27973.
20	Liu Q, Chen J, Fu B, et al. Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to chemotherapy and Ras activation[J]. Biochem Biophys Res Commun, 2018, 501(2): 556-562.
21	Xu J, Zhu Y, Wang F, et al. ICMT contributes to hepatocellular carcinoma growth, survival, migration and chemoresistance via multiple oncogenic pathways[J]. Biochem Biophys Res Commun, 2019, 518(3): 584-589.
22	Pan Q, Liu R, Banu H, et al. Inhibition of isoprenylcys-teine carboxylmethyltransferase sensitizes common chemotherapies in cervical cancer via Ras-dependent pathway[J]. Biomed Pharmacother, 2018, 99: 169-175.
23	Lau HY, Tang J, Casey PJ, et al. Isoprenylcysteine carboxylmethyltransferase is critical for malignant transformation and tumor maintenance by all RAS isoforms[J]. Oncogene, 2017, 36(27): 3934-3942.
24	Magkouta S, Pappas A, Moschos C, et al. Icmt inhibition exerts anti-angiogenic and anti-hyperpermeability activities impeding malignant pleural effusion[J]. Oncotarget, 2016, 7(15): 20249-20259.
25	Lau HY, Ramanujulu PM, Guo D, et al. An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo[J]. Cancer Biol Ther, 2014, 15(9): 1280-1291.
26	Wang M, Hossain MS, Tan W, et al. Inhibition of isoprenylcysteine carboxylmethyltransferase induces autophagic-dependent apoptosis and impairs tumor growth[J]. Oncogene, 2010, 29(35): 4959-4970.
27	Bergo MO, Gavino BJ, Hong C, et al. Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf[J]. J Clin Invest, 2004, 113(4): 539-550.
28	Manu KA, Chai TF, Teh JT, et al. Inhibition of isoprenylcysteine carboxylmethyltransferase induces cell-cycle arrest and apoptosis through p21 and p21-regulated BNIP3 induction in pancreatic cancer[J]. Mol Cancer Ther, 2017, 16(5): 914-923.
29	Bergo MO, Leung GK, Ambroziak P, et al. Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells[J]. J Biol Chem, 2000, 275(23): 17605-17610.
30	Olson MF, Ashworth A, Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1[J]. Science, 1995, 269(5228): 1270-1272.
31	Olson MF, Paterson HF, Marshall CJ. Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1[J]. Nature, 1998, 394(6690): 295-299.
32	Wang H, Wang B, Liao Q, et al. Overexpression of RhoGDI, a novel predictor of distant metastasis, promotes cell proliferation and migration in hepatocellular carcinoma[J]. FEBS Lett, 2014, 588(3): 503-508.
33	Zuckerbraun BS, Shapiro RA, Billiar TR, et al. RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cip1 expression[J]. Circulation, 2003, 108(7): 876-881.

名称	产品编号	产品名称	靶序列（5’-3’）
Icmt-siRNA-1	stB0010313A	genOFFTMst-h-ICMT-001	GAAGAAGAAATCTCACTAA
Icmt-siRNA-2	stB0010313B	genOFFTMst-h-ICMT-002	GTTAGAGTTCACACTTGAA
Icmt-siRNA-3	stB0010313C	genOFFTMst-h-ICMT-003	CTTCCGCGATCGAACAGAA

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体外沉默异戊二烯基半胱氨酸羧基甲基转移酶对人舌鳞状细胞癌细胞增殖和凋亡的影响

Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma

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