沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

doi:10.19438/j.cjoms.2021.02.001

中国口腔颌面外科杂志 ›› 2021, Vol. 19 ›› Issue (2): 97-104.doi: 10.19438/j.cjoms.2021.02.001

沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

陈正岗¹, 王奇民¹, 童磊¹, 王云英², 徐晓娜², 王莹³, 韩红钰¹, 盛善桂⁴, 王少如⁵

1.青岛大学附属青岛市市立医院口腔医学中心,
2.中心实验室,山东青岛 266071;
3.济南市第四人民医院口腔科,山东济南 250031;
4.青岛大学口腔医学院,山东青岛 266003;
5.大连医科大学口腔医学院,辽宁大连 116044

收稿日期:2020-05-28 修回日期:2020-08-17 出版日期:2021-03-20 发布日期:2021-05-11
通讯作者: 王少如,E-mail：wangxiaorukw@163.com
作者简介:陈正岗（1973-）,男,博士,主任医师E-mail：chenzhg1973@163.com
基金资助:
国家自然科学基金（81372908）

Effects of silencing Icmt on the proliferation, apoptosis, and cell cycle of CAL-27 and SCC-4 cells

CHEN Zheng-gang¹, WANG Qi-min¹, TONG Lei¹, WANG Yun-ying², XU Xiao-na², WANG Ying³, HAN Hong-yu¹, SHENG Shan-gui⁴, WANG Shao-ru⁵

1. Department of Stomatology,
2. Central Laboratory, Qingdao Municipal Hospital, Qingdao University. Qingdao 266071, Shandong Province;
3. Department of Stomatology, Fourth People’s Hospital of Jinan. Jinan 250031, Shandong Province;
4. School of Stomatology, Qingdao University. Qingdao 266003, Shandong Province;
5. School of Stomatology, Dalian Medical University. Dalian 116044, Liaoning Province, China

Received:2020-05-28 Revised:2020-08-17 Online:2021-03-20 Published:2021-05-11

摘要/Abstract

摘要： 目的探讨异戊二烯基半胱氨酸羧基甲基转移酶（isoprenylcysteine carboxyl methyltransferase,Icmt）对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡和细胞周期的影响及其相关机制。方法针对人Icmt基因序列设计并构建3条小干扰RNA（small interfering RNA,siRNA）,采用脂质体载体瞬时转染Icmt-siRNA抑制舌鳞癌CAL-27和SCC-4细胞Icmt表达,将实验组分为Icmt-siRNA-1组、Icmt-siRNA-2组、Icmt-siRNA-3组;同时将脂质体转染NC-siRNA作为阴性对照组,只加转染试剂作为空白对照组。采用实时荧光定量PCR（qRT-PCR）和蛋白质免疫印记法（Western blot）检测转染后各组细胞Icmt、K-Ras的mRNA和蛋白表达及K-Ras膜蛋白的表达;Western免疫印迹检测Cyclin D1、p21、Akt、p-Akt蛋白表达;细胞增殖活性检测试剂盒和流式细胞术检测细胞的增殖活性、周期变化和凋亡能力。应用GraphPad Prism 8.2.1 软件对实验数据进行统计学分析。结果 qRT-PCR和Western 免疫印迹检测结果显示,与对照组相比,实验组Icmt mRNA和蛋白表达显著下降（P<0.05）,K-Ras mRNA和蛋白表达无显著差异（P>0.05）,K-Ras膜蛋白表达显著下降（P<0.05）;周期相关蛋白Cyclin D1表达显著下调,p21表达显著上调（P<0.05）;Ras/PI3K/Akt/mTOR信号通路相关蛋白Akt表达无统计学差异（P>0.05）,但p-Akt表达显著下降（P<0.05）。细胞增殖活性检测结果表明,与对照组相比,实验组细胞增殖能力显著下降（P<0.05）;流式细胞术检测结果表明,实验组细胞凋亡水平较对照组显著增加,细胞周期被阻滞在G1/S期（P<0.05）。结论体外沉默Icmt基因可有效抑制CAL-27和SCC-4细胞增殖且诱导凋亡,其作用可能是通过影响K-Ras膜蛋白靶向膜定位,负性调控细胞周期和下调Ras/PI3K/Akt/mTOR信号通路而实现。

关键词: 异戊二烯基半胱氨酸羧基甲基转移酶, K-Ras, 舌鳞状细胞癌, CAL-27, SCC-4, 细胞增殖, 细胞周期, 细胞凋亡

Abstract: PURPOSE: This study was aimed to explore the effects and regulatory mechanisms of silencing Icmt on cell proliferation, apoptosis, and cell cycle of cell line CAL-27 and SCC-4 in vitro. METHODS: Three siRNAs were designed and constructed for Icmt gene sequence, and then transfected into CAL-27 and SCC-4 cells to silence Icmt expression. The tested cells were divided as follows: RNA interference groups including Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The mRNA and protein expression of Icmt and K-Ras were examined by real-time PCR and Western blot, respectively. The expression of Cyclin D1, p21, Akt, and p-Akt were ex-amined by Western blot. The proliferation abilities of CAL-27 and SCC-4 cells were determined by cell counting kit-8 assay. Cell cycle analysis and apoptosis abilities of CAL-27 and SCC-4 cells were detected by flow cytometry. Statistical analysis and presentation was performed using GraphPad Prism 8.2.1 software. RESULTS: The expression of Icmt mRNA and protein in CAL-27 and SCC-4 cells was reduced significantly after Icmt siRNAs were transfected (P<0.05). No significant difference in K-Ras mRNA and protein expression was detected(P>0.05), but the expression of K-Ras membrane protein was decreased significantly compared with the negative control group and the blank control group (P<0.05). Cyclin D1 expression was decreased, whereas p21 expression was increased significantly. The expression of Akt was invariant (P>0.05), but the expression of p-Akt was significantly decreased (P<0.05). The cell cycle was altered in G1/S, the growth-proliferative activity was inhibited and apoptosis was significantly induced (P<0.05). CONCLUSIONS: Silencing Icmt can effectively reduce the proliferation and induce apoptosis of CAL-27 and SCC-4 cells by affecting K-Ras membrane targeting localization, and then negatively regulating cell cycle and down-regulating K-Ras /PI3K/Akt/mTOR signaling pathway.

Key words: Isoprenylcysteine carboxyl methyltransferase, K-Ras, Tongue squamous cell carcinoma, CAL-27, SCC-4, Cell proliferation, Cell cycle, Cell apoptosis

中图分类号:

R739.8

陈正岗, 王奇民, 童磊, 王云英, 徐晓娜, 王莹, 韩红钰, 盛善桂, 王少如. 沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响[J]. 中国口腔颌面外科杂志, 2021, 19(2): 97-104.

CHEN Zheng-gang, WANG Qi-min, TONG Lei, WANG Yun-ying, XU Xiao-na, WANG Ying, HAN Hong-yu, SHENG Shan-gui, WANG Shao-ru. Effects of silencing Icmt on the proliferation, apoptosis, and cell cycle of CAL-27 and SCC-4 cells[J]. China J Oral Maxillofac Surg, 2021, 19(2): 97-104.

参考文献

[1] Li S, Chien C, Huang WT, et al.Prognostic significance and function of mammalian target of rapamycin in tongue squamous cell carcinoma[J]. Sci Rep, 2017, 7(1): 8178-8178.
[2] Feng Z, Xu QS, Qin LZ, et al.Predicting radiotherapy necessity in tongue cancer using lymph node yield[J]. Oral Maxillofac Surg, 2017, 75(5): 1062-1070.
[3] Prior IA, Lewis PD, Mattos C.A comprehensive survey of Ras mutations in cancer[J]. Cancer Res, 2012, 72(10): 2457-2467.
[4] Karnoub AE, Weinberg RA.Ras oncogenes: split personalities[J]. Nat Rev Mol Cell Biol, 2008, 9(7): 517-531.
[5] Winter-Vann AM, Casey PJ.Post-prenylation-processing enzymes as new targets in oncogenesis[J]. Nat Rev Cancer, 2005, 5(5): 405-412.
[6] 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]. Biol Chemi, 2000, 275(23): 17605-17610.
[7] Zhu YB, Hu QY, Li H.Isoprenylcysteine carboxylme thyltransferase is associated with nasopharyngeal carcinoma chemoresistance and Ras activation[J]. Biochem Biophys Res Commun, 2019, 516(3): 784-789.
[8] Liu Q, Chen J, Fu B, et al.Isoprenylcysteine carboxyl methyltransferase regulates ovarian cancer cell response to chemotherapy and Ras activation[J]. Biochem Biophys Res Commun, 2018, 501(2): 556-562.
[9] Court H, Amoyel M, Hackman M, et al.Isoprenylcysteine carboxyl methyltransferase deficiency exacerbates K-RAS-driven pancreatic neoplasia via Notch suppression[J]. J Clin Invest, 2013, 123(11): 4681-4694.
[10] 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.
[11] Tadokoro K, Ueda M, Ohshima T, et al.Activation of oncogenes in human oral cancer cells: a novel codon 13 mutation of c-H-ras-1 and concurrent amplifications of c-erbB-1 and c-myc[J]. Oncogene, 1989, 4(4): 499-505.
[12] Saranath D, Panchal RG, Nair R, et al.Oncogene amplification in squamous cell carcinoma of the oral cavity[J]. Jpn J Cancer Res, 1989, 80(5): 430-437.
[13] Das N, Majumder J, Dasgupta UB.Ras gene mutations in oral cancer in eastern India[J]. Oral Oncol, 2000, 36(1): 76-80.
[14] 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(1): 181-205.
[15] Dai Q, Choy E, Chiu V, et al.Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum[J]. Biol Chem, 1998, 273(24): 15030-15034.
[16] Wahlstrom AM, Cutts BA, Liu M, et al.Inactivating Icmt ameliorates K-Ras-induced myeloproliferative disease[J]. Blood, 2008, 112(4): 1357-1365.
[17] 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.
[18] 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.
[19] Pan Q, Liu R, Banu H, et al.Inhibition of isoprenylcysteine carboxylmethyltransferase sensitizes common chemotherapies in cervical cancer via Ras-dependent pathway[J]. Biomed Pharmacother, 2018, 99: 169-175.
[20] Murugan AK, Munirajan AK, Tsuchida N, et al.Ras oncogenes in oral cancer: the past 20 years[J]. Oral Oncol, 2012, 48(5): 383-392.
[21] Morgensztern D, Mcleod HL.PI3K/Akt/mTOR pathway as a target for cancer therapy[J]. Anti Cancer Drugs, 2005, 16(8): 797-803.
[22] Wang K, Lin C, Wang C, et al.Silencing Kif2 a induces apoptosis in squamous cell carcinoma of the oral tongue through inhibition of the PI3K/Akt signaling pathway[J]. Mol Med Rep, 2014, 9(1): 273-278.
[23] Pruitt K, Der CJ.Ras and Rho regulation of the cell cycle and oncogenesis[J]. Cancer Lett, 2001, 171(1): 1-10.
[24] Shvartsur A, Bonavida B.Trop2 and its overexpression in cancers: regulation and clinical/therapeutic implications[J]. Genes Cancer, 2015, 6(3-4): 84-105.
[25] Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities[J]. Nat Rev Cancer,2009,9(6):400-414.
[26] 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.

沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

Effects of silencing Icmt on the proliferation, apoptosis, and cell cycle of CAL-27 and SCC-4 cells

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	水文军, 高金星, 任蕾. 基质金属蛋白酶9抑制剂对人舌鳞癌SCC-25细胞侵袭、迁移能力的影响及机制探讨[J]. 中国口腔颌面外科杂志, 2021, 19(5): 424-428.
[2]	张莹, 吴利, 陈羽菱, 林云涛, 沈月洪, 杨宏宇. POLD1对口腔鳞癌细胞增殖、侵袭的影响及调控机制[J]. 中国口腔颌面外科杂志, 2021, 19(4): 302-308.
[3]	孔宪琛, 姜晓东, 芦昕, 禹雯怡, 薛娇, 张鹏, 赵璐, 袁荣涛. 核糖体蛋白L29在舌鳞状细胞癌中的表达及意义[J]. 中国口腔颌面外科杂志, 2021, 19(2): 110-115.
[4]	王子昱, 丁宇洁, 陈冰, 沈昊翀, 孟箭, 仝伟凤. α桐酸对舌鳞状细胞癌CAL-27细胞的体外抑制作用[J]. 中国口腔颌面外科杂志, 2020, 18(6): 490-494.
[5]	周光明, 梁衍灿, 黄子贤, 张彬. 长链非编码RNA FOXD2-AS1对舌鳞癌细胞迁移、侵袭的影响[J]. 中国口腔颌面外科杂志, 2020, 18(6): 495-500.
[6]	陈永菊, 陈伟良, 周斌, 钟江龙, 汪延, 陈睿. 面-颏下动脉岛状皮瓣修复年轻与老年舌癌患者的效果比较[J]. 中国口腔颌面外科杂志, 2020, 18(2): 117-121.
[7]	林志立, 梁建平, 周斌. lncRNA-HOTTIP在低氧诱导舌鳞癌细胞上皮-间质转化过程中的作用探讨[J]. 中国口腔颌面外科杂志, 2019, 17(1): 26-31.
[8]	王珊珊, XiaohuiRausch-Fan, OlehAndrukov, 林毅, 林李嵩, 施斌. Emdogain对共培养成骨样细胞及内皮细胞成骨及成血管基因表达的影响[J]. 中国口腔颌面外科杂志, 2019, 17(1): 32-39.
[9]	罗丹, 孙巧珍, 刘少华, 孙俊伟, 王升志. 热疗联合Id-1基因沉默对舌鳞癌CAL-27细胞增殖及侵袭的影响[J]. 中国口腔颌面外科杂志, 2018, 16(6): 488-492.
[10]	马利, 石冰, 张东升, 郑谦. 细胞凋亡及上皮-间充质转化在腭胚突融合过程中的作用探讨[J]. 中国口腔颌面外科杂志, 2018, 16(6): 493-497.
[11]	刘诗奇, 羊良慧, 陈国生, 王辉, 房芳, 廖显翔, 麦华明. β肾上腺素受体在舌癌CAL-27细胞系的表达及β受体阻滞剂对其增殖、迁移、侵袭的影响[J]. 中国口腔颌面外科杂志, 2018, 16(5): 391-396.
[12]	魏立, 周琦, 江莉婷, 李宁, 高益鸣. 利塞膦酸钠对骨质疏松大鼠牙槽骨Bcl-2、BAX表达的影响[J]. 中国口腔颌面外科杂志, 2018, 16(5): 402-408.
[13]	刘墨, 李劲松, 阮毅, 黄洪章. miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究[J]. 中国口腔颌面外科杂志, 2018, 16(4): 289-295.
[14]	孙俊伟, 罗丹, 刘浩, 王升志. 热疗对Cal27细胞Id-1、HSF1表达的影响[J]. 中国口腔颌面外科杂志, 2018, 16(2): 144-148.
[15]	刘墨, 阮毅, 李劲松, 黄洪章. Twist1调控上皮-间质转化促进舌鳞癌细胞顺铂耐药机制的研究[J]. 中国口腔颌面外科杂志, 2018, 16(1): 1-5.