生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理

doi:10.7518/hxkq.2016.03.008

华西口腔医学杂志 ›› 2016, Vol. 34 ›› Issue (3): 255-261.doi: 10.7518/hxkq.2016.03.008

生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理

付小娟,杨凯,李晗雪,赵钦,陈丹

重庆医科大学附属第一医院口腔颌面外科，重庆400016

收稿日期:2015-10-16 修回日期:2016-02-28 出版日期:2016-06-01 发布日期:2016-06-01
通讯作者: 杨凯，教授，博士，E-mail：cqfyyk@aliyun.com
作者简介:付小娟，硕士，E-mail：466133758@qq.com

Effects and mechanism of the circadian clock gene Per1 on the proliferation, apoptosis, cycle, and tumorigenicity in vivo of human oral squamous cell carcinoma

Fu Xiaojuan, Yang Kai, Li Hanxue, Zhao Qin, Chen Dan

Dept. of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China) Correspondence: Yang Kai, E-mail: cqfyyk@aliyun.com.

Received:2015-10-16 Revised:2016-02-28 Online:2016-06-01 Published:2016-06-01

摘要/Abstract

摘要： 目的探讨生物钟基因Per1对细胞周期相关基因的调控作用，以及对人口腔鳞状细胞癌细胞SCC15增殖、凋亡、细胞周期和体内成瘤的影响。方法构建3组针对Per1 RNA的短发夹RNA（shRNA）慢病毒重组质粒，转染SCC15细胞，用蛋白质印迹法（Western blot）及实时荧光定量PCR（qRT-PCR）检测筛选RNA干扰作用最强组为实验组，对照组（Control-shRNA）为对任何基因均无干扰效应的shRNA序列的质粒，空白组为未做任何处理的SCC15细胞。用qRT-PCR检测各组细胞中细胞周期相关基因Per1、p53、Cyclin D1、Cyclin E、Cyclin A2、Cyclin B1、CDK1、CDK2、CDK4、CDK6、p16、p21、Wee1、cdc25、E2F、Rb1 mRNA的表达；用流式细胞仪检测各组细胞增殖、凋亡和细胞周期分布；将实验组和空白组细胞分别接种于裸鼠背部皮下，观察成瘤情况。结果成功构建了3组Per1-shRNA慢病毒质粒，通过qRT-PCR和Western blot证明Per1-shRNA-Ⅰ组沉默效果最佳，作为实验组。Per1-shRNA-Ⅰ组中Cyclin D1、Cyclin E、Cyclin B1、CDK1和Wee1 mRNA的表达水平高于Control-shRNA组和SCC15组（P<0.05），p53、Cyclin A2、p16、p21和cdc25 mRNA的表达水平降低（P<0.05）；Control-shRNA组和SCC15组中各基因mRNA的表达水平无差异（P>0.05）。CDK2、CDK4、CDK6、E2F和Rb1 mRNA的表达水平在3组中均无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组中细胞增殖指数高于Control-shRNA组和SCC15组（P<0.05），凋亡指数降低（P<0.05），S期的细胞数降低（P<0.05），G2/M期的细胞数增加（P<0.05）。Control-shRNA组和SCC15组细胞的增殖指数和凋亡指数无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组细胞体内成瘤能力显著增强（P<0.05）。结论生物钟基因Perl是重要的抑癌基因，Perl能调控下游众多的细胞周期基因，其表达变化影响细胞周期进程、增殖和凋亡的平衡失调及体内成瘤能力，对Per1深入研究有可能进一步明确癌症的发生发展机制，为癌症的治疗提供新的有效分子靶点。

关键词: 生物钟, Per1, 细胞周期, 基因, 口腔癌

Abstract: Objective To determine the regulatory effects of the circadian clock gene Per1 on cell cycle-related genes and its influence on the proliferation, apoptosis, cycle, and tumorigenicity in vivo of human oral squamous cell carcinoma SCC15 cells. Methods Three groups of the short hairpin RNA (shRNA) of lentivirus recombinant plasmids were designed against the RNA of Per1 and then transfected to the SCC15 cells. The optimum interference group was screened through Western blot and quantitative real-time PCR (qRT-PCR) and assigned as the experimental group. The transfected lentivirus plasmid without an interference effect on any gene was set as the control group (Control-shRNA). Untreated SCC15 cells were set as the blank group. The mRNA expressions of cell cycle-related genes, namely, Per1, p53, Cyclin D1, Cyclin E, Cyclin A2, Cyclin B1, CDK1, CDK2, CDK4, CDK6, p16, p21, Wee1, cdc25, E2F, and Rb1, in each group were detected through qRT-PCR. The cell proliferation, apoptosis, and cell cycle distribution in each group were evaluated through flow cytometry. The cells of the experimental group and the blank group were subcutaneously inoculated in nude mice to observe tumorigenesis. Results Three groups of Per1-shRNA lentivirus plasmids were constructed successfully. Among the groups, the Per1-shRNA-Ⅰgroup exhibited the highest interference effect, as indicated by qRT-PCR and Western blot analysis. As such, this group was set as the experimental group. The mRNA expression levels of CyclinD1, CyclinE, CyclinB1, CDK1, and Wee1 gene in the Per1-shRNA-Ⅰgroup were significantly higher than those in the Control-shRNA group and the SCC15 group (P<0.05). By contrast, the mRNA expression levels of p53, Cyclin A2, p16, p21, and cdc25 in the Per1-shRNA-Ⅰgroup were significantly lower than those in the Control-shRNA group and the SCC15 group (P<0.05). The mRNA expression levels of each gene between the Control-shRNA group and the SCC15 group did not significantly differ (P>0.05). The mRNA expression levels of CDK2, CDK4, CDK6, E2F, and Rb1 did not significantly differed in the three groups (P>0.05). The proliferation index of the Per1-shRNA-Ⅰgroup was significantly higher than those of the Control-shRNA group and the SCC15 group (P<0.05). The apoptosis index of the Per1-shRNA-Ⅰgroup was significantly lower than those of the Control-shRNA group and the SCC15 group (P<0.05). The number of S-phase cells in the Per1-shRNA-Ⅰgroup was significantly lower than those of S-phase cells in the Control-shRNA group and the SCC15 group (P<0.05). The number of G2/M-phase cells in the Per1-shRNA-Ⅰgroup was significantly higher than those of G2/Mphase cells in the Control-shRNA group and the SCC15 group (P<0.05). Conversely, the proliferation index, apoptotic index, and cell cycle distribution of the cells in the Control-shRNA group did not significantly differ from those of the SCC15 group (P>0.05). The tumorigenic ability in vivo was significantly enhanced in the Per1-shRNA-Ⅰgroup (P<0.05). Conclusion Per1 is an important tumor suppressor gene. Perl can regulate a large number of downstream cell cycle-related genes. The alteration of its expression can affect cell cycle progression, proliferation, apoptosis imbalance, and tumorigenic ability in vivo. Further studies on Per1 may elucidate cancer development and provide novel effective molecular targets for cancer treatment.

Key words: circadian clock, Per1, cell cycle, gene, oral carcinoma

中图分类号:

R 739.8

付小娟,杨凯,李晗雪,赵钦,陈丹. 生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理[J]. 华西口腔医学杂志, 2016, 34(3): 255-261.

Fu Xiaojuan, Yang Kai, Li Hanxue, Zhao Qin, Chen Dan. Effects and mechanism of the circadian clock gene Per1 on the proliferation, apoptosis, cycle, and tumorigenicity in vivo of human oral squamous cell carcinoma[J]. West China Journal of Stomatology, 2016, 34(3): 255-261.

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生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理

Effects and mechanism of the circadian clock gene Per1 on the proliferation, apoptosis, cycle, and tumorigenicity in vivo of human oral squamous cell carcinoma

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