吉林大学学报(医学版)  2016, Vol. 42 Issue (03): 502-505

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温剑平, 崔永生, 张雄基
WEN Jianping, CUI Yongsheng, ZHANG Xiongji
转染EGFRL858R的肺腺癌细胞中IL-6和VEGF表达水平及其意义
Expression levels of IL-6 and VEGF in lung adenocarcinoma cells transfected with EGFRL858R and their significances
吉林大学学报(医学版), 2016, 42(03): 502-505
Journal of Jilin University (Medicine Edition), 2016, 42(03): 502-505
10.13481/j.1671-587x.20160316

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收稿日期: 2016-01-27
引用本文 0
温剑平, 崔永生, 张雄基. 转染EGFRL858R的肺腺癌细胞中IL-6和VEGF表达水平及其意义[J]. 吉林大学学报(医学版), 2016, 42(03): 502-505.
WEN Jianping, CUI Yongsheng, ZHANG Xiongji. Expression levels of IL-6 and VEGF in lung adenocarcinoma cells transfected with EGFRL858R and their significances[J]. Journal of Jilin University (Medicine Edition), 2016, 42(03): 502-505.
转染EGFRL858R的肺腺癌细胞中IL-6和VEGF表达水平及其意义
温剑平1, 崔永生2, 张雄基2     
1. 吉林大学基础医学院遗传学系, 吉林 长春 130021;
2. 吉林大学第一医院胸外科, 吉林 长春 130021
收稿日期: 2016-01-27
基金项目: 吉林省科技厅科研基金资助课题(20120708)
作者简介: 温剑平(1968-),男,吉林省长春市人,副教授,医学博士,主要从事药物基因组学方面的研究。
通信作者: 张雄基,教授,硕士研究生导师(Tel:0431-88782973,E-mail:lxzhxj@hotmail.com)
摘要: 目的: 探讨肿瘤细胞癌基因表皮生长因子受体(EGFR)L858R型突变与肺腺癌恶性转移患者癌细胞中白细胞介素6(IL-6)和血管内皮生长因子(VEGF)表达水平的关系及其意义。方法: 将含EGFRL858R全长基因质粒的载体转染EGFR野生型肺腺癌细胞系H460(EGFRL858R-H460组),空载体转染H460细胞作为对照组(Vec-H460组),QRT-PCR法检测2组细胞中IL-6和VEGFmRNA表达水平。将含IL-6siRNA的载体转染带有EGFRL858R型突变的细胞系H1975(IL-6siH1975组),转染空载体的H1975细胞作为对照组(Vec-H1975组),QRT-PCR法检测2组细胞中IL-6和VEGFmRNA表达水平,Western blotting法检测2组细胞中VEGF蛋白表达水平。结果: 与Vec-H460组比较,EGFRL858R-H460组细胞中IL-6和VEGFmRNA表达水平明显升高(P < 0.01);与Vec-H1975组比较,IL-6siH1975组细胞组IL-6和VEGFmRNA表达水平明显降低(P < 0.01),VEGF蛋白表达强度明显降低。结论: 肺腺癌细胞系中EGFRL858R型突变可升高肺腺癌患者癌细胞中IL-6和VEGFmRNA表达水平,其中IL-6mRNA水平升高可能是VEGFmRNA表达水平升高的原因,这些变化可能是EGFRL858R型突变参与肺腺癌恶性转移进程的途径之一。
关键词: 肺肿瘤    表皮生长因子受体    L858R突变    白细胞介素6    血管内皮生长因子    
Expression levels of IL-6 and VEGF in lung adenocarcinoma cells transfected with EGFRL858R and their significances
WEN Jianping1, CUI Yongsheng2, ZHANG Xiongji2     
1. Department of Genetic, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
2. Department of Thoracic Surgery, First Hospital, Jilin University, Changchun 130021, China
Abstract: Objective: To detect the association between carcinoma cell oncogene epidermal growth factor receptor (EGFR) with L858R mutation and the expression levels of interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) in the carcinoma cells of lung adenocarcinoma patients with malignant metastasis and their significances. Methods: The vetor contained EGFRL858R full gene was transfect into the EGFR wild type lung adenocarcinoma H460 cell line(EGFRL858R-H460 group), the empty vector was transfected into the H460 cell line as control group(Vec-H460 group),and the expression levels of IL-6 and VEGF mRNA in the cells in two groups were detected by QRT-PCR method.The IL-6 siRNA was transfected into the lung adenocarcinoma H1975 cell line with EGFRL858R mutation( IL-6 siH1975 group),the empty vector was also transfected into the H1975 cell line as control group(Vec-H1975 group);the expression levels of IL-6 and VEGF mRNA in the cells in two groups were detected by QRT-PCR method,and the expression level of VEGF protein was detected by Western blotting method. Results: Compared with Vec-H460 group,the expression levels of IL-6 and VEGF mRNA in the cells in EGFRL858R-H460 group were increased (P < 0.01); compared with Vec-H1975 group, the expression levels of IL-6 and VEGF mRNA in the cells in IL-6 siH1975 group were decreased (P < 0.01),and the expression level of VEGF protein in the cells in IL-6 siH1975 group were decreased. Conclusion: The EGFRL858R mutation can elevate the expression levels of IL-6 and VEGF mRNA in lung adenocarcinoma cell lines,and the increasing expression of IL-6 mRNA may be responsible for the increasing expression of VEGF mRNA.These changes may be the evidences to prove that the EGFRL858R mutation is involved in the malignant transfer process in lung adenocarcinoma.
Key words: ung neoplasms    epidermal growth factor receptor    L858R mutation    interleukin-6    vascular endothelial growth factor    

近年来，炎症因子参与肿瘤恶性转移进程的研究受到了人们的重视。有些细胞因子在微环境变得有利于肿瘤细胞生长和迁移时，可成为协助癌细胞扩散和转移的因素[1]。白细胞介素6(interleukin-6,IL-6)是一种与肺癌发生发展有关的细胞因子，其异常分泌与人肺腺癌细胞浸润及恶性胸膜转有密切关联[2]。IL-6的调控异常与非小细胞肺癌的发生发展和胸膜转移亦有密切关联[3, 4]。目前关于IL-6是否参与带有表皮生长因子受体(epidermal growth factor receptor,EGFR)突变型肺腺癌恶性进程尚未见报道。本研究构建带有L858R型突变的EGFR全长基因转染的肺腺癌细胞系，探讨EGFRL858R型突变与肿瘤细胞中IL-6表达及与IL-6相关的血管内皮生长因子(vascular endothelial growth factor,VEGF)之间是否存在关联。

1 材料与方法 1.1 细胞系、质粒、主要试剂和仪器

H1975和H460肺腺癌细胞系及EGFRL858R全长基因表达载体L858R-pCDNA3.1质粒均由吉林大学基础医学院遗传学教研室提供和构建。荧光定量检测试剂盒(杭州博日科技有限公司)，SiRNA-IL-6载体(北京合生基因技术有限公司)，抗人VEGF抗体(福州迈新生物科技有限公司)。JY600E型电泳仪(北京君意东方电泳设备有限公司)，荧光PCR仪FQD48a(杭州博日科技有限公司)。

1.2 稳定转染细胞系的构建和筛选

H460是表达野生型EGFR的肺腺癌细胞系，无EGFRL858R突变存在。将含有L858R-pCDNA3.1质粒稳定转染入H460细胞系中，G418压力筛选，获得稳定表达的EGFRL858R突变型细胞，命名为EGFRL858R-H460细胞(EGFRL858R-H460组)。提取细胞基因组DNA,等位基因PCR法筛选鉴定EGFRL858R-H460细胞。将空pCDNA3.1稳定转染的H460细胞系作为阴性对照，命名为Vec-H460细胞(Vec-H460组)。

根据人IL-6基因序列，设计带有互补发卡结构的siRNA片段，克隆入真核表达载体，委托北京合生基因技术有限公司完成。选取多个IL-6片段为靶片段。将上述IL-6 siRNA载体转染的H1975细胞系进行药物筛选，获得多个稳定转染的细胞后，采用qRT-PCR法鉴定细胞中IL-6表达情况，选择IL-6 mRNA表达水平最低的细胞用于下一步实验，该细胞命名为IL-6siH1975(IL-6siH1975组)。以转染空载体的H1975细胞为对照，命名为Vec-H1975细胞(Vec-H1975组)。

1.3 QRT-PCR法检测各组细胞中IL-6和VEGFmRNA表达水平

分别提取EGFRL858R-H460、Vec-H460、Vec-H1975和IL-6 siH1975组细胞总RNA,逆转录，以β-actin为内参对照，以人IL-6和VEGF特异性引物行qRT-PCR，采用2-ΔΔCt法计算实验结果。应用公式计算ΔΔCt 值，ΔΔCt=(实验组目的基因Ct-实验组内参基因Ct)-(对照组目的基因Ct- 对照组内参基因Ct)，2-ΔΔCt值即实验组相对于对照组ΔΔCt值的倍数。

1.4 Western blotting 法检测IL-6 siH1975和Vec-H1975组细胞中VEGF蛋白表达水平

提取IL-6 siH1975和Vec-H1975组细胞的蛋白，以β-actin为内参对照，Western blotting 法检测IL-6 siH1975和Vec-H1975组细胞中VEGF蛋白的表达强度。

1.5 统计学分析

采用SPSS 11.5统计软件对数据进行统计学分析。各组细胞中IL-6和VEGF mRNA 表达水平以 ± s 表示，组间比较采用t检验。以ɑ=0.05为检验水准。

2 结 果 2.1 各组细胞中IL-6 和VEGF mRNA的表达水平

与Vec-H460组(1.00±0.00)比较，EGFRL858R-H460组细胞中IL-6 mRNA表达水平(2.80 ± 0.33)升高(P < 0.01±0.00); 与Vec-H460组(1.00±0.00)比较，EGFRL858R-H460组细胞中VEGF mRNA表达水平(2.54 ± 0.41)升高(P < 0.01)。与Vec-H1975组(1.00±0.01)比较，IL-6 siH1975组细胞中IL-6 mRNA表达水平(0.22 ± 0.04)降低(P < 0.01); 与Vec-H1975组(1.00±0.01)比较，IL-6 siH1975组细胞中VEGF mRNA表达水平(0.20 ± 0.03)降低(P < 0.01)。

2.2 Vec-H1975和IL-6 siH1975组细胞中VEGF蛋白的表达水平

与Vec-H1975组比较，IL-6 siH1975组细胞中VEGF蛋白表达强度明显下降。见图 1。

图 1 Western blotting 法检测IL-6 siH1975和Vec-H1975组细胞中VEGF蛋白表达电泳图 Fig.1 Electrophoregram of expressions of VEGF protein in cells in IL-6 siH1975 and Vec-H1975 groups detected by Western blotting method Lane 1，2: IL-6 siH1975 group；Lane 3，4: Vec-H1975 group.
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3 讨 论

肺腺癌的发生和转移与EGFR基因突变有密切关联，在亚洲人群中，25% ~ 50%肺腺癌细胞中可以检测到EGFR突变[5]。突变后的EGFR发生了不依赖配体表皮生长因子(EGF)的激酶区持续激活，通过信号转导参与肿瘤生长、侵袭、转移和新生血管生成[6]。EGFR突变是否还通过其他途径参与上述肿瘤进程尚未见报道。

研究[7]显示：除免疫细胞外，多种肿瘤细胞也可以自分泌IL-6，IL-6介导的信号通路参与调控肿瘤的发生发展。IL-6自分泌增加的机制及其下游信号通路活化后对于肺腺癌胸膜转移的调节机制尚不清楚。研究[8, 9]显示：IL-6介导的信号转导及转录激活因子3(STAT3)持续激活与其他癌基因的突变或持续激活相互作用，共同促进肿瘤的发生发展。

肺腺癌细胞的浸润和恶性胸膜转移与IL-6的异常分泌有关联,提示作为肺腺癌发生发展的重要因素，癌基因EGFR的突变可能与IL-6的异常分泌有内在关联。

EGFR 21号外显子点突变EGFRL858R型突变是肺癌常见的EGFR突变类型之一。本研究选取该类型突变作为研究对象。本研究结果表明：在体外转染的肺腺癌细胞系中，EGFRL858R型突变可提高IL-6自分泌水平和VEGF水平。因此推测：在肺腺癌恶性转移进程中，EGFRL858R型突变可能是带有该型突变的肺腺癌患者IL-6和VEGF表达水平升高的原因之一。

在体外实验中，EGFR突变引起IL-6和VEGF表达水平升高的信号途径尚不清楚。为探讨IL-6表达水平升高与VEGF表达水平两者之间的关系，本研究构建了IL-6 siRNA转染细胞，观察该细胞中VEGF水平是否有变化。本研究结果表明：转染IL-6 siRNA载体使IL-6低表达的肺腺癌细胞中VEGF表达水平亦降低，说明与EGFRL858R型突变因素比较，IL-6自分泌水平升高是VEGF表达水平升高更为直接的因素。有关肿瘤细胞中IL-6和VEGF表达的关系问题，有研究者[10, 11, 12, 13, 14]在对其他多种肿瘤的研究中得到了相似的结论。

综上所述，在肿瘤发生发展和转移过程中，细胞中的癌基因持续激活且其是否与炎性细胞因子协调作用共同促进肿瘤的发生发展是一个值得深入探讨的问题[15, 16, 17]。

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