CCL18在胶质母细胞瘤中的表达及其对U87MG细胞增殖和迁移的影响

doi:10.11958/20221791

天津医药 ›› 2023, Vol. 51 ›› Issue (9): 915-921.doi: 10.11958/20221791

CCL18在胶质母细胞瘤中的表达及其对U87MG细胞增殖和迁移的影响

黄冠又¹(), 葛学成¹, 甘鸿川¹, 郝淑煜², 吴震²^,^△()

1.贵阳市第二人民医院神经外科（邮编550081）
2.首都医科大学附属北京天坛医院神经外科

收稿日期:2022-11-07 修回日期:2023-04-21 出版日期:2023-09-15 发布日期:2023-09-13
通讯作者: △E-mail：wuzhen1966@aliyun.com
作者简介:黄冠又（1982），男，副主任医师，主要从事颅脑肿瘤基础和临床方面研究。E-mail：hgy024410@126.com
基金资助:
国家自然科学基金资助项目(81672506);国家自然科学基金资助项目(81872052);贵州省卫健委科学技术基金(gzwkj2022-348)

Expression of CCL18 in glioblastoma and its effect on proliferation and migration of human U87MG cells

HUANG Guanyou¹(), GE Xuecheng¹, GAN Hongchuan¹, HAO Shuyu², WU Zhen²^,^△()

1. Department of Neurosurgery, the Second People’s Hospital of Guiyang, Guiyang 550081, China
2. Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University

Received:2022-11-07 Revised:2023-04-21 Published:2023-09-15 Online:2023-09-13
Contact: △E-mail：wuzhen1966@aliyun.com

黄冠又, 葛学成, 甘鸿川, 郝淑煜, 吴震. CCL18在胶质母细胞瘤中的表达及其对U87MG细胞增殖和迁移的影响[J]. 天津医药, 2023, 51(9): 915-921.

HUANG Guanyou, GE Xuecheng, GAN Hongchuan, HAO Shuyu, WU Zhen. Expression of CCL18 in glioblastoma and its effect on proliferation and migration of human U87MG cells[J]. Tianjin Medical Journal, 2023, 51(9): 915-921.

摘要/Abstract

摘要：

目的探讨趋化因子配体18（CCL18）在胶质母细胞瘤（GBM）中的表达与GBM临床预后的关系以及对U87MG细胞增殖和迁移的影响。方法基于癌症基因组图谱（TCGA）和基因型-组织表达（GTEx）数据库中GBM和正常脑组织数据，分析CCL18在GBM与正常脑组织中的表达差异，采用免疫组织化学染色进行验证。应用Kaplan-Meier生存分析、单因素和多因素Cox回归分析评估CCL18对GBM患者预后的影响。基于多因素Cox回归模型构建生存列线图，绘制校准曲线和受试者工作特征（ROC）曲线进行验证。将人胶质母细胞瘤U87MG细胞分为siRNA-CCL18组、siRNA-NC组和Mock-siRNA组，采用实时荧光定量PCR（qPCR）法检测细胞中CCL18 mRNA表达，CCK-8法和细胞划痕愈合实验检测U87MG细胞的增殖和迁移能力。结果 TCGA和GTEx数据库分析结果显示，GBM组织中CCL18基因表达水平高于正常脑组织；免疫组织化学染色结果显示，GBM组CCL18阳性表达率高于对照组（P<0.05）。CCL18高表达组患者预后较低表达组差（P<0.01）。Cox回归分析结果显示，IDH野生型和CCL18高表达是GBM患者死亡的独立危险因素（P<0.05）。校准曲线显示列线图预测模型与实际生存有较好一致性，ROC曲线表明该模型对GBM患者生存概率有较好预测能力。siRNA-CCL18组CCL18 mRNA表达水平、细胞增殖（48 h、72 h和96 h）和迁移能力（24 h）均低于siRNA-NC组和Mock-siRNA组（P<0.05）。结论 CCL18在GBM中表达上调，可促进GBM肿瘤细胞增殖和迁移，且可导致患者预后不良。

关键词: 胶质母细胞瘤, 趋化因子CCL18, 细胞增殖, 细胞运动, 预后

Abstract:

Objective To analyze the expression chemokine (C-C motif) ligand 18 (CCL18) on the clinical prognosis of glioblastoma (GBM) and its effects on the proliferation and migration of U87MG cells. Methods The differential expression of CCL18 gene in GBM and normal brain tissue was analyzed based on the GBM data from Cancer Genome Atlas (TGGA) database and the normal brain tissue data from Genotype-Tissue Expression (GTEx) database. Immunohistochemical staining was used to verification. Kaplan-Meier survival analysis, univariate and multivariable Cox regression analysis were used to evaluate the impact of CCL18 expression on the prognosis in GBM patients. A nomogram was established based on the results of multivariate Cox analysis. The calibration curve and receiver-operating characteristic (ROC) curve were draw for validation. Human glioblastoma U87MG cells were divided into the siRNA-CCL18 group, the siRNA-NC group and the Mock-siRNA group. qPCR was used to detect the content of CCL18 mRNA in the different U87MG cell groups. The CCK-8 assay and cell scratch test were used to detect the cell proliferation and migration ability of U87MG cells in each group. Results CCL18 was significantly upregulated in GBM tissue (P<0.05). The immunohistochemistry results suggested that CCL18 protein was significantly increased in GBM tissue (P<0.05). Survival analysis indicated that high expression of CCL18 was associated with poor prognosis in GBM patients (P<0.05). Multivariate Cox regression analysis revealed that IDH wild-type and high level of CCL18 expression were the two major risk factors affecting the poor prognosis of GBM (P<0.05). The calibration curve showed that the actual survival rate of patients was consistent with the predicted survival rate. The ROC curve demonstrated that the model had a good predictive for predicting the survival of GBM patients. siRNA reduced the expression of CCL18 in human U87MG cells. The results of CCK-8 assay showed that the proliferation of U87MG cells was significantly inhibited (P<0.05), and the wound healing scratch ability of U87MG cells with low expression of CCL18 decreased (P<0.01). Conclusion CCL18 is over-expressed in GBM tissue, and high CCL18 expression is associated with poor prognosis in GBM patients．CCL18 promotes GBM cell proliferation and migration and may act as a relevant predictive biomarker for GBM.

Key words: glioblastoma, chemokine CCL18, cell proliferation, cell movement, prognosis

中图分类号:

R739.41

图/表 12

表1 各组siRNA序列

Tab.1 Each groups of siRNA sequences

组别	引物序列（5′→3′）
siRNA-CCL18组	上游：AGUUCAUAGUUGACUAUUC
	下游：AAAGUUCAUAGUUGACUAU
Mock-siRNA组	上游：UAUCAAGAUGACGCUGCAA
	下游：AAUAUCAAGAUGACGCUGC
siRNA-NC组	上游：GAUGACGCUGCAAUGCAUA
	下游：AAGAUGACGCUGCAAUGCA

表2 qPCR引物序列

Tab.2 Primers for qPCR

基因名称	引物序列（5′→3′）	产物大小/ bp
CCL18	上游：TCGTCTATACCTCCTGGCAGATTCC	137
CCL18	下游：CCACTTCTTATTGGGGTCAGCACAG	137
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

图1 CCL18在GBM组织和正常脑组织中的表达 A：TCGA和GTEx数据库分析CCL18在GBM组和对照组的表达，＊＊P<0.01；B：正常脑组织CCL18表达阴性（免疫组织化学染色，×400）；C：GBM组织CCL18低表达（免疫组织化学染色，×400）；D：GBM组织CCL18高表达（免疫组织化学染色，×400）。

Fig.1 Expression of CCL18 in GBM and normal brain tissue

图2 CCL18高表达组与低表达组GBM患者Kaplan-Meier生存曲线

Fig.2 The expression level of CCL18 and Kaplan-Meier survival curve of GBM patients

表3 影响GBM患者术后生存的单因素Cox回归分析

Tab.3 Univariate Cox regression analysis on overall survival of GBM patients

变量	B	SE	Wald χ²	P	HR	HR 95%CI
年龄	0.311	0.173	3.241	0.072	1.365	0.973~1.915
性别	0.026	0.182	0.020	0.887	1.026	0.719~1.466
KPS评分	-0.177	0.226	0.611	0.434	0.838	0.538~1.305
IDH状态	-1.201	0.396	9.182	0.002	0.301	0.138~0.654
CCL18表达	0.464	0.174	7.114	0.008	1.870	1.303~2.683

表4 影响GBM患者术后生存的多因素Cox回归分析

Tab.4 Multivariate Cox regression analysis on overall survival of GBM patients

变量	B	SE	Wald χ²	P	HR	HR 95%CI
IDH状态	-1.241	0.419	8.772	0.003	0.289	0.127~0.657
CCL18表达	0.626	0.184	11.564	<0.001	1.870	1.303~2.683

图3 GBM患者1年、2年和3年生存概率的列线图预测模型

Fig.3 A nomogram prediction model of 1-year, 2-year and 3-year overall survival in GBM patients

图4 模型的校准曲线和时间依赖ROC曲线分析

Fig.4 The calibration curve of the model and time-dependent ROC curve

表5 CCL18表达对GBM患者1年、2年和3年生存概率的预测价值

Tab.5 The predictive value of CCL18 expression on overall survival of GBM patients at 1, 2 and 3 years

时间	Cut-off值	敏感度/ %	特异度/ %	约登指数	AUC（95%CI）
1年	0.59	65.2	70.1	0.353	0.700（0.641~0.759）
2年	0.58	55.2	72.1	0.273	0.648（0.595~0.701）
3年	0.54	54.5	77.2	0.317	0.662（0.607~0.717）

图5 抑制CCL18表达对U87MG细胞增殖能力的影响 24 h的F值为0.694，P=0.519；48 h、72 h和96 h的组间比较F值分别为7.207、24.012和27.252，均P<0.01；a与Mock-siRNA组比较，b与siRNA-NC组比较，P<0.05。

Fig.5 Effects of inhibition of CCL18 on the proliferation of U87MG cells

图6 抑制CCL18表达对U87MG细胞迁移能力的影响

Fig.6 Effects of inhibition of CCL18 on the migration of U87MG cells

表6 抑制CCL18表达后各组U87MG细胞迁移能力比较

Tab.6 Comparison of migration ability in U87MG cells after the inhibition of CCL18 expression between the three groups

组别	12 h迁移率/%	24 h迁移率/%
Mock-siRNA组	28.67±4.93	81.33±6.81
siRNA-NC组	25.67±4.16	56.67±8.02^a
siRNA-CCL18组	22.67±0.58	33.33±9.45^ab
F	0.257	25.927^＊＊

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CCL18在胶质母细胞瘤中的表达及其对U87MG细胞增殖和迁移的影响

Expression of CCL18 in glioblastoma and its effect on proliferation and migration of human U87MG cells

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