CBX3在肺腺癌中的表达、预后相关性及对癌细胞生物学行为的影响

doi:10.19401/j.cnki.1007-3639.2022.02.007

摘要/Abstract

摘要：

背景与目的：作为异染色质重要组分的染色体盒3（chromobox 3,CBX3）参与多种癌症的发生、发展过程,而CBX3基因在肺腺癌中的表达情况和作用尚不清楚。探讨CBX3在肺腺癌中的表达与肺腺癌患者预后的关系,并研究CBX3对A549肺腺癌细胞增殖、迁移和侵袭能力的影响。方法：下载癌症基因组图谱（The Cancer Genome Atlas,TCGA）中的肺腺癌转录组数据和对应的临床数据,应用R软件分析CBX3在肺腺癌中的表达差异和免疫浸润相关性,采用受试者工作特征（receiver operating characteristic,ROC）曲线和Kaplan-Meier分析CBX3在肺腺癌中的预后意义;收集北京大学深圳医院智能化生物样本库-80 ℃新鲜冻存的2020年8月—2021年1月行肺癌姑息性切除术或根治性切除术的肺腺癌样本20例（已获得知情同意）,采用实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）检测20例冻存的肺腺癌组织及配对正常肺组织中CBX3的表达;应用小干扰RNA（siRNA）转染A549细胞以沉默CBX3的表达,并根据siRNA转染的类型将A549细胞分为沉默组（si-CBX3组）和阴性对照组（si-NC组）,通过用细胞计数试剂盒（cell counting kit-8,CCK-8）实验、克隆形成实验、划痕实验和transwell实验分别研究CBX3对A549肺腺癌细胞增殖、迁移和侵袭能力的影响。结果：基于TCGA肺腺癌数据的差异分析与采集的肺腺样本RTFQ-PCR实验的结果显示,CBX3在肺腺癌中呈显著的高表达,差异有统计学意义（P<0.05）;辅助型T细胞2的浸润与CBX3表达呈正相关,相关系数（r）=0.437,而肥大细胞（r=-0.444）、嗜酸性粒细胞（r=-0.380）等免疫细胞与CBX3表达呈负相关;ROC曲线下面积（area under curve,AUC）值为0.912,表明CBX3对肺腺癌具有很好的诊断价值,Kaplan-Meier分析结果显示,CBX3高表达组生存期更短,提示预后较差;沉默CBX3在A549细胞中的表达后,CCK-8实验和克隆形成实验结果表明,A549细胞的增殖能力显著降低（P<0.01）;划痕实验结果表明沉默后的A549细胞的迁移率为（22.68±3.44）%,较阴性对照组迁移能力明显降低（P<0.05）;Transwell实验结果表明A549细胞的相对侵袭率为（53.94±5.39）%,侵袭能力显著降低（P<0.01）。结论：CBX3在肺腺癌中显著高表达并提示肺腺癌预后不良,可作为肺腺癌的预后标志物,且CBX3的高表达促进了A549细胞的增殖、迁移和侵袭能力。

关键词: CBX3, 肺腺癌, 免疫浸润, 预后, 细胞功能实验

Abstract:

Background and purpose: Chromobox 3 (CBX3), an important component of chromatin, is involved in the development and progression of various cancers. However, the expression and role of CBX3 in lung adenocarcinoma are still unclear. This study aimed to investigate the relationship between the expression of CBX3 in lung adenocarcinoma and prognosis of lung adenocarcinoma, and to study the effects of CBX3 on proliferation, migration and invasion of A549 lung adenocarcinoma cells. Methods: Transcriptome data of lung adenocarcinoma and corresponding clinical data were downloaded from the Cancer Genome Atlas (TCGA), and R software was used to analyze the expression difference of CBX3 and the correlation of immune infiltration in lung adenocarcinoma. The prognostic significance of CBX3 in lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve and Kaplan-Meier analysis. Twenty lung adenocarcinoma samples from patients (with informed consent obtained) who underwent palliative or radical lung cancer resection from August 2020 to January 2021 were collected from the intelligent Biobank of Peking University Shenzhen Hospital at -80 ℃. Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) was used to verify the expression of CBX3 in 20 frozen lung adenocarcinoma tissue samples and paired normal lung tissues. A549 cells were transfected with small interfering RNA (siRNA) to silence the expression of CBX3, and A549 cells were divided into silent group (si-CBX3 group) and negative control group (si-NC group) according to the type of siRNA transfection. The effects of CBX3 on proliferation, migration and invasion of A549 cells were investigated by cell counting kit-8 (CCK-8) assay, clone formation assay, scratch assay and transwell assay, respectively. Results: The difference analysis based on TCGA lung adenocarcinoma data and RTFQ-PCR results of lung adenocarcinoma samples showed that CBX3 was significantly overexpressed in lung adenocarcinoma (P<0.05). The infiltration of T helper 2 (Th2) cells was positively correlated with the expression of CBX3 [correlation coefficient (r)=0.437], while mast cells (r=-0.444), eosinophils (r=-0.380) and other immune cells were negatively correlated with the expression of CBX3. The area under ROC curve (AUC) value was 0.912, indicating that CBX3 has a good diagnostic value for lung adenocarcinoma. Kaplan-Meier analysis showed that the survival period of the group with high expression of CBX3 was shorter, suggesting a poor prognosis. After the expression of CBX3 was silenced in A549 cells, significantly decreased proliferation ability of A549 cells was detected by CCK-8 and clone formation assays (P<0.01). The results of scratch assay showed that the mobility of A549 cells was (22.68±3.44)% after silencing, which was significantly lower compared with the control group (P<0.05). Transwell assay results showed that the relative invasion rate of A549 cells was (53.94±5.39)%, and the invasion ability was significantly decreased (P<0.01). Conclusion: CBX3 is significantly overexpressed in lung adenocarcinoma, which predicts poor prognosis of lung adenocarcinoma and can be used as a prognostic marker of lung adenocarcinoma, and the high expression of CBX3 promotes the proliferation, migration and invasion of A549 cells.

Key words: CBX3, Lung adenocarcinoma, Immune infiltration, Prognosis, Cell function experiment

中图分类号:

R734.2

侯清华, 钟燕凤, 刘林壮, 吴柳盛, 刘继先. CBX3在肺腺癌中的表达、预后相关性及对癌细胞生物学行为的影响[J]. 中国癌症杂志, 2022, 32(2): 152-160.

HOU Qinghua, ZHONG Yanfeng, LIU Linzhuang, WU Liusheng, LIU Jixian. Expression, prognostic value of CBX3 in lung adenocarcinoma and its effect on biological behavior of cancer cells[J]. China Oncology, 2022, 32(2): 152-160.

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Characteristic^*	Low expression of CBX3	High expression of CBX3	P value
T stage n (%)			0.031
T₁	97 (19.0)	71 (13.9)
T₂	126 (24.7)	150 (29.4)
T₃	25 (4.9)	22 (4.3)
T₄	6 (1.2)	13 (2.5)
N stage n (%)			0.005
N₀	179 (35.7)	151 (30.1)
N₁	42 (8.4)	53 (10.6)
N₂	26 (5.2)	48 (9.6)
N₃	0 (0.0)	2 (0.4)
M stage n (%)			0.342
M₀	165 (44.7)	179 (48.5)
M₁	9 (2.4)	16 (4.3)
Pathologic stage n (%)			0.102
Ⅰ	148 (29.3)	126 (25.0)
Ⅱ	60 (11.9)	61 (12.1)
Ⅲ	34 (6.7)	50 (9.9)
Ⅳ	10 (2.0)	16 (3.2)
Gender n (%)			0.504
Female	142 (27.7)	134 (26.1)
Male	114 (22.2)	123 (24.0)
Age/year n (%)			0.024
≤65	105 (21.3)	133 (26.9)
>65	140 (28.3)	116 (23.5)
Smoker n (%)			0.368
No	41 (8.2)	33 (6.6)
Yes	208 (41.7)	217 (43.5)