2022, Vol. 51
基于公共数据库分析TNFAIP2在肾透明细胞癌中的表达及临床意义
文章信息
- 薛东炜, 李明山, 刘嘉, 祝兴旺
- XUE Dongwei, LI Mingshan, LIU Jia, ZHU Xingwang
- 基于公共数据库分析TNFAIP2在肾透明细胞癌中的表达及临床意义
- Analysis of the expression and clinical significance of TNFAIP2 in renal clear cell carcinoma by utilizing public databases
- 中国医科大学学报, 2022, 51(8): 695-700
- Journal of China Medical University, 2022, 51(8): 695-700
-
文章历史
- 收稿日期：2022-05-09
- 网络出版时间：2022-07-13 13:46
引用本文 |
薛东炜, 李明山, 刘嘉, 祝兴旺.
基于公共数据库分析TNFAIP2在肾透明细胞癌中的表达及临床意义[J]. 中国医科大学学报, 2022, 51(8): 695-700.
XUE Dongwei, LI Mingshan, LIU Jia, ZHU Xingwang. Analysis of the expression and clinical significance of TNFAIP2 in renal clear cell carcinoma by utilizing public databases[J]. Journal of China Medical University, 2022, 51(8): 695-700.
基于公共数据库分析TNFAIP2在肾透明细胞癌中的表达及临床意义
中国医科大学附属第四医院泌尿外科, 沈阳 110032
摘要：目的 探讨肿瘤坏死因子α诱导蛋白2(TNFAIP2) 在肾透明细胞癌(ccRCC) 中的表达情况及其临床意义。方法 通过TIMER2.0、癌症基因组图谱(TCGA)、ONCOMINE和UALCAN数据库, 分析TNFAIP2 mRNA在正常肾脏和ccRCC组织中的表达差异, 及其与临床指标和免疫细胞丰度的相关性; 利用Kaplan-Meier曲线数据库分析预后情况。采用Western blotting检测人肾脏正常细胞系HK-2和人肾癌细胞系786-O、ACHN中TNFAIP2的表达, 并应用实时PCR在24个配对的肾癌组织与癌旁肾脏正常组织样本中检测TNFAIP2的表达量。结果 公共数据库分析结果显示, TNFAIP2在ccRCC组织中异常高表达(P<0.001), 且TNFAIP2的高表达与肿瘤的分期、分级、淋巴结转移、免疫细胞Th1和Treg丰度密切相关(P<0.001); Kaplan-Meier曲线分析表明TNFAIP2表达与患者的预后有关, 其高表达提示病情恶化及总生存期缩短(P<0.001);受试者操作特征曲线显示TNFAIP2表达可用于评估患者的诊断预测效率(曲线下面积=0.814)。体外细胞系实验结果显示, 肾癌786-O、ACHN细胞中TNFAIP2表达水平均高于人肾脏正常细胞系HK-2;TNFAIP2 mRNA在ccRCC组织中的平均表达量显著高于正常组织。结论 TNFAIP2在ccRCC中高表达, 且与预后有关, 可作为ccRCC诊断和预后的生物标志物, 为肾癌的靶向治疗提供新方向。
Analysis of the expression and clinical significance of TNFAIP2 in renal clear cell carcinoma by utilizing public databases
Department of Urology, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China
Abstract: Objective To explore the expression and clinical significance of tumor necrosis factor-α-induced protein 2(TNFAIP2) in the prognosis of clear cell renal cell carcinoma(ccRCC). Methods We analyzed the differential expression of TNFAIP2 mRNA in normal kidney tissue and ccRCC tissues by using TIMER2.0, TCGA, ONCOMINE, and UALCAN databases, and its correlation with clinical indicators and immune cell abundance. Kaplan-Meier curves were used to analyze patient prognosis. Western blotting was used to detect the expression of TNFAIP2 in normal human kidney cell line, HK-2, and human renal cancer cell lines, 786-O and ACHN. Subsequently, cell line results were verified by assessing the TNFAIP2 mRNA expression using RT-PCR in 24 pairs of tissue samples, obtained from matched cancerous and normal patients. Results The sequencing analysis using public databases showed that TNFAIP2 was abnormally highly expressed in ccRCC tissues(P<0.001). The high expression of TNFAIP2 was closely related to tumor stage, grade, lymph node metastasis, immune cell Th1, and Treg abundance(P<0.001, each). Kaplan-Meier curve analysis showed that the expression of TNFAIP2 was related to the prognosis of cancer patients. High expression levels of TNFAIP2 indicated progression of the disease and reduced patient survival(P<0.001). The receiver operating characteristic curve analysis demonstrated that the predictive efficiency of TNFAIP2 expression can be used to evaluate patient disease progression(AUC=0.814). The in vitro cell line experiments showed that the expression of TNFAIP2 in renal cell carcinoma 786-O and ACHN cells was higher than that in the HK-2 cells. The average expression level of TNFAIP2 mRNA of cancer was significantly higher than that in normal tissues, which further confirmed our cell line results. Conclusion The high expression levels of TNFAIP2 in ccRCC is related to the prognosis of patients. Thus, TNFAIP2 levels may serve as a biomarker for the diagnosis and prognosis of ccRCC, and could provide new directions and strategies for targeted therapy of renal cell carcinoma.
Keywords:
renal clear cell carcinoma Cancer Genome Atlas tumor necrosis factor-α-induced protein 2 prognosis
肾细胞癌(renal cell carcinoma，RCC)是泌尿生殖系统的常见恶性肿瘤，占肾脏恶性肿瘤的80%~90%，占全身恶性肿瘤的2%~3%。自1975年以来，RCC的发病率以每年2%~4%的速度增长[1]。RCC的组织学亚型包括透明细胞RCC(clear cell renal cell carcinoma，ccRCC)、乳头状RCC和嫌色RCC，其中ccRCC是肾癌的最常见亚型[2]，其进展和预后受多种复杂基因的控制[3]。外科手术切除肿瘤是治疗ccRCC的有效方法，尽管如此，仍有20%~38%的ccRCC患者术后发生进展和转移[4]。因此，对ccRCC发生发展过程中基因表达变化的研究至关重要，有助于识别肾癌新的诊断和预后生物标志物，提供新的治疗靶点[5]。近年来研究发现，肿瘤坏死因子α诱导蛋白(tumor necrosis factor-α-induced protein，TNFAIPs)家族参与了细胞凋亡和分化、信号传导等多种生物学过程[6]，而TNFAIP2作为该家族中的重要一员，可能在肿瘤的发生和发展过程中发挥重要作用[7]。
1 材料与方法 1.1 材料 1.1.1 细胞正常肾小管上皮细胞系HK-2和人RCC细胞系786-O、ACHN(上海素尔生物科技有限公司)用含10%胎牛血清(美国Invitrogen公司)、10 U/mL青霉素和100 U/mL链霉素(美国Invitrogen公司)的DMEM培养基(美国Invitrogen公司)，培养于37 ℃、5%CO2培养箱。
1.1.2 组织正常肾脏组织和ccRCC组织(24对)来自中国医科大学附属第四医院泌尿外科手术切除的标本。
1.2 方法 1.2.1 数据库分析应用TIMER2.0数据库(http://timer.comp-genomics.org/)分析TNFAIP2在泛癌中的表达情况及其与免疫细胞表达的相关性[8]；应用UALCAN数据集(http://ualcan.path.uab.edu/index.html)下载癌症基因组图谱(The Cancer Genome Atlas，TCGA)中ccRCC组织的TNFAIP2 mRNA表达数据库，对TNFAIP2 mRNA的表达与临床病理参数之间的相关性进行分析，应用京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)进行ccRCC差异基因的富集分析。应用Kaplan-Meier曲线(Kaplan-Meier Plotter，http://www.kmplot.com)数据库网站分析TNFAIP2表达与ccRCC数据库中患者预后的关系。
1.2.2 Western blotting用裂解缓冲液裂解细胞，裂解物上样(40 µg/泳道)行SDS-PAGE电泳，蛋白转移至聚偏二氟乙烯膜，5%脱脂牛奶中室温封闭1 h，加入1：2 000稀释抗TNFAIP2抗体(美国Santa Cruz Biotechnology公司)、抗GAPDH抗体(美国Santa Cruz Biotechnology公司)4℃孵育过夜。TBST洗膜3次，5 min/次，加入二抗孵育90 min，TBST洗膜3次。用增强化学发光可视化系统(BeyoECL Plus，中国上海Beyotime生物技术研究所)进行检测。
1.2.3 实时PCR检测用TRIzol试剂(美国Invitrogen公司)从组织中分离总RNA。按照The master mix reagent(美国Applied Biosystems公司)说明书行实时PCR，反应体系15 μL，包括TB Green 7.5 μL，ROX 0.3μL，引物各0.3μL，ddH2O 5.6 μL，cDNA 1 μL。反应条件：95℃30s，95℃5s，60℃34s，40个周期。TNFAIP2正向引物序列为5’-CCCCAATGACATCATCAACA-3’，反向引物序列为5’-GCCTCACTGGACAGGAATGT-3’[9]；GAPDH作内参照，正向引物序列为5’-GAAGGTGAAGGTCGGAGTC-3’，反向引物序列为5’-GAAGATGGTGATGGGATTTC-3’。用2-ΔΔCt法计算mRNA的相对表达量。
1.3 统计学分析采用SPSS 13.0和Graphpad Prism 7.0软件进行统计分析。用Pearson χ2检验分析TNFAIP2表达水平与ccRCC的临床病理参数之间的相关性。用Kaplan-Meier曲线分析TNFAIP2表达水平与ccRCC患者预后之间的相关性。用TNFAIP2表达的受试者操作特征(receiver operating characteristic，ROC)曲线评估TNFAIP2对ccRCC的诊断效能，并计算曲线下面积(area under curve，AUC)。P<0.05为差异有统计学意义。
2 结果 2.1 TNFAIP2在ccRCC中的表达TIMER2.0数据库分析结果显示，TNFAIP2在ccRCC组织中异常高表达(P<0.001)，见图 1。ONCOMINE数据库分析结果显示，ccRCC TNFAIP2表达转录水平与正常组织比较有统计学差异[10-11]。TCGA、UALCAN、TIMER2.0数据库分析结果显示，TNFAIP2在ccRCC中高表达，并与肿瘤的分期、分级、淋巴结转移、免疫细胞Th1和Treg丰度密切相关，差异有统计学意义(P<0.001)，见图 2~3。KEGG通路的富集分析结果显示，ccRCC参与包括TNF信号通路在内的TOP20通路，见图 4。
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| *P<0.05；**P<0.01；***P<0.001. 图 1 TNFAIP2在泛癌中的表达情况 Fig.1 TNFAIP2 expression in pan-cancer |
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| A, expression of TNFAIP2 in KIRC based on sample types; B, expression of TNFAIP2 in KIRC based on individual cancer stages; C, expression of TNFAIP2 in KIRC based on tumor grade; D, expression of TNFAIP2 in KIRC based on nodal metastasis status. ***P<0.001. 图 2 TNFAIP2在ccRCC中的表达情况及其与分期、分级、淋巴转移的关系 Fig.2 The expression of TNFAIP2 in ccRCC and its relationship with staging, grade, and lymphatic metastasis |
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| 图 3 肾癌中TNFAIP2的表达与免疫Th1细胞、Treg细胞的相关性 Fig.3 Correlation between the expression of TNFAIP2 and immune Th1 cells and Treg cells in renal cell carcinoma |
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| 图 4 ccRCC差异免疫基因KEGG富集途径分析 Fig.4 KEGG enrichment pathway analysis of differential immune genes in ccRCC |
2.2 TNFAIP2与ccRCC患者预后的关系
Kaplan-Meier曲线分析表明TNFAIP2表达水平与患者的预后显著相关，TNFAIP2 mRNA高表达提示ccCRCC患者病情恶化及总生存期缩短(P<0.001)；通过ROC曲线评估TNFAIP2 mRNA表达对ccRCC的诊断效能，结果显示，AUC=0.814，95%CI：0.796~ 0.887(P<0.001)，见图 5。
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| A, survival curve; B, ROC curve. 图 5 TNFAIP2与ccRCC患者预后的关系 Fig.5 Relationship between TNFAIP2 and prognosis of ccRCC patients |
2.3 TNFAIP2在肾癌细胞系和肾癌组织中的表达水平
Western blotting结果显示，与人肾脏正常细胞系HK-2比较，肾癌786-O、ACHN细胞中TNFAIP2蛋白表达水平均明显升高，见图 6。实时PCR检测结果显示，肾癌组织中TNFAIP2 mRNA的表达量明显高于对应的癌旁正常组织，差异有统计学意义(P<0.001)，见图 7。
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| 图 6 TNFAIP2在肾癌细胞系中的表达情况 Fig.6 The expression of TNFAIP2 in renal cancer cell lines |
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| 图 7 TNFAIP2 mRNA在肾癌组织和正常肾组织中的表达 Fig.7 The expression of TNFAIP2 mRNA in kidney cancer tissues and normal kidney tissues |
3 讨论
ccCRCC被认为是免疫浸润性肿瘤，免疫治疗具有革命性的前景[12]。但肿瘤微环境的异质性促进了治疗抵抗，从而导致ccCRCC预后不良[13]。TNFAIPs包括TNFAIP1~TNFAIP8，均为促凋亡蛋白[14]，其中TNFAIP2最初被认为是人类内皮细胞中的一种新的肿瘤坏死因子α诱导基因，可能通过诱导TNFAIP2 mRNA表达，在细胞凋亡中发挥作用[15]。王斌等[16]研究发现，下调TNFAIP2可通过激活Wnt/β-catenin信号通路，抑制食管鳞状细胞癌的增殖和转移。但是TNFAIP2在ccRCC中的作用却鲜为人知，因此，本研究基于公共数据库分析TNFAIP2在ccCRCC中的表达情况及其与临床病理特征的相关性，并通过体外实验进行验证。
本研究中，利用公共数据库TIMER2.0、TCGA、ONCOMINE和UALCAN进行生物信息分析，发现TNFAIP2在ccRCC组织中异常高表达，且与肿瘤的分期、分级、淋巴结转移相关(P<0.001)，而TNFAIP2高表达亦能提示病情恶化及总生存期缩短，与其他研究[17]结果一致。
对人肾脏正常细胞系HK-2、肾癌细胞系786-O、ACHN中TNFAIP2表达水平的进一步研究发现，肾癌细胞中TNFAIP2蛋白表达水平显著增高，TNFAIP2 mRNA在ccCRCC组织中的表达量显著高于癌旁正常肾脏组织。提示TNFAIP2可能参与了肾癌的发生发展过程。虽然化疗、免疫治疗及靶向治疗是转移性和进展性肾癌重要的治疗手段，而ccRCC对化疗却具有高度耐药性[18-19]。文献[20]报道，TNFAIP2高表达可诱导上皮-间质转化，并导致尿路上皮癌细胞对化疗药物产生耐药。本研究发现TNFAIP2表达与免疫细胞Th1和Treg丰度呈正相关，这为今后肾癌的免疫治疗和化疗提供了新的思路，未来有待进一步研究探讨。
综上所述，TNFAIP2在ccCRCC中的高表达与患者的预后有关，因此，TNFAIP2有可能成为ccRCC早期诊断和预后评价的生物标志物之一，可能为肾癌的靶向治疗提供新的方向和策略。
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