基于生物信息学数据库分析肾透明细胞癌SKA1表达及临床意义

doi:10.3760/cma.j.cn371439-20200113-00085

摘要/Abstract

摘要：

目的探讨纺锤体和动粒相关蛋白复合体亚基1(SKA1)在肾透明细胞癌中的表达及临床意义。方法收集2018年1月至2018年12月在新疆医科大学附属肿瘤医院就诊的76例肾透明细胞癌患者术前和24例健康体检者的静脉血液标本,利用实时荧光定量PCR检测患者全血中SKA1的水平,分析SKA1的水平与临床病理特征的关系。挖掘Oncomine(v4.5)、The Human Protein Atlas(THPA)基因数据库、The Cancer Genome Atlas(TCGA)门户网站(cBioportal)和Gene Expression Omnibus(GEO)中关于SKA1的信息,基于cBioportal数据库中的肾透明细胞癌数据采用Kaplan-Meier法进行生存分析,log-rank检验进行生存率的比较,χ²检验分析SKA1水平与临床病理特征的关系。采用ROC曲线评价SKA1 mRNA在肾透明细胞癌中的诊断价值,并采用KOBAS 3.0版在线工具对SKA1基因进行富集分析。结果 Oncomine(v4.5)数据库中检索到2项关于SKA1 mRNA在人肾透明细胞癌组织中表达水平的研究,共38个样本,分析结果显示人SKA1 mRNA在肾透明细胞癌组织中呈高表达,进一步检测发现,与正常肾组织相比,SKA1 mRNA在肾透明细胞癌组织中的表达水平显著升高[-2.21(-3.56,-1.59) vs. -3.41(-4.55,-1.65)],差异具有统计学意义(Z=2.282,P=0.022)。利用THPA在线网站分析表明,SKA1蛋白在肾癌组织中呈明显中等强度染色,而在正常肾组织中极少部分弱阳性表达或不表达,SKA1主要定位于细胞质膜,这一检测结果与mRNA分析结果一致。cBioportal数据库分析结果表明,SKA1表达水平与AJCC分期(χ²=21.352,P<0.001)、T分期(χ²=19.967,P<0.001)、N分期(χ²=11.323,P=0.003)及M分期(χ²=27.248,P<0.001)显著相关。76例肾透明细胞癌患者外周血SKA1的水平为0.301±0.147,健康体检者为0.162 ± 0.052,差异具有统计学意义(t=7.360,P<0.001),其水平与AJCC分期(t=2.445,P=0.017)和淋巴结转移(t=2.242,P=0.028)相关,该结果与cBioportal数据库中组织分析结果相符。生存分析显示,cBioportal数据库中,肾透明细胞癌患者SKA1 mRNA的表达水平与总生存率和无瘤生存率相关(χ²=22.440,P<0.001;χ²=23.830,P<0.001)。GEO数据库中,肾透明细胞癌患者的SKA1 mRNA的表达水平与总生存率无关(χ²=0.241,P=0.632)。cBioportal数据库中ROC分析结果显示,选取诊断界值为-0.944时,SKA1 mRNA诊断肾透明细胞癌的敏感性为100%,特异性为98.7%,ROC曲线下面积(AUC)为0.991,95%CI为0.972~1.000。对76例肾透明细胞癌患者进行ROC分析结果显示,当诊断界值为0.235时,外周血中SKA1水平诊断肾透明细胞癌的敏感性为75.0%,特异性为95.8%,AUC为0.837,95%CI为0.761~0.914。KOBAS富集分析结果显示,SKA1高表达样本主要富集在染色体着丝粒、微管聚合与解聚调节、有丝分裂纺锤体检查点等基因集。结论 SKA1高表达于肾透明细胞癌组织中,并与患者预后显著相关,可作为肾透明细胞癌的诊断指标及潜在的治疗靶点。

关键词: 肾肿瘤, 预后, 计算生物学, SKA1

Abstract:

Objective To investigate the expression of spindle and kinetochore-associated complex subunit 1 (SKA1) gene in clear cell renal cell carcinoma (ccRCC) and its clinical significance. Methods The venous blood samples of 76 preoperative patients with ccRCC and 24 healthy subjects were collected from the Affiliated Tumor Hospital of Xinjiang Medical University from January 2018 to December 2018. The level of SKA1 in whole blood was detected by real-time fluorescence quantitative PCR, and the relationship between SKA1 level and clinicopathological characteristics was analyzed. SKA1 data were retrieved from Oncomine (v4.5), The Human Protein Atlas (THPA) gene databases, The Cancer Genome Atlas (TCGA) databasec (cBioportal) and Gene Expression Omnibus (GEO). The Kaplan-Meier method was used to perform patients' survival analysis based on cBioportal ccRCC data, and the survival rates were compared by log-rank method. The relationship between SKA1 expression level and clinicopathological characteristics was analyzed by χ² test. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of SKA1 mRNA in ccRCC, and enrichment analysis of SKA1 gene was carried out using KOBAS 3.0 online tool. Results Two studies on the expression level of SKA1 mRNA in ccRCC were retrieved from Oncomine (v4.5) database, and there were 38 samples. The results showed that SKA1 mRNA was highly expressed in ccRCC tissues. Further detection showed that the expression level of SKA1 mRNA in ccRCC tissues was significantly higher than that in normal renal tissues [-2.21(-3.56,-1.59) vs. -3.41(-4.55,-1.65)], and there was a statistically significant difference (Z=2.282, P=0.022). The analysis of THPA online website showed that SKA1 protein showed obvious moderate staining in ccRCC tissues, while weakly positive or no expression in normal renal tissues. SKA1 was mainly located in the plasma membrane, which was consistent with the results of mRNA analysis. The results of cBioportal showed that the expression level of SKA1 was significantly correlated with AJCC staging (χ²=21.352, P<0.001), T staging (χ²=19.967, P<0.001), N staging (χ²=11.323, P=0.003) and M staging (χ²=27.248, P<0.001). The relative level of SKA1 in peripheral blood of 76 patients with ccRCC was 0.301±0.147, and 0.162±0.052 in healthy subjects, with a statistically significant difference (t=7.360, P<0.001). The level of SKA1 was correlated with AJCC staging (t=2.445, P=0.017) and lymph node metastasis (t=2.242, P=0.028). The results were consistent with tissue analysis in cBioportal database. Survival analysis showed that in cBioportal database, the expression level of SKA1 mRNA was related to the overall survival rate and disease free survival rate of patients with ccRCC (χ²=22.440, P<0.001; χ²=23.830, P<0.001). In GEO database, the expression level of SKA1 mRNA was not related to the overall survival rate of patients with ccRCC (χ²=0.241, P=0.632). The results of ROC analysis in cBioportal database showed that when the cut-off value was -0.944, the sensitivity and specificity of SKA1 mRNA in the diagnosis of ccRCC were 100% and 98.7%. The area under the ROC curve (AUC) was 0.991 (95%CI: 0.972-1.000). The results of ROC analysis of 76 patients with ccRCC showed that when the cut-off value was 0.235, the sensitivity and specificity of peripheral blood SKA1 in the diagnosis of ccRCC were 75.0% and 95.8%, and the AUC was 0.837 (95%CI: 0.761-0.914). KOBAS enrichment analysis showed that SKA1 high expression samples were enriched in gene sets such as chromosomal centromeres, microtubule polymerization and depolymerization regulation and mitotic spindle check-up points. Conclusion SKA1 is highly expressed in ccRCC tissues, which is obviously related to the prognosis of patients. It can be used as a diagnostic indicator and potential therapeutic target for ccRCC.

Key words: Kidney neoplasms, Prognosis, Computational biology, SKA1

韩静, 袁帅, 蒲艳, 柳惠斌. 基于生物信息学数据库分析肾透明细胞癌SKA1表达及临床意义[J]. 国际肿瘤学杂志, 2020, 47(10): 598-605.

Han Jing, Yuan Shuai, Pu Yan, Liu Huibin. Analysis of SKA1 gene expression in clear cell renal cell carcinoma and its clinical significance based on bioinformatics database[J]. Journal of International Oncology, 2020, 47(10): 598-605.

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临床病理特征	例数	SKA1基因表达		χ²值	P值
临床病理特征	例数	高表达 (n=216)	低表达 (n=216)	χ²值	P值
性别
男	286	152(70.4)	134(62.0)	3.352	0.067
女	146	64(29.6)	82(38.0)
年龄(岁)
<59	205	108(50.0)	97(44.9)	1.123	0.289
≥59	227	108(50.0)	119(55.1)
Fuhrman分级
G1+G2	211	107(49.5)	104(48.1)
G3+G4	217	109(50.5)	108(50.0)	3.758	0.184
Gx	4	0(0.0)	4(1.9)
AJCC分期
Ⅰ~Ⅱ	278	116(53.7)	162(75.0)	21.352	<0.001
Ⅲ~Ⅳ	154	100(46.3)	54(25.0)
T分期
T₁	231	96(44.4)	135(62.5)
T₂	60	28(13.0)	32(14.8)	19.967	<0.001
T₃	138	90(41.7)	48(22.2)
T₄	3	2(0.9)	1(0.5)
N分期
N₀	193	107(49.5)	86(39.8)
N₁	12	10(4.6)	2(1.0)	11.323	0.003
N_x	227	99(45.9)	128(59.2)
M分期
M₀	353	174(80.6)	179(82.9)
M₁	53	39(18.1)	14(6.5)	27.248	<0.001
M_x	26	3(1.3)	23(10.6)

临床病理特征	例数	SKA1相对表达量	t值	P值
性别
男	62	0.309±0.145	0.129	0.898
女	14	0.266±0.154
年龄(岁)
>60	26	0.306±0.145	0.311	0.757
≤60	50	0.296±0.150
肿瘤直径(cm)
≥5.9	23	0.310±0.148	0.598	0.552
<5.9	53	0.293±0.147
AJCC分期
Ⅰ~Ⅱ	52	0.276±0.151	2.445	0.017
Ⅲ~Ⅳ	24	0.354±0.124
Fuhrman分级
G1+G2	50	0.288±0.137	1.159	0.250
G3+G4	26	0.329±0.164
淋巴结转移
有	11	0.286±0.141	2.242	0.028
无	65	0.391±0.151
远端转移
有	70	0.295±0.153	0.988	0.326
无	6	0.358±0.060

数据库	ID	注释	P值
Gene Ontology	GO:0000779	浓缩染色体,着丝粒区域	<0.001
	GO:0031110	微管聚合或解聚的调控	0.001
	GO:0099080	多分子复合物	0.003
	GO:0005856	细胞骨架	0.006
	GO:0031109	微管聚合或解聚	0.006
	GO:0032886	微管生物学过程的调控	0.007
	GO:0043228	非质膜结合细胞器	0.011
	GO:0070507	微管细胞骨架组织的调控	0.011
	GO:0033043	细胞器组织的调控	0.019
	GO:0007010	细胞骨架组织	0.024
	GO:0022402	细胞周期过程	0.035
Reactome	R-HSA-141444	通过MAD2抑制因子增强非着丝粒信号通路	0.002
	R-HSA-141424	着丝粒信号增强	0.002
	R-HSA-69618	有丝分裂纺锤体检查点	0.002
	R-HSA-2500257	姐妹染色单体凝聚力的分解	0.002
	R-HSA-5663220	激活肌动蛋白	0.002
	R-HSA-2467813	姐妹染色单体的分离	0.003
	R-HSA-68882	有丝分裂后期	0.003
	R-HSA-2555396	有丝分裂中期和后期	0.003
	R-HSA-68877	有丝分裂前中期	0.003
	R-HSA-69620	细胞周期检查点	0.005
	R-HSA-195258	效应器	0.006
	R-HSA-68886	M相	0.007
	R-HSA-194315	Rho GTPase 介导的信号通路	0.008
	R-HSA-69278	细胞周期,有丝分裂	0.009
	R-HSA-1640170	细胞周期	0.011