免疫相关基因CEBPB在肾透明细胞癌中的表达及其临床价值

Prognosis value of immune-related gene CEBPB in clear cell renal cell carcinoma

目的探讨CCAAT增强子结合蛋白(CEBPB)在肾透明细胞癌(ccRCC)中的表达情况及其与ccRCC临床特征的关系,并检测CEBPB基因对ccRCC细胞增殖和侵袭活性的影响。方法本研究于2020年3—12月完成。从癌症基因组图谱(TCGA)数据库中下载537例ccRCC患者的转录组数据和临床数据。男346例,女191例;≤60岁266例,>60岁271例;G_(1-2)级244例,G_(3-4)级285例,未确定分级8例;T_(1-2)期344例,T_(3-4)期193例;N0期240例,N1期17例,无法评估淋巴结转移情况280例;M0期426例,M1期79例,无法评估远处转移情况32例;生存367例,死亡170例。分析CEBPB mRNA在ccRCC组织中的表达及其与临床特征的相关性;单因素和多因素Cox回归分析CEBPB表达水平对ccRCC患者生存预后的影响;肿瘤免疫浸润评分(TIMER)数据库分析CEBPB表达与免疫细胞浸润的相关性。采用实时荧光定量PCR和蛋白质印迹法分别检测CEBPB mRNA和蛋白在人肾小管上皮细胞株HK2和ccRCC细胞株(Caki-1、ACHN、786O、769P和A498)中的表达;利用脂质体转染ACHN细胞和786O细胞,实验分为对照组、阴性对照siRNA(NC siRNA)组和CEBPB siRNA组。转染后48 h,分别通过MTT实验和侵袭实验检测各组ACHN细胞和786O细胞的增殖和侵袭活性。结果TCGA数据库资料分析结果显示,与正常肾脏组织比较,ccRCC组织中的CEBPB mRNA表达水平上调2.55倍(P<0.05)。CEBPB表达水平与ccRCC患者年龄、肿瘤分级、肿瘤分期、淋巴结转移及远处转移呈正相关(P<0.05),且肿瘤分级(HR=1.703,P=0.040)、肿瘤分期(HR=1.773,P=0.026)、远处转移(HR=3.080,P<0.001)和CEBPB高表达(HR=1.874,P=0.003)是影响ccRCC患者预后的独立危险因素。TIMER数据库分析结果表明,CEBPB表达水平与B细胞(Rho=0.168)、M2型巨噬细胞(Rho=0.373)、调节性T细胞(Rho=0.348)、中性粒细胞(Rho=0.194)和自然杀伤T细胞(Rho=0.421)的浸润程度呈正相关。Caki-1、ACHN、786O、769P、A498细胞中CEBPB mRNA表达水平分别是HK2细胞中的(9.43±1.25)、(5.44±0.82)、(4.50±0.52)、(4.88±0.73)、(7.50±1.04)倍,CEBPB蛋白表达水平分别是HK2细胞中的(6.22±0.45)、(5.84±0.85)、(6.51±0.55)、(6.23±0.62)、(3.84±0.45)倍,差异均有统计学意义(P<0.05)。MTT实验结果显示,NC siRNA组中ACHN细胞在培养24、48、72、96 h后细胞增殖率分别为(98.4±1.7)%、(97.8±2.1)%、(101.3±1.2)%、(97.5±2.0)%,786O细胞增殖率分别为(99.0±1.4)%、(98.5±1.5)%、(97.6±1.7)%、(99.1±1.3)%;CEBPB siRNA组中,ACHN细胞在培养24、48、72、96 h后细胞增殖率分别为(68.8±5.8)%、(57.9±6.1)%、(50.9±4.6)%、(43.2±5.0)%,786O细胞增殖率分别为(79.5±6.2)%、(70.8±5.1)%、(66.8±4.9)%、(60.5±5.3)%。与NC siRNA组比较,CEBPB siRNA组ACHN、786O细胞的增殖活性明显受到抑制(P<0.05)。细胞侵袭实验结果显示,NC siRNA组ACHN、786O细胞的侵袭活性分别为对照组的(95.0±5.2)%、(97.3±4.4)%,CEBPB siRNA组ACHN、786O细胞的侵袭活性分别为对照组的(35.2±5.4)%、(26.7±3.3)%。与NC siRNA组比较,CEBPB siRNA组ACHN、786O细胞的侵袭活性明显受到抑制(P<0.05)。结论CEBPB在ccRCC中高表达,且CEBPB表达水平与ccRCC患者生存预后及免疫浸润密切相关,沉默CEBPB表达显著抑制ccRCC细胞的增殖和侵袭。

Objective To explore the correlation between CCAAT enhancer binding protein beta(CEBPB)expression and clinical characteristics in ccRCC,and to investigate the effect of CEBPB on proliferation and invasion of ccRCC cells.Methods Between March 2020 to December 2020,the transcriptome and clinical data of 537 ccRCC cases were downloaded from TCGA database,and the correlation of CEBPB expression with clinical characteristics of ccRCC were analyzed.Univariate and multivariate Cox regression analysis were used to determine the effect of CEBPB expression on the prognosis of ccRCC patients.The correlation between CEBPB expression and immunocyte infiltration in ccRCC was investigated via TIMER database.The expression levels of CEBPB mRNA and protein in human renal tubular epithelial cell line HK2 and ccRCC cell lines(Caki-1,ACHN,786O,769P and A498)were determined by real-time PCR and western blot,respectively.After transfected with NC siRNA or CEBPB siRNA for 48 h,the proliferation and invasion of ACHN cells and 786O cells were determined by using MTT assay and invasion assay,respectively.Results TCGA databases analysis revealed that,compared with normal kidney tissue,the expression of CEBPB mRNA in ccRCC was up-regulated by 2.55-fold(P<0.05).CEBPB expression was positively correlated with age,tumor grade,tumor stage,lymph node metastasis and distant metastasis(P<0.05).The tumor grade(HR=1.703,P=0.040),tumor stage(HR=1.773,P=0.026),distant metastasis(HR=3.080,P<0.001)and the high expression of CEBPB(HR=1.874,P=0.003)were independent poor prognostic factors for ccRCC patients.The analysis results by using TIMER database showed that CEBPB expression was positively correlated with infiltrating levels of B cells(Rho=0.168),M2 macrophages(Rho=0.373),Tregs(Rho=0.348),neutrophils(Rho=0.194),and natural killer T cell(Rho=0.421)in ccRCC.The expression level of CEBPB mRNA in Caki-1,ACHN,786O,769P and A498 cells was(9.43±1.25)-fold,(5.44±0.82)-fold,(4.50±0.52)-fold,(4.88±0.73)-fold and(7.50±1.04)-fold of HK2 cells,respectively.The expression level of CEBPB protein was(6.22±0.45)-fold,(5.84±0.85)-fold,(6.51±0.55)-fold,(6.23±0.62)-fold and(3.84±0.45)-fold of HK2 cells,respectively(P<0.05).MTT assay showed that the proliferation rates of ACHN cells and 786O cells at 24,48,72,96 h were(98.4±1.7)%and(99.0±1.4)%,(97.8±2.1)%and(98.5±1.5)%,(101.3±1.2)%and(97.6±1.7)%,(97.5±2.0)%and(99.1±1.3)%in NC siRNA group,and(68.8±5.8)%and(79.5±6.2)%,(57.9±6.1)%and(70.8±5.1)%,(50.9±4.6)%and(66.8±4.9)%,(43.2±5.0)%and(60.5±5.3)%in CEBPB siRNA group.Compared with NC siRNA group,the proliferation activity of ACHN cells and 786O cells was significantly inhibited in the CEBPB siRNA group(P<0.05).Cell invasion assay showed that the invasion activity of ACHN cells and 786O cells were(95.0±5.2)%and(97.3±4.4)%in NC siRNA group,(35.2±5.4)%and(26.7±3.3)%in CEBPB siRNA group,respectively(P<0.05).Compared with NC siRNA group,the invasion activity of ACHN cells and 786O cells were significantly inhibited in the CEBPB siRNA group(P<0.05).Conclusions CEBPB was highly expressed in ccRCC,which was closely related to the prognosis and immunocyte infiltration of ccRCC patients.Silencing the expression of CEBPB significantly inhibited the proliferation and invasion of ccRCC cells.