泛素特异性蛋白酶41在乳腺癌中高表达并促进恶性生物学行为

Ubiquitin specific protease 41 is upregulated in breast cancer and and its correlation with malignant biological behaviors

目的探讨泛素特异性蛋白酶41(USP41)作为乳腺癌新型诊断/预后标志物的潜在价值,并观察USP41对乳腺癌细胞恶性生物学行为的影响。方法分别从癌症基因组图谱(TCGA)数据库、基因表达综合(GEO)数据库的GSE96058数据集中获取1086例乳腺癌样本、3273例乳腺癌样本的转录组数据及临床信息,利用R4.1.2分别进行基因表达分析、预后分析、功能富集分析及免疫浸润分析。分别用两种特异性小干扰RNA(siRNA)转染乳腺癌细胞系MCF-7,蛋白质印迹法(Westernblot)实验检测转染前后USP41蛋白表达水平,集落形成实验、细胞计数试剂盒(CCK-8)实验、划痕实验及Transwell实验检测转染后乳腺癌细胞增殖、侵袭与迁移能力并与对照组作比较。Cox回归用于比较预后差异,相对灰度值的比较由单因素方差分析和TukeyHSD事后检验完成,细胞集落数的比较由Welch方差分析和Games-Howell事后检验完成,同一时间下光密度值和相对宽度的比较由两因素重复测量数据方差分析和多重成对比较完成,细胞迁移数的比较由Kruskal-Wallis检验和Dunn事后检验完成,单因素和多因素Cox回归用于确定预后独立危险因素,采用Spearman分析进行相关性分析。结果USP41在10种癌症组织中高表达,作为乳腺癌诊断标志物的诊断效能一般(曲线下面积为0.717)。USP41高表达与乳腺癌患者更差的总生存期[P<0.01,风险比(HR)=1.93,95%可信区间(CI):1.40~2.66]和无病生存期相关(P<0.01,HR=2.10,95%CI:1.37~3.23)。Cox回归分析表明USP41是乳腺癌的独立危险因素(P<0.01,HR=1.406,95%CI:1.13~1.75)。基因集富集分析表明USP41高表达与多巴胺能的神经形成、多巴胺神经递质释放循环及胰岛素摄取等功能活动相关。免疫浸润分析表明USP41高表达与更丰富的免疫细胞浸润和多种免疫检查点表达呈明显正相关。siRNA1组和siRNA2组USP41蛋白表达水平(3.88±0.27、4.13±0.12)均显著低于对照组(6.03±0.40),差异有统计学意义(F=194.405,P<0.01)。siRNA1组和siRNA2组细胞集落数[(20.75±6.13)、(25.50±8.89)个]均显著低于对照组[(76.75±17.23)个],差异有统计学意义(F=16.720,P<0.01)。siRNA1组和siRNA2组24h吸光度值(0.43±0.01、0.44±0.01)均显著低于对照组(0.51±0.04),差异有统计学意义(F=11.933,P<0.05)。siRNA1组和siRNA2组24h侵袭宽度(2.53±0.12、2.67±0.12)均显著高于对照组(1.70±0.10),差异有统计学意义(F=67.364,P<0.01)。siRNA1组24h迁移细胞数[(190.50±19.09)个]显著低于对照组[(351.25±15.88)个],差异有统计学意义(F=-2.755,P<0.05)。结论USP41高表达与乳腺癌不良预后相关并促进细胞恶性生物学行为。

Objective To explore the potential value of ubiquitin specific protease 41(USP41)as novel diagnostic/prognostic biomarker for breast cancer and determine the correlation between USP41 and malignant biological behaviors.Methods Transcriptome data and clinical information of 1086 breast canc-er samples from The Cancer Genome Atlas(TCGA)database and 3273 breast cancer samples from the GSE96058 dataset in the Gene Expression Omnibus(GEO)database were obtained.Gene expression analysis,prognosis analysis,functional enrichment analysis,and immune infiltration analysis were conducted using R 4.1.2.Two specific small interfering RNAs(siRNAs)were transfected into the breast cancer cell line MCF-7,and Western bltting was used to detect the protein expression levels of USP41 before and afer transfection.Colony formation assay,cell counting kit-8(CCK-8)assay,wound healing assay,and Transwell assay were used to assess the proliferation,invasion,and migration abilities of breast cancer cells after transfection.Cox regression was used to compare prognostic differences.The comparison of relative grayscale values was performed by one-way analysis of variance and Tukey HSD post hoc test.The comparison of cell colony number was performed by Welch's analysis of variance and Games-Howell post hoc test.The comparison of absorbance values and relative widths at the same time was performed by two-factor repeated measures data analysis of variance and multiple pair comparisons.The comparison of cell migration number was performed by Kruskal-Wallis test and Dunn's post hoc test.Single-factor and multi-factor Cox regression was used to determine independent prognostic risk factors.Correlation analysis was performed using Spearman analysis.Results USP41 was highly expressed in 10 types of cancer tissues,and its diagnostic efficacy as a diagnostic marker for breast cancer was moderate(area under the curve=0.717).High expression of USP41 was associated with poorer overall survival in breast cancer patients[P<0.01,hazard ratio(HR)=1.93,95%confidence interval(CI):1.40-2.66]and disease-free survival[P<0.01,HR=2.10,95%Cl:1.37-3.23].Cox regression analysis indicated that USP41 was an independent risk factor for breast cancer(P<0.01,HR=1.406,95%ClI:1.13-1.75).Gene set enrichment analysis revealed that high expression of USP41 was associated with functional activities related to dopaminergic neurogenesis,dopamine neurotransmitter release cycle,and insulin uptake.Immune infiltration analysis showed that high expression of USP41 was significantly positively correlated with increased immune cell infiltration and expression of multiple immune checkpoints.The protein expression levels of USP41 in siRNA1 and siRNA2 groups(3.88±0.27,4.13±0.12)were significantly lower than those in the control group(6.03±0.40,F=194.405,P<0.01).The number of colonies in siRNA1 and siRNA2 groups[(20.75±6.13),(25.50±8.89)]was significantly less than that in the control group(76.75±17.23)(F=16.720,P<0.01).The absorbance values at 24 h for siRNA1 and siRNA2 groups(0.43±0.01,0.44±0.01)were significantly lower than those for the control group(0.51±0.04)(F=11.933,P<0.05).The invasion widths at 24 h for siRNA1 and siRNA2 groups(2.53±0.12,2.67±0.12)were significantly greater than those for the control group(1.70±0.10)(F=67.364,P<0.01).The number of migrating cells at 24 h for siRNA1 group(190.50±19.09)was significantly less than that for the control group(351.25±15.88,F=-2.755,P<0.05).Conclusion High expression of USP41 may be correlated with poor prognosis and enhance malignant biological behaviors of breast cancer.