摘要
目的:结直肠癌(colorectal cancer,CRC)是全球癌症相关死亡的第2大原因,其高转移率和治疗耐药性仍是临床面临的重大挑战。近年来,靶向调控肿瘤关键分子的治疗策略成为研究热点,其中泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)在肿瘤发生、发展中的作用日益受到关注。本研究旨在探索泛素羧基末端水解酶-1(ubiquitin carboxyl-terminal hydrolase L1,UCHL1)在CRC进展中的作用。方法:收集河南中医药大学第一附属医院和淮安市第一人民医院2016年6月至2018年6月收治的74例未接受化学治疗或放射治疗的CRC患者的肿瘤组织标本。通过免疫组织化学检测UCHL1蛋白表达水平并记录其阳性分类结果。采用CRC细胞系HCT 116和LoVo进行UCHL1基因功能丧失实验,将细胞分为转染UCHL1特异性小干扰RNA的Si-UCHL1组和转染阴性对照的Normal组。通过细胞克隆形成实验和噻唑蓝(thiazolyl blue,MTT)比色法评估细胞增殖能力;通过Transwell实验检测穿膜细胞数以评估细胞迁移和侵袭能力;通过蛋白质印迹法(Western blotting,WB)检测凋亡相关蛋白[B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、胱天蛋白酶(caspase)-3、caspase-9、细胞色素c(cytochrome c,cyt c)]和自噬相关蛋白[哺乳动物雷帕霉素靶蛋白复合物2(mechanistic target of rapamycin complex2,mTORC2)、磷酸化蛋白激酶B(phosphorylatedproteinkinaseB,pAKT)、叉头框蛋白O3(forkheadboxO3,FOXO3)、微管相关蛋白轻链3B-II(light chain 3B-II,LC3BII)]的表达变化。结果:免疫组织化学结果表明:CRC组织和转移性CRC组织的UCHL1蛋白表达水平显著升高,阳性率分别为89.10%(66/74)、90.00%(18/20),且UCHL1蛋白表达水平与CRC的肿瘤分期和患者不良临床结果相关。此外,癌巢周围的UCHL1蛋白水平显著高于肿瘤中心。细胞克隆形成实验结果显示:UCHL1沉默显著抑制CRC细胞的增殖能力(P<0.05)。MTT法检测结果显示:UCHL1沉默显著抑制CRC细胞的细胞活性(P<0.05)。Transwell检测结果表明:与Normal组相比,Si-UCHL1组HCT 116细胞迁移和侵袭细胞数分别由530.23±7.07和140.15±5.66下降至275.30±5.42和40.21±3.54,LoVo细胞迁移和侵袭细胞数分别由560.11±8.48和280.10±6.20下降至189.12±6.33和36.25±3.46。WB结果显示:与Normal组相比,Si-UCHL1组LoVo细胞的mTORC2、p-AKT及Bcl-2蛋白表达水平均显著下降(均P<0.05),而FoxO3、LC3BII、Bax、caspase-3、cyt c和caspase-9蛋白表达水平均显著上升(均P<0.05),并表现出显著的凋亡特征。结论:UCHL1通过抑制细胞凋亡和自噬促进CRC进展,其可作为CRC诊断和预后的分子标志物。靶向UCHL1治疗有望成为一种治疗CRC的新策略。
Objective:Colorectal cancer(CRC)is the second leading cause of cancer-related deaths worldwide,with high metastatic potential and therapeutic resistance remaining major clinical challenges.In recent years,targeted modulation of key tumor molecules has emerged as a research focus,particularly the role of the ubiquitin-proteasome system(UPS)in tumorigenesis and progression.This study aims to investigate the mechanistic role of ubiquitin carboxyl-terminal hydrolase L1(UCHL1)in CRC progression.Methods:Tumor tissue specimens were collected from 74 CRC patients(June 2016 to June 2018)at the First Affiliated Hospital of Henan University of Chinese Medicine and Huai’an First People’s Hospital,all untreated with radiotherapy or chemotherapy.The expression level of UCHL1 protein was analyzed by immunohistochemistry(IHC),and its positive classification results were records.Loss-of-function experiments were conducted in CRC cell lines(HCT 116 and LoVo),divided into a si-UCHL1(transfected with UCHL1-specific siRNA)group and a normal(negative control)group.Cell proliferation was evaluated using colony formation assays and thiazolyl blue tetrazolium bromide(MTT)assays,while cell migration and invasion capacities were assessed by quantifying transmembrane cells in Transwell assays.Expression changes of apoptosis-related proteins[B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3,caspase-9,cytochrome c(cyt c)]and autophagy-related proteins[mechanistic target of rapamycin complex 2(mTORC2),phosphorylated protein kinase B(pAKT),forkhead box O3(FOXO3),microtubule-associated protein light chain 3B-II(LC3BII)]were analyzed by Western blotting.Results:Immunohistochemical(IHC)analysis revealed significantly elevated UCHL1 protein expression in both primary CRC tissues(89.10%,66/74)and metastatic CRC lesions(90.00%,18/20).Notably,high UCHL1 expression levels showed significant correlation with advanced tumor stage and poorer clinical outcomes in CRC patients.The colony formation assay demonstrated that UCHL1 knockdown significantly inhibited the proliferative capacity of CRC cells(P<0.05).MTT assay results demonstrated that UCHL1 knockdown significantly inhibited cellular viability in CRC cells(P<0.05).Transwell assay results demonstrated that compared to the normal group,the Si-UCHL1 group exhibited significant reductions in migratory and invasive cell numbers.Specifically,in HCT 116 cells,migration decreased from 530.23±7.07 to 275.30±5.42;invasion declined from 140.15±5.66 to 40.21±3.54;in LoVo cells,migration decreased from 560.11±8.48 to 189.12±6.33;invasion dropped from 280.10±6.20 to 36.25±3.46.Western blotting analysis revealed that compared with the Normal group,the Si-UCHL 1 group showed significantly decreased protein expression levels of mTORC 2,p-AKT,and Bcl-2(all P<0.05),while demonstrating markedly increased expression of FoxO 3,LC 3 B-II,Bax,cleaved caspase-3,cytochrome c,and cleaved caspase-9(all P<0.05),accompanied by distinct apoptotic features.Conclusion:UCHL 1 promotes CRC progression by inhibiting apoptosis and autophagy,and it may serve as a molecular marker for CRC diagnosis and prognosis.Targeting UCHL 1 could be a novel therapeutic strategy for CRC treatment.
作者
杨凯丽
周武碧
牛德亮
郭地
成树贞
刘磊
周艳阳
任伟宏
YANG Kaili;ZHOU Wubi;NIU Deliang;GUO Di;CHENG Shuzhen;LIU Lei;ZHOU Yanyang;REN Weihong(Clinical Laboratory,First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000;Department of Pathology,Huai’an First People’s Hospital,Huai’an 223300;Department of Pathology,First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000;Department of Proctology,First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China)
出处
《临床与病理杂志》
2025年第4期405-415,共11页
Journal of Clinical and Pathological Research
基金
河南省高等学校重点科研项目计划基础研究专项(19zx009)。
