O-GlcNAc转移酶介导的Kruppel样因子5 O-GlcNAc糖基化修饰调控细胞周期素D1参与结直肠癌发生、发展实验研究  

O-GlcNAc transferase-mediated Kruppel-like factor 5 O-GlcNAc glycosylation participates in occurrence and development of colorectal cancer by regulating cyclin D1

作　　者：彭明沙 黎爽 周翼 黄世贵 舒子龙 彭洪[1] PENG Mingsha;LI Shuang;ZHOU Yi;HUANG Shigui;SHU Zilong;PENG Hong(Nanchong Central Hospital Affiliated to North Sichuan Medical College,Nanchong 637000,China)

机构地区：[1]川北医学院附属南充市中心医院,四川南充637000

出　　处：《陕西医学杂志》2023年第12期1648-1653,1659,共7页Shaanxi Medical Journal

基　　金：四川省中医药管理局科研专项课题(2020JC0078);四川省南充市市校科技战略合作专项资金资助项目(22SXQT0069,22SXQT0070);川北医学院校级科研发展计划项目(CBY22-QNA03,CBY22-QNA10)。

摘　　要：目的:探讨O-GlcNAc转移酶(OGT)通过O-GlcNAc糖基化修饰稳定Kruppel样因子5(KLF5)蛋白并上调细胞周期素D1(CCDN1)表达参与结直肠癌(CRC)发生、发展的分子机制。方法:采用生物信息学方法分析KLF5 mRNA和蛋白在CRC组织中的表达。采用实时荧光定量PCR(RT-qPCR)检测CRC细胞中KLF5的表达水平,CCK-8法检测细胞增殖,流式细胞仪检测细胞周期。通过生物信息学方法分析KLF5的靶基因和潜在结合位点,并通过双荧光素酶实验证实KLF5蛋白可与CCDN1结合,采用RT-qPCR检测KLF5对CCDN1 mRNA的影响。抑制KLF5并过表达CCDN1,分析KLF5是否通过正调控CCDN1促进细胞增殖和促进细胞进入G 1期。采用生物信息学分析KLF5蛋白是否存在O-GlcNAc糖基化位点,OGT mRNA和蛋白在结直肠癌组织中的表达以及OGT和KLF5表达的相关性。使用OGT抑制剂OSMI-1处理CRC细胞,蛋白质免疫印迹法(Western blot)分析KLF5蛋白表达,O-GlcNAc糖基化蛋白表达水平通过免疫荧光实验检测。并用Western blot法检测OSMI-1和放线菌酮(CHX)处理后KLF5蛋白的稳定性。最后抑制OGT并过表达KLF5,分析OGT是否通过正调控KLF5促进CCDN1表达。结果:KLF5 mRNA和蛋白在CRC中高表达。KLF5在CRC细胞中高表达并可促进细胞增殖和促进细胞进入G 1期。CCND1是KLF5的靶基因并可与CCND1靶向结合。KLF5通过正调控CCND1促进细胞增殖和促进细胞由G 1期向S期转化。生物信息学分析证实KLF5蛋白存在O-GlcNAc糖基化位点。OGT mRNA和蛋白在CRC中高表达,并且OGT和KLF5表达水平呈正相关。抑制OGT表达可降低O-GlcNAc糖基化蛋白和KLF5蛋白水平,并可降低KLF5蛋白的稳定性。OGT通过正调控KLF5上调CCND1表达。结论:KLF5通过正调控CCND1促进CRC细胞增殖,并促进细胞进入G 1期。OGT通过O-GlcNAc糖基化修饰稳定KLF5并上调CCND1表达。Objective:To investigate the molecular mechanism of O-GlcNAc transferase(OGT)involved in the occurrence and development of colorectal cancer(CRC)by O-GlcNAc glycosylation to stabilize Kruppel-like factor 5(KLF5)protein and up-regulate the expression of cyclin D1(CCDN1).Methods:The expression of KLF5 mRNA and protein in CRC was analyzed by bioinformatics.RT-qPCR was used to detect the expression of KLF5 in CRC cells.CCK-8 was used to analyze cell proliferation,and cell cycle was measured by FCM.Bioinformatics method was used to screen target gene which could bind to KLF5 and potential transcription factor binding sites,and dual luciferase reporter assay was utilized to confirm whether CCDN1 binds to KLF5.Influence of KLF5 on CCDN1 mRNA expression was detected by RT-qPCR.Inhibition of KLF5 and overexpression of CCDN1 to analyse whether KLF5 promotes cell proliferation and leads cell entering to G 1 phase through positive regulating CCDN1.Bioinformatics was used to analyze whether KLF5 protein has O-GlcNAc glycosylation site,the expression of OGT mRNA and protein in CRC and the relationship between the expression of OGT and KLF5.OGT inhibitor OSMI-1 was used to treat CRC cells,and expression of KLF5 protein was analyzed by Western blot and O-GlcNAc protein was observed by immunofluorescence chemistry.Western blot was used to measure the stability of KLF5 protein under OSMI-1 and actinomycin.At last inhibition of OGT and overexpression of KLF5 to analyse whether OGT promotes CCDN1 expression through positive regulating KLF5.Results:KLF5 mRNA and protein are overexpressed in CRC.KLF5 was significantly elevated in the CRC cell lines and could promote CRC cell proliferation and led cell entering to G 1 phase.CCDN1 might be target gene of KLF5 and CCDN1 could bind to KLF5.KLF5 promoted cell proliferation and led cell transition from G 1 to S phase through positive regulating CCDN1.Bioinformatics analysis showed KLF5 protein has O-GlcNAc glycosylation site.OGT mRNA and protein are overexpressed in CRC,and the expression b

关 键 词：结直肠癌 O-GlcNAc转移酶 Kruppel样因子5 细胞周期素D1 增殖 细胞周期 

分 类 号：R735.34[医药卫生—肿瘤]