ZDHHC5对结肠癌细胞Ⅰ型干扰素表达和细胞增殖及迁移的调控作用  

Regulatory role of ZDHHC5in IFN-ⅠExpression and cellular proliferation and migration in colorectal cancer cells

作　　者：邱晋 李帅广 姬美玲 杨心茹 叶辉 任浩 李若男 安华章 曹莉莉[1,2] QIU Jin;LI Shuaiguang;JI Meiling;YANG Xinru;YE Hui;REN Hao;LI Ruonan;AN Huazhang;CAO Lili(Oncology Center,The First Affiliated Hospital of Shandong First Medical University&Shandong Provincial Qianfoshan Hospital,Shandong Provincial University Laboratory of Clinical Immunology and Translational Medicine,Shandong Provincial Key Laboratory for Rheumatic Disease and Translational Medicine,Shandong Lung CancerInstitute,Jinan,Shandong250014,China;Second Clinical College of Shandong University of Traditional Chinese Medicine,Jinan,Shandong250014,China)

机构地区：[1]山东第一医科大学第一附属医院(山东省千佛山医院)肿瘤中心,山东省临床免疫转化医学高校实验室,山东省风湿免疫病转化医学重点实验室,山东省肺癌研究所,山东济南250014 [2]山东中医药大学第二临床医学院,山东济南250014

出　　处：《中华肿瘤防治杂志》2024年第18期1108-1115,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：山东省自然科学基金面上项目(ZR2020MH203)。

摘　　要：目的探讨ZDHHC5对Ⅰ型干扰素(IFN-Ⅰ)表达的调节作用以及对结肠癌细胞增殖和迁移能力的影响。方法利用慢病毒感染和质粒瞬转技术对小鼠结肠癌细胞株MC38ova进行ZDHHC5敲低和过表达,并进行ZDHHC5和Zeste同源物增强子2(EZH2)双重敲低;实时荧光定量聚合酶链式反应(qRT-PCR)实验检测MC38ova细胞株敲低和过表达ZDHHC5后IFN-Ⅰ表达水平,并检测同时敲低ZDHHC5和EZH2后IFN-Ⅰ表达水平变化;免疫共沉淀实验检测ZDHHC5与EZH2蛋白相互作用;细胞计数试剂盒(CCK 8)增殖实验、克隆形成实验和划痕实验检测MC38ova细胞株敲低ZDHHC5后细胞增殖和迁移能力变化。结果qRT-PCR结果显示,与NC-gfp组IFN-α(1.00±0.05)和IFN-β(1.00±0.06)mRNA水平相比,KD-ZDHHC5#1-gfp组IFN-α(5.72±0.27,t=29.56,P<0.001)和IFN-β(2.59±0.03,t=42.90,P<0.001)mRNA水平升高;与NC组IFN-α(1.00±0.13)和IFN-β(1.00±0.10)mRNA水平相比,KD-ZDHHC5#2组IFN-α(4.74±0.58,t=11.64,P=0.001)和IFN-β(3.85±0.34,t=13.97,P<0.001)mRNA水平升高;与OE-NC组IFN-α(1.00±0.07)和IFN-β(1.00±0.06)mRNA水平相比,OE-ZDHHC5组IFN-α(0.33±0.02,t=16.64,P<0.001)和IFN-β(0.47±0.03,t=14.29,P<0.001)mRNA水平降低;与KD-EZH2组IFN-α(1.00±0.16)mRNA水平相比,KD-EZH2+KD-ZDHHC5组IFN-α(1.82±0.06,t=8.15,P=0.001)mRNA水平升高。免疫共沉淀实验结果显示,FLAG-ZDHHC5能够结合HA-EZH2蛋白。CCK 8结果显示,与NC-gfp组相比,KD-ZDHHC5#1-gfp组细胞增殖能力降低(48 h,t=29.89,P<0.001;72 h,t=32.94,P<0.001);与NC组相比,KD-ZDHHC5#2组细胞增殖能力降低(48 h,t=20.95,P<0.001;72 h,t=28.67,P<0.001)。克隆形成实验结果显示,与NC-gfp组和NC组相比,KD-ZDHHC5#1-gfp组(t=11.44,P<0.001)和KD-ZDHHC5#2组(t=6.02,P=0.004)细胞增殖能力降低。划痕实验结果显示,与NC-gfp组和NC组相比,KD-ZDHHC5#1-gfp组(t=5.78,P=0.029)和KD-ZDHHC5#2组(t=15.22,P=0.004)细胞迁移能力降低。结论ZDHHC5通过与EZH2相互作用,抑制IFN-α和IFN-β表达,增强结肠癌细胞的增殖和迁移能�Objective To elucidate the regulatory role of zinc finger DHHC-type containing 5(ZDHHC5)in expression of typeⅠinterferon(IFN-Ⅰ)expression and its consequent impact on the proliferation and migration of colorectal cancer cells.Methods The mouse colon cancer cell line MC38ova underwent ZDHHC5knockdown and overexpression by using lentiviral infection and plasmid transient transfection.Simultaneous knockdown of ZDHHC5and EZH2was also conducted.Quantitative real-time PCR(qRT-PCR)assay was used to assess IFN-Ⅰexpression in MC38ova cell line post-ZDHHC5knockdown or overexpression.Additionally,the expression level of IFN-Ⅰfollowing concurrent knockdown of ZDHHC5and EZH2was determined.The interaction between ZDHHC5and EZH2proteins was examined through immunoprecipitation assays.Changes in cell proliferation and migration after ZDHHC5knockdown were evaluated by using Cell Counting Kit 8(CCK 8)assays,colony formation assays,and scratching assays.Results qRT-PCR results showed that,compared with the IFN-α(1.00±0.05)and IFN-β(1.00±0.06)mRNA levels of NC group,the KD-ZDHHC5#1-gfp group mRNA levels of IFN-α(5.72±0.27,t=29.56,P<0.001)and IFN-β(2.59±0.03,t=42.90,P<0.001)were increased.Similarly,the KD-ZDHHC5#2group exhibited a marked increase in IFN-α(4.74±0.58,t=11.64,P=0.001)and IFN-β(3.85±0.34,t=13.97,P<0.001)compared with the NC group,which had levels of 1.00±0.13and 1.00±0.10,respectively.In contrast,the OE-ZDHHC5group demonstrated significantly reduced mRNA levels of IFN-α(0.33±0.02,t=16.64,P<0.001)and IFN-β(0.47±0.03,t=14.29,P<0.001)as opposed to the OENC group,which had levels of 1.00±0.07and 1.00±0.06,respectively.Furthermore,the KD-EZH2+KD-ZDHHC5 group showed an increase in IFN-αmRNA level(1.82±0.06,t=8.15,P=0.001),when assessed against the KDEZH2group,which had levels of 1.00±0.16,respectively.Immunoprecipitation assays demonstrated the FLAG-ZDHHC5bind to the HA-EZH2protein.The CCK 8results revealed that the cell proliferation in the KD-ZDHHC5#1-gfp group was significantly reduced compare

关 键 词：ZDHHC5 Zeste同源物增强子2 Ⅰ型干扰素 结肠癌 

分 类 号：R735.35[医药卫生—肿瘤]