KLF4在肾透明细胞癌侵袭和迁移中的作用及其与SKA1的靶向调控关系探讨  

Role of KLF4 in invasion and migration of clear cell renal cell carcinoma and its targeted regulatory relationship with SKA1

作　　者：张萌萌[1] 刘盟雪 袁帅[2] 韩静[1] ZHANG Mengmeng;LIU Mengxue;YUAN Shuai;HAN Jing(Institute of GCP Office,Affiliated Tumor Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学附属肿瘤医院药物临床试验机构办公室,乌鲁木齐830011 [2]新疆医科大学附属肿瘤医院泌尿外科

出　　处：《山东医药》2023年第14期1-6,共6页Shandong Medical Journal

基　　金：新疆维吾尔自治区自然科学基金资助项目(2020D01C215)。

摘　　要：目的探讨肾透明细胞癌(ccRCC)组织中锌指转录调节因子4(KLF4)的表达变化,及其调控纺锤体和动粒相关复合体亚基1(SKA1)促进ccRCC细胞侵袭、迁移的作用。方法从癌症和肿瘤基因图谱(TCGA)数据库下载440例ccRCC原位癌组织和80例伴有远处转移的ccRCC组织的转录组测序数据,分析KLF4在原位癌组织和伴有远处转移癌组织中的表达差异。取人肾癌细胞786-O、ACHN,分为KLF4过表达组、空载体组、共表达KLF4/SKA1组,采用慢病毒载体技术,分别转染pCMV-KLF4、pCMV、pCMV-KLF4和pCMV-SKA1质粒;采用Transwell小室实验观察各组细胞侵袭和迁移能力,Western blotting法检测细胞上皮间质转化相关蛋白E-cadherin、N-cadherin、Vimentin表达。取786-0、ACHN细胞,分为KLF4干扰组及干扰对照组、KLF4过表达组及空载体组,KLF4干扰组和干扰对照组分别转染siKLF4和siNC,KLF4过表达组和空载体组分别转染pCMV-KLF4、pCMV质粒;采用实时荧光定量PCR法检测细胞SKA1 mRNA表达,Western blotting法检测细胞SKA1蛋白表达;利用Jaspar和ConTra V3在线工具分析预测KLF4在SKA1启动子区的结合位点,并采用双荧光素酶报告实验进行验证。结果KLF4在伴有远处转移的ccRCC组织中的表达低于ccRCC原位癌组织(P<0.01)。在786-O和ACHN细胞中,与空载体组相比,过表达KLF4组细胞侵袭和迁移能力降低(P均<0.01);与过表达KLF4组比较,共表达KLF4/SKA1组细胞侵袭和迁移能力增加(P均<0.01)。与空载体组相比,过表达KLF4组E-cadherin蛋白表达水平升高、N-cadherin和Vimentin蛋白表达水平降低;与过表达KLF4组比较,共表达KLF4/SKA1组E-cadherin蛋白表达水平降低、N-cadherin和Vimentin蛋白表达水平升高(P均<0.05)。与siNC组相比,siKLF4组SKA1 mRNA及蛋白表达水平均升高;与pCMV空载体组相比,pKLF4过表达组SKA1 mRNA及蛋白表达水平均下降(P均<0.01)。SKA1转录起始位点上游850~900 bp启动子序列中存在KLF4的结合位点。双荧�Objective To investigate the expression changes of zinc finger transcription factor Krüppel-like factor 4(KLF4)in clear cell renal cell carcinoma(ccRCC),and its role of regulating the kinetochore-associated complex 1(SKA1)in promoting the invasion and migration of ccRCC cells.Methods The transcriptome sequencing data of 440 cases of carcinoma in situ and 80 cases of ccRCC with distant metastasis were downloaded from the Cancer and Tumor Ge⁃nome Atlas(TCGA)database to analyze the expression difference of KLF4 between carcinoma in situ and distant metasta⁃sis.The ccRCC cells 786-O and ACHN were divided into KLF4 overexpression group,empty vector group,and KLF4/SKA1 co-expression group.Lentivirus vector technology was used to transfect the plasmids pCMV-KLF4,pCMV,pCMVKLF4 and pCMV-SKA1,respectively.The invasion and migration of cells in each group were observed by Transwell as⁃say,and the expression levels of E-cadherin,N-cadherin and Vimentin related to epithelial-mesenchymal transition were detected by Western blotting.The ccRCC cells were divided into KLF4 interference group,interference control group,KLF4 overexpression group,and empty vector group.Cells in the KLF4 interference group and interference control group were transfected with siKLF4 and siNC,respectively;cells in the KLF4 overexpression group and empty vector group were transfected with pCMV-KLF4 and pCMV plasmid,respectively.The SKA1 mRNA expression was detected by real-time fluorescence quantitative PCR,and the SKA1 protein expression was detected by Western blotting.The binding sites of KLF4 in SKA1 promoter region were predicted by Jaspar and ConTra V3 online tools,and verified by double luciferase re⁃porting assay.Results The expression of KLF4 in ccRCC tissues with distant metastasis was lower than that in carcino⁃ma in situ(P<0.01).In 786-O and ACHN cells,compared with the empty carrier group,the invasion and migration of cells in KLF4 overexpression group decreased(both P<0.01);compared with KLF4 overexpression group,the invasion an

关 键 词：锌指转录调节因子4 纺锤体和动粒相关复合体亚基1 细胞侵袭 细胞迁移 肾透明细胞癌 

分 类 号：R737.11[医药卫生—肿瘤]