SCL/TAL1中断基因座对膀胱癌增殖和迁移的影响及其作用机制  

Effects and mechanism of SCL/TAL1 interrupting locus on proliferation and migration of bladder cancer

作　　者：虞华 胡祎舜 丁勇杰 唐峰 王豪 万梓宇 刘涛[1] 曾金敏[2] 廖义翔[2] 彭建平[1,3] Yu Hua;Hu Yishun;Ding Yongjie;Tang Feng;Wang Hao;Wan Ziyu;Liu Tao;Zeng Jinmin;Liao Yixiang;Peng Jianping(Department of Urology,Wuhan University Zhongnan Hospital,Wuhan 430071,China;Department of Urology,Jingzhou Central Hospital,Jingzhou Hospital Affiliated to Yangtze University,Jingzhou 434020,China;Department of Urology,Xishui people’s Hospital,Xishui Hospital Affiliated to Hubei Institute of Science and Technology,Huanggang 438200,China)

机构地区：[1]武汉大学中南医院泌尿外科,430071 [2]荆州市中心医院,长江大学附属荆州医院泌尿外科,434020 [3]浠水县人民医院,湖北科技学院附属浠水医院泌尿外科,黄冈438200

出　　处：《中华实验外科杂志》2022年第12期2315-2318,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金项目(81970588,81902598)。

摘　　要：目的:探讨SCL/TAL1中断基因座(STIL)的表达对膀胱癌增殖和迁移的影响及其作用机制。方法:在Timer和UALCAN数据库中分析STIL在膀胱癌中的差异表达和分期。在T24细胞系中使用CRISPR/Cas9技术导入单链向导RNA(sgRNA)敲除STIL。设置组别为T24细胞株对照组和稳定敲除STIL实验组的T24细胞株(6#、30#)。通过细胞计数试剂盒(CCK-8)实验、平板克隆形成实验、Transwell实验和软琼脂克隆实验观察STIL对膀胱癌的增殖和迁移的影响。通过免疫荧光实验检测细胞增殖标志物细胞核增殖抗原(Ki-67)和5-乙炔基-2’-脱氧尿苷(EDU)验证敲除STIL抑制膀胱癌细胞的增殖。转录组测序技术(RNA-seq)和基因富集分析(GSEA)寻找STIL的相关信号通路。通过蛋白质印迹法(Western blot)实验观察STIL对实验观察STIL对细胞外信号调节激酶(ERK)、磷酸化ERK(p-ERK)、周期蛋白依赖性激酶1(CDK1)、细胞周期蛋白(Cyclin)B1的影响。组间比较采用独立样本t检验。结果:生信数据库发现STIL在多种癌症中高表达,其中包括膀胱癌,肿瘤组织组中STIL的表达高于正常组织组[4.411(2.322~7.133)比0.659(0.312~0.892),P<0.05]。STIL的表达与膀胱癌的分期显著相关。在敲除STIL后,CCK-8实验[对照组光密度明显高于敲除组(2.76±0.17比1.11±0.11),t=19.865,P<0.05和(2.76±0.17比1.30±0.17,t=14.720,P<0.05]、平板克隆形成实验结果显示,对照组集落数明显高于敲除组[(77.67±9.50)比(18.67±4.04),t=9.895,P<0.05]和[(77.67±9.50)比(20.67±3.79),t=9.650,P<0.05]、Transwell实验结果显示,对照组迁移细胞数明显高于敲除组[(171.00±13.53)比(56.33±13.5),t=10.391,P<0.05]和[(171.00±13.53)比(23.33±9.29),t=15.585,P<0.05],软琼脂克隆实验结果显示,对照组细胞数明显高于敲除组[(168.67±11.02)比(48.33±5.03),t=17.210,P<0.05]和[(168.67±11.02)比(23.33±5.69),t=20.307,P<0.05]。免疫荧光实验示,对照组Ki-67明显高于敲除组[(0.273±0.014)比(0.088±0.012),t=16.Objective To investigate the effect of SCL/TAL1 interrupting locus(STIL)expression on the proliferation and migration of bladder cancer and its mechanism.Methods The differential expression,staging and prognosis of STIL in bladder cancer were analyzed in Timer,GEPIA,UALCAN,and OncoLnc databases.Knockout of STIL by introducing single-stranded guide RNA(sgRNA)using CRISPR/Cas9 technology in T24 cell line.The groups were set as T24 cell line control group and T24 cell line(6#,30#)stably knocked out STIL experimental group.The effects of STIL on the proliferation and migration of bladder cancer were observed by cell counting kit-8(CCK-8)assay,plate colony formation assay,Transwell assay and soft agar cloning assay.The cell proliferation markers proliferation cell nuclear antigen(Ki-67)and 5-ethynyl-2-deoxyuridine(EDU)were detected by immunofluorescence assay to verify that knockout of STIL inhibited the proliferation of bladder cancer cells.RNA Sequencing(RNA-seq)and gene set enrichment analysis(GSEA)enrichment analysis searched for STIL-related signaling pathways.The effects of STIL on extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),cyclin-dependent kinase 1(CDK1)and Cyclin B1 were observed by Western blotting.Statistics were performed using independent samples t-test.Results The bioinformatics database found that STIL was highly expressed in a variety of cancers,including bladder cancer(P<0.05).The expression of STIL is significantly correlated with the stage of bladder cancer(P<0.05).After knockout of STIL,CCK-8 assay{The optical density of the control group was significantly higher than that of the knockout group(P<0.05),plate colony formation assay{The number of cell colonies in the control group was significantly higher than that in the knockout group(P<0.05),Transwell assay The number of migrated cells in the control group was significantly higher than that in the knockout group(P<0.05)and soft agar cloning assay.The number of cells in the control group was significantly higher than that i

关 键 词：SCL/TAL1中断基因座 膀胱癌 细胞周期相关基因 

分 类 号：R737.14[医药卫生—肿瘤]