沉默STC2对人结肠癌SW480细胞肝转移裸鼠模型的影响及作用机制研究  

Effect and mechanism of silencing STC2 on nude mouse model of human colon cancer SW480 cell liver metastasis

作　　者：李乾元[1] 周秀扣[1] 方征宇[1] 潘志芸[1] LI Qianyuan;ZHOU Xiukou;FANG Zhengyu;PAN Zhiyun(Department of Anorectal Surgery,Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,China)

机构地区：[1]浙江省中医院肛肠科,浙江杭州310006

出　　处：《国际消化病杂志》2021年第5期353-359,共7页International Journal of Digestive Diseases

基　　金：浙江省医药卫生科技计划项目(2020KY668)。

摘　　要：目的基于Wnt/β-连环蛋白(β-catenin)信号通路探讨沉默斯钙素2(STC2)对人结肠癌SW480细胞肝转移裸鼠模型的影响及作用机制。方法将30只SPF级雄性BALB/c裸鼠随机分为3组,即pLL3.7-shSTC2组(沉默STC2,以pLL3.7-shSTC2转染SW480细胞构建),pLL3.7-NC组(阴性对照,以阴性病毒载体转染SW480细胞构建)和pLL3.7-Con组(空白对照,仅以转染试剂处理的SW480细胞),每组各10只。建模3周后处死裸鼠,观察各组裸鼠肝脏标本的转移瘤情况,肝组织行H-E染色后在显微镜下观察各组的病理改变。采用RT-PCR、免疫组织化学法及蛋白质印迹法(Western blotting)检测STC2、上皮间质转化(EMT)及Wnt/β-catenin信号通路相关基因的表达。结果与pLL3.7-NC组、pLL3.7-Con组相比,pLL3.7-shSTC2组的STC2蛋白表达水平明显降低(P<0.05)。与pLL3.7-shSTC2组相比,pLL3.7-NC组和pLL3.7-Con组裸鼠的肝脏外形不规则程度、质地脆硬度及转移瘤的直径、数量、瘤体破溃等情况更为严重。Western blotting法检测结果显示,与pLL3.7-Con组、pLL3.7-NC组相比,pLL3.7-shSTC2组肝转移瘤组织中STC2、波形蛋白(Vimentin)、基质金属蛋白酶-9(MMP-9)、β-catenin、T细胞因子4(TCF-4)、C-myc及细胞周期蛋白D1(Cyclin D1)的蛋白表达水平均明显降低(P均<0.05),E-钙黏蛋白(E-cadherin)表达水平明显升高(P<0.05)。RT-PCR法检测结果显示,与pLL3.7-Con组、pLL3.7-NC组相比,pLL3.7-shSTC2组肝转移瘤组织中TCF-4、C-myc及Cyclin D1的mRNA表达水平均明显降低(P均<0.05)。免疫组织化学染色结果显示,与pLL3.7-Con组、pLL3.7-NC组相比,pLL3.7-shSTC2组肝转移瘤组织中β-catenin表达水平明显降低。结论沉默STC2可通过抑制Wnt/β-catenin信号通路来减少结肠癌细胞的增殖、侵袭,从而抑制肿瘤肝转移。Objective This paper intends to investigate the effect and mechanism of silencing stanniocalcin 2(STC2)on the nude mouse model of human colon cancer SW480 cell liver metastasis,based on the Wnt/β-catenin signaling pathway.Methods Thirty SPF male BALB/c nude mice were randomly assigned to the pLL3.7-shSTC2 group(silent STC2,constructed by transfecting SW480 cells with pLL3.7-shSTC2),the pLL3.7-NC group(negative control,constructed by transfecting SW480 cells with negative virus vector),and the pLL3.7-Con group(blank control,SW480 cells treated with transfection reagent only),10 in each group.The nude mice were sacrificed after 3 weeks of modeling,the liver metastases in the liver specimens of nude mice in each group were observed,the liver tissues were stained with H-E,and the pathological changes of each group were observed under a microscope.RT-PCR,immunohistochemistry and Western blotting were used to detect the expression of STC2,epithelial-mesenchymal transition(EMT),and Wnt/β-catenin signaling pathway related genes.Results Compared with the pLL3.7-NC and the pLL3.7-Con groups,the expression of STC2 protein in the pLL3.7-shSTC2 group is significantly reduced(P<0.05).Compared with the pLL3.7-shSTC2 group,the nude mice in the pLL3.7-NC and the pLL3.7-Con groups have more serious liver shape irregularities,fragile texture,size and number of tumor nodules,and tumor rupture.The Western blotting results show that compared with the pLL3.7-Con and the pLL3.7-NC groups,the STC2,Vimentin,matrix metalloproteinase-9(MMP-9),β-catenin,T cell factor 4(TCF-4),C-myc,and Cyclin D1(Cyclin D1)protein expression are significantly reduced(all P<0.05),but the E-cadherin(E-cadherin)expression is significantly increased(P<0.05).The RT-PCR results indicate that compared with the pLL3.7-Con and the pLL3.7-NC groups,the mRNA expressions of TCF-4,C-myc,and Cyclin D1 in the liver metastases of the pLL3.7-shSTC2 group are significantly reduced(all P<0.05).The immunohistochemistry results suggests that compared with the pLL3.7-Con and th

关 键 词：斯钙素2 WNT/Β-CATENIN信号通路 结肠癌 肝转移 

分 类 号：R73[医药卫生—肿瘤]