miR-32-5p通过靶向下调EZH2的表达抑制胰腺癌PANC-1细胞的恶性生物学行为与裸鼠移植瘤生长  

miR-32-5p inhibits the malignant biological behaviors and the growth of nude mice xenograft of pancreatic cancer PANC-1 cells by targeted down-regulation of EZH2 expression

作　　者：柳君君 张燕燕 雷衡阳 LIU Junjun;ZHANG Yanyan;LEI Hengyang(Department of General Surgery,Afiliated Hospital of Gansu Medical College,Pingliang 744000,Gansu,China;Department of Obstetrics and Gynecology,the Second People's Hospital of Pingliang City,Pingliang 744099,Gansu,China)

机构地区：[1]甘肃医学院附属医院普外科,甘肃平凉744000 [2]甘肃省平凉市第二人民医院妇产科,甘肃平凉744099

出　　处：《中国肿瘤生物治疗杂志》2022年第6期557-566,共10页Chinese Journal of Cancer Biotherapy

摘　　要：目的:探究miR-32-5p通过靶向zeste基因增强子人类同源物2(EZH2)对胰腺癌PANC-1细胞恶性生物学行为的影响及其分子机制。方法:利用GEPIA数据库分析胰腺癌组织中EZH2的表达水平及其与患者的预后生存期的关系,并分析miR-32-5p表达与患者临床病理因素的关系。qPCR法检测正常胰腺HPDE6-C7细胞和胰腺癌PANC-1、AsPC-1、SW1990细胞中miR-32-5p和EZH2 mRNA的表达,通过Lipofectamine^(TM)2000将miR-NC、miR-32-5p mimic、miR-32-5p inhibitor、pcDNA-NC、pcDNA EZH2质粒分别转染PANC-1细胞,其分为对照组(不转染)、miR-NC组(转染miR-NC)、miR-32-5p mimic组(转染miR-32-5p mimic)、Anti-miR-32-5p组(转染miR-32-5p inhibitor)、miR-NC+pcDNA-NC组(转染miR-NC+pcDNA-NC)、miR-NC+pcDNA EZH2组(转染miR-NC+pcDNA EZH2)、miR-32-5p mimic+pcDNA-NC组(转染miR-32-5p mimic+pcDNA-NC)、miR-32-5p mimic+pcDNA EZH2组(转染miR-32-5p mimic+pcDNA EZH2)。双荧光素酶报告基因实验验证miR-32-5p与EZH2的靶向关系;MTT法及克隆形成实验检测各组细胞的增殖能力,划痕愈合实验检测各组细胞的迁移能力;Transwell小室实验检测各组细胞的侵袭能力,WB法检测各组细胞EZH2、E-cadherin、N-cadherin的表达;裸鼠成瘤实验检测转染后各组PANC-1细胞移植瘤的生长情况,免疫组化染色法观察移植瘤组织中Ki67和MMP-2的表达。结果:GEPIA数据库显示胰腺癌组织中EZH2的表达水平高于癌旁组织,患者预后生存期与EZH2的表达水平呈负相关(均P<0.05);miR-32-5p表达水平与胰腺癌神经浸润、肿瘤分化程度、TNM分期、淋巴结转移有明显的关联(均P<0.05);与HPDE6-C7细胞相比,PANC-1、AsPC-1、SW1990细胞中miR-32-5p呈高表达、EZH2 mRNA呈低表达、miR-32-5p表达水平与EZH2表达水平呈负相关(均P<0.05)。miR-32-5p靶向EZH2且抑制其表达(均P<0.05);过表达miR-32-5p能够下调Ki67、MMP-2、N-cadherin表达水平、上调E-cadherin表达水平,抑制PANC-1细胞的增殖、迁移和侵袭能力,抑制移�Objective: To explore the effect of miR-32-5p on the malignant biological behaviors of pancreatic cancer PANC-1 cells via targeting zeste gene enhancer human homolog 2(EZH2) and its molecular mechanism. Methods: The GEPIA database was used to analyze the expression level of EZH2 in pancreatic cancer tissues and its relationship with the prognostic survival of patients. The association between miR-32-5p expression and clinicopathological features of patients was also analyzed. qPCR was used to detect the expression of miR-32-5p and EZH2 mRNA in pancreatic cancer cells(PANC-1, AsPC-1, SW1990) and normal pancreatic HPDE6-C7 cells. With Lipofectamine2000, miR-NC, miR-32-5p mimic, miR-32-5p inhibitor, pcDNA-NC and pcDNA EZH2 plasmids were separately or jointly transfected into pancreatic cancer PANC-1 cells, namely control group(untransfected),miR-NC group(transfected with miR-NC), miR-32-5p mimic group(transfected with miR-32-5p mimic), anti-miR-32-5p group(transfected with miR-32-5p inhibitor);miR-NC+pcDNA-NC group(transfected with miR-NC+pcDNA-NC), miR-NC+pcDNA EZH2 group(transfected with miR-NC+pcDNA EZH2), miR-32-5p mimic+pcDNA-NC group(transfected with miR-32-5p mimic+pcDNA-NC), and miR-32-5p mimic+pcDNA EZH2 group(transfected with miR-32-5p mimic+pcDNA EZH2). Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-32-5p and EZH2. MTT method and the clone formation assay were used to detect proliferation ability of the cells in each group;scratch healing test was used to detect cell migration ability;Transwell chamber test was used to detect cell invasion;Western blotting method was adopted to detect the expression of EZH2, epithelial cadherin(E-cadherin)and neural cadherin(N-cadherin) in cells. The tumorigenicity experiment in nude mice was used to detect the development of transplanted tumors;and the expression of Ki67 and metal matrix protease-2(MMP-2) in tumor tissues was observed by immunohistochemical staining. Results: The GEPIA database showed that the expression lev

关 键 词：miR-32-5p zeste基因增强子人类同源物2 胰腺癌 PANC-1细胞 增殖 迁移 侵袭 上皮间质转化 

分 类 号：R736.7[医药卫生—肿瘤] R730.2[医药卫生—临床医学]