摘要
旨在探讨微小RNA-502(microRNA-502/miR-502)靶向RNA结合基序单链相互作用蛋白1(RNA-binding motif,single-stranded-interacting protein 1,RBMS1)对犬乳腺肿瘤细胞增殖、迁移、上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响及其作用机制。通过实时荧光定量PCR(qRT-PCR)方法检测犬乳腺肿瘤细胞(CHMp和CHMm)和正常乳腺组织中miR-502和RBMS1的转录水平;利用脂质体转染法将miR-502模拟物(mimic)、抑制剂(inhibitor)、阴性对照(negative control,NC)分别转染至犬乳腺肿瘤CHMm和CHMp细胞,用qRT-PCR方法检测细胞中miR-502的表达水平,以MTT法和Transwell试验分别检测细胞的增殖和迁移能力;通过qRT-PCR和Western blot方法检测转染miR-502后对上皮细胞标志物E-钙黏蛋白(E-cadherin)、间质细胞标志物波形蛋白(vimentin)、细胞迁移标志物基质金属蛋白酶(MMP2)、增殖标志物增殖细胞核抗原(PCNA)mRNA和蛋白表达水平;利用qRT-PCR和双荧光素酶报告试验验证miR-502与RBMS1基因的靶向关系。结果显示:与正常乳腺组织组相比,miR-502在犬乳腺肿瘤细胞中表达显著上调,RBMS1在犬乳腺肿瘤细胞中转录下调;转染miR-502 mimic后miR-502的转录水平显著高于miR-NC组,反之转染miR-502 inhibitor后miR-502的转录水平显著下调;与miR-NC组相比,转染miR-502 mimic显著促进犬乳腺肿瘤细胞的增殖和迁移,抑制E-cadherin mRNA和蛋白的表达,促进vimentin、MMP2、PCNA mRNA和蛋白表达水平升高;反之,转染miR-502 inhibitor可显著抑制犬乳腺肿瘤细胞的增殖和迁移,促进E-cadherin mRNA和蛋白表达升高,降低vimentin、MMP2、PCNA mRNA和蛋白表达水平;qRT-PCR和荧光素酶报告试验显示,miR-502通过靶向RBMS13′-UTR抑制RBMS1 mRNA的表达。上述结果提示,miR-502在犬乳腺肿瘤细胞中高表达,干扰miR-502的表达可以影响犬乳腺肿瘤CHMm细胞的增殖、迁移及EMT,且miR-502靶向调控RBMS1 mRNA的表达。
This study aimed to investigate the effect and mechanism of microRNA-502(miR-502)targeting RBMS1 on proliferation,migration and epithelial-mesenchymal transition(EMT)of canine mammary tumor cells.The expression transcription level of miR-502 and RBMS1 mRNA in canine mammary tumor cell lines(CHMp and CHMm)and normal mammary gland tissues were detected by realtime quantitative PCR(qRT-PCR).The miR-502 mimic/inhibitor and negative control mimic/inhibitor NC were transfected into CHMp/CHMm cells using liposome transfection method,respectively,and the expression levels of miR-502 in cells were detected by qRT-PCR method.MTT assay and Transwell assay were used to detect the proliferation and migration ability of CHMm cells;the expression levels of epithelial cell marker E-cadherin,mesenchymal cell marker vimentin,cell migration markers matrix metalloproteinase(MMP2)and cell proliferation marker PCNA after transfection with miR-502 were detected by qRT-PCR and Western blot methods.qRT-PCR and double-luciferase reporter gene assay were adopted to verify the targeting relationship between miR-502 and RBMS1 gene.The results showed that compared with normal mammary tissues,the expression level of miR-502 in canine mammary tumor cells was significantly up-regulated,while RBMS1 was down-regulated.Transfection of miR-502 mimic up-regulated the expression level of miR-502,whereas miR-502 inhibitor down-regulated the expression level of miR-502.Compared with the miR-NC group,transfection of miR-502 mimic significantly promoted the proliferation and migration of CHMm cells,moreover,down-regulated the expression of E-cadherin mRNA and protein,and up-regulated the expression of vimentin,MMP2,PCNA mRNA and protein.On the contrary,transfection of miR-502 inhibitor significantly suppressed the proliferation and migration of CHMm cells,up-regulated the expression of E-cadherin and down-regulated the expression of vimentin,MMP2,PCNA mRNA and protein;qRT-PCR and luciferase report experiments showed that miR-502 inhibited the expression o
作者
任晓丽
范玉营
皇甫和平
王军
靳双星
刘云
石冬梅
REN Xiaoli;FAN Yuying;HUANGFU Heping;WANG Jun;JIN Shuangxing;LIU Yun;SHI Dongmei(College of Veterinary Medicine,Henan University of Animal Husbandry of Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2023年第10期4379-4388,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
郑州市重点培育项目(L4030008)
河南省科技攻关项目(222102110110)
河南省教育厅重点科研项目(22B230004)。
