miR-502调控犬乳腺肿瘤细胞增殖、迁移和EMT的作用机制

doi:10.11843/j.issn.0366-6964.2023.10.034

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4379-4388.doi: 10.11843/j.issn.0366-6964.2023.10.034

miR-502调控犬乳腺肿瘤细胞增殖、迁移和EMT的作用机制

任晓丽¹, 范玉营², 皇甫和平¹, 王军¹, 靳双星¹, 刘云², 石冬梅^1*

1. 河南牧业经济学院动物医药学院, 郑州 450046;
2. 东北农业大学动物医学院, 哈尔滨 150030

收稿日期:2022-11-08 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 石冬梅,主要从事动物疾病诊疗研究,E-mail:dongmeishi126@126.com
作者简介:任晓丽(1983-),女,河南安阳人,讲师,博士,主要从事小动物肿瘤学研究,E-mail:renxiaoli168@163.com
基金资助:
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）

Mechanism of miR-502 Regulating Proliferation, Migration, Invasion and EMT of Canine Mammary Tumor Cells

REN Xiaoli¹, FAN Yuying², HUANGFU Heping¹, WANG Jun¹, JIN Shuangxing¹, LIU Yun², SHI Dongmei^1*

1. College of Veterinary Medicine, Henan University of Animal Husbandry of Economy, Zhengzhou 450046, China;
2. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China

Received:2022-11-08 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

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RBMS1 3'-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。

关键词: miR-502, RBMS1, 犬乳腺肿瘤细胞, 增殖/迁移, 上皮-间质转化

Abstract: 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 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 of RBMS1 by targeting RBMS1 3'-UTR. Therefore, miR-502 is highly expressed in canine mammary tumor cells, and interfering with its expression might affected the proliferation, migration and EMT of the canine mammary tumor CHMm cells, and miR-502 targeted regulation the expression of RBMS1 mRNA.

Key words: miR-502, RBMS1, canine mammary tumor cells, cell proliferation/migration, epithelial-mesenchymal transition

中图分类号:

S858.2927.26

任晓丽, 范玉营, 皇甫和平, 王军, 靳双星, 刘云, 石冬梅. miR-502调控犬乳腺肿瘤细胞增殖、迁移和EMT的作用机制[J]. 畜牧兽医学报, 2023, 54(10): 4379-4388.

REN Xiaoli, FAN Yuying, HUANGFU Heping, WANG Jun, JIN Shuangxing, LIU Yun, SHI Dongmei. Mechanism of miR-502 Regulating Proliferation, Migration, Invasion and EMT of Canine Mammary Tumor Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4379-4388.

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miR-502调控犬乳腺肿瘤细胞增殖、迁移和EMT的作用机制

Mechanism of miR-502 Regulating Proliferation, Migration, Invasion and EMT of Canine Mammary Tumor Cells

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