miR-188对小鼠乳腺癌细胞增殖、迁移及凋亡的影响

doi:10.16431/j.cnki.1671-7236.2022.03.038

摘要/Abstract

摘要： 【目的】探讨microRNA-188-5p （miR-188）在乳腺癌细胞增殖、迁移和凋亡过程中的调控作用，以期为乳腺癌相关治疗药物的研发提供理论依据。【方法】培养小鼠乳腺癌细胞（4T1），建立4T1细胞小鼠移植瘤模型，分离肿瘤组织和瘤旁组织，用实时荧光定量PCR法检测miR-188的表达情况；在4T1细胞中分别转染miR-188的模拟物（miR-188 mimics）、模拟物对照（mimics-NC）、miR-188抑制物（miR-188 inhibitor）及抑制物对照（inhibitor-NC），不转染的细胞为空白对照（Con），用实时荧光定量PCR法检测各组细胞miR-188的表达水平，CCK-8法检测细胞增殖能力，细胞划痕试验检测细胞的迁移率，流式细胞术检测细胞的凋亡率，Western blotting检测细胞相关凋亡蛋白的表达情况。【结果】瘤旁组织中miR-188的相对表达量显著高于肿瘤组织（P<0.05）；细胞转染结果表明，与Con组相比，miR-188 mimics组miR-188的相对表达量显著上调（P<0.05），而miR-188 inhibitor组显著下调（P<0.05），mimics-NC、inhibitor-NC组均无显著差异（P>0.05），因此后续试验分别以mimics-NC、inhibitor-NC为miR-188 mimics、miR-188 inhibitor的对照进行分析。细胞增殖和划痕试验结果表明，与mimics-NC组相比，miR-188 mimics显著降低4T1细胞的增殖速率和迁移速率（P<0.05）；细胞凋亡试验结果显示，与mimics-NC组相比，miR-188 mimics显著促进4T1细胞凋亡（P<0.05）；与inhibitor-NC组相比，miR-188 inhibitor显著抑制细胞凋亡（P<0.05）；Western blotting结果表明，miR-188 mimics显著降低基质金属蛋白酶9（MMP-9）蛋白的表达水平（P<0.05），显著提高聚二磷酸腺苷核糖聚合酶（PARP1）、半胱氨酸蛋白酶-3（Caspase-3）和Bcl-2-associated X蛋白（Bax）的表达、抑制抑凋亡蛋白B淋巴细胞瘤-2（Bcl-2）的表达（P<0.05）。【结论】 miR-188可显著抑制4T1细胞的增殖和迁移，促进4T1细胞凋亡，说明miR-188可以作为抑制乳腺癌进展和转移潜能的肿瘤调节子。

关键词: 乳腺癌; 4T1细胞; miR-188; 增殖; 凋亡

Abstract: 【Objective】 This study was aimed to investigate the regulatory role of microRNA-188-5p (miR-188) on the proliferation, migration and apoptosis of breast cancer cells, in order to provide a theoretical basis for the development of breast cancer related treatment drugs.【Method】 The mouse breast cancer cells (4T1) was cultured to establish 4T1 mouse xenograft model.The tumour tissues and adjacent tissues were then separated, and the expression of miR-188 was detected by Real-time quantitative PCR.The oligos miR-188 mimics, mimics-NC, miR-188 inhibitor and inhibitor-NC were transferred into 4T1 cell lines, and the non transfected cells was blank control (Con), the expression level of miRNA-188 was detected by Real-time quantitative PCR, cell proliferation was detected by CCK-8 method, cell migration was detected by cell scratch test, the apoptosis rate was detected by flow cytometry, the expression of apoptosis related proteins was detected by Western blotting.【Result】 The expression of miR-188 in peritumoral tissues was significantly higher than that of tumour tissues (P<0.05).The results of cell transfection showed that compared with Con group, the relative expression of miR-188 in miR-188 mimics group was significantly up-regulated (P<0.05), and in miR-188 inhibitor group was significantly down regulated (P<0.05).There was no significant difference between mimics-NC and inhibitor-NC groups.Therefore, the follow-up tests were analyzed with mimics-NC and inhibitor-NC as the control of miR-188 mimics and miR-188 inhibitor, respectively.Cell proliferation test and cell scratch test showed that compared with mimics-NC group miR-188 significantly reduced the proliferation rate and migration rate of 4T1 cells (P<0.05).Cell apoptosis test results showed that compared with mimics-NC group miR-188 mimics significantly promoted 4T1 apoptosis (P<0.05);Compared with inhibitor-NC group, miR-188 inhibitor significantly inhibited apoptosis (P<0.05).Western blotting results showed that miR-188 mimics significantly decreased the expression of matrix metalloproteinase-9 (MMP-9) protein (P<0.05), significantly increased the expression of adenosine diphosphate ribose polymerase (PARP1), cysteine protease-3 (caspase-3) and Bcl-2-associated X protein (Bax), and significantly inhibited the expression of anti-apoptotic protein B lymphoma-2 (Bcl-2) (P<0.05).【Conclusion】 miR-188 could significantly inhibit the proliferation and migration of 4T1 cells and promote the apoptosis of 4T1 cells, indicating that miR-188 could be used as a tumor regulator to inhibit the progression and metastasis potential of breast cancer.

Key words: breast cancer; 4T1 cells; miR-188; proliferation; apoptosis

中图分类号:

Q527

朱新颖, 郭帅, 战晓燕, 钟登科, 郑江平, 滑志民, 李天顺. miR-188对小鼠乳腺癌细胞增殖、迁移及凋亡的影响[J]. 中国畜牧兽医, 2022, 49(3): 1153-1161.

ZHU Xinying, GUO Shuai, ZHAN Xiaoyan, ZHONG Dengke, ZHENG Jiangping, HUA Zhimin, LI Tianshun. Effects of miR-188 on the Proliferation, Migration and Apoptosis of Mouse Breast Cancer Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1153-1161.

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