miR-325-3p通过靶向FOXM1调控乳腺癌细胞增殖、侵袭、迁移及上皮-间质转化的实验研究

doi:10.20047/j.issn1673-7210.2023.17.04

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摘要目的探讨miR-325-3p通过靶向叉头框蛋白（FOX）M1调控乳腺癌细胞增殖、侵袭、迁移及上皮间质转化（EMT）。方法培养MCF-7细胞，根据转染质粒不同分为NC组（转染miRNA mimic阴性对照）、miR-325-3p组（转染miR-325-3p mimic）、miR-NC组（转染miRNA inhibitor阴性对照）、miR-325-3p inhibitor组（转染miR-325-3p inhibitor）、pcDNA3.1组（转染pcDNA3.1空质粒）、FOXM1组（转染pcDNA3.1-FOXM1）、si-NC组（转染si-NC无意义序列）、si-FOXM1组（转染si-FOXM1质粒）、miR-325-3p inhibitor+si-FOXM1组（转染miR-325-3p inhibitor+si-FOXM1质粒）及miR-325-3p inhibitor+si-NC组（转染miR-325-3p inhibitor+si-NC无意义序列）。CCK-8检测细胞24、48、72 h增殖能力，Transwell实验检测细胞侵袭能力，划痕实验检测细胞迁移能力，RT-qPCR检测细胞miR-325-3p、FOXM1 mRNA水平，Western blot检测细胞FOXM1、E-cadherin、N-cadherin及Vimentin蛋白水平，萤光素酶实验检测miR-325-3p与FOXM1的靶向关系。结果 miR-325-3p组miR-325-3p表达水平高于NC组（P＜0.01），miR-325-3p inhibitor组miR-325-3p水平低于miR-NC组（P＜0.01）。48、72 h，miR-325-3p组光密度（OD）值、侵袭细胞数及迁移率低于NC组（P＜0.01），miR-325-3p inhibitor组OD值，侵袭细胞数及迁移率高于miR-NC组（P＜0.01）。miR-325-3p组E-cadherin蛋白表达水平高于NC组，Vimentin、N-cadherin蛋白表达水平低于NC组（P＜0.01）；miR-325-3p inhibitor组E-cadherin蛋白表达水平低于miR-NC组，Vimentin、N-cadherin蛋白表达水平高于miR-NC组（P＜0.01）。FOXM1组细胞FOXM1蛋白、mRNA表达水平高于pcDNA3.1组（P＜0.01）；si-FOXM1组FOXM1蛋白、mRNA表达水平低于si-NC组（P＜0.01）。48、72 h，FOXM1组OD值、侵袭细胞数及迁移率高于pcDNA3.1组（P＜0.01）；si-FOXM1组OD值，侵袭细胞数及迁移率低于si-NC组（P＜0.01）。FOXM1组E-cadherin蛋白表达水平低于pcDNA3.1组，Vimentin、N-cadherin蛋白表达水平高于pcDNA3.1组（P＜0.01）；si-FOXM1组E-cadherin蛋白表达水平高于si-NC组，Vimentin、N-cadherin蛋白表达水平低于si-NC组（P＜0.01）。48、72 h，miR-325-3p inhibitor+si-FOXM1组OD值、侵袭细胞数及迁移率低于miR-325-3p inhibitor+si-NC组（P＜0.01）。miR-325-3p inhibitor+si-FOXM1组E-cadherin蛋白表达水平高于miR-325-3p inhibitor+si-NC组，Vimentin、N-cadherin蛋白表达水平低于miR-325-3p inhibitor+si-NC组（P＜0.01）。miR-325-3p组FOXM1蛋白、mRNA表达水平低于NC组（P＜0.01）；miR-325-3p inhibitor组FOXM1蛋白、mRNA表达水平高于miR-NC组（P＜0.01）。NC+WT-FOXM1组与NC+MUT-FOXM1组萤光素酶活性比较，差异无统计学意义（P＞0.05）；miR-325- 3p+WT-FOXM1组萤光素酶活性低于miR-325-3p+MUT-FOXM1组（P＜0.01）。结论 miR-325-3p在乳腺癌组织中低表达，过表达miR-325-3p可靶向FOXM1抑制乳腺癌细胞的增殖、侵袭、迁移及EMT。

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	吴沉昊　　王婧　　周蓓　　万珍琳

关键词 ： miR-325-3p, 叉头框蛋白M1, 乳腺癌, 上皮间质转化

Abstract：Objective To investigate the regulation of miR-325-3p on proliferation, invasion, migration, and epithelial mesenchymal transformation (EMT) of breast cancer cells by targeting forkhead box protein (FOXM1). Methods MCF-7 cells were cultured and divided into NC group (transfected miRNA mimic negative control), miR-325-3p group (transfected miR-325-3p mimic), miR-NC group (transfected miRNA inhibitor negative control), miR-325-3p inhibitor group (transfected with miR-325-3p inhibitor), pcDNA3.1 group (transfected with pcDNA3.1 empty plasmid), FOXM1 group (transfected with PCDNA3.1-FoxM1), si-NC group (transfected with si-NC meaningless sequence), si-FOXM1 group (transfected with si-FOXM1 plasmid), miR-325-3p inhibitor+si-FOXM1 group (transfected with miR-325-3p inhibitor+si-FOXM1 plasmid), and miR-325-3p inhibitor+si-NC group (transfected with miR-325-3p inhibitor+si-NC meaningless sequence). CCK-8 was used to detect cell proliferation at 24, 48 h, and 72 h, Transwell assay was used to detect cell invasion, scratch assay was used to detect cell migration, the levels of miR-325-3p and FOXM1 mRNA were detected by RT-qPCR, the levels of FOXM1, E-cadherin, N-cadherin, and Vimentin protein were detected by Western blot, the targeting relationship between miR-325-3p and FOXM1 was detected by luciferase assay. Results The expression level of miR-325-3p in miR-325-3p group was higher than that in NC group (P＜0.01), and the level of miR-325-3p in miR-325-3p inhibitor group was lower than that in miR-NC group (P＜0.01). At 48 h and 72 h, optical density (OD) value, invasion cell number, and migration of miR-325-3p group were lower than those of NC group (P＜0.01), while OD value, invasion cell number, and migration of miR-325-3p inhibitor group were higher than those of miR-NC group (P＜0.01). The expression level of E-cadherin protein in miR-325-3p group was higher than that in NC group, while the expression levels of Vimentin and N-cadherin protein in miR-325-3P group were lower than those in NC group (P＜0.01); the expression level of E-cadherin protein in miR-325-3p inhibitor group was lower than that in miR-NC group, while the expression levels of Vimentin and N-cadherin protein in miR-NC group were higher than those in miR -NC group (P＜0.01). The expression levels of FOXM1 protein and mRNA in FOXM1 group were higher than those in pcDNA3.1 group (P＜0.01); the expression levels of FOXM1 protein and mRNA in si-FOXM1 group were lower than those in si-NC group (P＜0.01). At 48 h and 72 h, OD value, invasion cell number, and migration in FOXM1 group were higher than those in pcDNA3.1 group (P＜0.01); OD value, invasion cell number, and migration of si-FOXM1 group were lower than those of si-NC group (P＜0.01). The expression level of E-cadherin protein in FOXM1 group was lower than that in pcDNA3.1 group, while the expression levels of Vimentin and N-cadherin protein in FXOM1 group were higher than those in pcDNA3.1 group (P＜0.01); the expression level of E-cadherin protein in si-FOXM1 group was higher than that in si-NC group, while the expression levels of Vimentin and N-cadherin protein were lower than those in Si-NC group (P＜0.01). At 48 h and 72 h, OD value, invasion cell number, and migration of miR-325-3p inhibitor+si-FOXM1 group were lower than those of miR-325-3p inhibitor+si-NC group (P＜0.01). The expression level of E-cadherin protein in miR-325-3p inhibitor+si-FOXM1 group was higher than that of miR-325-3p inhibitor+si-NC group, while the expression levels of Vimentin and N-cadherin protein were lower than those of miR-325-3p inhibitor+si-NC group (P＜0.01). The expression levels of FOXM1 protein and mRNA in miR-325-3p group were lower than those in NC group (P＜0.01); the expression levels of FOXM1 protein and mRNA of miR-325-3p inhibitor group were higher than those of miR-NC group (P＜0.01). There was no significant difference in luciferase activity between NC+WT-FOXM1 group and NC+MUT- FOXM1 group (P＞0.05); the luciferase activity of miR-325-3p+WT-FOXM1 group was lower than that of miR-325-3p+ MUT-FOXM1 group (P＜0.01). Conclusion miR-325-3p is underexpressed in breast cancer tissues, and overexpression of miR-325-3p can inhibit proliferation, invasion, migration, and EMT of breast cancer cells by targeting FOXM1.

Key words： miR-325-3p Forkhead box protein M1 Breast cancer Epithelial mesenchymal transition

基金资助:海南省卫生健康行业科研项目（琼卫科教〔2020〕9号）。

引用本文:

吴沉昊　　王婧　　周蓓　　万珍琳. miR-325-3p通过靶向FOXM1调控乳腺癌细胞增殖、侵袭、迁移及上皮-间质转化的实验研究[J]. 中国医药导报, 2023, 20(17): 19-25.
WU Chenhao WANG Jing ZHOU Bei WAN Zhenlin. Experimental study of miR-325-3p regulating the proliferation, invasion, migration, and epithelial mesenchymal transformation of breast cancer cells by targeting FOXM1. 中国医药导报, 2023, 20(17): 19-25.

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