雪松醇调节miR-503-5p/TCF3轴对乳腺癌细胞增殖、迁移和侵袭的影响

Effect of cedarol on proliferation，migration and invasion of breast cancer cells by regulating miR-503-5p/TCF3 axis

关键词:  雪松醇  miR-503-5p/TCF3轴  乳腺癌  增殖  迁移  侵袭

Key words:  cedarol  miR-503-5p/TCF3 axis  breast cancer  proliferation  migration  cell invasion

摘要:

[目的] 探讨雪松醇调节miR-503-5p/T细胞因子3（TCF3）轴对乳腺癌（BC）细胞增殖、迁移和侵袭的影响。[方法] 实时荧光定量聚合酶链式反应（qRT-PCR）、蛋白免疫印迹法（Western blot）分别检测BC组织和细胞中miR-503-5p及TCF3蛋白表达水平；以不同浓度的雪松醇（0、14、28、56、112、225 μmol/L）干预MCF7细胞，噻唑蓝比色法（MTT）法检测细胞增殖情况；将MCF7细胞分为：control组（正常培养）、雪松醇组（112 μmol/L）、inhibitor-NC（转染inhibitor-NC）、miR-503-5p inhibitor组（转染miR-503-5p inhibitor）。qRT-PCR检测细胞中miR-503-5p的表达水平；MTT法与胸腺嘧啶核苷类似物（Edu）染色和Transwell小室分别检测细胞增殖、迁移和侵袭；蛋白免疫印迹（Western blot）方法检测增殖细胞核抗原（PCNA）、基质金属蛋白酶-2（MMP-2）、TCF3蛋白的表达；双荧光素酶报告基因实验验证miR-503-5p和TCF3的靶向关系；小鼠体内肿瘤形成实验验证雪松醇对BC肿瘤生长及miR-503-5p/TCF3轴的影响。[结果] 在BC组织和细胞中，miR-503-5p表达水平降低，TCF3蛋白表达水平升高（P＜0.05）。雪松醇抑制MCF7细胞增殖、迁移和侵袭，降低MCF7细胞中PCNA、MMP-2、TCF3蛋白表达水平（P＜0.05），抑制miR-503-5p表达可逆转雪松醇对MCF7细胞增殖、迁移和侵袭的抑制作用（P＜0.05）；双荧光素酶报告基因实验证实miR-503-5p靶向调控MCF7的表达（P＜0.05）；小鼠荷瘤实验结果显示，雪松醇可降低移植瘤质量和体积，提高移植瘤中miR-503-5p水平，降低TCF3、MMP-2表达水平（P＜0.05）。[结论] 雪松醇能抑制BC细胞增殖、迁移和侵袭，其机制可能与调控miR-503-5p/TCF3轴有关，miR-503-5p/TCF3轴可能成为治疗BC的一种新靶点。

Abstract:

[Objective] To investigate the effect of cedarol on the proliferation，migration and invasion of breast cancer（BC） cells by regulating miR-503-5p/T cell factor 3（TCF3） axis. [Methods] The expression levels of miR-503-5p and TCF3 protein in BC tissues and cells were detected by real-time fluorescent quantitative PCR（qRT-PCR） and Western blot respectively；MCF7 cells were treated with different concentrations of cedarol（0，14，28，56，112，225 μmol/L）. Cell proliferation was detected by MTT method；MCF7 cells were grouped into control group（normal culture），cedarol group（112 μmol/L），inhibitor-NC（transfected with inhibitor-NC），miR-503-5p inhibitor group（transfected with miR-503-5p inhibitor）. The expression level of miR-503-5p in cells was detected by qRT-PCR；cell proliferation，migration and invasion were detected by MTT method，Edu staining and Transwell cell，respectively；Western blot was applied to detect the expression of proliferating cell nuclear antigen（PCNA），matrix metalloproteinase-2（MMP-2） and TCF3 proteins；the target relationship between miR-503-5p and TCF3 was verified by double luciferase reporter gene experiment；the effect of cedarol on the growth of BC tumor and the miR-503-5p/TCF3 axis was verified by the tumor formation experiment in mice. [Results] In BC tissues and cells，the expression level of miR-503-5p decreased and the expression level of TCF3 protein increased（P＜0.05）. Cedarol inhibited the proliferation，migration and invasion of MCF7 cells and decreased the expression of PCNA，MMP-2 and TCF3 proteins in MCF7 cells（P＜0.05），inhibition of miR-503-5p expression could reverse the inhibitory effects of cedarol on the proliferation，migration and invasion of MCF7 cells（P＜0.05）；the double luciferase reporter gene experiment confirmed that miR-503-5p targeted the expression of MCF7（P＜0.05）；the results of tumor-bearing experiment in mice showed that cedarol could reduce the weight and volume of transplanted tumors，increase the level of miR-503-5p in transplanted tumor，and reduce the level of TCF3 and MMP-2 expression（P＜0.05）. [Conclusion] Cedarol can inhibit the proliferation，migration and invasion of BC cells，and its mechanism may be related to the regulation of miR-503-5p/TCF3 axis，which may become a new target for the treatment of BC.