微小核糖核酸-205-5p靶向真核细胞翻译起始因子4E对人乳腺癌细胞MCF-7增殖、侵袭及上皮间质转化的影响

doi:10.19803/j.1672-8629.2022.03.06

摘要/Abstract

摘要： 目的探讨微小核糖核酸-205-5p（miR-205-5p）靶向真核细胞翻译起始因子4E（eIF4E）对人乳腺癌细胞MCF-7增殖、侵袭及上皮间质转化的影响,为乳腺癌（MC）新型靶向治疗药物的研发提供参考。方法对MCF-7进行培养,miR-205-5p转染mimic-NC及miR-205-5p mimic以验证miR-205-5p与eIF4E靶向关系,同时将培养好的MCF-7细胞分为对照组（正常培养的MCF-7细胞）、mimic-NC组、pcDNA组、miR-205-5p mimic组、miR-205-5p组、eIF4E-pcDNA组、miR-205-5p+eIF4E-pcDNA组,分析miR-205-5p靶向eIF4E对MCF-7细胞增殖、迁移和上皮间质转化的影响。结果 miR-205-5p mimic组的miR-205-5p相对表达量显著高于对照组和mimic-NC组,而eIF4E的mRNA表达水平则显著低于对照组和mimic-NC组（P<0.05）,而eIF4E野生型、mimic均为阳性的Lucifera-se activity（R/F ratio）更低。eIF4E-pcDNA组eIF4E蛋白和mRNA相对表达量较对照组、pcDNA组明显高（P<0.05）。miR-205-5p组MCF-7细胞阳性率、菌落分布率和Ki67、PCNA蛋白表达水平较对照组、eIF4E-pcDNA组、miR-205-5p+eIF4E-pcDNA组明显低（P<0.05）。miR-205-5p组侵袭性细胞阳性率和侵袭性细胞数量较对照组、miR-205-5p+eIF4E-pcDNA组、eIF4E-pcDNA组明显低（P<0.05）,而eIF4E-pcDNA组侵袭性细胞数量最高。miR-205-5p组MCF-7细胞上皮间质标志物E-Cadherin、N-Cadherin、Vimentin阳性表达率和蛋白表达水平明显低于对照组、miR-205-5p+eIF4E-pcDNA组、eIF4E-pcDNA组（P<0.05）,而eIF4E-pcDNA组上皮间质标志物E-Cadherin、N-Cadherin、Vimentin阳性表达率和蛋白表达水平显著高于其他组（P<0.05）。结论 miR-205-5p负靶向eIF4E能有效抑制MCF-7增殖、侵袭及上皮间质转化,有望为MC新型基因靶向治疗药物的研发提供重要参考。

关键词: miR-205-5p, eIF4E, 人乳腺癌细胞, MCF-7

Abstract: Objective To investigate the influence of micro ribonucleic acid-205-5p (miR-205-5p) targeting eukaryotic initiation factor 4E (eIF4E) on the proliferation, invasion and epithelial-mesenchymal transition of human breast cancer MCF-7 cells so as to provide reference for the development of new targeted therapeutic drugs against mammary cancer (MC). Methods MCF-7 cells were cultured, and miR-205-5p was transfected into mimic-NC and miR-205-5p mimic to verify the targeting relationship between miR-205-5p and eIF4E. Meanwhile, the cultured MCF-7 cells were divided into the control group (normally cultured MCF-7 cells), mimic-NC group, pcDNA group, miR-205-5p mimic group, miR-205-5p group, eIF4E-pcDNA group, and miR-205-5p+eIF4E-pcDNA group. The effect of miR-205-5p targeting eIF4E on the proliferation, migration and epithelial-mesenchymal transition of MCF-7 cells was analyzed. Results The relative expression level of miR-205-5p was significantly higher in the miR-205-5p mimic group than in the control group and mimic-NC group, while the mRNA expression level of eIF4E was significantly lower (P<0.05). The luciferase activity (R/F ratio) of eIF4E wild-type and mimic positive group was lower. The relative expressions of eIF4E protein and mRNA were significantly higher in the eIF4E-pcDNA group than in the control group and pcDNA group (P<0.05). The positive rate of MCF-7 cells, colony distribution rate, Ki67 and PCNA protein expression levels in the miR-205-5p group were significantly lower than those in the control group, eIF4E-pcDNA group, and miR-205-5p+eIF4E-pcDNA group (P<0.05). The positive rate and number of aggressive cells in the miR-205-5p group were significantly lower or smaller than those in the control group, miR-205-5p+eIF4E-pcDNA group, and eIF4E-pcDNA group (P<0.05), but the number of aggressive cells was the largest in eIF4E-pcDNA group. The positive expression rates and protein expression levels of the epithelial mesenchymal markers E-Cadherin, N-Cadherin, and Vimentin of MCF-7 cells were significantly lower in the miR-205-5p group than in the control group, miR-205-5p+eIF4E-pcDNA group, and eIF4E-pcDNA group (P<0.05), but significantly higher in the eIF4E-pcDNA group than in the other groups (P<0.05). Conclusion miR-205-5p can effectively inhibit the proliferation, invasion and epithelial-mesenchymal transition of human breast cancer MCF-7 cells by negatively targeting eIF4E, which is expected to provide reference for the development of new gene-targeted therapeutic drugs against MC.

Key words: miR-205-5p, eIF4E, human breast cancer cell, MCF-7

中图分类号:

R979.1

张娜贤, 刘柳, 李刚, 刘琳瑞, 李元颖. 微小核糖核酸-205-5p靶向真核细胞翻译起始因子4E对人乳腺癌细胞MCF-7增殖、侵袭及上皮间质转化的影响[J]. 中国药物警戒, 2022, 19(3): 259-264.

ZHANG Naxian, LIU Liu, LI Gang, LIU Linrui, LI Yuanying. Influence of miR-205-5p targeting eIF4E on proliferation, invasion and epithelial-mesenchymal transition of MCF-7 cells[J]. Chinese Journal of Pharmacovigilance, 2022, 19(3): 259-264.

参考文献

[1] TAN LJ, WANG M, ZHU Y.Research progress of adverse reactions of traditional Chinese medicine injections[J]. China Journal of Chinese Medica(中国中药杂志), 2014, 39(20): 3889-3898.
[2] LE XC, WANG Y.Analytical methods in toxicology[J]. Anal Chem, 2014, 86(24): 11929.
[3] YAN HZ, MA HX, YANG XD.Current research progress on the calculation methods of LD50 and similar biological effect indicators[J]. World Notes on Antibiotics[国外医药(抗生素分册)], 2012, 33(2): 62-66.
[4] VAN GERVEN JM, COHEN AF.Integrating data from the investigational medicinal product dossier/investigator's brochure. a new tool for translational integration of preclinical effects[J]. British Journal of Clinical Pharmacology, 2018, 84(7): 1457-1466.
[5] BUCKLEY LA, DORATO MA.High dose selection in general toxicity studies for drug development: a pharmaceutical industry perspective[J]. Regulatory Toxicology and Pharmacology, 2009, 54(3): 301-307.
[6] National Toxicology Program.Guidelines for carcinogen bioassay in small rodents[J]. Natl Cancer Inst Carcinog Tech Rep Ser, 1976, 1:1-65.
[7] MO ZH, CHEN MX, WANG PC, et al. Acute toxicity of huoxin pill in mice[J/OL]. Chinese Journal of Pharmacovigilance(中国药物警戒):1-12[2021-01-26].http://kns.cnki.net/kcms/detail/11.5219.R.20210115.1631.030.html.
[8] HUANG HH, CHEN JH.Screening of the effective part of ficus microcarpa l.f. on relieving cough and reducing sputum and analysis of chemical constituents by UHPLC-MS[J]. Guiding Journal of Traditional Chinese Medicine and Pharmacy(中医药导报), 2017, 23(1): 54-58.
[9] HUANG HH, CHEN JH.Simultaneous determination of three flavo-noids in different parts of ficus microcarpa L.f. by UPLC[J]. Chinese Journal of Pharmaceutical Analysis(药物分析杂志), 2017, 37(1): 161-165.
[10] YUAN R, SUN LL, GUAN SY, et al.Fingerprints of dry extracts of ficus herba and keteling capsules by hplc and analysis of chemical constituents by LC-Q-TOF-MS[J]. Chinese Journal of Experimental Traditional Medical Formulae(中国实验方剂学杂志), 2016, 22(3): 57-62.
[11] YE ZG.Research thoughts on toxicology of traditional Chinese medicine[J]. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology(世界科学技术),2003(6):1-5,77.
[12] LV XJ, WANG LY, ZHANG J, et al.Single-dose toxicity experiment of Changbai Mountain Ginseng in mice[J]. Ginseng Research(人参研究), 2012, 24(4): 8-10.
[13] LIU YM, JIANG DW, LI KX, et al.Experimental studies on the acute toxicity and chronic toxicity of Yinshen capsules[J]. China Pharmacist(中国药师), 2020, 23(7): 1457-1461.
[14] XIA LJ, YIN XH, ZHANG CD, et al.Study on 6-month repeated dose toxicity of sanqi yaojiu via oral administration in rats[J]. Chinese Journal of Pharmacovigilance(中国药物警戒), 2020, 17(10): 653-658.
[15] CHEN B. Observation on the clinical effect of Keteling in treating chronic cough and asthma[J]. Electronic Journal of Clinical Medical Literature(临床医药文献电子杂志), 2017, 4(60): 11861, 11864.
[16] LIANG JX.Clinical study on Keteling in the treatment of chronic bronchitis and cough[J]. Electronic Journal of Clinical Medical Literature(临床医药文献电子杂志), 2017, 4(59): 11644-11645.
[17] HUANG FH, WANG QL.Interpretation of guidance on single dose toxicity study for pharmaceuticals[J]. Chinese Journal of New Drugs(中国新药志杂志), 2015, 24(4): 386-389, 399.
[18] YANG JP.Improvement of traditional paraffin section preparation methods[J] Journal of Biology(生物学杂志), 2006, 23(1):45-46.
[19] WU CQ, WEI QZ, WANG JX et al. Study on the toxicity of single-dose Zhuang medicine Guangshiwei and its effect on blood coagulation and antitussive effect[J]. Chinese Journal of Traditional Medical Science and Technology(中国中医药科技), 2020, 27(1): 28-31.
[20] HU JW, LU SM, JUN CP, et al.A probe into normal levels of hematological and biochemical indexes in 10 kinds of common spf rats and mice[J]. Laboratory Animal Science(实验动物科学), 2007(2): 5-10.
[21] QIAO YL.Curative effect of salvia miltiorrhiza phenolic acid combined with ulinastatin on acute respiratory distress syndrome and its effect on biochemical indices of patients[J]. China Pharmacist(中国药师), 2016, 19(9): 1713-1715.
[22] GAO Z, YU C, LIANG H, et al.Andrographolide derivative CX-10 ameliorates dextran sulphate sodium-induced ulcerative colitis in mice: Involvement of NF-κB and MAPK signalling pathways[J]. Int Immunopharmacol, 2018, 57: 82-90.
[23] LIU X, WANG J.Anti-inflammatory effects of iridoid glycosides fraction of Folium syringae leaves on TNBS-induced colitis in rats[J]. Journal of Ethnopharmacology, 2011, 133(2): 780-787.