miR-485-5p通过下调FOSL2表达影响鼻咽癌细胞增殖和凋亡

Experimental study on miR-485-5p affecting proliferation and apoptosis of nasopharyngeal carcinoma cells by down-regulating FOSL2 expression

目的探讨微小RNA-485-5p(miR-485-5p)对鼻咽癌细胞增殖和凋亡的影响及作用机制。方法选取2016年5月~2018年4月本院行手术治疗的36例鼻咽癌患者癌组织为研究对象,对应癌旁组织距离肿瘤边缘>3cm作为对照组。RT-qPCR检测鼻咽癌组织和对应的癌旁组织中miR-485-5p和FOS样抗原2(FOSL2)mRNA水平,Pearson相关性分析鼻咽癌组织中miR-485-5p和FOSL2 mRNA表达相关性。以人永生化鼻咽上皮细胞系NP69为对照,RT-qPCR检测鼻咽癌细胞系6-10B和5-8F中miR-485-5p和FOSL2 mRNA表达水平,Western Blot检测FOSL2蛋白水平。将5-8F细胞分为miR-NC组、miR-485-5p组、si-NC组、si-FOSL2组、miR-485-5p+pcDNA3.0组和miR-485-5p+pcDNA3.0-FOSL2组,MTT实验、流式细胞术、Western Blot分别检测各组5-8F细胞增殖、凋亡及Cyclin D1、p21、Bcl-2和Bax表达。双荧光素酶报告基因实验验证miR-485-5p与FOSL2调控关系。结果与癌旁组织比较,鼻咽癌组织中miR-485-5p水平降低(P<0.05),FOSL2 mRNA表达水平升高(P<0.05),且鼻咽癌组织中miR-485-5p与FOSL2 mRNA表达水平呈负相关(P<0.05)。与NP69细胞比,鼻咽癌细胞系6-10B和5-8F中miR-485-5p表达水平显著降低(P<0.05),FOSL2 mRNA和蛋白表达水平显著升高(P<0.05)。与miR-NC组比较,miR-485-5p组5-8F细胞存活率、CyclinD1和Bcl-2蛋白水平降低(P<0.05),细胞凋亡率、p21和Bax蛋白水平升高(P<0.05)。与si-NC组比较,si-FOSL2组5-8F细胞存活率及CyclinD1和Bcl-2蛋白水平降低(P<0.05),细胞凋亡率、p21和Bax蛋白水平升高(P<0.05)。miR-485-5p在5-8F细胞中负调控FOSL2表达。与miR-485-5p+pcDNA3.0组比较,miR-485-5p+pcDNA3.0-FOSL2组5-8F细胞存活率及CyclinD1和Bcl-2蛋白水平升高(P<0.05),细胞凋亡率、p21和Bax蛋白水平降低(P<0.05)。结论miR-485-5p在鼻咽癌组织和细胞中表达降低,FOSL2表达升高;过表达miR-485-5p通过靶向负调控FOSL2抑制鼻咽癌细胞增殖,并促进其凋亡。

Objective To investigate the effects and mechanism of microRNA-485-5p(miR-485-5p)on proliferation and apoptosis of nasopharyngeal carcinoma cells.Methods Real-time quantitative PCR(qRT-PCR)was used to detect levels of miR-485-5p and FOS-like antigen 2(FOSL2)mRNA in nasopharyngeal carcinoma tissues and cell lines 6-10B and 5-8F,Western Blot was used to detect levels of FOSL2 protein in cells,and Pearson correlation was used to analyze the correlation between miR-485-5p and FOSL2 mRNA expression in nasopharyngeal carcinoma tissues.To construct 5-8F cells overexpressing miR-485-5p or low expressing FOSL2,miR-485-5p mimic or FOSL2 small interfering RNA was transfected into 5-8F cells.MTT,flow cytometry,and Western Blot were used to detect the effects of overexpression of miR-485-5p or low expression of FOSL2 on 5-8F cell proliferation,apoptosis and expression of Cyclin D1,p21,Bcl-2 and Bax protein,respectively.The dual luciferase reporter gene assay verified the relationship between miR-485-5p and FOSL2.Results Compared with adjacent cancer tissues,the expression level of miR-485-5p reduced in nasopharyngeal carcinoma tissues(P<0.05),while the expression level of FOSL2 mRNA increased(P<0.05).There was a negative correlation between miR-485-5p and FOSL2 mRNA expression in nasopharyngeal carcinoma tissues(P<0.05).Compared with NP69 cells,the expression levels of miR-485-5p in nasopharyngeal carcinoma cell lines 6-10B and 5-8F decreased(P<0.05),while the expression levels of FOSL2 mRNA and protein increased(P<0.05).Compared with the miR-NC group,the survival rate of 5-8F cells and the protein levels of CyclinD1 and Bcl-2 in the miR-485-5p group decreased(P<0.05),while the apoptosis rate and the protein levels of p21 and Bax increased(P<0.05).Compared with the si-NC group,the survival rate of 5-8F cells and the protein levels of CyclinD1 and Bcl-2 in the si-FOSL2 group decreased(P<0.05),while the apoptosis rate and the protein levels of p21 and Bax increased(P<0.05).miR-485-5p negatively regulated FOSL2 expression in 5-8F cells.Compared with the miR-485-5p+pcDNA3.0 group,the survival rate of 5-8F cells and the protein levels of CyclinD1 and Bcl-2 in the miR-485-5p+pcDNA3.0-FOSL2 group increased(P<0.05),while the apoptosis rate and the protein levels of p21 and Bax decreased(P<0.05).Conclusion The expression of miR-485-5p decreased in nasopharyngeal carcinoma tissues and cells,while the expression of FOSL2 increased;overexpression of miR-485-5p inhibits the proliferation of nasopharyngeal carcinoma cells and promotes their apoptosis through targeted negative regulation of FOSL2.Over-expressing miR-485-5p may inhibit proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by targeting negatively regulated FOSL2.