长链非编码RNA MEG3对鼻咽癌细胞增殖和侵袭的影响

Influences of long chain non-coded RNA MEG3 on the proliferation and invasion of nasopharyngeal carcinoma cells

目的探究长链非编码RNA母系表达基因3(MEG3)对鼻咽癌细胞增殖和侵袭的影响。方法体外培养鼻咽癌细胞系5-8F、CNE-2,转染MEG3分为空白对照组(转染试剂转染)、阴性转染组(MEG3阴性对照质粒转染)、MEG3转染组(MEG3 mimis质粒转染)。实时定量PCR(qRT-PCR)检测MEG3 mRNA表达;MTT法检测细胞增殖情况;平板细胞克隆实验检测菌落形成情况;流式细胞仪检测细胞凋亡以及周期分布情况;裸鼠模型肿瘤形成实验评估体内肿瘤生长情况;蛋白免疫印迹法(WB)检测细胞中Snail、N-钙黏着蛋白(N-cadherin)、波形蛋白(Vimentin)、E-钙黏着蛋白(E-cadherin)蛋白表达情况。结果与正常鼻咽上皮细胞系NP69相比,5-8F、CNE-2细胞中MEG3 mRNA表达均降低(P<0.05)。在5-8F、CNE-2细胞中,与空白对照组、阴性转染组相比,MEG3转染组细胞中MEG3mRNA表达升高(P<0.05)。在5-8F、CNE-2细胞中,随着细胞培养时间延长,MEG3转染组细胞抑制率逐渐升高;同一时间点,与空白对照组、阴性转染组相比,MEG3转染组细胞抑制率均升高(P<0.05)。与空白对照组、阴性转染组相比,MEG3转染组细胞菌落数量、细胞迁移、侵袭数量均降低(P<0.05)。与空白组、阴性转染组对比,MEG3转染组细胞凋亡率、G1期DNA含量均升高,差异有统计学意义(P<0.05)。与空白组、阴性转染组对比,在1、2周MEG3转染组肿瘤体积均变小(P<0.05)。与空白组、阴性转染组相比,MEG3转染组Snail、N-cadherin、Vimentin蛋白表达均降低,E-cadherin蛋白表达升高(P<0.05)。结论 MEG3在鼻咽癌中表达降低,MEG3表达升高后可抑制鼻咽癌细胞的增殖、侵袭、迁移,使细胞周期停留在G1期,促进细胞凋亡,其机制可能与抑制上皮细胞-间充质转化有关。

Objective To investigate the influences of long chain non-coded RNA matrilineal expression gene 3（ MEG3） on the proliferation and invasion of nasopharyngeal carcinoma cells. Methods Cultured the nasopharyngeal carcinoma cell line 5-8F and CNE-2 in vitro,and they were divided into blank control group（ transfection reagent transfection）,negative transfection group（ MEG3 negative control plasmid transfection）,MEG3 transfection group（ MEG3 MIMIS plasmid transfection） after the transfection of MEG3,real-time quantitative PCR（ qRT-PCR） was used to detect MEG3 mRNA expression,MTT assay was used to detect cell proliferation; the clone formation test of flat cell was used to detect the colony formation; the flow cytometry was used to detect the cell apoptosis and the cycle distribution; the tumor formation experiment in nude mice model was used to evaluate the tumor growth in the body; Western blotting（ WB） was used to detect the expressions of Snail,N-cadherin,Vimentin and E-cadherin in cells. Results Compared with the normal nasopharyngeal epithelial cell line NP69,the expression of MEG3 mRNA in 5-8 F and CNE-2 cells was decreased（ P〈0. 05）. Compared with the blank control group and the negative transfection group,In 5-8 F and CNE-2 cells,compared with the blank control group and the negative transfection group,the expression of MEG3 mRNA in the cells of MEG3 transfected group was increased（ P〈0. 05）. In 5-8 F and CNE-2 cells,with the prolongation of cell culture time,the cell inhibition rate of MEG3 transfected group increased gradually,at the same time point,compared with the blank control group and negative transfection group,the cell inhibition rate of MEG3 transfected group was increased（ P〈0. 05）. Compared with the blank control group and the negative transfection group,the numbers of cell colonies,cell migration and invasion in the MEG3 transfected group were decreased（ P〈0. 05）. Compared with the blank group and negative transfection group,the apoptosis rate and the content of DNA in the G1 phase of MEG3 transfected group were increased,and the differences were statistically significant（ P〈0. 05）. Compared with the blank group and negative transfection group,the tumor volume in the MEG3 transfected group was smaller in the 1 and 2 weeks（ P〈0. 05）. Compared with the blank group and the negative transfection group,the expressions of Snail,N-cadherin and vimentin protein in MEG3 transfection group were decreased,and the expression of E-cadherin protein was increased（ P〈0. 05）. Conclusion The expression of MEG3 in nasopharyngeal carcinoma is reduced,the increase of MEG3 expression can inhibit the proliferation,invasion and migration of nasopharyngeal carcinoma cells,can make the cell cycle stay in the G1 phase,promote cell apoptosis,the mechanism may be related to the inhibition of epithelial cell mesenchymal transition（ EMT）.