黄癸素通过ZFPM2-AS1/miR-519e-5p对神经母细胞瘤细胞增殖和凋亡的影响

The effects of luteolin on the proliferation and apoptosis of neuroblastoma cells through ZFPM2-AS1/miR-519e-5p

目的探讨黄癸素(luteolin)是否通过长链非编码RNA(long noncoding RNA,LncRNA)ZFPM2反义RNA 1(ZFPM2-AS1)/微小RNA(miRNA)-519e-5p轴影响神经母细胞瘤细胞的增殖和凋亡。方法将神经母细胞瘤细胞分为对照组,黄癸素低、中、高剂量组(1、2、8 mg·L^(-1)黄癸素),si-NC组(转染si-NC),si-ZFPM2-AS1组(转染si-ZFPM2-AS1),黄癸素高剂量+pcDNA组(8 mg·L^(-1)黄癸素处理前转染pcDNA),黄癸素高剂量+pcDNA-ZFPM2-AS1组(8 mg·L^(-1)黄癸素处理前转染pcDNA-ZFPM2-AS1),miR-NC组(转染miR-NC),miR-519e-5p组(转染miR-519e-5p mimic)。采用细胞计数试剂盒8(CCK-8)法、集落形成实验、流式细胞术、免疫印迹实验(Western blotting)和定量逆转录聚合酶链反应(qRT-PCR)检测神经母细胞瘤细胞的活力、集落形成、凋亡率、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)和ZFPM2-AS1、miR-519e-5p表达。生物信息学StarBase软件与双荧光素酶报告实验分析ZFPM2-AS1与miR-519e-5p之间的靶向结合。结果与对照组或黄癸素低剂量组相比,黄癸素中剂量组和黄癸素高剂量组神经母细胞瘤细胞OD值、集落形成数、Bcl-2蛋白、ZFPM2-AS1表达量降低,细胞凋亡率、Bax蛋白、miR-519e-5p表达量升高(P<0.05),对照组与黄癸素低剂量组各指标比较差异不显著。si-ZFPM2-AS1组神经母细胞瘤细胞中miR-519e-5p表达量、凋亡率和Bax蛋白表达量比si-NC组高,OD值、集落形成数、Bcl-2蛋白表达量比si-NC组低(P<0.05)。与黄癸素高剂量+pcDNA组相比,黄癸素高剂量+pcDNA-ZFPM2-AS1组神经母细胞瘤细胞miR-519e-5p表达量、凋亡率、Bax蛋白表达量降低,OD值、集落形成数、Bcl-2蛋白表达量升高(P<0.05)。ZFPM2-AS1可以靶向miR-519e-5p。miR-519e-5p组神经母细胞瘤细胞OD值、集落形成数、Bcl-2蛋白表达量比miR-NC组低,凋亡率、Bax蛋白表达量比miR-NC组高(P<0.05)。结论黄癸素可以通过调控ZFPM2-AS1/miR-519e-5p轴抑制神经母细胞瘤细胞的增殖并诱导其凋亡。

Objective To investigate whether luteolin affects the proliferation and apoptosis of neuroblastoma cells via the long non-coding RNA(LncRNA)ZFPM2 antisense RNA 1(ZFPM2-AS1)/microRNA(miRNA)-519 e-5 p axis.Methods The neuroblastoma cells were divided into control groups,low,medium and high doses of luteolin group(1,2,8 mg·L^(-1)luteolin),si-NC group(transfected with si-NC),si-ZFPM2-AS1 group(transfected with si-ZFPM2-AS1),high-dose luteolin+pcDNA group(transfected with pcDNA before 8 mg·L^(-1)luteolin treatment),high dose luteolin+pcDNA-ZFPM2-AS1 group(transfected with 8 mg·L^(-1)luteolin before treatment pcDNA-ZFPM2-AS1),miR-NC group(transfected miR-NC),miR-519 e-5 p group(transfected miR-519 e-5 p mimic).Cell counting kit 8(CCK-8)method,colony formation experiment,flow cytometry,western blotting and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)were used to detect the neuroblastoma cell viability,colony formation,apoptosis rate,expression levels of Bcl-2 related X protein(Bax),B-cell lymphoma/leukemia-2(Bcl-2),ZFPM2-AS1 and miR-519 e-5 p.Bioinformatics StarBase software and dual luciferase report experiment were employed to analyze the targeted binding between ZFPM2-AS1 and miR-519 e-5 p.Results Compared with the control group or the low-dose flavonoid group,the OD value,colony formation number,Bcl-2 protein,and ZFPM2-AS1 expression of neuroblastoma cells in the flavonoid medium-dose group and the flavonoid high-dose group decreased,and the apoptosis rate,Bax protein,miR-519 e-5 p expression increased(P<0.05).There was no significant difference in each index between the control group and the low-dose flavonoids group.The expression of miR-519 e-5 p,apoptosis rate and Bax protein expression in neuroblastoma cells of the si-ZFPM2-AS1 group were higher than those of the si-NC group,and the OD value,the number of colonies formed,and the expression of Bcl-2 protein were lower than those of si-NC group(P<0.05).Compared with the high-dose of luteolin+pcDNA group,miR-519 e-5 p expression,apoptosis rate,and Bax protein expression of neuroblastoma cell in the high-dose of luteolin+pcDNA-ZFPM2-AS1 group decreased,while the OD value,the number of colonies formed,and the expression of Bcl-2 protein increased(P<0.05).ZFPM2-AS1 can target miR-519 e-5 p.The OD value,colony formation number,and Bcl-2 protein expression of neuroblastoma cell in the miR-519 e-5 p group were lower than those in the miR-NC group,and the apoptosis rate and Bax protein expression were higher than those in the miR-NC group(P<0.05).Conclusion Luteolin can inhibit the proliferation and induce the apoptosis of neuroblastoma cells by regulating the ZFPM2-AS1/miR-519 e-5 p axis.