LncRNA NORAD靶向抑制miR-363-3p对多发性骨髓瘤细胞生物学行为的影响及机制
| 作者: |
1苑军伟,
1吴静,
1林静,
1罗广立
1 漯河市中心医院血液科，河南 漯河 462000 |
| 通讯: |
苑军伟
Email: wux0783@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2020.07.004 |
摘要
目的：探索长链非编码RNA NORAD(lncRNA NORAD)对多发性骨髓瘤细胞增殖和凋亡的影响及其与微小RNA-363-3p(miR-363-3p)的靶向关系。方法：RT-qPCR检测健康人成骨细胞hFOB1.19和多发性骨髓瘤细胞U266，LP-1，H929中NORAD和miR-363-3p表达。在U266细胞中转染si-NORAD，或共转染si-NORAD和anti-miR-363-3p，噻唑蓝(MTT)法检测细胞增殖，流式细胞术检测细胞凋亡，蛋白质印迹法检测细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶4(CDK4)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达。starBase软件预测结合双荧光素酶报告实验分析NORAD与miR-363-3p的靶向结合。结果：多发性骨髓瘤细胞系U266，LP-1和H929中NORAD表达明显上调(P<0.05)，miR-363-3p表达显著下调(P<0.05)。沉默NORAD表达显著降低48，72 h的U266细胞活性和cyclin D1，CDK4，Bcl-2蛋白水平(P<0.05)，明显提高细胞凋亡率和cleaved caspase-3，Bax蛋白表达量(P<0.05)。NORAD靶向调控miR-363-3p表达。抑制miR-363-3p表达逆转沉默NORAD表达对U266细胞增殖、cyclin D1，CDK4和Bcl-2表达的抑制作用，以及逆转沉默NORAD表达对细胞凋亡、cleaved caspase-3和Bax蛋白表达的促进作用。结论：LncRNA NORAD通过靶向miR-363-3p表达影响多发性骨髓瘤细胞增殖和凋亡。
关键词：
多发性骨髓瘤；lncRNA NORAD；miR-363-3p；增殖；凋亡
Effects of lncRNA NORAD targeted inhibition of miR-363-3p on biological behavior of multiple myeloma cells and its mechanisms
CorrespondingAuthor: YUAN Junwei Email: wux0783@163.com
DOI: 10.3978/j.issn.2095-6959.2020.07.004
Abstract
Objective: To explore the effects of long noncoding RNA NORAD (lncRNA NORAD) on proliferation and apoptosis of multiple myeloma cells and its targeting relationship with microRNA-363-3p (miR-363-3p). Methods: The expression of NORAD and miR-363-3p in healthy human osteoblasts hFOB1.19 and multiple myeloma cells U266, LP-1 and H929 were detected by RT-qPCR. The U266 cells were transfected with si-NORAD, or co-transfected with si-NORAD and anti-miR-363-3p. MTT assay was used to determined cell proliferation. Flow cytometry was applied to test cell apoptosis. Western blotting was employed to assay the expression of cyclin D1, CDK4, cleaved caspase-3, Bcl-2 and Bax. StarBase software prediction combined with dual luciferase reporting experiment was used to analyze the targeted binding of NORAD to miR-363-3p. Results: The expression of NORAD was greatly up-regulated in multiple myeloma cell lines U266, LP-1 and H929 (P<0.05), while the expression of miR-363-3p was obviously down-regulated (P<0.05). Silencing NORAD expression significantly decreased the activity of U266 cells at 48 and 72 h and cyclin D1, CDK4, bcl-2 protein levels (P<0.05), and evidently increased the apoptotic rate and the expression of cleaved caspase-3 and Bax protein (P<0.05). NORAD targeting regulated the expression of miR-363-3p. Inhibiting the expression of miR-363-3p reversed the inhibition of silenced NORAD expression on the proliferation of U266 cells, the expression of cyclin D1, CDK4 and Bcl-2, and the promotion of silenced NORAD expression on cell apoptosis, cleaved caspase-3 and Bax protein expression. Conclusion: LncRNA NORAD affects the proliferation and apoptosis of multiple myeloma cells by targeting the expression of miR-363-3p.
Keywords：
multiple myeloma; lncRNA NORAD; miR-363-3p; proliferation; apoptosis