Department of Neurosurgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China (mainland).
Med Sci Monit. 2020 Jul 21;26:e922659. doi: 10.12659/MSM.922659.
BACKGROUND We aimed to investigate the functions of long non-coding RNA (lncRNA) non-coding RNA activated by DNA damage (NORAD) in glioma and identify the potential mechanisms. MATERIAL AND METHODS The expression of NORAD and AKR1B1 in human glioma cell lines were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, cell proliferation, invasion, and migration were tested by Cell Counting Kit-8 (CCK-8), colony formation assay, Transwell, and scratch wound healing assay after NORAD silencing. Meanwhile, western blotting was utilized to measure the expression of migration-related proteins. Apoptosis of glioma cells was detected using flow cytometry and apoptosis-related proteins expression was determined. Moreover, the correlation between NORAD and AKR1B1 was verified by RNA-binding protein immunoprecipitation (RIP assay). After co-transfection with AKR1B1 overexpressed plasmid and NORAD siRNA, cell proliferation, invasion, migration, and apoptosis were examined again. Furthermore, the expression of proteins in extracellular signal-regulated kinase (ERK) signaling was tested using western blotting. RESULTS The results revealed that NORAD and AKR1B1 were highly expressed in glioma cells. NORAD silencing inhibited proliferation, invasion and migration but promoted apoptosis of glioma cells, accompanied by the expression changes of migration- and apoptosis-related proteins. However, after co-transfection with AKR1B1 pcDNA3.1 in NORAD silencing cells, the effects of NORAD silencing on proliferation, invasion, migration, and apoptosis were attenuated. Consistently, the expression of phosphorylated ERK (p-ERK) was decreased after NORAD silencing, which were reversed following AKR1B1 overexpression. CONCLUSIONS These findings demonstrated that NORAD silencing suppressed proliferation, invasion, and migration and boosted apoptosis of glioma cells via downregulating the AKR1B1 expression, which may provide a potential therapeutic target for glioma treatment.
本研究旨在探讨长链非编码 RNA（lncRNA）DNA 损伤激活的非编码 RNA（NORAD）在脑胶质瘤中的作用，并确定潜在的机制。
采用逆转录定量聚合酶链反应（RT-qPCR）检测人脑胶质瘤细胞系中 NORAD 和 AKR1B1 的表达。然后，通过细胞计数试剂盒-8（CCK-8）、集落形成实验、Transwell 和划痕愈合实验检测 NORAD 沉默后细胞的增殖、侵袭和迁移。同时，采用 Western blot 检测迁移相关蛋白的表达。通过流式细胞术检测胶质瘤细胞的凋亡，测定凋亡相关蛋白的表达。此外，通过 RNA 结合蛋白免疫沉淀（RIP 实验）验证 NORAD 与 AKR1B1 的相关性。在共转染 AKR1B1 过表达质粒和 NORAD siRNA 后，再次检测细胞增殖、侵袭、迁移和凋亡。进一步采用 Western blot 检测细胞外信号调节激酶（ERK）信号通路中蛋白的表达。
结果显示，NORAD 和 AKR1B1 在脑胶质瘤细胞中高表达。NORAD 沉默抑制了脑胶质瘤细胞的增殖、侵袭和迁移，促进了细胞凋亡，同时迁移和凋亡相关蛋白的表达也发生了变化。然而，在 NORAD 沉默细胞中转染 AKR1B1 pcDNA3.1 后，NORAD 沉默对增殖、侵袭、迁移和凋亡的影响减弱。同样，NORAD 沉默后磷酸化 ERK（p-ERK）的表达降低，而 AKR1B1 过表达后则逆转。
这些发现表明，NORAD 沉默通过下调 AKR1B1 的表达抑制脑胶质瘤细胞的增殖、侵袭和迁移，促进细胞凋亡，为脑胶质瘤的治疗提供了一个潜在的治疗靶点。
