Department of Pediatrics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, Hubei, China.
Mol Cell Biochem. 2021 Apr;476(4):1871-1879. doi: 10.1007/s11010-020-04042-9. Epub 2021 Jan 21.
The aim of this study was to determine the expression of long-chain non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) in children with neuroblastoma and its effect on cancer cell growth. A polymerase chain reaction assay was carried out to quantify lncRNA CCAT2 miRNA in neuroblastoma cells, corresponding paracancerous cells, SH-SY5Y and SK-N-SH cells, and human umbilical vein endothelial cells (HUVEC), and two groups of children with different lncRNA CCAT2 expression were compared in clinical pathological parameters and prognosis. CCAT2-NC and si-CCAT2 were transfected into SH-SY5Y cells, separately. Then a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was carried out to analyze the cell proliferation, migration, and invasion ability, a flow cytometry to detect cell apoptosis, and a Western blotting (WB) assay to quantify p53 and Bcl-2 proteins. lncRNA CCAT2 expression in cancer tissues of children with neuroblastoma was notably higher than that in corresponding paracancerous tissues (P < 0.05), and children with different tissue differentiation, tumor staging, and lymph node metastasis (LNM) showed notably different lncRNA CCAT2 expression (P < 0.05). In addition, children with neuroblastoma in the high lncRNA CCAT2 expression group showed lower 3-year survival rate than those in the low expression group (P < 0.05). Multivariate analysis revealed that tissue differentiation, tumor-node-metastasis staging, LNM, and lncRNA CCAT2 expression were all independent risk factors affecting the prognosis of children with neuroblastoma (all P < 0.05). Compared with HUVEC cells, SH-SY5Y and SK-N-SH cells showed notably up-regulated lncRNA CCAT2, and the expression of it in SH-SY5Y was higher than that in SK-N-SH cells (P < 0.05). Compared with the CCAT2-NC group, the si-CCAT2 group presented notably down-regulated CCAT2 (P < 0.05). Moreover, according to the MTT assay, the si-CCAT2 group showed notably weakened cell viability and proliferation than the CCAT2-NC group (both P < 0.05), and SH-SY5Y cells in the former group were less active than those in the latter group in terms of migration and invasion. The cell apoptosis rate of SH-SY5Y cells in the si-CCAT2 was higher than that in the CCAT2-NC. The results suggested that knock down of lncRNA CCAT2 could improve the apoptosis activity of neuroblastoma cells in children. According to the WB assay, the si-CCAT2 group showed notably higher p53 expression and notably lower Bcl-2 protein expression than the CCAT2-NC group (both P < 0.05). LncRNA CCAT2 can inhibit the proliferation of neuroblastoma cells and promote their apoptosis, which provides a basis for the treatment of neuroblastoma.
本研究旨在探讨长链非编码 RNA（lncRNA）结肠癌相关转录物 2（CCAT2）在神经母细胞瘤患儿中的表达及其对癌细胞生长的影响。采用聚合酶链反应（PCR）法检测神经母细胞瘤细胞、相应癌旁细胞、SH-SY5Y 细胞和 SK-N-SH 细胞及人脐静脉内皮细胞（HUVEC）中的 lncRNA CCAT2 miRNA，并比较两组不同 lncRNA CCAT2 表达水平的患儿临床病理参数和预后。将 CCAT2-NC 和 si-CCAT2 分别转染至 SH-SY5Y 细胞。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法分析细胞增殖、迁移和侵袭能力，流式细胞术检测细胞凋亡，Western blot 法检测 p53 和 Bcl-2 蛋白表达。神经母细胞瘤患儿癌组织中 lncRNA CCAT2 的表达明显高于相应癌旁组织（P<0.05），不同组织分化、肿瘤分期和淋巴结转移（LNM）的患儿 lncRNA CCAT2 表达明显不同（P<0.05）。此外，lncRNA CCAT2 高表达组患儿的 3 年生存率明显低于低表达组（P<0.05）。多因素分析显示，组织分化、肿瘤-淋巴结-转移分期、LNM 和 lncRNA CCAT2 表达均是影响神经母细胞瘤患儿预后的独立危险因素（均 P<0.05）。与 HUVEC 细胞相比，SH-SY5Y 和 SK-N-SH 细胞明显上调 lncRNA CCAT2，且 SH-SY5Y 细胞的表达水平高于 SK-N-SH 细胞（P<0.05）。与 CCAT2-NC 组相比，si-CCAT2 组明显下调 CCAT2（P<0.05）。此外，根据 MTT 检测结果，si-CCAT2 组细胞活力和增殖明显低于 CCAT2-NC 组（均 P<0.05），且前者 SH-SY5Y 细胞的迁移和侵袭能力均弱于后者。si-CCAT2 组 SH-SY5Y 细胞的细胞凋亡率明显高于 CCAT2-NC 组。结果提示，敲低 lncRNA CCAT2 可提高儿童神经母细胞瘤细胞的凋亡活性。根据 Western blot 检测结果，si-CCAT2 组 p53 表达明显升高，Bcl-2 蛋白表达明显降低（均 P<0.05）。lncRNA CCAT2 可抑制神经母细胞瘤细胞的增殖并促进其凋亡，为神经母细胞瘤的治疗提供了依据。
