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[淋巴细胞白血病缺失基因1通过调控miR-513a-5p和RANBP2通路促进肾母细胞瘤细胞增殖和凋亡]

[Deleted in lymphocytic leukemia 1 promoted proliferation and apoptosis of nephroblastoma cells through regulating miR-513a-5p and RANBP2 pathway].

作者信息

Zhao J L, Zhao L L, Niu W Z, Ding X C, Zhang W L

机构信息

Department of Pediatrics, Nanyang Central Hospital, Nanyang 473000, China.

Department of Children Hematology and Oncology, Henan Cancer Hospital, Zhengzhou 450000, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2020 Oct 23;42(10):849-855. doi: 10.3760/cma.j.cn112152-20200311-00194.

Abstract

To study the regulatory effects and mechanisms of deleted in lymphocytic leukemia 1 (DLEU1), microRNA-513a-5p (miR-513a-5p), and RAN binding protein 2 (RANBP2) in nephroblastoma. The GHINK-1 cells were transfected with pcDNA (pcDNA group), pcDNA-DLEU1 (pcDNA-DLEU1 group), miR-NC (miR-NC group), miR-513a-5p mimics (miR-513a-5p group), pcDNA-RANBP2 (pcDNA-RANBP2 group), pcDNA-DLEU1 and miR-NC (pcDNA-DLEU1+ miR-NC group), pcDNA-DLEU1 and miR-513a-5p mimics (pcDNA-DLEU1+ miR-513a-5p group), miR-513a-5p mimics and pcDNA (miR-513a-5p+ pcDNA group), miR-513a-5p mimics and pcDNA-RANBP2 (miR-513a-5p + pcDNA-RANBP2 group). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expressions of DLEU1, miR-513a-5p, RANBP2 in nephroblastoma tissues, normal adjacent tissues, normal kidney cell HK2, and hemangioblastoma cell GHINK-1. Western blot was used to detect the expressions of proliferating cell nuclear antigen (PCNA), B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X (Bax). Cell counting kit 8 (CCK-8) was used to detect the cell survival rate. Flow cytometry was used to detect the apoptosis rate. Dual luciferase report test was used to detect the luciferase activity of cells. The expression levels of DLEU1, miR-513a-5p and RANBP2 in adjacent tissues were 1.02±0.08, 1.01±0.06, 1.00±0.05, respectively, significantly lower than 5.16±0.24, 0.23±0.02, 1.67±0.09 in nephroblasts tumor tissues (<0.05). Their expression levels in HK2 cells were 1.00±0.06, 1.00±0.08, 1.02±0.09, respectively, significantly lower than 3.15±0.21, 0.18±0.01, 1.54±0.10 in GHINK-1 cells (<0.05). Overexpression of DLEU1 significantly reduced the apoptosis rate (7.35±0.41 vs 12.35±1.12, <0.05). Overexpression of RANBP2 significantly reduced the apoptosis rate (8.89±0.48 vs 12.64±1.12, <0.05). Compared with the miR-NC group (1.01±0.06, 0.99±0.06), the luciferase activity of DLEU1-WT (0.43±0.04) and RANBP2-WT (0.61±0.07) in miR-513a-5p group were significantly reduced (<0.05). Compared with anti-miR-NC group (0.99±0.07, 0.98±0.05), the luciferase activity of DLEU1-WT (1.34±0.11) and RANBP2-WT (1.39 ±0.13) in anti-miR-513a-5p group was significantly increased (<0.05). Simultaneous overexpression of pcDNA-DLEU1 and miR-513a-5p in GHINK-1 cells significantly reduced the apoptosis rate (11.34±1.03 vs 8.51±0.69, <0.05). Simultaneous overexpression of miR-513a-5p and RANBP2 in GHINK-1 cells significantly reduced the apoptosis rate (9.96±0.72 vs 15.94±1.00, <0.05). The long-chain non-coding RNA (lncRNA) DLEU1 can promote the proliferation and inhibit the apoptosis of nephroblastoma cells. The mechanism is related to the targeted regulation of miR-513a-5p and RANBP2 function, which will provide theoretical support for the nephroblastoma treatment.

摘要

研究淋巴细胞白血病缺失基因1(DLEU1)、微小RNA-513a-5p(miR-513a-5p)和RAN结合蛋白2(RANBP2)在肾母细胞瘤中的调控作用及机制。将GHINK-1细胞分别转染pcDNA(pcDNA组)、pcDNA-DLEU1(pcDNA-DLEU1组)、miR-NC(miR-NC组)、miR-513a-5p模拟物(miR-513a-5p组)、pcDNA-RANBP2(pcDNA-RANBP2组)、pcDNA-DLEU1和miR-NC(pcDNA-DLEU1 + miR-NC组)、pcDNA-DLEU1和miR-513a-5p模拟物(pcDNA-DLEU1 + miR-513a-5p组)、miR-513a-5p模拟物和pcDNA(miR-513a-5p + pcDNA组)、miR-513a-5p模拟物和pcDNA-RANBP2(miR-513a-5p + pcDNA-RANBP2组)。采用实时定量逆转录聚合酶链反应(RT-qPCR)检测肾母细胞瘤组织、癌旁正常组织、正常肾细胞HK2和血管母细胞瘤细胞GHINK-1中DLEU1、miR-513a-5p、RANBP2的表达。采用蛋白质免疫印迹法检测增殖细胞核抗原(PCNA)、B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达。采用细胞计数试剂盒8(CCK-8)检测细胞存活率。采用流式细胞术检测细胞凋亡率。采用双荧光素酶报告试验检测细胞的荧光素酶活性。DLEU1、miR-513a-5p和RANBP2在癌旁组织中的表达水平分别为1.02±0.08、1.01±0.06、1.00±0.05,显著低于肾母细胞瘤组织中的5.16±0.24、0.23±0.02、1.67±0.09(<0.05)。它们在HK2细胞中的表达水平分别为1.00±0.06、1.00±0.08、1.02±

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