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  • Title: [Deleted in lymphocytic leukemia 1 promoted proliferation and apoptosis of nephroblastoma cells through regulating miR-513a-5p and RANBP2 pathway].
    Author: Zhao JL, Zhao LL, Niu WZ, Ding XC, Zhang WL.
    Journal: Zhonghua Zhong Liu Za Zhi; 2020 Oct 23; 42(10):849-855. PubMed ID: 33113626.
    Abstract:
    Objective: 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. Methods: 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. Results: 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 (P<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 (P<0.05). Overexpression of DLEU1 significantly reduced the apoptosis rate (7.35±0.41 vs 12.35±1.12, P<0.05). Overexpression of RANBP2 significantly reduced the apoptosis rate (8.89±0.48 vs 12.64±1.12, P<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 (P<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 (P<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, P<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, P<0.05). Conclusions: 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. 目的： 探讨淋巴细胞白血病缺失基因1(DLEU1)、miR-513a-5p和RANBP2在肾母细胞瘤中的调控作用及机制。 方法： 采用脂质体法转染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＋miR-NC(pcDNA-DLEU1＋miR-NC组)、pcDNA-DLEU1＋miR-513a-5p(pcDNA-DLEU1＋miR-513a-5p mimics组)、miR-513a-5p＋pcDNA(miR-513a-5p mimics＋pcDNA组)和miR-513a-5p＋pcDNA-RANBP2(miR-513a-5p mimics＋pcDNA-RANBP2组)至肾母细胞瘤GHINK-1细胞中。实时荧光定量逆转录聚合酶链反应检测肾母细胞瘤组织、癌旁组织、正常肾细胞HK2和肾母细胞瘤细胞GHINK-1中DLEU1、miR-513a-5p和RANBP2的表达水平，Western blot检测PCNA、Bcl-2和Bax蛋白的表达水平，细胞计数试剂盒8法检测细胞的存活率，流式细胞术检测细胞的凋亡率，双荧光素酶报告实验检测细胞的荧光素酶活性。 结果： 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和1.00±0.05，与肾母细胞瘤组织(分别为5.16±0.24、0.23±0.02和1.67±0.09)比较，差异均有统计学意义(均P<0.05)；DLEU1、miR-513a-5p和RANBP2在HK2细胞中的表达水平分别为1.00±0.06、1.00±0.08和1.02±0.09，与GHINK-1细胞(分别为3.15±0.21、0.18±0.01和1.54±0.10)比较，差异均有统计学意义(均P<0.05)。过表达DLEU1后，pcDNA组GHINK-1细胞的凋亡率[(12.35±1.12)%]与pcDNA-DLEU1组[(7.35±0.41)%]比较，差异有统计学意义(P<0.05)。过表达RANBP2后，pcDNA组GHINK-1细胞的凋亡率[(12.64±1.12)%]与pcDNA-RANBP2组[(8.89±0.48)%]比较，差异有统计学意义(P<0.05)。miR-513a-5p组DLEU1-WT和RANBP2-WT细胞的荧光素酶活性分别为0.43±0.04和0.61±0.07，与miR-NC组(分别为1.01±0.06和0.99±0.06)比较，差异均有统计学意义(均P<0.05)。anti-miR-513a-5p组DLEU1-WT和RANBP2-WT细胞的荧光素酶活性分别为1.34±0.11和1.39±0.13，与anti-miR-NC组(分别为0.99±0.07和0.98±0.05)比较，差异均有统计学意义(均P<0.05)。pcDNA-DLEU1＋miR-NC组和pcDNA-DLEU1＋miR-513a-5p组GHINK-1细胞的凋亡率分别为(8.51±0.69)%和(11.34±1.03)%，miR-513a-5p＋pcDNA组和miR-513a-5p＋pcDNA-RANBP2组GHINK-1细胞的凋亡率分别为(15.94±1.00)%和(9.96±0.72)%，差异均有统计学意义(均P<0.05)。 结论： 长链非编码RNA DLEU1可促进肾母细胞瘤细胞的增殖，其机制与靶向调控miR-513a-5p和RANBP2功能有关，可为肾母细胞瘤的治疗提供理论支持。.
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