摘要
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<
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
作者
赵静丽
赵丽丽
牛文忠
丁显春
张文林
Zhao Jingli;Zhao Lili;Niu Wenzhong;Ding Xianchun;Zhang Wenlin(Department of Pediatrics,Nanyang Central Hospital,Nanyang 473000,China;Department of Children Hematology and Oncology,Henan Cancer Hospital,Zhengzhou 450000,China)
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2020年第10期849-855,共7页
Chinese Journal of Oncology
