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Title: [The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p].
Author: Xing F, Li YM, Gao MM.
Journal: Zhonghua Zhong Liu Za Zhi; 2023 Mar 23; 45(3):230-237. PubMed ID: 36944544.
Abstract:
Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p. 目的： 探讨长链非编码RNA二磷酸腺苷依赖的葡萄糖激酶反义RNA1(lnc RNA ADPGK－AS1)对视网膜母细胞瘤细胞增殖及凋亡的影响及其可能的作用机制。 方法： 收集2020年2—6月河南省立眼科医院收治的31例视网膜母细胞瘤患者的肿瘤组织及其相应癌旁正常组织，采用实时荧光定量聚合酶链反应(qRT－PCR)法检测视网膜母细胞瘤组织和癌旁正常组织中lncRNA ADPGK－AS1、miR－200b－5p的表达水平。体外培养人视网膜上皮细胞ARPE－19、人视网膜母细胞瘤细胞Y－79和WERI－Rb－1，qRT－PCR法检测lncRNA ADPGK－AS1、miR－200b－5p的表达水平。以Y－79细胞为研究对象，随机分组si－con组、si－lncRNA ADPGK－AS1组、miR－con组、miR－200b－5p组、si－lncRNA ADPGK－AS1+anti－miR－con组、si－lncRNA ADPGK－AS1+anti－miR－200b－5p组。四甲基偶氮唑蓝法、平板克隆形成实验与流式细胞术分别检测各组细胞的增殖、克隆及凋亡情况，双荧光素酶报告实验检测lncRNA ADPGK－AS1与miR－200b－5p的靶向关系，Western blot法检测Cleaved－caspase－3蛋白的表达水平。 结果： 视网膜母细胞瘤组织中lncRNA ADPGK－AS1的表达水平高于癌旁正常组织，miR－200b－5p的表达水平低于癌旁正常组织(均P<0.05)；Y－79、WERI－Rb－1细胞中lncRNA ADPGK－AS1的表达水平高于ARPE－19细胞，miR－200b－5p的表达水平低于ARPE－19细胞(均P<0.05)。si－lncRNA ADPGK－AS1组细胞吸光度(A)值(0.53±0.05)、细胞克隆形成数[(57.00±4.13)个]低于si－con组[分别为1.06±0.09和(114.00±8.03)个，均P<0.05]，细胞凋亡率[(25.43±1.94)%]高于si－con组[(7.93±0.68)%，P<0.05]，Cleaved－caspase－3蛋白水平高于si－con组(P<0.05)。miR－200b－5p组细胞A值(0.57±0.05)、细胞克隆形成数[(64.00±5.13)个]低于miR－con组[分别为1.05±0.08和(118.00±10.02)个，均P<0.05)]，细胞凋亡率[(23.15±1.62)%]高于miR－con组[(7.89±0.71)%，P<0.05]，Cleaved－caspase－3蛋白水平高于miR－con组(P<0.05)。lncRNA ADPGK－AS1可靶向调控miR－200b－5p的表达。si－lncRNA ADPGK－AS1+anti－miR－200b－5p组细胞A值(1.25±0.10)、细胞克隆形成数[(125.00±10.03)个]高于si－lncRNA ADPGK－AS1+anti－miR－con组[分别为0.53±0.04和(54.00±4.39)个，均P<0.05]，细胞凋亡率[(9.76±0.71)%]低于si－lncRNA ADPGK－AS1+anti－miR－con组[(25.38±1.53)%，P<0.05]，Cleaved－caspase－3蛋白水平低于si－lncRNA ADPGK－AS1+anti－miR－con组(P<0.05)。 结论： 抑制lncRNA ADPGK－AS1表达可通过靶向调控miR－200b－5p表达而抑制视网膜母细胞瘤细胞增殖及克隆形成，并可诱导细胞凋亡。.

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