摘要
目的探讨高糖诱导的人视网膜色素上皮细胞(ARPE-19)中含有卵巢肿瘤结构域的6B反义RNA 1(OTUD6B-AS1)的功能及其作用机制。方法取ARPE-19细胞,加入含30 mmol·L^(-1)葡萄糖的培养基培养,设为高糖组,另取ARPE-19细胞不做任何处理设为对照组。用Lipofectamine^(TM) 2000转染试剂分别将过表达空质粒(pcDNA-NC组)、OTUD6B-AS1过表达质粒(pcDNA-OTUD6B-AS1组)和OTUD6B-AS1过表达质粒+miR-21-5p模拟物(pcDNA-OTUD6B-AS1+miR-21-5p mimics组)转染进高糖诱导的ARPE-19细胞。实时荧光定量PCR(qRT-PCR)检测细胞中OTUD6B-AS1和miR-21-5p的表达;用CCK-8法检测各组细胞增殖能力;流式细胞术检测细胞凋亡情况;细胞划痕实验检测各组细胞迁移能力;荧光素酶报告基因实验验证OTUD6B-AS1与miR-21-5p的靶向关系。结果与对照组比较,高糖组ARPE-19细胞中OTUD6B-AS1表达量显著降低、miR-21-5p表达量显著升高,差异均有统计学意义(均为P<0.05)。与对照组比较,高糖组细胞凋亡率显著升高,细胞增殖率和迁移率均显著降低,差异均有统计学意义(均为P<0.05)。与pcDNA-NC组比较,pcDNA-OTUD6B-AS1组中细胞增殖率和迁移率显著升高,细胞凋亡率显著降低,差异均有统计学意义(均为P<0.05)。双荧光素酶报告基因实验显示,OTUD6B-AS1可靶向miR-21-5p。与pcDNA-OTUD6B-AS1组比较,pcDNA-OTUD6B-AS1+miR-21-5p mimics组细胞凋亡率显著升高,细胞增殖率和迁移率显著降低,差异均有统计学意义(均为P<0.05)。结论OTUD6B-AS1可抑制高糖诱导的ARPE-19细胞凋亡,并促进细胞增殖和迁移,其作用机制与miR-21-5p有关。
Objective To investigate the function and mechanism of ovarian tumor domain containing 6B antisense RNA1(OTUD6B-AS1)in retinal pigment epithelial(RPE)cells induced by high glucose(HG).Methods ARPE-19 cells cultured in medium containing 30 mmol·L^(-1) glucose were set as the HG group,and ARPE-19 cells without any treatment were set as the control group.Overexpressed empty plasmids(pcDNA-NC group),OTUD6B-AS1 overexpressed plasmids(pcDNA-OTUD6B-AS1 group),and OTUD6B-AS1 overexpressed plasmids+miR-21-5p mimics(pcDNA-OTUD6B-AS1+miR-21-5p group)were transfected into HG-induced ARPE-19 cells using Lipofectamine^(TM) 2000 transfection reagent,respectively.The expression of OTUD6B-AS1 and miR-21-5p was detected by quantitative real-time polymerase chain reaction,the cell proliferation was detected by CCK-8 assay,the cell apoptosis was detected by flow cytometry,the cell migration was detected by wound-healing assay,and the targeting relationship between OTUD6B-AS1 and miR-21-5p was verified by luciferase reporter gene assay.Results Compared with the control group,OTUD6B-AS1 expression was significantly decreased while miR-21-5p expression was significantly increased in the HG group(both P<0.05).Compared with the control group,the cell apoptosis was significantly increased while the cell proliferation and migration were significantly reduced in the HG group(all P<0.05).Compared with the pcDNA-NC group,the cell proliferation and migration in the pcDNA-OTUD6B-AS1 group were significantly increased while the cell apoptosis was significantly reduced(all P<0.05).Dual-luciferase reporter gene assay showed that OTUD6B-AS1 could target miR-21-5p.Compared with the pcDNA-OTUD6B-AS1 group,the cell apoptosis in the pcDNA-OTUD6B-AS1+miR-21-5p group was significantly increased while the cell proliferation and migration were significantly decreased(all P<0.05).Conclusion OTUD6B-AS1 can inhibit the apoptosis of HG-induced ARPE-19 cells and promote their proliferation and migration,which is related to miR-21-5p.
作者
李敏
李霞
王征
李印
王亚丽
LI Min;LI Xia;WANG Zheng;LI Yin;WANG Yali(Department of Ophthalmology,Enshi Central Hospital,Enshi 445400,Hubei Province,China;Central South Hospital of Wuhan University,Wuhan 430000,Hubei Province,China)
出处
《眼科新进展》
CAS
北大核心
2022年第5期364-368,共5页
Recent Advances in Ophthalmology
