LncRNA LUCAT1靶向miR-502-5p对人视网膜母细胞瘤细胞生物学行为的影响

Effect of LncRNA LUCAT1 targeting miR-502-5p on the biological behavior of human retinoblastoma cells

目的研究长链非编码RNA(LncRNA)肺癌相关转录产物1(LUCAT1)靶向微小RNA(miR)-502-5p对人视网膜母细胞瘤(RB)细胞增生、迁移和侵袭的影响。方法收集2019年5月至2021年1月在河南省立眼科医院确诊并行眼球摘除术的27例RB患者的RB组织样本,正常视网膜组织标本(12例眼球破裂伤、7例眼球萎缩、8例眼球穿通伤合并有色素膜嵌顿)取自同期在河南省立眼科医院行眼球摘除术的27例患者。采用实时荧光定量PCR检测LncRNA LUCAT1和miR-502-5p在RB组织、细胞系(Y-79、WERI-Rb-1、HXO-RB44)及人视网膜上皮细胞(ARPE-19)中的表达。将RB细胞Y-79分为对照组、小干扰RNA(si)-LncRNA LUCAT1组、si-对照(con)组、pcDNA组、pcDNA-LncRNA LUCAT1组、miR-con组、miR-502-5p组、si-LncRNA LUCAT1+anti-miR-con组和si-LncRNA LUCAT1+anti-miR-502-5p组,分别转染相应试剂。采用Western blot法检测MMP2和MMP9蛋白表达;采用细胞计数试剂盒8法检测细胞增生活力;采用克隆形成实验检测细胞增生能力;采用Transwell实验检测细胞迁移和侵袭能力。采用双荧光素酶报告实验和实时荧光定量PCR验证LncRNA LUCAT1对miR-502-5p的靶向调控作用。结果RB组织中LncRNA LUCAT1表达量为2.73±0.34,明显高于正常视网膜组织的1.00±0.15;miR-502-5p表达量为0.42±0.06,明显低于正常视网膜组织的1.00±0.13,差异均有统计学意义(t=24.190、21.049,均P<0.001)。人RB细胞系Y-79、WERI-Rb-1和HXO-RB44中LncRNA LUCAT1表达水平明显高于ARPE-19细胞,miR-502-5p表达水平明显低于ARPE-19细胞,差异均有统计学意义(均P<0.05)。si-LncRNA LUCAT1组LncRNA LUCAT1表达量,MMP2、MMP9蛋白相对表达量,吸光度(A)值,细胞增生、迁移、侵袭数量较对照组均明显减少,差异均有统计学意义(均P<0.05)。miR-502-5p组miR-502-5p表达量高于miR-con组,MMP2、MMP9蛋白相对表达量,细胞活力A值,细胞增生、迁移、侵袭数量少于miR-con组,差异均有统计学意义(t=20.274、14.884、14.181、12.692、17.749、20.889、21.913,均P<0.001)。si-LncRNA LUCAT1+anti-miR-502-5p组miR-502-5p表达量低于si-LncRNA LUCAT1+anti-miR-con组,MMP2、MMP9蛋白相对表达量,细胞活力A值,细胞增生、迁移、侵袭数量高于si-LncRNA LUCAT1+anti-miR-con组,差异均有统计学意义(t=14.097、15.839、15.757、11.860、16.235、16.565、16.487,均P<0.001)。与LncRNA LUCAT1-野生型共转染时,miR-502-5p组相对荧光素酶活性值低于miR-con组,差异有统计学意义(t=16.379,P<0.001)。pcDNA-LncRNA LUCAT1组LncRNA LUCAT1表达量高于pcDNA组,miR-502-5p表达量低于pcDNA组,si-LncRNA LUCAT1组LncRNA LUCAT1表达量低于si-con组,miR-502-5p表达量高于si-con组,差异均有统计学意义(均P<0.05)。结论抑制LncRNA LUCAT1表达通过靶向上调miR-502-5p可减弱人RB细胞的增生、迁移和侵袭能力。

Objective To investigate the effects of long noncoding RNA(LncRNA)lung cancer associated transcript 1(LUCAT1)targeting microRNA(miR)-502-5p on the proliferation,migration and invasion of human retinoblastoma(RB)cells.Methods RB tissue samples were collected from 27 RB patients who underwent eyeball enucleation in Henan Eye Hospital from May 2019 to January 2021.Another 27 normal retinal tissue specimens were collected from 12 patients with eyeball rupture,7 with eyeball atrophy,and 8 with eyeball penetrating injury combined with pigment film incarceration who underwent the eyeball enucleation in Henan Eye Hospital during the same period.The expressions of LncRNA LUCAT1 and miR-502-5p in RB tissues,cell lines(Y-79,WERI-Rb-1,HXo-RB44)and human retinal epithelial cells(ARPE-19)were detected by real-time quantitative PCR.Y-79 RB cell was divided into control group,small interfering RNA(si)-LncRNA LUCAT1 group,si-control(con)group,pcDNA group,pcDNA-LncRNA LUCAT1 group,miR-con group,miR-502-5p group,si-LncRNA LUCAT1+anti-miR-con group and si-LncRNA LUCAT1+anti-miR-502-5p group,and cells in different groups were transfected with corresponding reagents.The expressions of MMP2 and MMP9 proteins were detected by Western blot.Cell proliferation activity was assayed by cell counting kit 8.Cell proliferation capability was detected by colony formation assay.Cell migration and invasion ability were determined by Transwell assay.The targeting regulation of LncRNA LUCAT1 against miR-502-5p was confirmed by dual luciferase reporter assay and real-time quantitative PCR.The study protocol was approved by the Ethics Committee of Henan Eye Hospital(No.HNEECKY-2021[32]).Written informed consent was obtained from guardians of subjects.Results LncRNA LUCAT1 expression level in RB tissue was 2.73±0.34,which was significantly higher than 1.00±0.15 in normal retinal tissue(t=24.190,P<0.001).The miR-502-5p expression level in RB tissues was 0.42±0.06,which was significantly lower than 1.00±0.13 in normal retinal tissue(t=21.049,P<0.001).LncRNA LUCAT1 expression level was significantly higher and the miR-502-5p expression level was significantly lower in human RB cell lines Y-79,WERI-Rb-1 and HXO-RB44 than those in ARPE-19 cells,with statistically significant differences(all at P<0.05).The LncRNA LUCAT1 expression,the relative expressions of MMP2 and MMP9 proteins,the absorbance(A)value,and the number of proliferated,migrating and invading Y-79 cells in si-LncRNA LUCAT1 group were significantly reduced in comparison with control group,and the differences were statistically significant(all at P<0.05).The miR-502-5p expression level was higher,and the relative expression levels of MMP2 and MMP9,A value,as well as the number of proliferated,migrating and invading Y-79 cells were lower in miR-502-5p group than in miR-con group,showing statistically significant differences(t=20.274,14.884,14.181,12.692,17.749,20.889,21.913;all at P<0.001).The miR-502-5p expression level was lower and the relative expression levels of MMP2 and MMP9,A value as well as the number of proliferated,migrating and invading Y-79 cells were higher in si-LncRNA LUCAT1+anti-miR-502-5p group than in si-LncRNA LUCAT1+anti-miR-con group,showing statistically significant differences(t=14.097,15.839,15.757,11.860,16.235,16.565,16.487;all at P<0.001).When co-transfected with LncRNA LUCAT1-wild type,the relative luciferase activity of miR-502-5p group was lower than that of miR-con group,and the difference was statistically significant(t=16.379,P<0.001).The LncRNA LUCAT1 expression level was higher and the miR-502-5p expression level was lower in pcDNA-LncRNA LUCAT1 group than in pcDNA group,and the differences were statistically significant(both at P<0.05).The LncRNA LUCAT1 expression level was lower and the miR-502-5p expression level was higher in si-LncRNA LUCAT1 group than in si-con group,and the differences were statistically significant(both at P<0.05).Conclusions Inhibition of LncRNA LUCAT1 can attenuate the proliferation,migration and invasion ability of human RB cells by the targeting up-regulation of miR-502-5p.