下调微小RNA-183表达对肾母细胞瘤SK-NEP-1细胞增殖凋亡的影响及机制

Effect of down-regulation of miR-183 expression on proliferation and apoptosis of nephro⁃blastoma SK-NEP-1 cells and its mechanism

目的探讨下调微小RNA(miR)-183表达对肾母细胞瘤SK-NEP-1细胞增殖凋亡的影响及机制。方法2018年12月至2019年7月,体外培养SK-NEP-1细胞,分为对照组(细胞正常培养)、抑制剂阴性对照组(Anti-NC组)(转染Anti-NC至SKNEP-1细胞)、miR-183抑制剂组(Anti-miR-183组)(转染Anti-miR-183至SK-NEP-1细胞)、Anti-miR-183+小干扰RNA阴性序列(si-NC)组(共转染Anti-miR-183和si-NC至SK-NEP-1细胞)和Anti-miR-183+程序性细胞死亡4小干扰RNA(si-PDCD4)组(共转染Anti-miR-183和si-PDCD4至SK-NEP-1细胞),实时荧光定量逆转录聚合酶链反应(qRT-PCR)测定miR-183,MTT法检测细胞增殖变化,平板克隆实验检测细胞克隆能力,流式细胞术测定细胞凋亡变化,蛋白质印迹法(Westernblotting)检测细胞中活化胱天蛋白酶-3(cleaved-caspase-3)和PDCD4蛋白水平。双荧光素酶报告基因实验验证miR-183与PDCD4的靶向关系。结果Anti-miR-183组miR-183表达水平为(0.32±0.04),显著低于Anti-NC组(0.98±0.11)(P<0.05)。Anti-miR-183组细胞增殖活性为(0.20±0.02)、克隆形成数为(213.69±20.94)个,均低于Anti-NC组[(0.40±0.03)、(312.87±22.28)个](P<0.05);Anti-miR-183组细胞凋亡率为(16.47±1.58)%、cleaved-caspase-3蛋白水平为(0.69±0.09),均高于Anti-NC组[(2.36±0.32)%、(0.27±0.04)](P<0.05)。对照组与Anti-NC组各检测指标比较均差异无统计学意义(P>0.05)。PDCD4是miR-183的靶基因,且Anti-miR-183组细胞中DCD4蛋白水平为(0.89±0.07),显著高于Anti-NC组(0.37±0.06)(P<0.05)。Anti-miR-183+si-PDCD4组细胞增殖活性为(0.32±0.04),克隆形成数为(286.47±20.65)个,PDCD4蛋白水平为(0.45±0.05),均高于Anti-miR-183+si-NC组[(0.21±0.03)、(216.20±13.68)个,(0.86±0.09)](P<0.05);Anti-miR-183组细胞凋亡率为(10.79±1.28)%、cleaved-caspase-3蛋白水平为(0.30±0.03),均低于Anti-miR-183+si-NC组[(17.93±1.64)%、(0.72±0.08)](P<0.05)。结论下调miR-183通过靶向调控PDCD4抑制肾母细胞瘤SK-NEP-1细胞增殖并诱导凋亡。

Objective To investigate the effect of down-regulation of miR-183 on proliferation and apoptosis of nephroblastoma SK-NEP-1 cells and its mechanism.Methods From December 2018 to July 2019,SK-NEP-1 cells were cultured in vitro and assigned in⁃to control group(SK-NEP-1 cells were cultured normally),inhibitor negative control(Anti-NC)group(SK-NEP-1 cells were transfected with Anti-NC),miR-183 inhibitor(Anti-miR-183)group(SK-NEP-1 cells were transfected with Anti-miR-183),Anti-miR-183+small interfering RNA negative sequence(si-NC)group(SK-NEP-1 cells were co-transfected with Anti-miR-183 and si-NC)and Anti-miR-183+programmed cell death 4 small interfering RNA(si-PDCD4)group(SK-NEP-1 cells were co-transfected with Anti-miR-183 and si-PDCD4).Real-time fluorescence quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect miR-183.Tetrathiazolyl blue(MTT)assay was used to detect cell proliferation.Plate cloning assay was used to detect cell cloning ability.Apoptotic changes were measured by flow cytometry.Western blotting was used to detect the protein levels of cleaved-caspase-3 and PDCD4.The dual luciferase reporter gene experiment verified the targeting relationship between miR-183 and PDCD4.Results The expres⁃sion level of miR-183 in the Anti-miR-183 group was(0.32±0.04),which was significantly lower than that in the Anti-NC group(0.98±0.11)(P<0.05).The cell proliferation activity of the Anti-miR-183 group was(0.20±0.02)and the number of clones was(213.69±20.94),which were lower than those of the Anti-NC group[(0.40±0.03),(312.87±22.28)](P<0.05).The apoptotic rate of the Anti-miR-183 group was(16.47±1.58)%and the level of cleaved-caspase-3 protein was(0.69±0.09),which were higher than those of the Anti-NC group[(2.36±0.32)%,(0.27±0.04),respectively](P<0.05).There was no statistically significant difference in the detection indicators between the control group and the Anti-NC group(P>0.05).PDCD4 was identified as the target gene of miR-183,and the level of DCD4 protein in the Anti-miR-183 group was(0.89±0.07),which was significantly higher than that of the Anti-NC group(0.37±0.06)(P<0.05).The cell proliferation activity of Anti-miR-183+si-PDCD4 group was(0.32±0.04),the number of clones was(286.47±20.65),and the level of PDCD4 protein was(0.45±0.05),all higher than those in the Anti-miR-183+si-NC group[(0.21±0.03),(216.20±13.68),(0.86±0.09),respectively](P<0.05);the apoptosis rate of Anti-miR-183 group was(10.79±1.28)%,and the level of cleaved-caspase-3 protein was(0.30±0.03),which were both lower than those in the Anti-miR-183+si-NC group[(17.93±1.64)%,(0.72±0.08),respectively](P<0.05).Conclusion Downregulation of miR-183 inhibits proliferation and induces apoptosis of nephroblastoma SK-NEP-1 cells by targeting PDCD4.