miRNA-4497靶向PEA15对视网膜母细胞瘤细胞增殖及药物敏感性研究

miRNA-4497 promotes the proliferation and reduces the drug sensitivity of retinoblastoma cells by targeting PEA15

目的探讨miRNA-4497通过靶向PEA15对视网膜母细胞瘤(Rb)Y79细胞株增殖及对奥沙利铂药物敏感性的影响。方法收集2017年1月—2019年12月病理确诊的Rb患者27例,取其肿瘤组织及瘤旁组织,实时荧光定量PCR(RT-qPCR)检测miRNA-4497表达。使用慢病毒感染的方法构建过表达组及阴性对照组Y79细胞。RT-qPCR检测感染效果。CCK8法检测2组细胞的增殖能力及对奥沙利铂的药物敏感性。荧光素酶报告基因验证miRNA-4497的潜在靶点。RT-qPCR和Western blot检测2组细胞中靶基因的表达水平。结果(1)miRNA-4497表达:在Rb组织中表达升高,差异有统计学意义(t=14.590,P=0.000)。miRNA-4497在过表达组细胞中的表达水平显著高于阴性对照组(t=16.150,P=0.000)。(2)对Y79细胞增殖的影响:相对于阴性对照组,miRNA-4497过表达组细胞增殖速度从第3 d开始明显增高,2组细胞增殖速度的差异具有统计学意义(t_(3 d)=6.500,t_(4 d)=7.691,均P=0.000)。(3)药物敏感性:奥沙利铂浓度为0.125μM时,2组细胞生长抑制率的差异无统计学意义(P>0.05),在浓度为0.25、0.5、1、2μM时,过表达组细胞的生长抑制率比对照组低,且差异具有统计学意义(t_(0.25μM)=4.277,t_(0.5μM)=7.547,t_(1μM)=8.302,t_(2μM)=8.302;均P=0.000)。(4)荧光素酶活性检测:双荧光素酶报告基因显示miRNA-4497能够与PEA15靶向结合,与阴性对照组比较有统计学意义(t=5.514,P=0.000)。(5)PEA15的蛋白及mRNA表达:与阴性对照组相比,miRNA-4497过表达组细胞中PEA15 mRNA表达水平显著降低(t=77.856,P=0.000),PEA15蛋白水平明显降低。结论miRNA-4497可通过靶向PEA15促进人Rb细胞株Y79的增殖水平,降低对奥沙利铂的药物敏感性。

OBJECTIVE To investigate the effect of miRNA-4497 on the proliferation and sensitivity to oxaliplatin of retinoblastoma cell line Y79 by targeting PEA15.METHODS Neoplastic tissues and peritumoral tissues of 27 cases with retinoblastoma were collected from January 2017 to December 2019.The expression of miRNA-4497 in these tissues was detected by real-time quantitative PCR(RT-qPCR).The overexpression group and the negative control group of Y79 retinoblastoma cell lines were constructed by lentivirus infection.RT-qPCR was used to detect the effect of infection.The proliferation ability and drug sensitivity to oxaliplatin of the two groups were detected by CCK8 method.Luciferase reporter gene was used to identify potential targets for miRNA-4497.RT-qPCR and Western blot were used to detect the expression level of target genes in the two group cells.RESULTS(1)The expression of miRNA-4497:The expression was increased in retinoblastoma tissue,and the difference was statistically significant(t=14.590,P=0.000).The expression level of miRNA-4497 in the overexpression group was significantly higher than that in the negative control group(t=16.150,P=0.000).(2)The effect of Y79 cells proliferation:Compared with the negative control group,the cell proliferation rate of the overexpression group increased significantly from the 3rd day.The difference in cell proliferation rate was statistically significant(t_(3 d)=6.500,t_(4 d)=7.691,all P=0.000).(3)Drug sensitivity:When the concentration of oxaliplatin was 0.125μM,there was no significant difference in cell growth inhibition rate between the two groups(P>0.05).When the concentrations were 0.25,0.5,1,2μM,the growth inhibition rate of the cells in the overexpression group was lower than that of the control group,and the difference was statistically significant(t_(0.25μM)=4.277,t_(0.5μM)=7.547,t_(1 M)=8.302,t_(2μM)=8.302,all P=0.000).(4)Luciferase activity detection:The dual luciferase reporter gene showed that miRNA-4497 could bind PEA15 by target(t=5.514,P=0.000).(5)Protein expression and mRNA of PEA15:Compared with the negative control group,the PEA15 expression level in the cells of the overexpression group was significantly reduced(t=77.856,P=0.000).The PEA15 protein level detected by Western blot was significantly reduced.CONCLUSIONS The miRNA-4497 can promote the proliferation of retinoblastoma cell line Y79 by targeting PEA15 and reducing the drug sensitivity to oxaliplatin.