miR-590-5p与信号传导及转录激活因子3的靶向作用关系及其对视网膜母细胞瘤Y-79细胞生长和转移能力的影响

Targeted relationship of miR-590-5p to signal transducers and activators of transcription 3 and influence of miR-590-5p on growth and metastasis of retinoblastoma Y-79 cells

目的探讨miR-590-5p与信号传导及转录激活因子3(signal transducers and activators of transcription 3,STAT3)的靶向作用关系及其对视网膜母细胞瘤(retinoblastoma,RB)Y-79细胞生长和转移能力的影响。方法临床收集人RB组织样本和癌旁组织样本各20份,RT-PCR检测miR-590-5p和STAT3基因的转录水平,并分析两者的相关性。RT-PCR检测RB细胞株Y-79和SO-RB50及正常视网膜细胞株HREC中miR-590-5p基因的转录水平。荧光素酶报告基因试验检测miR-590-5p和STAT3的相互作用关系。将Y-79细胞分为Y-79、miR-NC、miR-590-5p mimic和mi R-590-5p mimic+STAT3组,转染相应的miRNA及载体,RT-PCR检测mi R-590-5p和STAT3基因转录水平,Western blot检测STAT3、增殖蛋白Ki67、活化半胱天冬酶(cleave caspase-3,cl-caspase-3)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-9(matrix metalloprotein-9,MMP-9)蛋白表达水平,CCK8法检测细胞增殖,流式细胞术检测细胞凋亡。结果与正常视网膜组织比较,RB组织中mi R-590-5p基因转录水平显著降低,STAT3基因转录水平显著升高(P<0.01),且miR-590-5p与STAT3基因转录水平呈负相关(r=-0.8835,P<0.001)。与正常视网膜细胞比较,SO-RB50和Y-79细胞miR-590-5p转录水平显著降低(P<0.01),从中选取miR-590-5p转录水平最低的Y-79细胞进行后续试验。与Y-79组比较,miR-590-5p mimic组mi R-590-5p表达水平显著升高,STAT3基因转录和蛋白表达水平显著降低,细胞增殖水平显著降低,细胞凋亡率显著升高,Ki67、VEGF、MMP-9蛋白表达水平显著降低,cl-caspase-3蛋白表达水平显著升高(P<0.01);与miR-590-5p mimic组比较,miR-590-5p mimic+STAT3组STAT3蛋白表达水平显著升高,细胞增殖水平显著升高,细胞凋亡率显著降低,Ki67、VEGF、MMP-9蛋白表达水平显著升高,cl-caspase-3蛋白表达水平显著降低(P<0.01)。与STAT3 WT+miR-NC组比较,STAT3 WT+miR-590-5p mimic组荧光素酶活性显著降低(P<0.01)。结论miR-590-5p可通过靶向作用于STAT3,抑制RB Y-79细胞的生长和转移能力。

Objective To investigate the targeted relationship of mi R-590-5 p to signal transducers and activators of transcription 3(STAT3)and the influence of miR-590-5 p on growth and metastasis of retinoblastoma(RB)Y-79 cells.Methods Twenty specimens of human RB tissue and 20 specimens of paracancerous tissue were collected in clinic and determined for the transcription levels of miR-590-5 p and STAT3 genes by RT-PCR,between which the relationship was analyzed.The miR-590-5 p transcription levels in Y-79,SO-RB50 and normal retina cells were determined by RT-PCR.The relationship between miR-590-5 p and STAT3 was analyzed by luciferase reporter assay.Y-79 cells were divided into Y-79(control),mi R-NC,miR-590-5 p mimic and miR-590-5 p mimic+STAT3 groups.The cells in control group were untreated,while those in the other groups were transfected with the corresponding miRNAs and vectors,and determined for the transcription levels of miR-590-5 p and STAT3 genes by RT-PCR.The expression levels of STAT3,Ki67,cleave caspase-3,vascular endothelial growth factor(VEGF),matrix metalloprotein-9(MMP-9)were determined by Western blot,while the cell growth by CCK8 assay,and the cell apoptosis by flow cytometry.Results Compared with those in normal retina tissue,the transcription level of miR-590-5 p gene in RB tissue decreased significantly,while that of STAT3 increased significantly(P<0.01),and the miR-590-5 p gene transcription was negatively correlated with the STAT3 gene transcription(r=-0.8835,P<0.001).Compared with those in normal retina cells,the transcription levels of miR-590-5 p gene in SO-RB50 and Y-79 cells decreased significantly(P<0.01).The Y-79 cells with the lowest transcription level of miR-590-5 p gene were selected for subsequent experiments.Compared with those in Y-79(control)group,the expression level of miR-590-5 p in Y-79 cells of miR-590-5 p mimic group increased significantly,while the gene transcription and protein expression levels of STAT3 decreased significantly,the cell growth fold decreased significantly,the cell apoptosis rate increased significantly,the protein expression levels of Ki67,VEGF and MMP-9 decreased significantly,the expression level of cl-caspase-3 protein increased significantly(P<0.01).Compared with those in miR-590-5 p group,the protein expression level of STAT3 in miR-590-5 p mimic+STAT3 group increased significantly,while the cell growth fold increased significantly,the cell apoptosis rate decreased significantly,the protein expression levels of Ki67,VEGF and MMP-9 increased significantly,and the expression level of cl-caspase-3 protein decreased significantly(P<0.01).However,compared with that in STAT3 WT+miR-NC group,the luciferase activity in STAT3 WT+miR-590-5 p mimic group decreased significantly(P<0.01).Conclusion The miR-590-5 p may inhibit the growth and metastasis of RB Y-79 cells by targeting STAT3.