萝卜硫素通过miR-520a-3p/EGFR信号通路诱导人肾腺癌细胞凋亡  

Sulforaphane induces the apoptosis of renal carcinoma cells through the inhibition of miR-520a-3p/EGFRpathway

作　　者：吴敏 李丽 顾文燕 Wu Min;Li Li;Gu Wenyan(Operating Room,the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture,Enshi 445000,China)

机构地区：[1]恩施土家族苗族自治州中心医院手术室,恩施445000

出　　处：《国际泌尿系统杂志》2022年第6期1010-1015,共6页International Journal of Urology and Nephrology

摘　　要：目的探讨萝卜硫素(SFP)对人肾腺癌细胞凋亡的影响及其潜在的作用机制。方法选取人肾小管上皮细胞系(HK-2)和视网膜母细胞瘤细胞系(ACHN、UOK146、786-0和GRC-1)为研究对象,采用CCK-8方法和流式细胞术分析细胞增殖和凋亡情况;应用TargetScan软件预测miR-520a-3p与EGFR的关系,并采用双荧光素酶报告基因系统检测其相互作用;采用实时荧光定量PCR法(qRT-PCR)和Western blot法检测miR-520a-3p和人表皮生长因子受体(EGFR)mRNA和蛋白的表达水平。结果SFP呈时间和浓度依赖性的抑制ACHN细胞增殖(均P<0.05),SFP处理细胞36 h后,SFP组细胞凋亡率相对于对照组明显升高(均P<0.05)。与对照组比较,SFP组的Cleaved-Caspase-3的表达水平增高,而Pro-Caspase-3的表达水平则降低(P<0.05)。20、40、80μg/mL SFP处理ACHN细胞36 h后,miR-520a-3p表达水平均较对照组明显升高(P<0.05)。与对照组比较,转染WT-EGFR质粒组中miR-520a-3p荧光素酶活性明显降低(P<0.05),而MUT-EGFR质粒组中miR-520a-3p荧光素酶活性无变化(P>0.05)。转染miR-520a-3p mimics质粒能明显诱导miR-520a-3p基因表达(P<0.05),而抑制EGFR mRNA和蛋白表达(P<0.05)。80μg/mL SFP处理ACHN细胞36 h后,EGFR mRNA和蛋白的表达水平均较对照组降低(均P<0.05)。与SFP组比较,SFP+miR-520a-3p inhibitor组的miR-520a-3p表达、细胞凋亡率明显降低,而EGFR表达、细胞活力明显升高,差异均有统计学意义(均P<0.05)。结论SFP通过上调miR-520a-3p水平降低EGFR表达,进而抑制肾腺癌细胞增殖、诱导其凋亡,miR-520a-3p/EGFR信号通路可能是肾腺癌治疗的新靶点。Objective To investigate the effect of sulforaphane(SFP)on apoptosis of human renal adenocarcinoma cells and its potential mechanism.Methods Human renal tubular epithelial cell line(HK-2)and retinoblastoma cell lines(ACHN,UOK146,786-0 and GRC-1)were used in our study.CCK-8 assay and flow cytometry assay were used to measure cell viability and apoptosis.The relationship between miR-520a-3p and human epidermal growth factor receptor(EGFR)was predicted and confirmed by TargetScan and dual-luciferase reporter assay.The mRNA and protein expression levels of miR-520a-3p and human EGFR were detected by quantitative real-time PCR(qRT-PCR)and Western blot.Results SFP inhibited the proliferation of ACHN cells in a time-and concentration--dependent manner(all P<0.05).After 36 h of SFP treatment,the apoptosis rate of ACHN cells in the SFP group was significantly higher than that in the control group(allP<0.05).Compared with the control group,the expression level of Cleaved-Caspase-3 in the SFP group was increased,while the expression level of Pro-Caspase-3 was decreased(P<0.05).After treatment with 20,40,and 80μg/mL SFP for 36 h,the expression levels of miR-520a-3p in ACHN cells were significantly higher than those in the control group(P<0.05).Compared with the control group,the luciferase activity of miR-520a-3p in the WT-ECFR plasmid group was significantly decreased(P<0.05),while there was no change in the luciferase activity of miR-520a-3p in the MUT-EGFR plasmid group(P>0.05).Transfection of miR-520a-3p mimics plasmid significantly induced the expression of miR-520a-3p gene(P<0.05),but inhibited the expression of ECFR mRNA and protein(P<0.05).After treatment of ACHN cells with 80μg/mL SFP for 36 h,the expression levels of ECFR mRNA and protein were lower than those in the control group(all P<0.05).Compared with the SFP group,the miR-520a-3p expression and apoptosis rate in the SFP+miR-520a-3p inhibitor group were significantly decreased,while the EGFR expression and cell viability were significantly increased(all P<0.

关 键 词：肾肿瘤 萝卜硫素 miR-520a-3p 人类表皮生长因子受体 细胞凋亡 

分 类 号：R737.11[医药卫生—肿瘤]