MiR-30b通过靶向CCL22调控人NK/T细胞淋巴瘤细胞株SNK-6和YTS顺铂耐受的研究  

MiR-306 Regulates the Cisplatin-Resistance of Human NK/T Cell Lypnphoma Cell Lines SNK-6 and YTS by Targeting the CCL22

作　　者：王健红 刘芳[1] 段晓辉 郝彩霞 刘祥祥 鲁英娟 王哲[1] 梁蓉 WANG Jian-Hong;LIU Fang;DUAN Xiao-Hui;HAO Cai-Xia;LIU Xiang-Xiang;LU Ying-Juan;WANG Zhe;LIANG Rong(Department of Hematology,Xijing Hospital,Air Force Medical University of PLA,Xi'an,710032,Shaanxi Province,China)

机构地区：[1]中国人民解放军空军军医大学西京医院血液内科

出　　处：《中国实验血液学杂志》2019年第6期1838-1844,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(8136064)

摘　　要：目的:探讨miR-30b对人NK/T细胞淋巴瘤(NKTCL)SNK-6和YTS细胞对顺铂耐受的调控及其作用机制。方法:培养正常NK细胞以及SNK-6和YTS细胞,应用real-time PCR法和Western blot分别检测miR-30b和巨噬细胞来源的趋化因子(CCL22)mRNA和蛋白的表达。以miR-30b过表达和miR-30b抑制剂分别转染SNK-6和YTS细胞,MTT法检测SNK-6和YTS细胞对顺铂耐受性的影响;caspase-3试剂盒检测caspase-3活性;流式细胞术检测细胞凋亡水平。双荧光素酶报告基因检测miR-30b与CCL22的靶向关系。Western blot法检测CCL22表达,评估CCL22对细胞耐受性和caspase-3活性的影响。结果:与正常NK细胞相比,SNK-6和YTS细胞中miR-30b表达均显著降低(P<0.01),CCL22 mRNA表达均显著升高(P<0.01)。miR-30b过表达可降低细胞活性,顺铂耐受性显著降低(P<0.05),细胞凋亡比例显著增高(P<0.05),Caspase-3活性增加(P<0.05)。miR-30b抑制剂作用与miR-30b过表达作用相反。荧光素酶活性分析显示,miR-30b过表达显著降低荧光素酶报告基因的荧光强度,而结合位点突变后荧光素酶活性不变,表明CCL22是miR-30b的直接靶分子。此外,miR-30b过表达显著降低CCL22表达(P<0.05)。而CCL22过表达可增加SNK-6细胞活性,降低Caspase-3活性,逆转miR-30b的作用,增加对顺铂的耐受性。结论:miR-30b可通过靶向CCL22抑制人NKTCL SNK-6和YTS细胞的顺铂耐受性。Objective:To explore the effect and mechanism of miR-30 b on cisplatin-resistance of human NK/T cell lymphoma lines SNK-6 and YTS cells.Methods:Normal NK cells,SNK-6 and YTS cells were cultured,the expression levels of miR-30 b and macrophage-derived chemokine(CCL22)were detected by real-time PCR assay,and the CCL22 expression was detected by Western blot.The SNK-6 and YTS cells were transfected with miR-30 b mimics and inhibitor respectively,then the effect of cisplatin resistance in SNK-6 and YTS cells was measured by MTT assay,the activity of caspase-3 was detected by caspase-3 assay kit,and the cell apoptosis was analyzed by flow cytometry.Dual-luciferase reporter gene assay was used to determine the targeting relationship between miR-30 b and CCL22.Furthermore,the effect of CCL22 on cisplatin-resistance and caspase-3 actirity was also evaluated.Results:Compared with the normal NK cells,the expression levels of miR-30 b significantly decreased in both SNK-6 and YTS cells(P<0.01),but CCL22 mRNA expression increase in both cells(P<0.01).MiR-30 b mimics decreased the cell activity(P<0.05),down-regulated the cisplatin-resistance(P<0.05),and increased cell apoptosis and caspase-3 activity(P<0.05).The effects of miR-30 b inhibitor were contrary to the mimics.Up-regulation of miR-30 b expression significantly decreased the luciferase activity in CCL223'-UTR-transfected NK cells,but not in Mut-CCL223'UTR group,suggesting that CCL22 could act as a direct target of miR-30 b.The expressions of CCL22 pathway proteins were down-regulated after SNK-6 cells transfected with miR-30 b mimics(P<0.05),while this effect was restored by overexpression of CCL22.Moreover,CCL22 overexpression also increased the cell activity and decreased caspase-3 activity when SNK-6 cells were transfected with miR-30 b mimics.Conclusion:MiR-30 b inhibits cisplatin-resistance of human NK/TCL SNK-6 and YTS cells by targeting CCL22.

关 键 词：miR-30b 顺铂耐受 巨噬细胞来源的趋化因子 NK/T细胞淋巴瘤 

分 类 号：R733.1[医药卫生—肿瘤]