Objective: To explore the effect and mechanism of miR-30b 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-30b 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-30b 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-30b 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-30b significantly decreased in both SNK-6 and YTS cells (P＜0.01), but CCL22 mRNA expression increase in both cells (P＜0.01). MiR-30b 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-30b inhibitor were contrary to the mimics. Up-regulation of miR-30b expression significantly decreased the luciferase activity in CCL22 3'-UTR-transfected NK cells, but not in Mut-CCL22 3'UTR group, suggesting that CCL22 could act as a direct target of miR-30b. The expressions of CCL22 pathway proteins were down-regulated after SNK-6 cells transfected with miR-30b 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-30b mimics.
Conclusion: MiR-30b inhibits cisplatin-resistance of human NK/TCL SNK-6 and YTS cells by targeting CCL22.
题目: 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.
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。.