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miR-218-2-3P靶向SIN3A基因影响NK/T细胞淋巴瘤的增殖和凋亡

Effect of miR-218-2-3P on proliferation and apoptosis of NK/T-cell lymphoma by targeting SIN3A
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摘要 目的·探讨miR-218-2-3P在NK/T细胞淋巴瘤中的表达情况,及其通过靶向SIN3A基因对NK/T细胞淋巴瘤增殖、凋亡及周期的影响。方法·采用实时荧光定量PCR(quantitative real-time PCR,qPCR)检测miR-218-2-3P在正常人NK细胞和NK/T细胞淋巴瘤细胞NK92MI、NKYS中的表达。采用脂质体3000瞬时转染法向NK92MI细胞分别转染含无义序列的抑制剂(inhibitor NC)、miR-218-2-3P抑制剂(mi R-218-2-3P inhibitor)及不含片段的转染试剂,并采用qPCR和蛋白质印迹法(Western blotting)分别检测上述3组即inhibitor NC组、miR-218-2-3P inhibitor组及空白对照组细胞中miR-218-2-3P及SIN3A蛋白的表达水平。通过CCK8法检测3组细胞的增殖活力。利用流式细胞仪检测3组细胞的凋亡率和周期。采用双荧光素酶报告基因实验检测SIN3A是否为miR-218-2-3P的靶基因。向NK92MI细胞分别转染mi R-218-2-3P inhibitor+SIN3A干扰小RNA(si-SIN3A)、miR-218-2-3P inhibitor+含无义序列的干扰小RNA(si-NC),并采用CCK8法检测该2组细胞的增殖活力。结果·与正常人NK细胞相比,NK92MI细胞、NKYS细胞中miR-218-2-3P的表达显著升高(均P<0.05)。与空白对照组和inhibitor NC组相比,miR-218-2-3P inhibitor组NK92MI细胞的增殖活力减弱(均P<0.05)、凋亡率增加(均P<0.05)且发生了G0/G1期细胞周期阻滞(均P<0.05)。双荧光素酶报告基因实验显示SIN3A为miR-218-2-3P的靶基因。与空白对照组和inhibitor NC组相比,miR-218-2-3P inhibitor组的SIN3A蛋白表达水平升高(均P<0.05)。下调SIN3A表达,能够恢复由转染miR-218-2-3P inhibitor减弱的细胞增殖活力(均P<0.05)。结论·miR-218-2-3P在NK92MI、NKYS细胞株中表达较高,其可能通过靶向调控SIN3A对NK/T细胞淋巴瘤的增殖、凋亡及周期产生影响。 Objective·To investigate the expression of miR-218-2-3P in NK/T-cell lymphoma,and the effect of miR-218-2-3P on the proliferation,apoptosis and cycle of NK/T-cell lymphoma by targeting SIN3A.Methods·Quantitative real-time PCR(qPCR)was used to detect the expressions of miR-218-2-3P in normal NK cells and NK/T-cell lymphoma cells NK92MI and NKYS.Lipofectamine 3000 was used to transfect the inhibitor containing nonsense sequences(inhibitors NC),miR-218-2-3P inhibitor and the same dose of transfection reagent without any fragment into NK92MI cells,which were divided into three groups.qPCR and Western blotting were used to detect the expression levels of miR-218-2-3P and SIN3A protein in the inhibitor NC group,the miR-218-2-3P inhibitor group and the blank control group,respectively.The cell proliferation activities of the three group were measured by CCK8 method.The apoptosis rates and cell cycles of the three group were determined by flow cytometry.The double luciferase reporter gene assay was performed to detect whether SIN3A was a target gene of miR-218-2-3P.NK92MI cells were transfected with miR-218-2-3P inhibitor+SIN3A small interfering RNA(si-SIN3A)and miR-218-2-3P inhibitor+nonsense sequences small interfering RNA(si-NC),respectively,which were divided into two groups.The cell proliferation activities of the two groups were detected by CCK8 method.Results·Compared with the normal NK cells,the expressions of miR-218-2-3P in NK92MI and NKYS cells significantly increased(both P<0.05).Compared with the blank control group and the inhibitor NC group,the proliferation activity of NK92MI cells in the miR-218-2-3P inhibitor group decreased(both P<0.05),the apoptosis rate increased(both P<0.05)and cell cycle arrest occurred in G0/G1 phase(both P<0.05).The double luciferase reporter gene assay showed that SIN3A was the target gene of miR-218-2-3P.Compared with the blank control group and the inhibitor NC group,the SIN3A protein expression of the miR-218-2-3P inhibitor group was increased(both P<0.05).The down-regulation of SIN3A expression could restore the proliferation activity of cells weakened by miR-218-2-3P inhibitor(both P<0.05).Conclusion·miR-218-2-3P is highly expressed in the NK92MI and NKYS cell lines.miR-218-2-3P may affect the proliferation,apoptosis and cycle of NK/T-cell lymphoma through targeted regulation of SIN3A.
作者 王佳琳 季迪 陈祥 杨博 余林 WANG Jia-lin;JI Di;CHEN Xiang;YANG Bo;YU Lin(Department of Otorhinolaryngology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处 《上海交通大学学报(医学版)》 CAS CSCD 北大核心 2019年第12期1394-1401,共8页 Journal of Shanghai Jiao tong University:Medical Science
基金 重庆市科学技术委员会科技惠民计划项目(cstc2015jcsf10001-02-03)~~
关键词 NK/T细胞淋巴瘤 miR-218-2-3P SIN3A基因 增殖 凋亡 细胞周期 NK/T-cell lymphoma miR-218-2-3P SIN3A proliferation apoptosis cell cycle
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