摘要
目的探讨长链非编码RNA(lncRNA)-MIAT通过调控微小RNA(miR)-584/ZESTE同源物增强子2(EZH2)轴对弥漫性大B细胞淋巴瘤(DLBCL)细胞自噬的影响。方法将DLBCL细胞系Raji分为siMIAT组[lncRNA-MIAT的小干扰RNA(siRNA)构建沉默组]、沉默阴性对照组(siNC组)、miR-584的模拟物组(mimic组)、模拟物阴性对照组(mimic-NC组)。将siMIAT组的Raji继续分为miR-584的inhibitor组(inhibitor组)、inhibitor的阴性对照组(inhibitor-NC组)、EZH2过表达组(pcDNA-EZH2组)以及过表达空载体组(pcDNA-null组)。用实时定量PCR法(qRT-PCR)检测lncRNA-MIAT和miR-584的表达,用荧光原位杂交(FISH)实验检测lncRNA-MIAT在Raji中的定位。用Western blot法检测EZH2和细胞自噬标志物LC3-I、LC3-II、Atg5、Beclin-1和p62的表达。CCK-8法检测细胞的增殖活性。用双荧光素酶报告基因分析miR-584与EZH2的靶向调控作用以及miR-584与lncRNA-MIAT的靶向调控作用。结果与B淋巴细胞系MAO比,lncRNA-MIAT在Raji中高表达(P<0.05),lncRNA-MIAT的表达主要定位在MAO和Raji的细胞质中。沉默lncRNA-MIAT抑制细胞的增殖活性并抑制细胞的自噬(均P<0.05),沉默lncRNA-MIAT还抑制EZH2的表达并促进miR-584的表达(均P<0.05)。生信分析预测lncRNA-MIAT与miR-584具有多个结合位点,以及EZH2与miR-584也具有多个结合位点,双荧光素酶报告基因实验证实EZH2是miR-584的靶基因,且miR-584与lncRNA-MIAT也具有靶向调控作用。在沉默lncRNA-MIAT的细胞中,与inhibitor-NC组比,inhibitor组中细胞的增殖活性以及细胞的自噬均增加(P<0.05)。在沉默lncRNA-MIAT的细胞中,与pcDNA-null组比,pcDNA-EZH2组中细胞的增殖活性以及细胞的自噬均增加(P<0.05)。结论lncRNA-MIAT通过靶向调控miR-584/EZH2轴促进DLBCL的增殖,增强DLBCL细胞的自噬。
Objective To investigate the effect of long non-coding RNA(lncRNA)-MIAT on autophagy in diffuse large B-cell lymphoma(DLBCL)cells by regulating micRNA(miR)-584/ZESTE homolog enhancer 2(EZH2)axis.Methods DLBCL cell line Raji was divided into a silenced group for lncRNA-MIAT using small interfering RNA(siMIAT group),a silenced negative control group(siNC group),a mimic group for miR-584(mimic group),and a mimic negative control group(mimic-NC group).The siMIAT group of Raji was further divided into an inhibitor group for miR-584(inhibitor group),an inhibitor negative control group(inhibitor-NC group),an EZH2 overexpression group(pcDNA-EZH2 group),and an overexpression empty vector group(pcDNA-null group).Real-time quantitative PCR(qRT-PCR)was used to detect the expression of lncRNA-MIAT and miR-584,while fluorescence in situ hybridization(FISH)experiment was used to detect the localization of lncRNA-MIAT in Raji.Western blot was used to detect the expression of EZH2 and autophagy markers LC3-I,LC3-II,Atg5,Beclin-1,and p62.CCK-8 assay was used to detect cell proliferation activity.Dual luciferase reporter gene analysis was used to analyze the targeted regulation of miR-584 and EZH2,as well as the targeted regulation of miR-584 and lncRNA-MIAT.Results The results showed that compared with normal human B lymphocyte cell line MAO,lncRNA-MIAT was highly expressed in Raji(P<0.05)and was mainly located in the cytoplasm.Silencing lncRNA-MIAT inhibited cell proliferation activity and autophagy(both P<0.05),while also inhibiting EZH2 expression and promoting miR-584 expression(both P<0.05).Bioinformatics analysis predicted that lncRNA-MIAT and miR-584 had multiple binding sites,as well as EZH2 and miR-584.Dual luciferase reporter gene experiments confirmed that EZH2 was a target gene of miR-584,and that lncRNA-MIAT also had a targeted regulatory effect on miR-584.In cells with silenced lncRNA-MIAT,cell proliferation activity and autophagy increased in the inhibitor group compared with the inhibitor negative control group(both P<0.05).In cells with silenced lncRNA-MIAT,cell proliferation activity and autophagy increased in the pcDNA-EZH2 group compared with the pcDNA-null group(both P<0.05).Conclusion lncRNA-MIAT promotes autophagy in lymphoma cells by targeting the miR-584/EZH2 axis.
作者
张红莉
徐晓玮
阿迪娜·乌提库尔
石雨薇
ZHANG Hongli;XU Xiaowei;ADINA Wutikuer;SHI Yuwei(Department of Hematology,The Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830062,China)
出处
《西部医学》
2024年第10期1412-1418,1426,共8页
Medical Journal of West China
基金
新疆神经系统疾病研究重点实验室项目(XJDX1711-2247)。
