lncRNA ENST00000655811.1在胸腺瘤中的表达及对细胞增殖、迁移和凋亡能力的影响

Expression of lncRNA ENST00000655811.1 in thymoma and its effect on proliferation,migration and capacity for apoptosis

目的检测lncRNA ENST00000655811.1在胸腺瘤组织中的表达水平,探讨其对细胞增殖、迁移和凋亡的影响及其作用机制。方法收集2例胸腺瘤组织和癌旁组织,高通量测序技术检测差异表达lncRNA,以实时定量荧光聚合酶链反应(quantitative real time polymerase chain reaction,qPCR)对上调表达最显著的10个lncRNA进行验证;收集15例胸腺瘤组织、癌旁组织和外周血样本,qPCR检测lncRNA ENST00000655811.1的相对表达量,分析其与临床指标相关性;构建lncRNA ENST00000655811.1过表达质粒以及特异性敲降siRNA转染HEK293、MTEC1细胞,分别使用CCK8、EdU、划痕、transwell、蛋白印迹法和细胞流式实验分析lncRNA ENST00000655811.1对细胞增殖、迁移和凋亡的作用;最后运用生物信息学软件预测lncRNA ENST00000655811.1的靶标miRNA,并以双荧光素酶报告基因实验验证其具体结合位点。结果高通量测序筛选出6436个差异表达lncRNA(上调2404个,下调4032个)。其中上调最为显著的lncRNA ENST00000655811.1在胸腺瘤组织的表达水平明显高于癌旁组织[0.024(0.009,0.058)vs.0.005(0.002,0.030),P=0.0266]。与正常人外周血相比,lncRNA ENST00000655811.1在胸腺瘤患者外周血中表达显著增加[0.025(0.012,0.133)vs.0.002(0.001,0.006),P<0.0001]。胸腺瘤组织中lncRNA ENST00000655811.1表达水平与胸腺瘤分期、病理学分型及是否伴有重症肌无力无明显相关性。在HEK293、MTEC1细胞中,lncRNA ENST00000655811.1显著促进细胞增殖和迁移,并抑制其凋亡。lncRNA ENST00000655811.1通过其951-972碱基位点与miR-619-5p特异性结合。结论lncRNA ENST00000655811.1在胸腺瘤患者癌组织和外周血中表达水平显著上升,lncRNA ENST00000655811.1可能通过miR-619-5p在胸腺瘤细胞中发挥促进细胞增殖、迁移以及抑制凋亡的作用。

Objective To investigate the expression level of lncRNA ENST00000655811.1 in thymoma and explore its effect on cell proliferation,migration,apoptosis and its underlying mechanism.Method High-throughput sequencing technology was used to detect differentially expressed lncRNAs of 2 collected thymoma tissues and adjacent tissues,then quantitative real time polymerase chain reaction(qPCR)was used to verify the top 10 upregulated lncRNAs.The relative expression levels of lncRNA ENST00000655811.1 in thymoma,adjacent tissue and blood from 15 patients were detected by qPCR,and the correlations between lncRNA ENST00000655811.1 and clinical indicators were analyzed.lncRNA ENST00000655811.1 overexpression plasmid and specific knockdown siRNA were constructed and transfected to HEK293 and MTEC1 cells.CCK8,EdU,wound healing,transwell,western blotting and flow cytometry were used to analyze the effects of lncRNA ENST00000655811.1 on cell proliferation,migration and apoptosis.Finally,bioinformatic software was used to predict the target miRNA of lncRNA ENST00000655811.1,then dual luciferase reporter gene assay was used to verify this specific binding site.Result A total of 6436 differentially expressed lncRNAs were screened by high-throughput sequencing(2404 up-regulated and 4032 down-regulated).The expression level of lncRNA ENST00000655811.1 in thymoma tissue was significantly higher than that in adjacent tissue[0.024(0.009,0.058)vs.0.005(0.002,0.030),P=0.0266].The expression of lncRNA ENST00000655811.1 in the peripheral blood of thymoma patients was significantly increased compared with that of normal controls[0.025(0.012,0.133)vs.0.002(0.001,0.006),P<0.0001].However,the expression levels of lncRNA ENST00000655811.1 in thymoma tissues were not correlated with thymoma stage,pathological type,and myasthenia gravis.lncRNA ENST00000655811.1 significantly promoted cell proliferation and migration and inhibited apoptosis in HEK293 and MTEC1 cells.lncRNA ENST00000655811.1 specifically binded to miR-619-5p through its 951-972 base site.Conclusion The expression level of lncRNA ENST00000655811.1 may significantly increase in cancer tissue and peripheral blood of thymoma patients.lncRNA ENST00000655811.1 may play a role in promoting cell proliferation,migration and inhibiting apoptosis through binding with miR-619-5p.