长链非编码RNA ZEB1反义RNA1通过靶向微小RNA-224调控淋巴瘤细胞增殖、侵袭、迁移的分子机制研究OACSTPCD
Molecular mechanisms of long non-coding RNA ZEB1-antisense RNA1 in regulating lymphoma cell proliferation,invasion,and migration by targeting microRNA-224
目的 探讨长链非编码RNA ZEB1反义RNA1(LncRNA ZEB1-AS1)通过靶向微小RNA(miR)-224调控淋巴瘤细胞增殖、侵袭、迁移的分子机制.方法 体外培养淋巴瘤细胞系Raji,分别转染si-NC、si-ZEB 1-AS1、miR-NC、miR-224 mimics、si-ZEB1-AS1+anti-miR-NC、si-ZEB1-AS1+anti-miR-224.采用实时荧光定量聚合酶链式反应(RT-qPCR)评估 LncRNA ZEB1-AS1和miR-224在淋巴瘤细胞中的表达量;采用细胞计数试剂盒(CCK-8)检测细胞活力;采用Transwell实验检测细胞迁移和侵袭能力;通过双荧光素酶报告实验确定LncRNA ZEB1-AS1对miR-224的调控作用;采用Western blot检测细胞周期素D1(CyclinD1)、基质金属蛋白酶(MMP)-2、MMP-9蛋白的表达水平.结果 与对照组比较,淋巴瘤组LncRNA ZEB1-AS1水平升高,miR-224表达水平降低,差异有统计学意义(P＜0.05).与si-NC组比较,si-ZEB1-AS1组中LncRNA ZEB1-AS1的表达水平、光密度(OD)值、迁移细胞和侵袭细胞数量,以及CyclinD1、MMP-2和MMP-9表达水平均降低,差异有统计学意义(P＜0.05).与miR-NC组比较,miR-224组miR-224表达水平提高,而OD值、细胞迁移和侵袭数量以及CyclinD1、MMP-2和MMP-9表达水平降低,差异有统计学意义(P＜0.05).上调miR-224水平可降低WT-ZEB1-AS1的荧光素酶活性,但不影响MUT-ZEB1-AS1的荧光素酶活性.与pcDNA-NC组相比,pcDNA-ZEB1-AS1组miR-224表达水平降低,差异有统计学意义(P＜0.05);与si-NC组比较,si-ZEB1-AS1组miR-224表达升高,差异有统计学意义(P＜0.05).与si-ZEB1-AS1+anti-miR-NC组比较,si-ZEB1-AS1+anti-miR-224组的OD值、迁移和侵袭数量,以及CyclinD1、MMP-2和MMP-9蛋白水平提高,差异有统计学意义(P＜0.05).结论 抑制LncRNA ZEB1-AS1从而靶向上调miR-224的表达,能够有效降低淋巴瘤细胞的增殖、侵袭及迁移能力.
Objective To investigate the molecular mechanisms of long non-coding RNA ZEB1-antisense RNA1(LncRNA ZEB1-AS1)in regulating lymphoma cell proliferation,invasion,and mi-gration by targeting microRNA(miR)-224.Methods The lymphoma cell line Raji was cultured in vitro and transfected with si-NC,si-ZEB1-AS1,miR-NC,miR-224 mimics,si-ZEB1-AS1+anti-miR-NC,and si-ZEB1-AS1+anti-miR-224,respectively.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to assess the expression levels of LncRNA ZEB1-AS1 and miR-224 in lymphoma cells.Cell viability was detected using the Cell Counting Kit-8(CCK-8).Cell migra-tion and invasion abilities were evaluated by Transwell experiments.Dual luciferase reporter experiments were conducted to confirm the regulatory effect of LncRNA ZEB1-AS1 on miR-224.Western blot was performed to detect the expression levels of cyclin D1(CyclinD1),matrix metalloproteinase(MMP)-2,and MMP-9 proteins.Results Compared with the control group,the lymphoma group showed increased LncRNA ZEB1-AS1 levels and decreased miR-224 expression levels(P＜0.05).Compared with the si-NC group,the si-ZEB1-AS1 group exhibited decreased expression levels of LncRNA ZEB1-AS1,optical density(OD)values,the number of migrated and invaded cells,and expression levels of CyclinD1,MMP-2,and MMP-9 protein(P＜0.05).Compared with the miR-NC group,the miR-224 group showed increased miR-224 expression levels but decreased OD val-ues,the number of migrated and invaded cells,and expression levels of CyclinD1,MMP-2,and MMP-9 proteins(P＜0.05).Upregulation of miR-224 levels reduced the luciferase activity of WT-ZEB1-AS1 but had no effect on MUT-ZEB1-AS1 luciferase activity.Compared with the pcDNA-NC group,the pcDNA-ZEB1-AS1 group showed decreased miR-224 expression levels(P＜0.05);compared with the si-NC group,the si-ZEB1-AS1 group showed increased miR-224 expression levels(P＜0.05).Compared with the si-ZEB1-AS1+anti-miR-NC group,the si-ZEB1-AS1+anti-miR-224 group showed increased OD values,the number of migrated and invaded cells,and expression levels of CyclinD1,MMP-2,and MMP-9 proteins(P＜0.05).Conclusion Inhibition of ZEB1-AS1 to targetedly upregulate of miR-224 expression can effectively reduce lymphoma cell prolifera-tion,invasion,and migration abilities.
施丹;沈超
上海健康医学院附属崇明医院,血液内科,上海,202150上海健康医学院附属崇明医院,宁养院,上海,202150
临床医学
长链非编码RNA ZEB1反义RNA1淋巴瘤微小RNA-224增殖迁移侵袭
long non-coding RNA ZEB1-antisense RNA1lymphomamicroRNA-224pro-liferationmigrationinvasion
《实用临床医药杂志》 2024 (022)
35-40 / 6
上海市崇明区"可持续发展科技创新行动计划"项目(CKY2022-20)
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