长链非编码RNA CRNDE通过下调miR-136-5p表达并上调MCM5表达促进U937细胞增殖并抑制其凋亡  

lncRNA CRNDE promotes proliferation and inhibits apoptosis of U937 cells by downregulating miR-136-5p and upregulating MCM5

作　　者：刘晨[1,2] 刘北忠 沈陈兰[2] 楚璇 罗栩 余莉华 叶娇[1] 熊玲 但文冉 李健 钟梁[1] LIU Chen;LIU Beizhong;SHEN Chenlan;CHU Xuan;LUO Xu;YU Lihua;YE Jiao;XIONG Ling;DAN Wenran;LI Jian;ZHONG Liang(Key Laboratory of Clinical Laboratory Diagnostics of Chongqing,Key Laboratory of Clinical Laboratory Diagnostics Designated by the Ministry of Education,Chongqing Medical University,Chongqing 00016;Central Laboratory,Yongchuan Hospital,Chongqing Medical University,Chongqing 402160;Department of Laboratory Medicine,Yongchuan Hospital,Chongqing Medical University,Chongqing 402160,China)

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室,重庆市重点实验室,重庆400016 [2]重庆医科大学附属永川医院中心实验室,重庆402160 [3]重庆医科大学附属永川医院检验科,重庆402160

出　　处：《细胞与分子免疫学杂志》2021年第11期987-995,共9页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81772280);生物治疗国家重点实验室资助课题(SKLB202008)。

摘　　要：目的 探究长链非编码RNA结直肠肿瘤差异表达基因(CRNDE)对U937细胞增殖、凋亡和细胞周期的影响及其作用机制。方法 首先通过基因表达谱交互分析(GEPIA)数据库分析CRNDE在急性髓系白血病(AML)患者骨髓细胞中的表达水平;实时荧光定量PCR检测AML细胞系miR-136-5p、 CRNDE及微小染色体维持蛋白5(MCM5)的mRNA表达水平。构建敲低CRNDE的慢病毒载体并转染U937细胞,设为敲低CRNDE组(sh-CRNDE组)和阴性对照组(sh-NC组);分别转染miR-136-5p模拟物(miR-136-5p mimic)和miR-136-5p抑制物(miR-136-5p inhibitor)对U937细胞中的miR-136-5p进行过表达和敲低,设为miR-136-5p过表达组(miR-136-5p-mimic)、过表达阴性对照组(NC-mimic)、 miR-136-5p敲低组(miR-136-5p-inhibitor)和敲低阴性对照组(NC-inhibitor)。CCK-8法和细胞计数试验检测CRNDE和miR-136-5p对细胞增殖的影响,流式细胞术检测CRNDE和miR-136-5p对细胞周期和凋亡的影响。实时荧光定量PCR检测miR-136-5p、 CRNDE及MCM5的mRNA表达,Western blot法检测MCM5、 P53、 B细胞淋巴瘤因子2(Bcl2)、细胞周期蛋白D1(cyclin D1)和细胞周期蛋白A2(cyclin A2)的蛋白表达水平。结果 CRNDE在AML患者骨髓和细胞系中高表达。敲低CRNDE可以上调miR-136-5p,抑制MCM5 mRNA和蛋白表达,抑制细胞增殖,促进凋亡并使细胞周期阻滞在G1期。过表达miR-136-5p也可抑制MCM5核酸和蛋白水平表达。而敲低miR-136-5p则可以逆转以上作用。结论 CRNDE通过下调miR-136-5p和上调MCM5表达,进而促进U937细胞增殖并抑制其凋亡。Objective To investigate the effect of lncRNA CRNDE on proliferation, apoptosis, and cell cycle of U937 cells and its mechanism. Methods The expression level of CRNDE in bone marrow cells of AML patients was analyzed by GEPIA database;the mRNA expression levels of miR-136-5p, CRNDE, and minichromosome maintenance 5(MCM5) in AML cell lines were detected by quantitative real-time PCR(qRT-PCR). The lentiviral vector with CRNDE knocked down was constructed and transfected into U937 cells which were randomized into CRNDE knockdown group(sh-CRNDE group) and negative control group(sh-NC group);miR-136-5p mimic and miR-136-5p inhibitor were transfected respectively to overexpress and knock down miR-136-5p in U937 cells which were randomized into miR-136-5p-mimic group, NC-mimic group, miR-136-5p-inhibitor group, and NC-inhibitor group. The effect of CRNDE and miR-136-5p on proliferation was detected by CCK-8 assay and cell counting assay, and the effect of them on cell cycle and apoptosis was detected by flow cytometry. The mRNA expressions of miR-136-5p, CRNDE, and MCM5 were detected by qRT-PCR, and the protein expressions of MCM5, Bcl2, cyclin D1, and cyclin A2 weredetected by Western blotting. Results CRNDE was highly expressed in the bone marrow and cell lines of AML patients. Knockdown of CRNDE upregulated miR-136-5p, inhibited the MCM5 mRNA and protein expressions and the cell proliferation, promoted the cell apoptosis, and blocked the cell cycle in G1 phase. Overexpression of miR-136-5p also inhibited the expression of MCM5 at both mRNA and protein levels, while knockdown of miR-136-5p reversed those effects. Conclusion CRNDE promotes the proliferation and inhibits the apoptosis of U937 cells by downregulating miR-136-5p and upregulating MCM5.

关 键 词：急性髓系白血病 长链非编码RNA(lncRNA) 结直肠肿瘤差异表达基因(CRNDE) miR-136-5p 微小染色体维持蛋白5(MCM5) 

分 类 号：R733.71[医药卫生—肿瘤] R733.73[医药卫生—临床医学]