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 400016; Central Laboratory, Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China.
Central Laboratory, Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2021 Nov;37(11):987-995.
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 were detected 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.
目的 探讨长链非编码RNA CRNDE对U937细胞增殖、凋亡及细胞周期的影响及其机制。方法 通过GEPIA数据库分析急性髓系白血病（AML）患者骨髓细胞中CRNDE的表达水平；采用定量实时荧光定量PCR（qRT-PCR）检测AML细胞系中微小RNA-136-5p（miR-136-5p）、CRNDE及微小染色体维持蛋白5（MCM5）的mRNA表达水平。构建CRNDE敲低的慢病毒载体并转染至U937细胞，将其随机分为CRNDE敲低组（sh-CRNDE组）和阴性对照组（sh-NC组）；分别转染miR-136-5p模拟物和miR-136-5p抑制剂，使U937细胞中miR-136-5p过表达和敲低，将其随机分为miR-136-5p模拟物组、NC模拟物组、miR-136-5p抑制剂组和NC抑制剂组。采用CCK-8法和细胞计数法检测CRNDE和miR-136-5p对细胞增殖的影响，采用流式细胞术检测其对细胞周期和凋亡的影响。通过qRT-PCR检测miR-136-5p、CRNDE及MCM5的mRNA表达，采用蛋白质印迹法检测MCM5、Bcl2、细胞周期蛋白D1及细胞周期蛋白A2的蛋白表达。结果 CRNDE在AML患者的骨髓及细胞系中高表达。敲低CRNDE可上调miR-136-5p，抑制MCM5的mRNA和蛋白表达及细胞增殖，促进细胞凋亡，并使细胞周期阻滞于G1期。miR-136-5p过表达也可抑制MCM5的mRNA和蛋白表达，而敲低miR-136-5p可逆转这些作用。结论 CRNDE通过下调miR-136-5p、上调MCM5促进U937细胞增殖并抑制其凋亡。
