摘要
目的探讨长链非编码RNA(long non-coding RNA,LncRNA)LINC01140对U937细胞增殖及周期的影响。方法收集陆军军医大学第一附属医院血液科2016年7-12月20例急性髓细胞白血病(acute myeloid leukemia,AML)患者骨髓标本和2017年5-6月在血液科进行骨髓检查提示为正常的10例骨髓标本,采用qRT-PCR检测AML患者骨髓及白血病细胞中LINC01140的表达情况。通过构建LncRNA LINC01140-shRNA慢病毒干扰载体转染U937细胞(对照组为shR-NC,实验组为shR-1、shR-2),采用qRT-PCR检测LINC01140的表达。细胞计数法、细胞克隆成球实验分别检测干扰LINC01140表达对U937细胞增殖和克隆成球能力的影响。流式细胞术、蛋白免疫印迹分别检测U937细胞周期和周期相关蛋白的变化。结果 (1)LncRNA LINC01140在白血病患者和细胞中表达显著升高(P<0.01)。(2)下调LINC01140表达,U937细胞增殖减慢,细胞克隆成球数量减少,表明细胞增殖能力显著降低(P<0.01)。(3)流式细胞术检测结果显示干扰LINC01140表达可导致U937细胞G_1期阻滞,其中shR-1和shR-2的G_1期细胞的百分比分别为(62.89±1.66)%、(59.03±1.00)%,相对于对照组的(46.25±1.06)%,差异有统计学意义(P<0.01)。Western blot检测结果显示干扰LINC01140表达可下调U937细胞中G_1期相关蛋白Cyclin E1的表达,并可升高G_1期相关蛋白p21和p27的表达。结论LncRNA LINC01140在AML患者中高表达,在U937细胞中敲降LINC01140可导致细胞周期G_1期阻滞,从而显著抑制细胞增殖。
Objective To investigate the effect of long non-coding RNA （LncRNA） LINC01140 on proliferation and cell cycle in U937 cells. Methods Bone marrow samples from 20 acute myeloid leukemia （AML） patients identified in the hematological department of our university from July to December 2016, and from 10 healthy individuals for examination during May and June 2017 were collected in this study. Quantitative real-time PCR （qRT-PCR） was used to detect the expression of LINC01140 in the bone marrow tissues and leukemia cells. Lentiviral vector LncRNA LINC01140 was constructed, and then transfected into U937 cells （shR-NC in the control group, shR-1 and shR-2 in the experimental group）. The expression of LINC01140 was detected by qRT-PCR. Cell proliferation and colony formation were detected by cell counting and tumor sphere formation assay. Cell cycle distribution and the expression of related proteins were determined by flow cytometry and Western blot analyses, respectively. Results ① LncRNA LINC01140 was highly expressed in the bone marrow tissues from AML patients and U937 cells （P〈0.01）. ② Knockdown of LINC01140 with siRNA significantly inhibited cell proliferation and colony formation in U937 cells （P〈0.01）. ③ Flow cytometry indicated that knockdown of LINC01140 resulted in cell cycle arrested at G1 phase, with the proportion of G1 phase cells accounting for （62.89±1.66）% and （59.03±1.00）% respectively in shR-1 and shR-2 cells, which were significantly higher than that in the control cells [（46.25±1.06）%, P〈0.01]. Western blotting showed that the knockdown of LINC01140 decreased the expression level of G1 phase related protein Cyclin E1 and increased those of p21 and p27. Conclusion LncRNA LINC01140 is highly expressed in AML patients. Its knockdown induces cell cycle arrested in G1 phase and thus leads inhibition of cell proliferation in U937 cells.
作者
骆青松
胡晓烨
朱垚
高宁
LUO Qingsong;HU Xiaoye;ZHU Yao;GAO Ning(Department of Pharmacognosy, Faculty of Pharmacy ＆ Laboratory Medicine, Army Medical University （Third Military Medical University）, Chongqing, 400038, Chin)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第9期768-773,共6页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(31571425)~~
