应用慢病毒shRNA文库结合二代测序筛选白血病细胞系增殖相关lncRNA

Screening of proliferation related lncRNAs in leukemia cell lines by lentivirus shRNA library combined with second-generation sequencing

摘要:

长链非编码RNA (long non-coding RNA,lncRNA)是包括细胞增殖在内的许多细胞过程的重要调节因子。虽然已有研究表明多种lncRNA在造血系统恶性肿瘤的发生发展过程中发挥重要作用，但是缺少一个更全面和无偏倚的方法同时研究多个lncRNA中对白血病细胞系产生功能性影响的lncRNA。在此，我们利用短发夹RNA (short hairpin RNA,shRNA)文库结合高通量测序的方法，筛选对白血病细胞系增殖有影响的lncRNA，确定了74个候选lncRNAs。从中选取lncRNA C20orf204-203作为验证研究对象，发现C20orf204-203在K562和THP-1细胞系中均定位于胞质，敲降C20orf204-203的K562和THP-1细胞系增殖能力降低，早期凋亡细胞增加，BAD基因在mRNA水平上表达量增加，TP53、BCL2蛋白表达量下降，在THP-1细胞系中Caspase 3蛋白表达量减少，激活型Caspase 3蛋白表达量上升，但是二者变化在两种细胞系中不一致。结果表明，在白血病细胞系中敲降lncRNA C20orf204-203会使细胞增殖能力降低。但其在不同细胞系作用途径和机制可能存在差异。这一研究表明了利用shRNA文库结合高通量测序大规模研究lncRNA在白血病细胞系中发挥作用的可行性。

Abstract:

Long non-coding RNA (lncRNA) has become an important regulator of many cellular processes, including cell proliferation. Although studies have shown that a variety of lncRNAs play an important role in the occurrence and development of hematopoietic malignancies, a more comprehensive and unbiased method to study the function of lncRNAs in leukemia cell lines is lacking. Here, we used short hairpin RNA (shRNA) library combined with high-throughput sequencing to screen lncRNAs that may affect the proliferation of leukemia cell lines, and identified lncRNA C20orf204-203 among 74 candidate lncRNAs in this study. Further experiments showed that C20orf204-203 was localized in the cytoplasm in both K562 and THP-1 cell lines. C20orf204-203 knockdown decreased the proliferation of K562 and THP-1 cell lines accompanied with the increased proportion of early apoptotic cells. We observed the increased mRNA level of BAD gene while decreased protein level of TP53 and BCL2. The expression of Caspase 3 decreased and Caspase 3-cleaved protein increased in THP-1 cell line. However, their changes were inconsistent in the two cell lines. Our experimental results showed that knockdown of lncRNA C20orf204-203 in leukemia cell lines affected cell proliferation although the mechanism of action in different cell lines may differ. Importantly, our research demonstrated the feasibility of using shRNA library combined with high-throughput sequencing to study the role of lncRNA in leukemia cell lines on a large scale.