Study On The Function And Mechanism Of Lnc-MC And RBP-ZFP36L1 In Monocyte/macrophage Differentiation

Hematopoiesis is a highly orchestrated process wherein the pluripotent self-renewing hematopoietic stem cells (HSCs) give rise to all blood cell lineages, including monocytes/macrophages. Control of hematopoietic differentiation is a complex process requiring the coordinated expression of stage-specific transcription factors, cytokines, and noncoding RNAs. Monocytes/macrophages, which belong to white blood cells and innate immune cells, are mononuclear phagocytes that play crucial roles in the maintenance of homeostasis and the initiation of immune response. Monocyte/ macrophage differentiation is eleborately regulated by a complex regulatory network and the defects in their biogenesis and function can contribute to a broad spectrum of pathologies, such as acute myeloid leukemia (AML) and imbalance of organism homeostasis.Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Accumulating evidences have demonstrated that lncRNAs function as versatile regulators through interaction with DNA, RNA and proteins to modulate gene expression on many levels, such as chromatin remodeling, gene transcription and post-transcriptional regulation. LncRNAs are emerging as important regulators in mammalian development, but little is known about their roles in monocyte/macrophage differentiation. RNA binding proteins (RBPs) are proteins that bind to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes to regulate RNA splicing, localization, transport, decay, translation and other RNA metabolic processes. RBPs-mediated post-transcriptional control has been implicated in playing important roles in mammalian development and pathological diseases. However, the functions of specific RBP and the molecular mechanisms through which they act in monocyte/macrophage differentiation remain to be determined. In this study, through bioinformatics analysis and experimental validation, we identified a long noncoding monocytic RNA (lnc-MC) and a RNA binding protein (ZFP36L1), both of which promote monocyte/macrophage differentiation.To identify lncRNAs involved in myeloid differentiation, we comprehensively analyzed the RNA-Seq data for white blood cells and the PU.1l-ChIP-seq data from ChIPBase, and we finally obtained 390 candidate lncRNAs. Lnc-MC was chosen for further investigation on the basis of its relatively high abundance in 22 tissues and cell lines. Lnc-MC is also known as Inc-TRIP10/TCONS 00026873/XLOC 012931; it is annotated in LNCipedia, and its gene is located at chromosome 19, positions 6656385 to 6662832. Its conservation was analyzed using the UCSC Genome Browser (http://genome.ucsc.edu/index.html), which shows that lnc-MC is conserved only among some primates (such as the chimpanzee). The coding potential of lnc-MC was analyzed using ORF Finder and the Coding Potential Assessment Tool (CPAT), which suggested that lnc-MC tends to be a noncoding RNA. According to the experimental results, lnc-MC exhibits increased expression during monocyte/macrophage differentiation of THP-1 and HL-60 cells as well as CD34+ hematopoietic stem/progenitor cells (HSPCs). Gain- and loss-of-function assays demonstrate that lnc-MC promotes monocyte/macrophage differentiation of THP-1 cells and CD34+HSPCs. During the differentiation, PU.1 binds to the 1826 site upstream of lnc-MC loci and transactivate lnc-MC expression. Mechanistic investigation reveals that lnc-MC acts as a competing endogenous RNA to sequester microRNA-199a-5p (miR-199a-5p) and alleviate repression on the expression of activin A receptor typelB (ACVR1B), an important regulator of monocyte/macrophage differentiation. We also noted a repressive effect of miR-199a-5p on lnc-MC expression and function, but PU.1-dominant downregulation of miR-199a-5p weakens the role of miR-199a-5p in the reciprocal regulation between miR-199a-5p and lnc-MC. Altogether, our work demonstrates that two PU.1-regulated noncoding RNAs, lnc-MC and miR-199a-5p, have opposing roles in monocyte/macrophage differentiation and that lnc-MC facilitates the differentiation process, enhancing the effect of PU.1, by soaking up miR-199a-5p and releasing ACVR1B expression. Thus, we reveal a novel regulatory mechanism, comprising PU.1, lnc-MC, miR-199a-5p and ACVR1B in monocyte/macrophage differentiation.To systematically screen the RBPs involved in myeloid differentiation, we performed bioinformatics analysis using the expression profiling data of AML patients and in vitro myeloid differentiation from HSPCs annotated in the GEO DataSets, and obtained twenty-three RBPs with differential expression in both AML patients and myeloid differentiation. Finally we focused on ZFP36L1 which exhibits decreased expression in most of the AML patients and time-course increased expression during the monocytic differentiation according to the the array data. The experimental validation results also unraveled that ZFP36L1 expression exhibited significant down-regulation in AML patients compared with the normal controls both at the mRNA and protein levels, and also remarkable time-course up-regulation during PMA-induced moncytic differentiation of THP-1 cells and HL-60 cells as well as in vitro monocytic induction culture of CD34+ HSPCs, all in accordance with array data analyzed before. Lentivirus-mediated gain and loss of function assays demonstrate that ZFP36L1 acts as a positive regulator to participate in monocyte/macrophage differentiation. Mechanistic investigation further reveals that ZFP36L1 binds to the CDK6 mRNA 3’untranslated region bearing adenine-uridine rich elements and negatively regulates the expression of CDK6 which is subsequently demonstrated to impede the in vitro monocyte/macrophage differentiation of CD34+HSPCs. Collectively, our work unravels a ZFP36L1-mediated regulatory circuit through repressing CDK6 expression during monocyte/macrophage differentiation, which may also provide a therapeutic target for AML therapy.