Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.

Long noncoding RNAs (lncRNAs) are emerging as important regulators in mammalian development, but little is known about their roles in monocyte/macrophage differentiation. Here we identified a long noncoding monocytic RNA (lnc-MC) that exhibits increased expression during monocyte/macrophage differentiation of THP-1 and HL-60 cells as well as CD34؉ hematopoietic stem/progenitor cells (HSPCs) and is transcriptionally activated by PU.1. Gain-and loss-of-function assays demonstrate that lnc-MC promotes monocyte/macrophage differentiation of THP-1 cells and CD34 ؉ HSPCs. 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 type 1B (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.H ematopoiesis is a highly orchestrated process wherein the pluripotent self-renewing hematopoietic stem cells (HSCs) give rise to all blood cell lineages, including monocytes/macrophages (1). Monocytes/macrophages are mononuclear phagocytes that play crucial roles in innate immunity and the inflammatory response, and defects in their biogenesis and function can contribute to a broad spectrum of pathologies (2, 3). Control of monocyte/macrophage differentiation is a complex process requiring the coordinated expression of stage-specific transcription factors, cytokines, and noncoding RNAs (4, 5).PU.1 is a hematopoiesis-specific transcription factor that binds to a purine-rich sequence (GAGGAA) and regulates lineage-specific gene expression (6). Homozygous PU.1-deficient mice died at a late gestational stage, and PU.1 mutant embryos exhibited a defect in the generation of progenitors for monocytes and granulocytes (7). High expression of PU.1 in granulocyte-macrophage progenitors (GMPs) antagonizes C/EBP␣ function and favors monocyte development. Conversely, GMPs with low expression of PU.1 commit to granulocyte differentiation (8). In addition, transcription factors RUNX1, KLF4, and MafB are important regulators in monocyte/macrophage development (9-11). Colonystimulating factors (CSF), including granulocyte-macrophage CSF, granulocyte CSF, and CSF-1, also play fundamental roles in the early and late stages of the monocyte/macrophage differentiation process (12).MicroRNAs (miRNAs) are short (20-to 24-nucleotide [nt]) noncoding RNAs tha...

Pancreatic cancer patients are often resistant to chemotherapy treatment, which results in poor prognosis. The objective of this study was to delineate the mechanism by which miR‐21 induces drug resistance to 5‐fluorouracil (5‐FU) in human pancreatic cancer cells (PATU8988 and PANC‐1). We report that PATU8988 cells resistant to 5‐FU express high levels of miR‐21 in comparison to sensitive primary PATU8988 cells. Suppression of miR‐21 expression in 5‐Fu‐resistant PATU8988 cells can alleviate its 5‐FU resistance. Meanwhile, lentiviral vector‐mediated overexpression of miR‐21 not only conferred resistance to 5‐FU but also promoted proliferation, migration, and invasion of PATU8988 and PANC‐1 cells. The proresistance effects of miR‐21 were attributed to the attenuated expression of tumor suppressor genes, including PTEN and PDCD4. Overexpression of PTEN and PDCD4 antagonized miR‐21‐induced resistance to 5‐FU and migration activity. Our work demonstrates that miR‐21 can confer drug resistance to 5‐FU in pancreatic cancer cells by regulating the expression of tumor suppressor genes, as the target genes of miR‐21, PTEN and PDCD4 can rescue 5‐FU sensitivity and the phenotypic characteristics disrupted by miR‐21.

MicroRNA-22 (miR-22) is emerging as a critical regulator in organ development and various cancers. However, its role in normal hematopoiesis and leukaemogenesis remains unclear. Here, we detected its increased expression during monocyte/macrophage differentiation of HL-60, THP1 cells and CD34+ hematopoietic stem/progenitor cells, and confirmed that PU.1, a key transcriptional factor for monocyte/macrophage differentiation, is responsible for transcriptional activation of miR-22 during the differentiation. By gain- and loss-of-function experiments, we demonstrated that miR-22 promoted monocyte/macrophage differentiation, and MECOM (EVI1) mRNA is a direct target of miR-22 and MECOM (EVI1) functions as a negative regulator in the differentiation. The miR-22-mediated MECOM degradation increased c-Jun but decreased GATA2 expression, which results in increased interaction between c-Jun and PU.1 via increasing c-Jun levels and relief of MECOM- and GATA2-mediated interference in the interaction, and thus promoting monocyte/macrophage differentiation. We also observed significantly down-regulation of PU.1 and miR-22 as well as significantly up-regulation of MECOM in acute myeloid leukemia (AML) patients. Reintroduction of miR-22 relieved the differentiation blockage and inhibited the growth of bone marrow blasts of AML patients. Our results revealed new function and mechanism of miR-22 in normal hematopoiesis and AML development and demonstrated its potential value in AML diagnosis and therapy.

miRNAs are short, noncoding RNAs that regulate expression of target genes at post-transcriptional levels and function in many important cellular processes, including differentiation, proliferation, etc. In this study, we observed down-regulation of miR-199a-5p during monocyte/macrophage differentiation of HL-60 and THP-1 cells, as well as human CD34(+) HSPCs. This down-regulation of miR-199a-5p resulted from the up-regulation of PU.1 that was demonstrated to regulate transcription of the miR-199a-2 gene negatively. Overexpression of miR-199a-5p by miR-199a-5p mimic transfection or lentivirus-mediated gene transfer significantly inhibited monocyte/macrophage differentiation of the cell lines or HSPCs. The mRNA encoding an ACVR1B was identified as a direct target of miR-199a-5p. Gradually increased ACVR1B expression level was detected during monocyte/macrophage differentiation of the leukemic cell lines and HSPCs, and knockdown of ACVR1B resulted in inhibition of monocyte/macrophage differentiation of HL-60 and THP-1 cells, which suggested that ACVR1B functions as a positive regulator of monocyte/macrophage differentiation. We demonstrated that miR-199a-5p overexpression or ACVR1B knockdown promoted proliferation of THP-1 cells through increasing phosphorylation of Rb. We also demonstrated that the down-regulation of ACVR1B reduced p-Smad2/3, which resulted in decreased expression of C/EBPα, a key regulator of monocyte/macrophage differentiation, and finally, inhibited monocyte/macrophage differentiation.

RNA binding proteins (RBPs)-mediated post-transcriptional control has been implicated in influencing various aspects of RNA metabolism and playing important roles in mammalian development and pathological diseases. However, the functions of specific RBPs 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 identify that ZFP36L1, a member of ZFP36 zinc finger protein family, exhibits significant decrease in acute myeloid leukemia (AML) patients compared with normal controls and remarkable time-course increase during monocyte/macrophage differentiation of PMA-induced THP-1 and HL-60 cells as well as induction culture of CD34+ hematopoietic stem/progenitor cells (HSPCs). 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.

In China, the traditional Chinese medicine "YiSui ShenXu Granule" has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from "YiSui ShenXu Granule" are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.

Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine and cellular therapy due to their multi-lineage differentiation potential and immunomodulatory function. The applicability of MSCs also depends on their cellular sources and in vivo functions. Here in this study, we systematically compared the morphologic characteristics, immunophenotypes and the adipogenic differentiation of MSCs derived from umbilical cord (UC), adipose tissue (Ad) and bone marrow (BM). We found that the three tissues-derived MSCs displayed decreased adipogenic capacity in the order: Ad-MSC > BM-MSC > UC-MSC, and no morphologic and immunophenotypic differences were observed. Mechanistic investigation revealed a miR-301b~miR-130b—PPARγ axis, whose expression pattern in UC-MSC, Ad-MSC and BM-MSC significantly correlates with their adipogenic capacity. Our results come up with a potential mechanism to elucidate the differential adipogenesis of Ad-MSC, BM-MSC and UC-MSC, which would provide instructional advice for which source of MSCs to choose according to a certain clinical purpose. Furthermore, the miR-301b~miR-130b—PPARγ axis may also be used as a potential therapeutic target for the disorders associated with MSCs-mediated abnormal adipogenesis.