Beiyan Zhou

miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely.

MicroRNAs (miRNAs) are a family of ≈22-nt noncoding RNAs that can posttranscriptionally regulate gene expression. Several miRNAs are specifically expressed in hematopoietic cells. Here we show that one such miRNA, miR-150, is mainly expressed in the lymph nodes and spleen and is highly up-regulated during the development of mature T and B cells; expression of miR-150 is sharply up-regulated at the immature B cell stage. Overexpression of miR-150 in hematopoietic stem cells, followed by bone marrow transplantation, had little effect on the formation of either mature CD8- and CD4-positive T cells or granulocytes or macrophages, but the formation of mature B cells was greatly impaired. Furthermore, premature expression of miR-150 blocked the transition from the pro-B to the pre-B stage. Our results indicate that miR-150 most likely down-regulates mRNAs that are important for pre- and pro-B cell formation or function, and its ectopic expression in these cells blocks further development of B cells.

Micromanagement of the immune system by microRNAs

MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved small non-coding RNAs that are thought to control gene expression by targeting mRNAs for degradation or translational repression. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programmes at the post-transcriptional level and has diverse functional roles in animals. Here, we provide an overview of the mechanisms by which miRNAs regulate gene expression, with specific focus on the role of miRNAs in regulating the development of immune cells and in modulating innate and adaptive immune responses.

MicroRNA miR-125b causes leukemia

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL–induced leukemia and to independently induce leukemia in a mouse model.

microRNA-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets

ABSTRACT MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

A Novel Regulator of Macrophage Activation miR-223 in Obesity-Associated Adipose Tissue Inflammation

Background— Macrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. Methods and Results— We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulin resistance. miR-223−/− mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell–mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow–derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic proinflammatory pathways and enhances the alternative antiinflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. Conclusion— For the first time, this study demonstrates that miR-223 acts to inhibit Pknox1, suppressing proinflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.Background— Macrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. Methods and Results— We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulin resistance. miR-223−/− mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell–mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow–derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic proinflammatory pathways and enhances the alternative antiinflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. Conclusion— For the first time, this study demonstrates that miR-223 acts to inhibit Pknox1, suppressing proinflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance. # Clinical Perspective {#article-title-46}

Integrated microRNA and mRNA expression profiling in a rat colon carcinogenesis model: effect of a chemo-protective diet

We have recently demonstrated that nutritional bioactives (fish oil and pectin) modulate microRNA molecular switches in the colon. Since integrated analysis of microRNA and mRNA expression at an early stage of colon cancer development is lacking, in this study, four computational approaches were utilized to test the hypothesis that microRNAs and their posttranscriptionally regulated mRNA targets, i.e., both total mRNAs and actively translated mRNA transcripts, are differentially modulated by carcinogen and diet treatment. Sprague-Dawley rats were fed diets containing corn oil ± fish oil with pectin ± cellulose and injected with azoxymethane or saline (control). Colonic mucosa was assayed at an early time of cancer progression, and global gene set enrichment analysis was used to obtain those microRNAs significantly enriched by the change in expression of their putative target genes. In addition, cumulative distribution function plots and functional network analyses were used to evaluate the impact of diet and carcinogen combination on mRNA levels induced via microRNA alterations. Finally, linear discriminant analysis was used to identify the best single-, two-, and three-microRNA combinations for classifying dietary effects and colon tumor development. We demonstrate that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs (miR-16, miR-19b, miR-21, miR26b, miR27b, miR-93, and miR-203) linked to canonical oncogenic signaling pathways. Complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels.

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Pregnane X receptor suppresses proliferation and tumourigenicity of colon cancer cells

Background:Pregnane X receptor (PXR) is a nuclear receptor that regulates the metabolism and disposition of various xenobiotics and endobioitics. We investigated a novel PXR function in regulating colon tumourigenesis in this study.Methods:Histochemistry, transfection, cell proliferation assay, anchorage-α-dependent assay, xenograft, immunohistochemistry, immunofluorescence flow cytometry.Results:Using histochemistry analysis, we found that PXR expressions were lost or greatly diminished in many colon tumours. Ectopic expression of human PXR through stable transfection of PXR into colon cancer cell line HT29 significantly inhibited cell proliferation as determined by cell proliferation assay and anchorage-independent assay. Pregnane X receptor suppressed significantly HT29 xenograft tumour growth in nude mice compared with control (310±6.2 vs 120±6 mg, P<0.01). Immunohistochemistry and immunofluorescence analysis of Ki-67 on excised xenograft tumour tissues showed that PXR inhibited cancer cell proliferation. Furthermore, expressions of PXR and Ki-67 were mutually exclusive. The flow cytometry analysis indicated that PXR caused G0/G1 cell-cycle arrest. p21WAF1/CIP1 expression was markedly elevated whereas E2F1 expression was inhibited by PXR.Conclusion:PXR inhibits the proliferation and tumourigenicity of colon cancer cells by controlling cell cycle at G0/G1 cell phase by regulating p21WAF1/CIP1 and E2F/Rb pathways.

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs). Methodology/Principal Findings The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10−8 to 10−5 M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium. Conclusion GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.

MicroRNA-223 is a crucial mediator of PPARγ-regulated alternative macrophage activation

Polarized activation of adipose tissue macrophages (ATMs) is crucial for maintaining adipose tissue function and mediating obesity-associated cardiovascular risk and metabolic abnormalities; however, the regulatory network of this key process is not well defined. Here, we identified a PPARγ/microRNA-223 (miR-223) regulatory axis that controls macrophage polarization by targeting distinct downstream genes to shift the cellular response to various stimuli. In BM-derived macrophages, PPARγ directly enhanced miR-223 expression upon exposure to Th2 stimuli. ChIP analysis, followed by enhancer reporter assays, revealed that this effect was mediated by PPARγ binding 3 PPARγ regulatory elements (PPREs) upstream of the pre-miR-223 coding region. Moreover, deletion of miR-223 impaired PPARγ-dependent macrophage alternative activation in cells cultured ex vivo and in mice fed a high-fat diet. We identified Rasa1 and Nfat5 as genuine miR-223 targets that are critical for PPARγ-dependent macrophage alternative activation, whereas the proinflammatory regulator Pknox1, which we reported previously, mediated miR-223-regulated macrophage classical activation. In summary, this study provides evidence to support the crucial role of a PPARγ/miR-223 regulatory axis in controlling macrophage polarization via distinct downstream target genes.