scholarly journals MicroRNA-200b Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting p27 and KLF9

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2417
Author(s):  
Xiayang Jin ◽  
Jiqing Wang ◽  
Jiang Hu ◽  
Xiu Liu ◽  
Shaobin Li ◽  
...  
Keyword(s):  
Target Genes ◽  
Expression Profiles ◽  
Adipogenic Differentiation ◽  
Ovis Aries ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Expression Levels ◽  
Viability Analysis ◽  
Proliferation And Differentiation

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.

scholarly journals MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 820
Author(s):  
Xiayang Jin ◽  
Zhiyun Hao ◽  
Mengli Zhao ◽  
Jiyuan Shen ◽  
Na Ke ◽  
...  
Keyword(s):  
Adipogenic Differentiation ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Over Expression ◽  
Proliferation And Differentiation ◽  
Reporter Assays ◽  
Luciferase Reporter Vector ◽  
Differentiation And Proliferation ◽  
Subcutaneous Adipose

MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

scholarly journals Elevated miR-10a-5p facilitates cell cycle and restrains adipogenic differentiation via targeting Map2k6 and Fasn, respectively

2020 ◽  
Vol 52 (11) ◽  
pp. 1227-1235
Author(s):  
Xiaoyu Wang ◽  
Huifang Zhang ◽  
Meixue Xu ◽  
Xin’E Shi ◽  
Gongshe Yang ◽  
...  
Keyword(s):  
Target Genes ◽  
Adipogenic Differentiation ◽  
Luciferase Reporter ◽  
Proliferative Stage ◽  
Primary Mouse ◽  
Direct Targeting ◽  
Treatment Of Obesity ◽  
The Stability ◽  
Oil Red O Staining

Abstract miRNAs are a small class of noncoding RNAs that perform biological functions by regulating the stability or translation of target genes in various biological processes. This study illustrated the role of miR-10a-5p, which is relatively enriched in adipose tissues, using primary mouse preadipocytes as model. With elevated miR-10a-5p expression, the proliferative ability of mouse preadipocytes was significantly enhanced, indicated by increased EdU+ cells and G1/S transition, accompanied by upregulated Cyclin B, Cyclin D and PCNA and downregulated p21 and p27. Meanwhile, the adipogenic differentiation was significantly attenuated by elevated miR-10a-5p, supported by Oil Red O staining and suppressed PPARγ and aP2 expression. Furthermore, Map2k6 and Fasn were predicted to be the target genes of miR-10a-5p in silico, and dual luciferase reporter assay confirmed the direct targeting effects. Western blot analysis results showed that miR-10a-5p specially reduced Map2k6 expression at the proliferative stage without affecting Fasn expression, while significantly restrained Fasn expression with unchanged Map2k6 expression during adipogenic differentiation. Taken together, these results revealed a potential role of miR-10a-5p in adipogenesis and in the treatment of obesity.

Homozygous Mutation of the ETS DNA-Binding Domain of FLI1 Causes a Bleeding Disorder with Giant Alpha Granules Similar to Paris-Trousseau Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 78-78
Author(s):  
William S Stevenson ◽  
David John Rabbolini ◽  
Timothy Brighton ◽  
Joel Mackay ◽  
Christopher M Ward ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Bleeding Disorder ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Homozygous Mutation ◽  
Jacobsen Syndrome ◽  
Ets Domain

Abstract Deletion of a variable region on chromosome 11q containing FLI1 causes a platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. We report a kindred with the autosomal recessive inheritance of an ETS domain mutation of FLI1, c.970C>T, that causes macrothrombocytopenia with large alpha granule fusion similar to that observed in Paris-Trousseau thrombocytopenia but with no other syndromal features of Paris-Trousseau or Jacobsen syndromes. Affected individuals are moderately thrombocytopenic (mean = 71 x109/L), have absent collagen-induced platelet aggregation and a lifelong mucosal bleeding history. Platelet MYH10 levels were increased in affected members of the kindred consistent with previous reports of elevated MYH10 in Paris-Trousseau thrombocytopenia. Luciferase reporter assays in HEK293 cells demonstrate that the mutant FLI1 transcript is associated with decreased transcription at the FLI target genes GP6, GP9 and ITGA2B compared to the wild-type transcript (23 vs 31, n=9, P<0.01; 3.8 vs 6.5, P<0.01, n=12; 11 vs 14, n=9, P=0.01, respectively). This transcriptional change was consistent with reduced expression of GPVI (P<0.01), GPIbIX (P<0.01) and GPIIbIIIa (P=0.04) observed on western blotting of platelet lysates from affected family members. This mutation replaces the conserved Arg324 with a tryptophan in the DNA-binding loop between the alpha-2 and alpha-3 helices of the FLI1 ETS domain. Protein modelling suggests that Arg324 does not directly bind DNA but may instead make direct contact with an N-terminal autoinhibitory domain of FLI1 that is considered to regulate DNA-binding affinity through a transition between a folded and unfolded state. This mutation may disrupt this important conformational change. These data suggest abnormalities of FLI1 function may be responsible for the complex platelet defect observed in Paris-Trousseau thrombocytopenia and Jacobsen Syndrome and confirm the role of FLI1 as an important transcriptional regulator of normal platelet development. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation

2005 ◽  
Vol 168 (7) ◽  
pp. 1065-1076 ◽  
Author(s):  
Alka Mansukhani ◽  
Davide Ambrosetti ◽  
Greg Holmes ◽  
Lizbeth Cornivelli ◽  
Claudio Basilico
Keyword(s):  
Osteoblast Differentiation ◽  
Target Genes ◽  
Expression Profiles ◽  
Constitutive Expression ◽  
Growth Factor Receptor ◽  
Gene Expression Profiles ◽  
Down Regulation ◽  
Activating Mutations ◽  
Proliferation And Differentiation

Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with β-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt–β-catenin pathway.

scholarly journals Role of miR-10b-5p in the prognosis of breast cancer

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7728 ◽  
Author(s):  
Junmin Wang ◽  
Yanyun Yan ◽  
Zhiqi Zhang ◽  
Yali Li
Keyword(s):  
Breast Cancer ◽  
Target Genes ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Cancer Development ◽  
Clinicopathological Parameters ◽  
Aberrant Expression ◽  
Expression Levels ◽  
Ppi Networks

Breast cancer is the leading cause of cancer-related death in women worldwide. Aberrant expression levels of miR-10b-5p in breast cancer has been reported while the molecular mechanism of miR-10b-5p in tumorigenesis remains elusive. Therefore, this study was aimed to investigate the role of miR-10b-5p in breast cancer and the network of its target genes using bioinformatics analysis. In this study, the expression profiles and prognostic value of miR-10b-5p in breast cancer were analyzed from public databases. Association between miR-10b-5p and clinicopathological parameters were analyzed by non-parametric test. Moreover, the optimal target genes of miR-10b-5p were obtained and their expression patterns were examined using starBase and HPA database. Additionally, the role of these target genes in cancer development were explored via Cancer Hallmarks Analytics Tool (CHAT). The protein–protein interaction (PPI) networks were constructed to further investigate the interactive relationships among these genes. Furthermore, GO, KEGG pathway and Reactome pathway analyses were carried out to decipher functions of these target genes. Results demonstrated that miR-10b-5p was down-regulated in breast cancer and low expression of miR-10b-5p was significantly correlated to worse outcome. Five genes, BIRC5, E2F2, KIF2C, FOXM1, and MCM5, were considered as potential key target genes of miR-10b-5p. As expected, higher expression levels of these genes were observed in breast cancer tissues than in normal tissues. Moreover, analysis from CHAT revealed that these genes were mainly involved in sustaining proliferative signaling in cancer development. In addition, PPI networks analysis revealed strong interactions between target genes. GO, KEGG, and Reactome pathway analysis suggested that these target genes of miR-10b-5p in breast cancer were significantly involved in cell cycle. Predicted target genes were further validated by qRT-PCR analysis in human breast cancer cell line MDA-MB-231 transfected with miR-10b mimic or antisense inhibitors. Taken together, our data suggest that miR-10b-5p functions to impede breast carcinoma progression via regulation of its key target genes and hopefully serves as a potential diagnostic and prognostic marker for breast cancer.

scholarly journals An Intronic Risk SNP rs12454712 for Central Obesity Acts as an Allele-Specific Enhancer to Regulate BCL2 Expression

2021 ◽  
Author(s):  
Shan-Shan Dong ◽  
Dong-Li Zhu ◽  
Xiao-Rong Zhou ◽  
Yu Rong ◽  
Mengqi Zeng ◽  
...  
Keyword(s):  
Central Obesity ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Adipogenic Differentiation ◽  
Significant Negative Correlation ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Allele Specific

Genome-wide association studies (GWASs) have reproducibly associated the SNP rs12454712 with waist-to-hip ratio adjusted for body mass index (WHRadjBMI), but the functional role underlying this intronic variant is unknown. Integrative genomic and epigenomic analyses supported rs12454712 as a functional independent variant. We further demonstrated that rs12454712 acted as an allele-specific enhancer regulating expression of its located gene <i>BCL2</i> by using dual-luciferase reporter assays and CRISPR-Cas9. Specifically, rs12454712-C allele can bind transcription factor ZNF329, which efficiently elevates the enhancer activity and increases <i>BCL2</i> expression. Knocking down Bcl2 in 3T3-L1 cells led to the down-regulation of adipogenic differentiation marker genes and increased cell apoptosis. Significant negative correlation between <i>BCL2</i> expression in subcutaneous adipose tissues and obesity was observed. Our findings illustrate the molecular mechanisms behind the intronic SNP rs12454712 for central obesity, which would be a potential and promising target for developing appropriate therapies.

scholarly journals DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors

Life ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 59
Author(s):  
Joanna Boresowicz ◽  
Paulina Kober ◽  
Natalia Rusetska ◽  
Maria Maksymowicz ◽  
Agnieszka Paziewska ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mirna Expression ◽  
Target Genes ◽  
Expression Profiles ◽  
Pituitary Tumors ◽  
Target Prediction ◽  
Aberrant Methylation ◽  
Expression Levels ◽  
Putative Target ◽  
Encoding Genes

microRNAs are involved in pathogenesis of cancer. DNA methylation plays a role in transcription of miRNA-encoding genes and may contribute to changed miRNA expression in tumors. This issue was not investigated in pituitary neuroendocrine tumors (PitNETs) previously. DNA methylation patterns, assessed with HumanMethylation450K arrays in 34 PitNETs and five normal pituitaries, were used to determine differentially methylated CpGs located at miRNA genes. It showed aberrant methylation in regions encoding for 131 miRNAs. DNA methylation data and matched miRNA expression profiles, determined with next-generation sequencing (NGS) of small RNAs, were correlated in 15 PitNETs. This showed relationship between methylation and expression levels for 12 miRNAs. DNA methylation and expression levels of three of them (MIR145, MIR21, and MIR184) were determined in the independent group of 80 tumors with pyrosequencing and qRT-PCR and results confirmed both aberrant methylation in PitNETs and correlation between methylation and expression. Additionally, in silico target prediction was combined with analysis of established miRNA profiles and matched mRNA expression pattern, assessed with amplicon-based NGS to indicate putative target genes of epigenetically deregulated miRNAs. This study reveals aberrant DNA methylation in miRNA-encoding genes in gonadotroph PitNETs. Methylation changes affect expression level of miRNAs that regulate putative target genes with tumorigenesis-relevant functions.

scholarly journals MicroRNA Profiling Reveals an Abundant ssc-miRNA-148a-3p that Promotes Proliferation and Differentiation during Intramuscular Adipogenesis in Chinese Guizhou Congjiang Pigs Breeds by targeting PPARGC1A

2021 ◽  
Author(s):  
Lulin Tan ◽  
Zhaojun Chen ◽  
Mingde Teng ◽  
Bin Chen ◽  
Houqiang Xu
Keyword(s):  
Expression Profiles ◽  
Critical Role ◽  
Differentially Expressed ◽  
Marker Genes ◽  
Preadipocyte Differentiation ◽  
Longissimus Dorsi Muscle ◽  
Non Coding Rna ◽  
Proliferation And Differentiation ◽  
G2 Phase

Abstract BackgroundIntramuscular fat development is regulated by a series of complicated processes, and non-coding RNA (ncRNA) such as microRNA (miRNA) plays a critical role during intramuscular preadipocyte proliferation and differentiation development in pigs. In present research, we detected the expression profiles of miRNA during different differentiation stages, namely, day 0 (D0), day 4 (D4), and day 8 (D8), of intramuscular preadipocytes from the longissimus dorsi muscle of Chinese Guizhou Congjiang pigs to provide first insights into their potential involvement in intramuscular preadipocyte development. And we investigated the function of miR-148a-3p in adipocyte proliferation, apoptosis, and differentiation. ResultsA total of 67, 95, and 16 differentially expressed (DE) miRNAs were detected between D4 and D0, between D8 and D0, and between D8 and D4, respectively. We further characterized the role of miR-148a-3p which was differentially expressed and highest expressed abundance in D0, D4, and D8. To explore the role of miR-148a-3p in porcine intramuscular preadipocyte, miR-148a-3p mimics and inhibitors were used to perform miR-148a-3p overexpression and knockdown, respectively. Overexpression of miRNA-148a-3p increased the number of intramuscular preadipocytes in the S/G2 phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted proliferation by regulation of cyclin B, cyclin G1, cyclin D1, CDK2, CDK3, and CDK4 and inhibited apoptosis of intramuscular preadipocyte by regulating the expression of Caspase-3, Bax, and Bcl-2. Meanwhile, the mimics of miR-148a-3p dramatically promoted intramuscular preadipocyte differentiation and upregulated the expression levels of adipogenic marker genes PPARγ, FASN, FABP4, HSL, APOE, LPL, and CEBPα. Furthermore, miR-148a-3p promoted intramuscular preadipocyte differentiation via restraining the AMPK/ACC/CPT1C signaling pathway. PPARGC1A was identified as a target gene of miR-148a-3p by luciferase activity and western blotting assays. ConclusionOur study provides novel insights into the regulatory mechanisms underlying intramuscular preadipocyte development and identified amount of miRNAs whose regulatory potential will need to be explored in the future. Our results establish that miR-148a-3p promoted adipocyte differentiation by targeting PPARGC1A.

Differential Gene and MicroRNA Expression In a HOXA9-MEIS1 Model of Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 64-64
Author(s):  
Joanne Margaret Ramsey ◽  
Glenda Joanne Dickson ◽  
Janetta Jacoba Bijl ◽  
Ken I. Mills ◽  
Terence R.J. Lappin ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Normal Karyotype ◽  
Retroviral Infection ◽  
Appreciable Change ◽  
Inhibition Of Proliferation ◽  
Altered Expression ◽  
Expression Levels ◽  
Blast Cells

Abstract Abstract 64 Altered expression of the HOX-MEIS axis is associated with leukemia, particularly AML, where it plays a role in increased proliferation and impaired differentiation. Particular focus has been placed on the role of HOX-MEIS in leukemia with MLL gene rearrangements and MLL-fusion induced models that represent this poor prognostic subgroup. However more recently, we and others have identified altered expression of HOX-MEIS in AML with normal karyotype (AML-NK) that accounts for the vast majority of patients with this disease. The prognostic outcome of this large subgroup of patients is varied and may depend on other molecular aberrations such as NPM-1 mutation, FLT3-ITD or CEBPB status. A murine model of leukemia using co-overexpression of HOXA9-MEIS1 was developed to investigate their role in an AML-NK setting. Expression levels of the collaborating oncogenes was limited by combination of a bicistronic ires targeting vector, low titre retroviral infection and limiting dilution assay (LDA). HOXA9 and MEIS1 expression was measured using quantitiative PCR (qPCR) and levels obtained in the mouse were similar to those observed for AML-NK patients. The resultant leukemia was transplantable leading to premature death in non-irradiated recipient mice within 30 days (104 cells transplanted). Observed pathology included circulating blasts in the periphery, splenomegaly, tissue infiltration of blast cells (kidney, lung, liver) and enlarged lymph nodes, indicative of leukemia. Blast cells were identified as Kit+, Gr-1+, Mac-1+, CD150lo consistent with a myeloid progenitor phenotype. To evaluate downstream events associated with the leukaemic phenotype TaqMan-based Low Density Arrays (TLDAs) and Open Arrays (Biotrove) were used to obtain gene and microRNA (mIR) expression profiles. The majority of genes assayed 699/821 were either not expressed or showed no appreciable change in expression (ΔΔCT < 2) between the leukaemic and normal bone marrow control samples. Of those genes that showed a measurable change in expression, 111/122 assessed in the pluripotent stem cell, transcription factor and immune arrays were downregulated in the HOXA9-MEIS1 induced leukemia samples compared to control mice (n=4 per group). Individual assays were designed for a subset of candidate genes and validated against the TLDA results. Several genes identified as consistently downregulated in the leukaemic cells, including Smad1 and E2f7, were associated with negative regulation of cell proliferation using DAVID Bioinformatics Resources 6.7. Consistent increased expression of HOXA9 and MEIS1 was confirmed by individual assays as was elevated Elf5, Cd34 and Cd28 levels. mIRs regulate the expression of protein-coding genes in part through cleavage of targeted transcripts by partial or complete base pairing. A growing number of mIRs are differentially regulated in normal and malignant hematopoiesis, where increased mIR levels are associated with downregulation of target gene subsets. mIR expression profiles were compared between leukemic and control mice to investigate their association with the global downregulation of gene expression observed in the leukaemic model. Approximately 16% (48/303) of the mIRs showed measurable changes in expression levels (ΔΔCT >2) between the two models, with >70% (34/48) demonstrating increased expression, up to 360-fold, in the leukemia samples. Candidate mIRs were further examined using the mirDB and/or miRecords target prediction databases. Several of the leukaemia-associated upreglated mIRs putatively target members of the validated subset of downregulated genes e.g. mIR-466c and mIR-181c are predicted to target Smad1 and E2F7 respectively. This study suggests that HOXA9-MEIS1 leukemogenesis occurs in part due to increased expression of a subset of mIRs that are predicted to target genes associated with inhibition of proliferation in an AML-NK model. Disclosures: No relevant conflicts of interest to declare.

Equivalence testing in microarray analysis: similarities in the transcriptome of human atherosclerotic and nonatherosclerotic macrophages

2010 ◽  
Vol 41 (3) ◽  
pp. 212-223 ◽  
Author(s):  
Wouter J. Eijgelaar ◽  
Anton J. G. Horrevoets ◽  
Ann-Pascale J. J. Bijnens ◽  
Mat J. A. P. Daemen ◽  
Wim F. J. Verhaegh
Keyword(s):  
Gene Expression ◽  
Microarray Analysis ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
New Method ◽  
Expression Level ◽  
Marker Genes ◽  
Equivalence Testing ◽  
Expression Levels ◽  
Macrophage Populations

We focus on similarities in the transcriptome of human Kupffer cells and alveolar, splenic, and atherosclerotic plaque-residing macrophages. We hypothesized that these macrophages share a common expression signature. We performed microarray analysis on mRNA from these subsets (4 patients) and developed a novel statistical method to identify genes with significantly similar expression levels. Phenotypic and functional diversity between macrophage subpopulations reflects their plasticity to respond to microenvironmental signals. Apart from detecting differences in expression profiles, the comparison of the transcriptomes of different macrophage populations may also allow the definition of molecular similarities between these subsets. This new method calculates the maximum difference in gene expression level, based on the estimated confidence interval on that gene's expression variance. We listed the genes by equivalence ranking relative to expression level. FDR estimation was used to determine significance. We identified 500 genes with significantly equivalent expression levels in the macrophage subsets at 5.5% FDR using a confidence level of α = 0.05 for equivalence. Among these are the established macrophage marker CD68, IL1 receptor antagonist, and MHC-related CD1C. These 500 genes were submitted to IPA and GO clustering using DAVID. Additionally, hierarchical clustering of these genes in the Novartis human gene expression atlas revealed a subset of 200 genes specifically expressed in macrophages. Equivalently expressed genes, identified by this new method, may not only help to dissect common molecular mechanisms, but also to identify cell- or condition-specific sets of marker genes that can be used for drug targeting and molecular imaging.

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