PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells

<p>Abstract</p> <p>Background</p> <p>For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data.</p> <p>Results</p> <p>The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt ± SD, 23.66 ± 0.86) and RPL32 (18.65 ± 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (<it>p </it>= 0.004); no change was observed using GAPDH/ACTB (<it>p </it>> 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (<it>p </it>< 0.03).</p> <p>Conclusion</p> <p>PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.</p

Hypomethylation of IL1RN and NFKB1 genes is linked to the dysbalance in IL1β/IL-1Ra axis in female patients with type 2 diabetes mellitus.

Inflammation has received considerable attention in the pathogenesis of type 2 diabetes mellitus (T2DM). Supporting this concept, enhanced expression of interleukin (IL)-1β and increased infiltration of macrophages are observed in pancreatic islets of patients with T2DM. Although IL-1 receptor antagonist (IL-1Ra) plays a major role in controlling of IL-1β-mediated inflammation, its counteraction effects and epigenetic alterations in T2DM are less studied. Thus, we aimed to analyze the DNA methylation status in IL1RN, RELA (p65) and NFKB1 (p50) genes in peripheral blood mononuclear cells (PBMCs) from treated T2DM patients (n = 35) and age-/sex- matched healthy controls (n = 31). Production of IL-1β and IL-1Ra was analyzed in plasma and supernatants from LPS-induced PBMCs. Immunomodulatory effects of IL-1β and IL-1Ra were studied on THP-1 cells. Average DNA methylation level of IL1RN and NFKB1 gene promoters was significantly decreased in T2DM patients in comparison with healthy controls (P< 0.05), which was associated with the increased IL-1Ra (P< 0.001) and IL-1β (P = 0.039) plasma levels in T2DM patients. Negative association between average methylation of IL1RN gene and IL-1Ra plasma levels were observed in female T2DM patients. Methylation of NFKB1 gene was negatively correlated with IL-1Ra levels in the patients and positively with IL-1β levels in female patients. LPS-stimulated PBMCs from female patients failed to raise IL-1β production, while the cells from healthy females increased IL-1β production in comparison with unstimulated cells (P< 0.001). Taken together, the findings suggest that hypomethylation of IL1RN and NFKB1 gene promoters may promote the increased IL-1β/IL-1Ra production and regulate chronic inflammation in T2DM. Further studies are necessary to elucidate the causal direction of these associations and potential role of IL-1Ra in anti-inflammatory processes in treated patients with T2DM

Correlation Network Analysis Reveals Relationships between MicroRNAs, Transcription Factor T-bet, and Deregulated Cytokine/Chemokine-Receptor Network in Pulmonary Sarcoidosis

Sarcoidosis is an inflammatory granulomatous disease with unknown etiology driven by cytokines and chemokines. There is limited information regarding the regulation of cytokine/chemokine-receptor network in bronchoalveolar lavage (BAL) cells in pulmonary sarcoidosis, suggesting contribution of miRNAs and transcription factors. We therefore investigated gene expression of 25 inflammation-related miRNAs, 27 cytokines/chemokines/receptors, and a Th1-transcription factor T-bet in unseparated BAL cells obtained from 48 sarcoidosis patients and 14 control subjects using quantitative RT-PCR. We then examined both miRNA-mRNA expressions to enrich relevant relationships. This first study on miRNAs in sarcoid BAL cells detected deregulation of miR-146a, miR-150, miR-202, miR-204, and miR-222 expression comparing to controls. Subanalysis revealed higher number of miR-155, let-7c transcripts in progressing (n=20) comparing to regressing (n=28) disease as assessed by 2-year follow-up. Correlation network analysis revealed relationships between microRNAs, transcription factor T-bet, and deregulated cytokine/chemokine-receptor network in sarcoid BAL cells. Furthermore, T-bet showed more pronounced regulatory capability to sarcoidosis-associated cytokines/chemokines/receptors than miRNAs, which may function rather as “fine-tuners” of cytokine/chemokine expression. Our correlation network study implies contribution of both microRNAs and Th1-transcription factor T-bet to the regulation of cytokine/chemokine-receptor network in BAL cells in sarcoidosis. Functional studies are needed to confirm biological relevance of the obtained relationships

PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells-1

Ents with involvement of parenchyma: CXR stages II/III)-Mean(patients without involvement of parenchyma: CXR stage I), Mean(Löfgren's syndrome patients)-Mean(non-Löfgren's syndrome patients), Mean(multi-organ involvement)-Mean(involvement of lung only), Mean(smokers)-Mean(non-smokers), Mean(males)-Mean(females), and Mean(pathological BAL cell count)-Mean(normal BAL cell count) for macrophages, lymphocytes, neutrophils and eosinophils. For more details see the legend to Fig. 2.<p><b>Copyright information:</b></p><p>Taken from "PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells"</p><p>http://www.biomedcentral.com/1471-2199/9/69</p><p>BMC Molecular Biology 2008;9():69-69.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2529339.</p><p></p

PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells-2

Zed to 1); the whiskers on each box represent the SD values. For details see Methods section Gene expression measurements by qRT-PCR. *< 0.05.<p><b>Copyright information:</b></p><p>Taken from "PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells"</p><p>http://www.biomedcentral.com/1471-2199/9/69</p><p>BMC Molecular Biology 2008;9():69-69.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2529339.</p><p></p

PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells-0

Ker box plots; the box represents the 25th–75th percentiles, the median is indicated by a bar across the box, the whiskers on each box represent the minimum and maximum values.<p><b>Copyright information:</b></p><p>Taken from "PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells"</p><p>http://www.biomedcentral.com/1471-2199/9/69</p><p>BMC Molecular Biology 2008;9():69-69.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2529339.</p><p></p