Expression Stability of Common Housekeeping Genes Is Differently Affected by Bowel Inflammation and Cancer

Krzystek−Korpacka, Małgorzata; Diakowska, Dorota; Bania, Jacek; Gamian, Andrzej

doi:10.1097/mib.0000000000000067

Cited by 39 publications

(34 citation statements)

References 24 publications

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“…All duodenal biopsy extracts were used at a common concentration (1 g/l). Fifty microliters were used to assay INADL, MAPKAPK5, and MAGI1, (MyBio-Source, San Diego, CA) whereas 100 l was used for the housekeeping gene phosphoglycerate kinase 1 (PGK1; LifeSpan Biosciences, Seattle, WA), which has been recommended as the preferred gene [over ␤-actin (ACTB) and ␤2-microglobulin (␤2M)] in the context of inflammatory bowel diseases (29). Enzyme-linked immunosorbent assay (ELISA) kits were used for all these protein assays following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Pilot study of small bowel mucosal gene expression in patients with irritable bowel syndrome with diarrhea

Camilleri

Carlson

Valentin

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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Prior studies in with irritable bowel syndrome with diarrhea (IBS-D) patients showed immune activation, secretion, and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2((-ΔΔCT)) formula. Nominal P values (P < 0.05) were interpreted with false detection rate (FDR) correction (q value). Cluster analysis with Lens for Enrichment and Network Studies (LENS) explored connectivity of mechanisms. Upregulated genes (uncorrected P < 0.05) were related to ion transport (INADL, MAGI1, and SONS1), barrier (TJP1, 2, and 3 and CLDN) or immune functions (TLR3, IL15, and MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1β, TGF-β1, and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated (q < 0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, and IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, and MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4 ng/ml) in patients with IBS-D relative to controls (median 5.8 ng/ml, P > 0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune, and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.

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Section: Methodsmentioning

confidence: 99%

Pilot study of small bowel mucosal gene expression in patients with irritable bowel syndrome with diarrhea

Camilleri

Carlson

Valentin

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…The obtained values are referred to as a normalized relative quantity (NRQ) [72] and subjected to statistical analysis. Reference gene selection was based on previous results, showing PPIA and RPLP0 the most suitable pair of genes for studies on bowel tissues [73].…”

Section: Reverse-transcription Quantitative (Real-time) Polymerase Chmentioning

confidence: 99%

Transcriptional and Metabolomic Analysis of L-Arginine/Nitric Oxide Pathway in Inflammatory Bowel Disease and Its Association with Local Inflammatory and Angiogenic Response: Preliminary Findings

Krzystek−Korpacka

Fleszar

Bednarz−Misa

et al. 2020

IJMS

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L-arginine/nitric oxide pathway in Crohn's disease (CD) and ulcerative colitis (UC) is poorly investigated. The aim of current study is to quantify pathway serum metabolites in 52 CD (40 active), 48 UC (33 active), and 18 irritable bowel syndrome patients and 40 controls using mass spectrometry and at determining mRNA expression of pathway-associated enzymes in 91 bowel samples. Arginine and symmetric dimethylarginine decreased (p < 0.05) in active-CD (129 and 0.437 µM) compared to controls (157 and 0.494 µM) and active-UC (164 and 0.52 µM). Citrulline and dimethylamine increased (p < 0.05) in active-CD (68.7 and 70.9 µM) and active-UC (65.9 and 73.9 µM) compared to controls (42.7 and 50.4 µM). Compared to normal, CD-inflamed small bowel had downregulated (p < 0.05) arginase-2 by 2.4-fold and upregulated dimethylarginine dimethylaminohydrolase (DDAH)-2 (1.5-fold) and arginine N-methyltransferase (PRMT)-2 (1.6-fold). Quiescent-CD small bowel had upregulated (p < 0.05) arginase-2 (1.8-fold), DDAH1 (2.9-fold), DDAH2 (1.5-fold), PRMT1 (1.5-fold), PRMT2 (1.7-fold), and PRMT5 (1.4-fold). Pathway enzymes were upregulated in CD-inflamed/quiescent and UC-inflamed colon as compared to normal. Compared to inflamed, quiescent CD-colon had upregulated DDAH1 (5.7-fold) and ornithine decarboxylase (1.6-fold). Concluding, the pathway is deregulated in CD and UC, also in quiescent bowel, reflecting inflammation severity and angiogenic potential. Functional analysis of PRMTs and DDAHs as potential targets for therapy is warranted.

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“…Instability of HKG expression has already been demonstrated for normal and cancerous tissue samples obtained from CRC patients (Sørby et al 2010; Kheirelseid et al 2010) as well as normal and inflamed bowel of patients with inflammatory bowel disease (Krzystek-Korpacka et al 2014). However, the issue has not been systematically addressed in colon adenocarcinoma cell cultures yet.…”

Section: Introductionmentioning

confidence: 96%

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

et al. 2016

Self Cite

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Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A, TP53, and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA, RPLP0, YWHAZ, and IPO8 were repeatedly highly ranked while ACTB, B2M, UBC, and PGK1 were ranked poorly. Normalization against PPIA/RPLP0/SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.

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Expression Stability of Common Housekeeping Genes Is Differently Affected by Bowel Inflammation and Cancer

Cited by 39 publications

References 24 publications

Pilot study of small bowel mucosal gene expression in patients with irritable bowel syndrome with diarrhea

Pilot study of small bowel mucosal gene expression in patients with irritable bowel syndrome with diarrhea

Transcriptional and Metabolomic Analysis of L-Arginine/Nitric Oxide Pathway in Inflammatory Bowel Disease and Its Association with Local Inflammatory and Angiogenic Response: Preliminary Findings

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

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