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Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization.

ABSTRACT: AIM:To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs. METHODS:We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays) to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WI/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70, Hsp27, Gadd45a and IL-1rI). RESULTS:Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context. CONCLUSION:This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70 and Gadd45a might represent promising new candidates indicating WI/R liver damage.

SUBMITTER: Fallsehr C

PROVIDER: S-EPMC4250677 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization.

Fallsehr Christine C Zapletal Christina C Kremer Michael M Demir Resit R von Knebel Doeberitz Magnus M Klar Ernst E

World journal of gastroenterology 20050301 9

<h4>Aim</h4>To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs.<h4>Methods</h4>We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays) to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WI/R; 1 h/2 h) and analyzed in more ...[more]

PMID: 15761968

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Project description:Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma.To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay.Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways.Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

Project description:The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

Project description:BackgroundTo understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes.MethodsTwo cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR) and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression.ResultsTwo libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL) and the reverse-subtracted library (RSL) contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P < 0.05). The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells, and expression of ERGIC3 was correlated with the differentiated degree and histological type of lung cancer. The up-regulation of ERGIC3 could promote cellular migration and proliferation in vitro.ConclusionsThe two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer-related gene. ERGIC3 may play an active role in the development and progression of lung cancer.

Project description:BackgroundTumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model.ResultsWe established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients.ConclusionThe comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.

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Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization.

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Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization.

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