Unknown,Transcriptomics,Genomics,Proteomics

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Salmonella enterica subsp. Enteric sv. Typhimurium in vivo induced enterocyte transcriptional response

ABSTRACT: 1-day-old C57BL/6 mice were left untreated (co) or orally infected with 10E2 CFU wildtype (wt) or delta invC SPI1 mutant Salmonella Typhimurium (ATCC14028). Four biological replicates obtained from individual animals were exmained; each group contained animals from at least 2 different litters. On day 4 p.i., animals were sacrificed and intestinal epithelial cells were isolated from total small intestine (protocol according to: Lotz et al., J. Exp. Med. 2006). Total RNA was isolated by TriZol and its purity was examined using a Bioanalyzer. We used microarrays to detail the global gene expression in primary total isolated intestinal epithelial cells. Four biological replicates of isolated intestinal epithelial cells obtained 4 daysp.i. from neonate mice infected at the age of 1 day (each group with animals from at least two litteres). Single color array.

ORGANISM(S): Mus musculus

SUBMITTER: Mathias Hornef

PROVIDER: E-GEOD-51160 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Evidence has recently accumulated suggesting that mature hepatocytes have the unique capacity to autonomously decide their replication fate. The molecular mechanism by which hepatocytes switch from an essentially quiescent state to rapidly proliferation after liver injury is not fully understood. To provide molecular insights into the self-renewal of mature hepatocytes, we have applied an ex vivo liver slice culture system. Results: After liver injury, activation of p38 and Erk1/2 began in mature hepatocytes within 5 min. Erk1/2 was activated at the edge of the cut as well as on the surface of liver slices. The number of hepatocytes that contain activated Erk1/2 increased within 1 h and then decreased. Concomitantly, immediate early genes (IEGs), such as Jun, early gene response 1 (Egr1) and Myc were induced and Ki67 positive hepatocytes appeared 24 h after liver injury in the slices. In agreement with in vivo studies, transcriptome analysis using 2 h slice culture reveals that 63% of upregulated genes were downstream of lipopolysaccharide (LPS) stimulation which induces interleukin 6 (IL 6) or tumor necrosis factor (TNF) alpha pathways. Furthermore, circadian clock regulator, such as members of the basic helix-loop-helix family, Bhlhe40 and Bhlhe41, were highly upregulated for 4 h after injury. Although upon injury Jun or Egr1 were induced in liver slices from proliferation-defective liver, no circadian clock regulator genes were upregulated. Moreover, using an in vitro cell culture system we show that Bhlhe40 is required for the G0-G1 transition. Conclusion: Our data suggest that the circadian clock regulator, Bhlhe40, is involved in the G0-G1 transition. An ex vivo system using normal and proliferation defective KO liver is a useful tool for identification of genes that trigger cell proliferation shortly after liver injury. This method may also be applied for measurement of the liver regeneration potential of individual livers at the priming phase. In one dual-color microarray hybridization, mRNA expression changes after 2h ex vivo incubation of liver slices were examined.

Project description:Hepatocellular carcinoma (HCC) is a frequent cancer with poor prognosis and with limited possibilities for anti-cancer treatment. Evidence has recently accumulated suggesting that multiple signaling pathways are activated in human cancer. It has been recently shown by exosome sequencing of HCC that 161 putative driver genes are associated with 11 recurrently altered pathways, suggesting that we need to inhibit these multiple pathways for HCC therapy. THOC5, a member of transcription/export (TREX) complex, that plays a role in less than 1% of mRNA processing in normal cells such as fibroblasts or macrophages, is not required for maintenance of mature hepatocytes. In this study, we have examined the role of THOC5 in human HCC. Enhanced THOC5 expression was not observed in differentiation grading 1 (G1) human HCC, but 78% of G2 and G3 exhibited increased levels of THOC5. Furthermore, the 50% depletion of THOC5 in hepatocellular carcinoma cell lines, Huh7 and HepG2 causes lipid accumulation and apoptosis. Transcriptome analysis using THOC5 depleted Huh7 and HepG2 cells revealed that 1049 and 837 genes were downregulated upon depletion of THOC5 in Huh7 and HepG2 cells, respectively. Among these genes, 396 were commonly downregulated in both cell lines. Some of these, such as tissue inhibitor of metalloproteinase 3 (TIMP3), tripartite motif containing 24 (TRIM24), ribosomal protein S6 kinase polypeptide 3 (RPS6KA3) and transmembrane emp24-like trafficking protein 10 (TMED10) are known to be fine tuners for several signal transduction pathways. The expression of these proteins was correlated with the THOC5 expression level in HCC. To inhibit multiple signaling pathways we are currently examining the effects of a combination of siRNAs against THOC5 target genes in HCC cell lines. Our data suggest that THOC5, which controls a set of genes that are involved in HCC malignancy can be a potential biomarker for HCC. In addition, THOC5 target gene, TMED10 may also be a novel biomarker of HCC. Furthermore, the suppression of the THOC5 gene per se or multiple THOC5 target genes may represent a novel strategy for cancer therapy. Huh7 and HepG2 cells were infected with lentiviral THOC5 and control shRNAs. Five days after infection, total RNAs were isolated and subjected to microarray analysis.

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Salmonella enterica subsp. Enteric sv. Typhimurium in vivo induced enterocyte transcriptional response

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