Whole-genome Gene Expression Analysis Research Articles

Mechanism of Androgen-Independent Stromal Proliferation in Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH) is a chronic proliferative disease showing stromal-dominant proliferation. However, the detailed proliferation mechanism has remained unclear. Although aging and androgen have been reported as definitive risk factors for BPH, recent studies have focused on the involvement of androgen-independent factors. Androgen-independent factors include ischemia, oxidative stress, metabolic syndrome, infection, autoimmune reactions, and inflammation, with inflammation in BPH tissues playing a central role in the BPH proliferative process. Inflammation in BPH tissues by various factors finally leads to tissue remodeling and stromal proliferation through the wound healing process of the prostate. To elucidate the proliferative mechanism of BPH, a study using whole-genome gene expression analysis in a stromal-dominant BPH rat model was performed and showed that immune response-related pathways and complement classical pathways are activated. Furthermore, expression analysis using this BPH rat model showed that the autoimmune reaction triggered complement pathway activation in the proliferative process of BPH. BPH is a multifactorial disease, and understanding the role of androgen-independent factors including immune responses contributes to elucidating the pathogenesis of BPH. Androgen-independent factors may lead to new therapeutic targets for BPH, and further development of this research is expected.

Abstract 4901: The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones

Abstract α-N-Heterocyclic thiosemicarbazones (TSCs) have long been investigated as anticancer compounds. Triapine is one of the best-known TSCs for anticancer therapy and is currently tested in a clinical phase III trial. To further improve the anticancer activity of TSCs, novel derivatives (such as DpC and COTI-2) have been developed and clinically investigated for their activity against solid tumors. These novel TSCs belong to a subclass (incl. the tetra-methylated Triapine derivative, Me2NNMe2) that are terminally disubstituted and demonstrated enhanced anticancer activity in vitro and in vivo. The improved activity of these TSCs correlated with their ability to form stable copper complexes as well as induce paraptosis, a form of programmed but caspase-independent cell death, that is characterized by the formation of vesicles originating from the endoplasmic reticulum (ER). However, the molecular events behind paraptosis induction are still not fully elucidated. Consequently, the aim of this study was to investigate the molecular signaling behind paraptosis as well as the reasons behind the induction of this quite unknown form of cell death by TSCs. A whole-genome gene expression analysis of cells treated with a terminally disubstituted TSC revealed an upregulation of thiol homeostasis-related genes, copper metabolism, and metallothionein genes as well as ER stress response genes. In addition, we found an upregulation of the copper-sensitive and thiol-containing protein disulfide isomerase (PDI), which was confirmed on protein level. In cell-free activity studies, PDI was indeed inhibited by terminally disubstituted TSCs, although only in form of their copper complexes. In agreement, removal of copper ions by specific chelators greatly reduced their anticancer activity as well as paraptotic potential in cell culture. This inhibition was hypothesized to be induced by a general disruption of the thiol redox homeostasis affecting also other responsive proteins and resulting in a more oxidative environment, especially in the ER. In confirmation, thiol-containing small molecules could reduce the compound’s anticancer activity in viability assays. Furthermore, glutathione as well as PDI were detected predominantly in their oxidized form in cells treated with terminally disubstituted TSCs. Consequently, terminally disubstituted TSCs, which are characterized by higher anticancer activity, induce paraptosis due to their high copper complex stability. The formation of the complex results in the interaction with thiol-containing proteins and subsequently in the disruption of the thiol-redox homeostasis, which leads to paraptotic cell death. Overall this work shed light on the paraptotic cell death and will be of interest for future clinical development of anticancer TSCs, as paraptosis is hypothesized to overcome apoptosis-resistance of cancer cells. Citation Format: Sonja Hager, Walter Berger, Christian R. Kowol, Eva A. Enyedy, Petra Heffeter. The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4901.

Tidal emersion effects on universal metrics, elemental contents, and health conditions of Pacific oyster (Crassostrea gigas) on artificial reefs in Yellow River Delta, China

Oysters form natural reefs and are important coastal foundation species that provide a wide range of ecosystem services. Clarification of the mechanisms by which tidal emersion affects oyster reef performance will help guide and direct future oyster reef restoration efforts. In the present study, we constructed artificial Pacific oyster (Crassostrea gigas) reefs in the coastal zones of the Yellow River Delta and quantified the effects of tidal emersion on the growth, density, biomass, element (carbon, nitrogen, phosphorus) content within tissues, and health conditions of oysters. The results showed that temperature change driven by the tidal emersion effect was one of the major forces affecting reef performance. In the experimental field, fully immersed oysters performed better than partially exposed ones in terms of growth and density. The mean biomass of the fully immersed oysters was 2.88-fold higher than that of the partially exposed oysters (p < 0.05). However, we found that tidal emersion had no significant effect on the carbon, nitrogen, and phosphorus content within oyster tissues. Oyster health condition revealed by the biochemical marker and whole-genome gene expression analyses demonstrated that the oysters partially exposed to air presented enhanced oxidative and lipid metabolism and were forced to allocate metabolic energy towards defense against stress rather than growth, which suggested sub-optimal health conditions. Overall, the study indicated that full immersion is optimal for oyster reef restoration in the experimental field. The results of this research will provide future conservationists, aquaculturists, and other specialists with a better understanding of tidal emersion effects on the success of oyster reef restoration to achieve region-specific restoration goals and improve protection of the coastal environment.

Integrated whole-genome gene expression analysis reveals an atlas of dynamic immune landscapes after myocardial infarction.

Myocardial infarction (MI) is a deadly medical condition leading to irreversible damage to the inflicted cardiac tissue. Elevated inflammatory response marks the severity of MI and is associated with the development of heart failure (HF), a long-term adverse outcome of MI. However, the efficacy of anti-inflammatory therapies for MI remains controversial. Deciphering the dynamic transcriptional signatures in peripheral blood mononuclear cells (PBMCs) is a viable and translatable route to better understand post-MI inflammation, which may help guide post-MI anti-inflammatory treatments. In this work, integrated whole-genome gene expression analysis was performed to explore dynamic immune landscapes associated with MI. GSEA and GSVA showed that pathways involved in the inflammatory response and metabolic reprogramming were significantly enriched in PBMCs from MI patients. Based on leukocyte profiles generated by xCell algorithm, the relative abundance of monocytes and neutrophils was significantly increased in PBMCs from MI patients and had positive correlations with typical inflammation-associated transcripts. Mfuzz clustering revealed temporal gene expression profiles of PBMCs during the 6-month post-MI follow-up. Analysis of DEGs and gene sets indicated that PBMCs from HF group were characterized by elevated and lasting expression of genes implicated in inflammation and coagulation. Consensus clustering generated 4 metabolic subtypes of PBMCs with molecular heterogeneity in HF patients. In summary, integrated whole-genome gene expression analysis here outlines a transcriptomic framework that may improve the understanding of dynamic signatures present in PBMCs, as well as the heterogeneity of PBMCs in MI patients with or without long-term clinical outcome of HF. Moreover, the work here uncovers the diversity and heterogeneity of PBMCs from HF patients, providing novel bioinformatic evidence supporting the mechanistic implications of metabolic reprogramming and mitochondrial dysfunction in the post-MI inflammation and HF. Therefore, our work here supports the notion that individualized anti-inflammatory therapies are needed to improve the clinical management of post-MI patients.

Host Transcriptome Analysis of Ferret Tissues Following Henipavirus Infection.

Ferrets are commonly used as experimental models of infection for a variety of viruses due to their susceptibility to human respiratory viruses and the close resemblance of pathological outcomes found in human infections. Even though ferret-specific reagents are limited, the use of ferrets as a preclinical experimental model of infection has gained considerable interest since the publication of the ferret transcriptome and draft ferret genome. These advances have made it feasible to easily perform whole-genome gene expression analysis in the ferret infection model. Here, we describe methods for genome-wide gene expression analysis using RNA sequence (RNAseq) data obtained from the lung and brain tissues obtained from experimental infections of Hendra (HeV) and Nipah (NiV) viruses in ferrets. We provide detailed methods for RNAseq and representative data for host gene expression profiles of the lung tissues that show early activation of interferon pathways and later activation of inflammation-related pathways.

Genome-Wide Identification and Expression Analysis of Chitinase-like Genes in Petunia axillaris.

Chitinase (EC 3.2.1.14) is a kind of chitin-degrading glycosidase, which plays important roles in the abiotic and biotic defense of plants. In this study, we conducted whole-genome annotation, molecular evolution, and gene expression analyses on the chitinase-like (CTL) gene family members of Petunia axillaris. Thirty-three Petunia axillaris chitinase-like genes (PaCTLs) were identified from the latest Petunia genome database. According to the phylogenetic analyses, these genes were divided into GH18 and GH19 subgroups and further subdivided into five classes (Class I to Class V). Conserved motif arrangements indicated their functional relevance within each group. The expansion and homeology analyses showed that gene replication events played an important role in the evolution of PaCTLs and the increase of the GH18 subgroup members was the main reason for the expansion of the PaCTL gene family in the evolution progress. By qRT-PCR analysis, we found that most of the PaCTLs showed a very low expression level in the normal growing plants. But lots of PaCTLs showed upregulated expression profiles when the plants suffered different abiotic stress conditions. Among them, five PaCTLs responded to high temperature and exhibited significantly upregulate expression level. Correspondingly, many hormone responses, as well as biotic and abiotic stress elements were found in the promoters of PaCTLs by using cis-acting element analysis. These results provide a foundation for the exploration of PaCTLs’ function and enrich the evolutionary process of the CTL gene family.

Exploration of the developmental toxicity of TCS and PFOS to zebrafish embryos by whole-genome gene expression analyses.

Triclosan (TCS) and perfluorooctane sulfonate (PFOS) are known to have both endocrine disrupting and developmental toxicity effects on zebrafish embryos. Currently, potential molecular mechanisms underlying these toxicological phenomena require further studies. To address this gap in the literature, we used whole transcriptome microarrays to being to address the potential molecular mechanisms underlying developmental toxicity of TCS and PFOS on zebrafish embryos. Zebrafish embryos were exposed to 300 μg/L TCS and 500 μg/L PFOS from 4 to 120 h post fertilization (hpf). Phenotypically, the hatching rate of zebrafish embryos was significantly reduced after TCS exposure at 72 hpf. Additionally, body length was significantly decreased in the TCS treatment group at 120 hpf. Gene ontology analysis of differentially expressed genes revealed that lipid metabolism, steroid metabolism, and organ development-related biological processes were significantly enriched in TCS- and PFOS-treated zebrafish embryos. Furthermore, signaling network analysis indicated that the steroid biosynthesis process was the most significant biological process disrupted by TCS in 120 hpf zebrafish embryos, while organ development was the most significant biological process disrupted by PFOS exposure. Our findings enhance the understanding of the specific types of embryotoxicity elicited by TCS and PFOS, and also provide information that can be used to inform future mechanistic studies.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI’s hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

ALDH2 deficiency induces atrial fibrillation through dysregulated cardiac sodium channel and mitochondrial bioenergetics: A multi-omics analysis

Point mutation in alcohol dehydrogenase 2 (ALDH2), ALDH2*2 results in decreased catalytic enzyme activity and has been found to be associated with different human pathologies. Whether ALDH2*2 would induce cardiac remodeling and increase the attack of atrial fibrillation (AF) remains poorly understood. The present study evaluated the effect of ALDH2*2 mutation on AF susceptibility and unravelled the underlying mechanisms using a multi-omics approach including whole-genome gene expression and proteomics analysis. The in-vivo electrophysiological study showed an increase in the incidence and reduction in the threshold of AF for the mutant mice heterozygous for ALDH2*2 as compared to the wild type littermates. The microarray analysis revealed a reduction in the retinoic acid signals which was accompanied by a downstream reduction in the expression of voltage-gated Na+ channels (SCN5A). The treatment of an antagonist for retinoic acid receptor resulted in a decrease in SCN5A transcript levels. The integrated analysis of the transcriptome and proteome data showed a dysregulation of fatty acid β-oxidation, adenosine triphosphate synthesis via electron transport chain, and activated oxidative responses in the mitochondria. Oral administration of Coenzyme Q10, an essential co-factor known to meliorate mitochondrial oxidative stress and preserve bioenergetics, conferred a protection against AF attack in the mutant ALDH2*2 mice. The multi-omics approach showed the unique pathophysiology mechanisms of concurrent dysregulated SCN5A channel and mitochondrial bioenergetics in AF. This inspired the development of a personalized therapeutic agent, Coenzyme Q10, to protect against AF attack in humans characterized by ALDH2*2 genotype.

An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student’s t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

P0517RENAL STEM CELLS (ARPCS) AS A NEPHROPROTECTIVE APPROACH DURING CISPLATIN-INDUCED ACUTE KIDNEY INJURY: A DEFENSE MECHANISM BY EXTRACELLULAR VESICLES CARRYING THE CYP1B1 GENE

Abstract Background and Aims Cisplatin, is a nonspecific cytotoxic agent that primarily interferes with cellular DNA replication and the cell cycle, nevertheless it lacks tumor selectivity and acts also in normal cells. The most serious adverse reaction of cisplatin is Acute Kidney Injury (AKI), limiting its use and efficacy in chemotherapy. Cisplatin nephrotoxicity is observed in more than 30% of older patients, however the mechanism of nephrotoxicity remains unclear and specific preventive measures are not available. Today, there is an urgent need for specific nephroprotective strategies to be used during cisplatin chemotherapy. Recently, we found that tubular stem/progenitor cells (tARPC) are able to protect the tubular epithelial (RPTEC) from cisplatin induced injury, preserving their proliferation and inhibiting apoptosis. The aim of this study was to identify the molecular mechanisms involved in tARPC-mediated resistance to cisplatin. Method Co-cultures of RPTEC cells and tARPCs were exposed to cisplatin (2.5 µM) for 6 h and then kept in culture for 96 h. Gene expression profile was obtained from tARPCs and RPTECs by Agilent SurePrint G3 Human Gene Expression Microarrays. Genespring and R software were used for the analysis. Gene expression data were validated by Real-time PCR. Extracellular vesicles were isolated from cell culture supernatant by miRCURY Exosome Cell/Urine/CSF Kit (Qiagen) and RNA contained in extracellular vesicles was purified, analyzed in quality by Bioanalyzer (RNA nano) and evaluated by qPCR. The BrdU assay and caspase 3 were used to measure proliferation and apoptosis levels. Immunohistochemical expression of activated caspase-3 was used as a marker of apoptosis in RPTECs. Results By a whole-genome gene expression analysis, we found 107 genes specifically modulated by RPTECs in response to cisplatin and, among these, 30 genes induced by ARPCs following the cisplatin damage. In particular, we found a strong upregulation of the CYP1B1 gene (false discovery rate corrected p value &amp;lt;0.05; fold change=4,1). The qPCR confirmed the increase in CYP1B1 levels in the co-cultures with respect to the respective basal conditions (p &amp;lt;0.05). Interestingly, the CYP1B1 mRNA was also enveloped in Extracellular Vesicles released in the cell co-culture media by tARPC and RPTEC after cisplatin exposition. The CYP1B1 gene encodes a member of the cytochrome P450 superfamily of enzymes and the produced enzyme metabolizes procarcinogens, such as polycyclic aromatic hydrocarbons. CYP1B1 has been shown to be active within tumors and is also capable of metabolizing a structurally diverse range of anticancer drugs. It is responsible for the resistance to docetaxel, cisplatin, tamoxifen and nucleoside analogues. CYP1B1 is involved in the detoxification of the body by various exogenous toxic agents, including cisplatin. We found that CYP1B1 gene was expressed at low levels in RPTECs and in cisplatin-damaged RPTECs. Moreover, 96 h days after 2.5 μM exposure to cisplatin, RPTECs reduced the proliferation and underwent in apoptosis, as showed by caspase 3. However, in co-culture with ARPCs, ARPC cellular and extracellular vesicles CYP1B1 gene expression significantly increased, the apoptotic process was stopped and RPTECs increased their proliferation rate. These data support the hypothesis that ARPCs are sensor of cisplatin damaged-RPTEC and confers cisplatin resistance by transferring CYP1B1 gene in extracellular vesicles. Conclusion This is the first evidence of a cisplatin-induced overexpression of CYP1b1 in renal epithelial cells as a defense mechanism against cisplatin toxicity. This is consistent with our previous data showing that renal progenitors are resistant to cisplatin. The findings may have biological and clinical significance in terms of their implications in cellular communications and potential use of CYP1B1 as biomarkers for AKI induced by cisplatin or as protective agent.

Early Response to the Plant Toxin Stenodactylin in Acute Myeloid Leukemia Cells Involves Inflammatory and Apoptotic Signaling.

Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Adenia stenodactyla Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt’s lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.

Gastric Corpus Mucosal Hyperplasia and Neuroendocrine Cell Hyperplasia, but not Spasmolytic Polypeptide-Expressing Metaplasia, Is Prevented by a Gastrin Receptor Antagonist in H+/K+ATPase Beta Subunit Knockout Mice.

Proton pump inhibitor use is associated with an increased risk of gastric cancer, which may be mediated by hypergastrinemia. Spasmolytic polypeptide-expression metaplasia (SPEM) has been proposed as a precursor of gastric cancer. We have examined the effects of the gastrin receptor antagonist netazepide (NTZ) or vehicle on the gastric corpus mucosa of H+/K+ATPase beta subunit knockout (KO) and wild-type (WT) mice. The gastric corpus was evaluated by histopathology, immunohistochemistry (IHC), in situ hybridization (ISH) and whole-genome gene expression analysis, focusing on markers of SPEM and neuroendocrine (NE) cells. KO mice had pronounced hypertrophy, intra- and submucosal cysts and extensive expression of SPEM and NE cell markers in the gastric corpus, but not in the antrum. Numerous SPEM-related genes were upregulated in KO mice compared to WT mice. NTZ reduced hypertrophia, cysts, inflammation and NE hyperplasia. However, NTZ neither affected expression of SPEM markers nor of SPEM-related genes. In conclusion, NTZ prevented mucosal hypertrophy, cyst formation and NE cell hyperplasia but did not affect SPEM. The presence of SPEM seems unrelated to the changes caused by hypergastrinemia in this animal model.

Autologous fat transplantation alters gene expression patterns related to inflammation and hypoxia in the irradiated human breast.

Radiation-induced fibrosis, an adverse effect of breast cancer treatment, is associated with functional and cosmetic impairment as well as surgical complications. Clinical reports suggest improvement following autologous fat transplantation, but the mechanisms underlying this effect are unknown. A global gene expression analysis was undertaken to identify genetic pathways dysregulated by radiation and evaluate the impact of autologous fat transplantation on gene expression. Adipose tissue biopsies were taken synchronously from irradiated and contralateral non-irradiated breasts, before and 1 year after autologous fat transplantation. Whole-genome gene expression analyses were performed, and Hallmark gene set analysis used to explore the effect of radiotherapy and autologous fat transplantation on gene expression. Forty microarrays were analysed, using bilateral biopsies taken from ten patients before and after autologous fat transplantation. Forty-five pathways were identified among the 3000 most dysregulated transcripts after radiotherapy in irradiated compared with non-irradiated breast (P ≤ 0·023; false discovery rate (FDR) no higher than 0·026). After autologous fat transplantation, 575 of the 3000 genes were again altered. Thirteen pathways (P ≤ 0·013; FDR 0·050 or less) were identified; the top two canonical pathways were interferon-γ response and hypoxia. Correlative immunohistochemistry showed increased macrophage recruitment in irradiated tissues. The present findings contribute to understanding of how autologous fat transplantation can ameliorate radiation-induced fibrosis. This further supports the use of autologous fat transplantation in the treatment of radiation-induced fibrosis. Surgical relevance Clinical studies have indicated that autologous fat transplantation (AFT) stimulates regression of chronic inflammation and fibrosis caused by radiotherapy in skin and subcutaneous fat. However, there is a paucity of biological evidence and the underlying processes are poorly understood. Human data are scarce, whereas experimental studies have focused mainly either on the effect of irradiation or AFT alone. The present results indicate that radiotherapy causes dysregulated gene expression in fibrosis-related pathways in adipose tissues in humans. They also show that AFT can cause a reversal of this, with several dysregulated genes returning to nearly normal expression levels. The study provides biological evidence for the impact of AFT on radiation-induced dysregulated gene expression in humans. It supports the use of AFT in the treatment of radiation-induced fibrosis, associated with severe morbidity and surgical challenges.

Potential biomarkers of acute myocardial infarction based on weighted gene co-expression network analysis

BackgroundAcute myocardial infarction (AMI) is the common cause of mortality in developed countries. The feasibility of whole-genome gene expression analysis to identify outcome-related genes and dysregulated pathways remains unknown. Molecular marker such as BNP, CRP and other serum inflammatory markers have got the notice at this point. However, these biomarkers exhibit elevated levels in patients with thyroid disease, renal failure and congestive heart failure. In this study, three groups of microarray data sets (GES66360, GSE48060, GSE29532) were collected from GEO, a total of 99, 52 and 55 samples, respectively. Weighted gene co-expression network analysis (WGCNA) was performed to obtain a classifier which composed of related genes that best characterize the AMI.ResultsHere, this study obtained three groups of microarray data sets (GES66360, GSE48060, GSE29532) on AMI blood samples, a total of 99, 52 and 24 samples, respectively. In all, 4672 genes, 3185 genes, 3660 genes were identified in GSE66360, GSE48060, GSE60993 modules, respectively. We preformed WGCNA, GO and KEGG pathway enrichment analysis on these three data sets, finding function enrichment of the differential expression gene on inflammation and immune response. Transcriptome analysis were performed in AMI patients at four time points compared to CAD patients with no history of MI, to determine gene expression profiles and their possible changes during the recovery from myocardial infarction.ConclusionsThe results suggested that three overlapping genes (FGFBP2, GFOD1 and MLC1) between two modules could be a potential use of gene biomarkers for the diagnose of AMI.

Genome-wide transcriptional response to altered levels of the Rpb7 subunit of RNA polymerase II identifies its role in DNA damage response in Schizosaccharomyces pombe.

Transcription of protein-coding genes is a highly regulated process. In eukaryotes, it involves cross-talk between the RNA polymerase II enzyme and different proteins of the transcriptional machinery. Twelve different subunits, Rpb1 to Rpb12, constitute RNA polymerase II. The sequence of the Rpb7 subunit is highly conserved across organisms. However, our knowledge and understanding of the role of Rpb7 in Schizosaccharomyces pombe is still limited. Therefore, in the present study we have studied the transcriptional response of S. pombe cells expressing reduced levels of rpb7+. Our global transcriptional analysis revealed that expression of genes belonging to different DNA repair pathways was downregulated by reduced rpb7+ expression. It was observed that survival of S. pombe cells expressing low rpb7+ levels was compromised under genotoxic stress conditions. Rpb7 also exhibited genetic interaction with genes of various DNA repair pathways. Furthermore, the growth sensitivity of S. pombe cells with low rpb7+ levels under DNA-damaging conditions was completely rescued by human Rpb7, indicating a functional conservation between these proteins. In summary, results from our whole-genome level gene expression analysis, as well as phenotypic and genetic experiments suggest a role for Rpb7 in DNA damage response in S. pombe.

IL6/STAT3 axis mediates resistance to BRAF inhibitors in thyroid carcinoma cells

Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 26–53% of human thyroid malignancies and, differently from melanomas, are poorly sensitive to BRAF inhibitors (BRAFi), and develop acquired resistance through activation of alternative signaling pathways. A whole-genome gene expression analysis of TC BRAF V600E cells exposed to PLX4032 identified JAK/STAT among the most significantly modulated signaling pathways. Interestingly, both transient exposure and chronic adaptation to PLX4032 resulted in upregulation of IL6/STAT3 axis and this impaired the cytostatic activity of PLX4032. Mechanistically, exposure to PLX4032 enhanced IL6 secretion and this, in turn, was responsible for STAT3 upregulation, activation of ERK signaling and poor sensitivity to BRAF inhibition. Consistently, the dual blockade of STAT3 (by siRNA or pharmacological inhibition) or IL6 signaling (by the humanized anti-human IL6 receptor antibody, tocilizumab) and BRAF (by PLX4032) improved the inhibition of cell cycle progression compared to PLX4032 single agent. These data support the role of IL6/STAT3 signaling pathway in modulating TC cell response to PLX4032 and candidate IL6 targeting as a strategy to improve the activity of PLX4032 in BRAF V600E TC cells.

Photodynamic therapy corrects abnormal cancer-associated gene expression observed in actinic keratosis lesions and induces a remodeling effect in photodamaged skin

BackgroundActinic keratoses (AK) are proliferations of neoplastic keratinocytes in the epidermis resulting from cumulative exposure to ultraviolet radiation (UVR), which are liable to transform into squamous cell carcinoma (SCC). Organ Transplant Recipients (OTR) have an increased risk of developing SCC as a consequence of long-term immunosuppressive therapy. The aim of this study was to determine the molecular signature of AKs from OTR prior to treatment with methyl aminolevulinate-photodynamic therapy (MAL-PDT), and to assess what impact the treatment has on promoting remodeling of the photo-damaged skin. MethodsSeven patients were enrolled on a clinical trial to assess the effect of MAL-PDT with biopsies taken at screening prior to the first treatment session (week 1), and six weeks after completion of final treatment (week 18). Whole-genome gene expression analysis was carried out on skin biopsies isolated from an AK lesion, an area surrounding the lesion, and a non-sun exposed region of the body. Quantitative PCR was utilized to confirm the differential expression of key genes. ResultsMAL-PDT treatment corrected abnormal proliferation-related gene profiles, corrected aberrantly expressed cancer-associated genes and induced expression of dermal extracellular matrix genes in photo-exposed skin. ConclusionThe efficacy of the MAL-PDT on AK lesions was confirmed at whole-genome gene expression level. A transcriptional signature of remodeling, identified through assessing the effect of MAL-PDT on photodamaged skin, supports the use of MAL-PDT for treating photodamaged skin and field cancerized areas.

Whole-genome gene expression analysis in urine samples of patients with prostate cancer and benign prostate hyperplasia

ObjectiveThere is an urgent need to find new biomarkers with higher specificity and sensitivity for using early detection of prostate cancer (PrCa) and reducing recurrent unnecessary biopsy rates, psychological and physical stress on the patient, and costs. Being noninvasive, urine-based tests might be suitable in routine practice. The aim of this study was to report the first whole-genome gene expression analysis in urine samples, as noninvasive method, that were obtained from PrCa, benign prostate hyperplasia (BPH), and control groups by using the microarray system from Turkey, to our knowledge. MethodsWhole-genome gene expression profiling was conducted in urine samples of 25 patients with PrCa, 24 patients with BPH, and 11 healthy males by using the Illumina Hi Scan microarray system. ResultsThe number of probes showing a significant change at the level of expression were 101 and 75 in PrCa-control and BPH-control comparison groups, respectively. Further, 51 of them were the same in both comparison groups. There was no significant change at the level of expression in PrCa-BPH comparison group. ConclusionThis study revealed several candidate biomarkers for early diagnosis of PrCa and contributed to the literature by detecting the differences of gene expression profiles in urine samples of PrCa-control and BPH-control comparison groups using the microarray. However, further studies are needed in larger groups.