Differential network analysis by simultaneously considering changes in gene interactions and gene expression.

IntroductionPancreatic cancer (PaCa) is one of the most aggressive and deadliest cancer with 6 months average survival rate. Recent studies indicate that the tumour microenvironment (TME) plays an important role in all stages of tumorigenesis in PaCa. Therefore, both tumour cells and their TME should be targeted for an effective treatment. Eukaryotic elongation factor 2 kinase (EF2K) is an enzyme which is overexpressed in cancer cells and plays a key role in cancer cell survival under stress conditions. There is no study that have shown the relationship between EF2K and TME. Thus, we investigated that the effects of EF2K expression on PaCa cell line (PANC1) and the TME.Material and methodsPANC1 cells were cocultured with monocytic THP-1 cells and macrophages were polarised from THP-1 cells. The changes in EF2K expression, cell migration and invasion were analysed using western blot, migration and invasion assays, respectively. The effects of coculture on monocyte chemoattractant protein-1 (MCP-1) levels, which is one of the most important chemokines in TME were analysed using ELISA. The role of MCP-1 on both EF2K and migration of PANC1 cells were investigated. The role of MCP-1 on monocyte-macrophage differentiation was also investigated. To determine the relationship between EF2K and MCP-1, EF2K was stably overexpressed in PANC1 cells and MCP-1 levels were measured using western blot. Then, EF2K was silenced using siRNA and the changes in MCP-1 expression levels, the ability of colony formation, migration and invasion of PANC1 cells were investigated. Pancreatic orthotopic tumour model was used to determine the in vivo effects of EF2K inhibition. In tumour tissues, the changes in expression levels of MCP-1 was measured using western blot and the presence of tumor-infiltrated pro-tumorigenic macrophages were shown using immunohistochemical staining.Results and discussionsThe interaction between PANC1 cells and macrophages caused an increase in EF2K and MCP-1 proteins and this interaction accelarated cell migration and invasion. In addition, it was found a bidirectional interaction between MCP-1 and EF2K. MCP-1 also caused differentiation of monocytes to pro-tumorigenic macrophages. In vitro silencing of EF2K decreased MCP-1 expression, cell invasion and migration. In vivo inhibition of EF2K also decreased MCP-1 expression, tumour volume and the number of tumor-infiltrated pro-tumorigenic macrophages.ConclusionTargeting both tumour cells and macrohages through EF2K inhibition might be a promising strategy for PaCa treatment.

Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

Relationship between Altered Gene Expression and DNA Methylation of the DLK1-DIO3 region in Azacitidine-Treated Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia with Myelodysplasia-Related Changes

The vast majority of primary renal tumors were malignant and had a greater damage to the kidneys. The cause and development of tumor diseases were not only related to a single gene, but also caused by molecular aberrations in a network of complex genes or their generations. In this paper, we used differential network analysis method. The gene chip data of renal tumor related gene from Affymetrix hgu13b was firstly screened to obtain differential expressed genes, and the gene network was constructed separately under disease samples and normal samples. Secondly, the difference between disease network and normal genes was constructed, and the renal tumor related gene was analyzed. In our differential network model, we used a more accurate method than before. What’s more, we used Bayesian information criteria to adjust the parameters, and the gene network under two different conditions was coded as the difference between the precision covariance matrices. More than two hundred pairs of gene pairs were identified from the differential network. Based on the differential network analysis of the central gene, GO enrichment analysis showed that the gene TBX3 had the highest degree of enrichment, indicating that it may be related to the formation of renal tumors. In the KEGG pathway enrichment analysis, it was found that p<0.05 was mainly enriched as a chemokine pathway, and chemokines had an important influence on the occurrence, metastasis and treatment of renal tumors.

Balancing type one and two errors in multiple testing for differential expression of genes

Taurine is one of the most abundant amino acids in the brain. It plays various important physiological functions as a neuromodulator and antioxidant. Taurine is suspected to be involved in depression as families that suffer from a hereditary taurine deficiency have a tendency to develop depression. However, knowledge regarding its function in relation to depression is limited. Previously, we reported that the chronic oral administration of taurine has an anti-depressant-like effect in rats and changes the phosphorylation levels of hippocampal signal transduction molecules including ERK and CaMKII. In this study, we studied the global changes of expression in hippocampal proteins and genes under chronic taurine supplementation. Wistar male rats were fed taurine-contained diet for 4 weeks. Hippocampal gene, protein expression and their phosphorylation levels were evaluated by the commercial microarrays. Some prominent changes of protein phosphorylation and expression levels in the taurine-fed rats were observed by the antibody microarray. Especially, hippocampal p53, Jun, PKCs and HDAC5 were overexpressed and/or phosphorylated in the taurine-fed rats compared to control. Also, DNA microarray showed some changes of hippocampal gene expression in the taurine-fed rats. In conclusion, our findings suggest that chronic taurine supplementation has an antidepressant-like effect via some changes of expression and phosphorylation of the hippocampal proteins.

Proteome Analysis of Hepatocellular Carcinoma

Understanding how populations adapt to changing environmental conditions is a long-standing theme in evolutionary biology. Gene expression changes have been recognized as an important driver of local adaptation, but relatively little is known regarding the direction of change and in particular, about the interplay between plastic and evolutionary gene expression. We have previously shown that the gene expression profiles of European grayling (Thymallus thymallus) populations inhabiting different thermal environments include both plastic and evolutionary components. However, whether the plastic and evolutionary responses were in the same direction was not investigated in detail, nor was the identity of the specific genes involved. In this study, we show that the plastic changes in protein expression in response to different temperatures are highly correlated with the evolutionary response in grayling subpopulations adapted to different thermal environments. This finding provides preliminary evidence that the plastic response most likely facilitates adaptation during the early phases of colonization of thermal environments. The proteins that showed significant changes in expression level between warm and cold temperature treatments were mostly related to muscle development, which is consistent with earlier findings demonstrating muscle mass differentiation between cold and warm grayling populations.

Photoperiodic regulation of cold tolerance and expression levels of regucalcin gene in Drosophila montana

Simple SummaryThe extracellular matrix comprises an important collection of proteins that aids in the body’s response to pain. Evaluating the effect of electrical neuromodulation treatments on expression levels in neural tissues associated with neuropathic pain is important for improving such treatments. Here, we analyzed extracellular matrix proteins in spinal cords of a rodent model of neuropathic pain. We evaluated two spinal cord stimulation (SCS) therapies, one based on differential target multiplexed programming (DTMP) and another based on a conventional low-rate program (LR-SCS), for their ability to reverse the effects associated with the pain model. Of the 186 proteins identified as extracellular-matrix-related, DTMP reversed expression levels of 83% of them back to levels seen in uninjured animals, whereas LR-SCS reversed 67%. Protein phosphorylation is indicative of activation/deactivation of signaling pathways. Phosphorylation can occur at multiple locations on a protein and multiple times as well. There were 883 unique phosphorylated states found among 93 ECM-related proteins. Relative to the pain model, DTMP had a reversal effect on 76% versus 58% by LR-SCS on phosphorylated proteins. This study helps up to better understand pain pathways and also highlights a more robust reversal by DTMP relative to LR-SCS.There is limited research on the association between the extracellular matrix (ECM) and chronic neuropathic pain. The objective of this study was twofold. Firstly, we aimed to assess changes in expression levels and the phosphorylation of ECM-related proteins due to the spared nerve injury (SNI) model of neuropathic pain. Secondly, two modalities of spinal cord stimulation (SCS) were compared for their ability to reverse the changes induced by the pain model back toward normal, non-injury levels. We identified 186 proteins as ECM-related and as having significant changes in protein expression among at least one of the four experimental groups. Of the two SCS treatments, the differential target multiplexed programming (DTMP) approach reversed expression levels of 83% of proteins affected by the pain model back to levels seen in uninjured animals, whereas a low-rate (LR-SCS) approach reversed 67%. There were 93 ECM-related proteins identified in the phosphoproteomic dataset, having a combined 883 phosphorylated isoforms. DTMP back-regulated 76% of phosphoproteins affected by the pain model back toward levels found in uninjured animals, whereas LR-SCS back-regulated 58%. This study expands our knowledge of ECM-related proteins responding to a neuropathic pain model as well as providing a better perspective on the mechanism of action of SCS therapy.

High risk neuroblastoma tumors (HR-NB) are characterized by the presence of undifferentiated histopathology, while tumors from low risk patients have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Determining the cellular and molecular underpinnings that contribute to this undifferentiated state will lead to better understanding of the disease pathogenesis. Retinoic acid (RA) is known to prime embryonic stem cells for lineage specific differentiation. To elucidate the early events important for reprogramming of NB cells leading to changes in the degree of cellular differentiation, we treated HR-NB cells (KCNR : MYCN amplified) with 5µM RA for 24 hrs. RNA-Seq analysis, to assess early changes in the transcriptome, showed significant changes in gene expression [2fold-up-regulated-446, 2fold-down-regulated-250, FDR≤0.01] which were enriched for biological functions like neuron projection, neuron differentiation, indicating onset of neurogenesis as a marker of differentiation. Gene set enrichment analysis identified significant positive enrichment of NB differentiation (p=0.001) and PRC2 (Polycomb Repressor Complex2) gene sets (p=0.02) along with downregulation of MYCN signature gene set (p=0.001). PRC2 component EZH2 (Histone Methyltransferase) is highly expressed in NB and represses differentiation genes. Though, EZH2 protein levels did not change after treatment, RA caused 40% reduction in H3 bound EZH2. To gain insight into how this reduction affects subsequent gene expression; we evaluated changes in genome wide binding of EZH2 and H3K27me3 after treatment. Indeed, significant correlation was observed between the changes in expression levels of genes after RA treatment and relative binding of EZH2 (p=3.88e-6) and H3K27me3 (p=3.88e-6) at their promoter regions. One-third (n=147) of the upregulated genes (eg. HEY1, RARB) and half (n=117) of the downregulated genes (eg. INSM2, CAMK4) were EZH2 targets. Upregulated genes were related to biological functions like negative regulation of cell growth and transcription, nervous system development. To probe the underlying mechanism, in-silico analysis indicated RA decreased Wnt/ß-catenin, eNOS and PTEN pathways while stimulating cAMP, Integrin linked kinase and G-coupled protein pathways. Evaluation of the cAMP pathway candidates identified 13 genes as EZH2 targets. eg CNR1, DUSP6, PKIA, RSG4. In primary NB tumors, microarray studies indicate that low levels of CNR1 and DUSP6 are associated with poor prognosis. RA stimulates 10-fold and 2-fold increases in CNR1 and DUSP6 levels, respectively. RA treatment inhibits EZH2 binding to its target genes thereby activating pathways involved in reprogramming of HR-NBs to a more differentiated state. Studies are underway to further evaluate the molecular mechanism leading to decreased EZH2 binding upon RA treatment. Citation Format: Deblina Banerjee, Zhihui Liu, Doo-Yi Oh, Maggie Cam, Bong-Hyun Kim, Carol Thiele. Retinoic acid (RA) relieves EZH2-mediated epigenetic suppression in high-risk neuroblastoma (HR-NB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2017-3872

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans. Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

ABSTRACTThe Long-Evans Cinnamon (LEC) rat, an animal model of human Wilson’s disease, spontaneously develops fulminant hepatitis associated with severe jaundice at about 4 months of age. In this study, we examined the changes in gene expression during progression of acute hepatic injury. When levels of gene expression in the liver of LEC rats at 13 weeks of age were compared to those in rats at 4 weeks of age using oligonucleotide arrays, 1,620 genes out of 7,700 genes analyzed showed more than 2-fold differences. Expression levels of 11 of 29 genes related to stress-activating protein kinase (SAPK) changed by more than 2-fold in the liver of LEC rats, but none of the SAPK-related genes showed changes in expression levels in the liver of control rats. Activity of p38 mapk in the liver of LEC rats at 13 weeks of age was about 8.1-fold higher than that in rats at 4 weeks of age. When LEC rats were administered SB203580, a p38 mapk-specific inhibitor, by s.c. injection twice a week from 10 to 13 weeks of age, activities of p38 mapk in the liver, activities of AST and ALT and concentrations of bilirubin in sera of rats administered SB203580 significantly decreased compared to those in rats not administered. These results showed that the increase in activities of p38 mapk was related to the occurrence of acute hepatic injury in LEC rats.

PD19-12 TEMPORAL EFFECTS OF CHRONIC BLADDER OVER-DISTENSION ON HIF-1α AND VEGF EXPRESSION IN MOUSE UROTHELIUM

Study question To investigate whether erzhi-tiangui granules (ETG) can reduce granulosa cells (GCs) apoptosis levels by regulating NEAT1 expression, thereby improving the cycle outcomes of patients. Summary answer ETG significantly improved oocyte quality and decreased apoptotic factor levels in GCs, which may be related to its intervention in NEAT1 expression. What is known already The mechanism by which diminished ovarian reserve (DOR) occurs is mainly due to extensive apoptosis of GCs, which leads to follicular atresia and reduced follicle numbers. It has been found that changes in NEAT1 expression in ovarian tissues can exert influence the extent of apoptosis of GCs, which leads to diseases such as POF and PCOS. While ETG, a clinical formula used to improve female fertility, can improve the quality of oocytes and embryos by down-regulating the expression of apoptosis-related effector proteins, thus improving clinical outcomes. Whether ETG improves clinical outcomes in DOR patients via NEAT1 hasn’t been elucidated. Study design, size, duration This study was conducted in two separate parts from January 2020 to September 2021. Experiment I included 15 women each with normal ovarian reserve (NOR) and DOR, aiming to investigate the expression differences of NEAT1, key apoptosis proteins (Bcl-2 and Caspase-3). Experiment II, a randomized controlled trial, recruited 70 DOR patients, aimed to investigate if ETG could regulate the expression levels of Bcl-2 and Caspase-3 through NEAT1, thus acting to improve clinical outcomes. Participants/materials, setting, methods We detected expression levels of lncRNA NEAT1 and apoptotic proteins Bcl-2 and Caspase-3 of DOR and NOR women using RT-qPCR and Western-blotting, respectively. DOR patients were randomly assigned to ETG and control group, treated with ETG and placebo respectively. Clinical efficacy was confirmed by comparing two groups regarding changes in clinical and laboratory data. We also probed the changes in expression levels of NEAT1, Bcl-2 and Caspase-3 to investigate effects of ETG on GCs’ apoptosis. Main results and the role of chance In Experiment I, compared to the NOR group, the expression levels of NEAT1 and Bcl-2 were down-regulated while those of Caspase-3 were up-regulated in the GCs of DOR patients (P &amp;lt; 0.05). In Experiment II, a total of 65 of 70 patients completed the study and were included in the outcome analysis. There were no significant differences between the two groups regarding baseline characteristics. Before treatment, there was no significant difference in the Kidney-deficiency syndrome scores between the two groups. After treatment, the ETG group had significantly lower Kidney-deficiency syndrome scores compared to the control group (P &amp;lt; 0.01). Also, Kidney-deficiency syndrome scores were significantly lower in the ETG group post-treatment compared with that pre-treatment (P &amp;lt; 0.001); there was no significant change in the control group (P &amp;gt; 0.05). There were no significant differences between the two groups in the duration of gonadotrophin stimulation, serum estrogenand progesterone levels on the trigger day, and normal fertilization rate. Compared to the control group, the ETG group had lower gonadotropin dosage and increased number of oocytes, transferable embryos and top embryos obtained (P &amp;lt; 0.05). Furthermore, the expression of NEAT1 was upregulated (P &amp;lt; 0.05), Caspase-3 expression was decreased (P &amp;lt; 0.01) in GCs from the ETG group than those in the control group (P &amp;lt; 0.05). Limitations, reasons for caution The small number of observed cases and the lack of long-term efficacy may lead to biased study conclusions. Further studies are also expected in the future to understand the relevant pathway mechanisms. Wider implications of the findings In this study, not only to judge the therapeutic effects of ETG by using clinical symptoms and clinical laboratory indexes as observations, but also to explore the molecular mechanism of its treatment of Kidney-deficiency type DOR based on NEAT1, which may be related to affecting the apoptotic process. Trial registration number No. 82274573, No. 82174429, No. ZR2021MH255, No. tsqn202103182