Whole-genome Gene Expression Profiling Research Articles

Exploring the dynamic three-dimensional chromatin architecture and transcriptional landscape in goose liver tissues underlying metabolic adaptations induced by a high-fat diet

BackgroundGoose, descendants of migratory ancestors, have undergone extensive selective breeding, resulting in their remarkable ability to accumulate fat in the liver and exhibit a high tolerance for significant energy intake. As a result, goose offers an excellent model for studying obesity, metabolic disorders, and liver diseases in mammals. Although the impact of the three-dimensional arrangement of chromatin within the cell nucleus on gene expression and transcriptional regulation is widely acknowledged, the precise functions of chromatin architecture reorganization during fat deposition in goose liver tissues still need to be fully comprehended.ResultsIn this study, geese exhibited more pronounced changes in the liver index and triglyceride (TG) content following the consumption of the high-fat diet (HFD) than mice without significant signs of inflammation. Additionally, we performed comprehensive analyses on 10 goose liver tissues (5 HFD, 5 normal), including generating high-resolution maps of chromatin architecture, conducting whole-genome gene expression profiling, and identifying H3K27ac peaks in the livers of geese and mice subjected to the HFD. Our results unveiled a multiscale restructuring of chromatin architecture, encompassing Compartment A/B, topologically associated domains, and interactions between promoters and enhancers. The dynamism of the three-dimensional genome architecture, prompted by the HFD, assumed a pivotal role in the transcriptional regulation of crucial genes. Furthermore, we identified genes that regulate chromatin conformation changes, contributing to the metabolic adaptation process of lipid deposition and hepatic fat changes in geese in response to excessive energy intake. Moreover, we conducted a cross-species analysis comparing geese and mice exposed to the HFD, revealing unique characteristics specific to the goose liver compared to a mouse. These chromatin conformation changes help elucidate the observed characteristics of fat deposition and hepatic fat regulation in geese under conditions of excessive energy intake.ConclusionsWe examined the dynamic modifications in three-dimensional chromatin architecture and gene expression induced by an HFD in goose liver tissues. We conducted a cross-species analysis comparing that of mice. Our results contribute significant insights into the chromatin architecture of goose liver tissues, offering a novel perspective for investigating mammal liver diseases.

Preliminary study on whole genome methylation and transcriptomics in age-related cataracts

DNA methylation plays an important role in the occurrence and development of age-related cataracts (ARC). This study aims to reveal potential epigenetic biomarkers of ARC by detecting modifications to the DNA methylation patterns of genes shown to be related to ARC by transcriptomics. The MethylationEPIC BeadChip (850 K) was used to analyze the DNA methylation levels in ARC patients and unaffected controls, and the Pearson correlation test was used to perform genome-wide integration analysis of DNA methylation and transcriptome data. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to perform functional analysis of the whole genome, promoter regions (TSS1500/TSS200), and the associated differentially methylated genes (DMG). Pyrosequencing was used to verify the methylation levels of the selected genes. The results showed that, compared with the control group, a total of 52,705 differentially methylated sites were detected in the ARC group, of which 13,858 were hypermethylated and 38,847 were hypomethylated. GO and KEGG analyses identified functions related to the cell membrane, the calcium signaling pathway, and their possible molecular mechanisms. Then, 57 DMGs with negative promoter methylation correlations were screened by association analysis. Pyrosequencing verified that the ARC group had higher methylation levels of C3 and CCKAR and lower methylation levels of NLRP3, LEFTY1, and GPR35 compared with the control group. In summary, our study reveals the whole-genome DNA methylation patterns and gene expression profiles in ARC, and the molecular markers of methylation identified herein may aid in the prevention, diagnosis, treatment, and prognosis of ARC.

Whole-genome DNA methylation and gene expression profiling in the livers of mice with nonalcoholic steatohepatitis

AimsNon-alcoholic fatty liver disease (NAFLD) has emerged as one of the major causes of liver-related morbidity and mortality. It ranges simple steatosis to non-alcoholic steatohepatitis (NASH). Previous studies have shown that epigenetic factors, such as DNA methylation, can contribute to the development and progression of simple steatosis. However, the profiling of whole-genome DNA methylation remains poorly characterized in NASH. Main methodsIn this study, we established a mouse model of diet-induced NASH, by maintaining male mice on a high-fructose-high-cholesterol diet (HFHC), to generate hepatic steatosis, inflammation and injury. We profiled hepatic gene expression by RNA-Sequencing and locus-specific 5-methylcytosine level, using Whole Genome Bisulfite Sequencing (WGBS). Key findingsWe identified >1000 differentially methylated regions in NASH versus control group, indicating that NASH diet could modulate the liver methylome. Furthermore, integrated analysis of methylome and transcriptome identified certain key methylated genes and pathways, which may be involved in steroid metabolism and inflammation response. The liver methylation levels of key genes especially Tgfb, Msn, Iqgap1, Cyba, Fcgr1 decreased, and their consequent increased expression may lead to NASH development. SignificanceWe found that HFHC diet-induced NASH could induces genome-wide differential DNA methylation changes. Thus, we proposed that DNA methylation profiles of genomes may be a useful signature of gene transcription and may play an important role in the development of NASH. We also screened and validated the changes of key genes, which may provide new perspectives for the mechanistic study of NASH in future.

Transcriptomic study of gastrointestinal stromal tumors with liver metastasis.

Introduction: GIST (gastrointestinal stromal tumor) is the most prominent mesenchymal neoplasms of the gastrointestinal tract, and liver is the most common metastasis site for GIST. The molecular mechanism leading to liver metastasis of GIST is currently unclear. Methods: With the goal of revealing the underlying mechanism, we performed whole-genome gene expression profiling on 18 pairs of RNA samples comprised of GIST tissues (with liver metastasis) and corresponding non-tumor tissues. After identifying differentially expressed gene, functional annotation and signal pathway analyses were conducted. GSE13861, datasets that compare GIST (without liver metastasis) with adjacent tissues, served as a comparison. Results: A total of 492 up-regulated genes and 629 down-regulated genes were identified as differentially expressed genes between liver metastasis tissues and non-tumor tissues. We characterized expression patterns of DEGs identified from our cohort and GSE13861 that show signatures of enrichment for functionality. In subsequent gene set enrichment analysis, differentially expressed genes were mainly enriched in Epithelial Mesenchymal Transition in both datasets. 493 genes were overlapped among our whole-genome gene expression profiling results and GSE13861, consisting 188 up-regulated genes and 305 down-regulated genes. By using CytoHubba plugin of Cytoscape, CDH1, CD34, KIT, PROM1, SOX9, FGF2, CD24, ALDH1A1, JAG1 and NES were identified as top ten hub genes in tumorigenesis and liver metastasis of GIST. higher expression levels of FGF2, JAG1, CD34, ALDH1A1 and the lower expression level of CDH1 were respectively associated with unfavorable overall survival. Meanwhile higher expression levels of CD34, FGF2, KIT, JAG1, ALDH1A were correlated with worse disease-free survival. Discussion: The present study may help to provide candidate pathways and targets for treatment of GIST and prevention methods to liver metastasis.

Integrative Methylome and Transcriptome Characterization Identifies SERINC2 as a Tumor-Driven Gene for Papillary Thyroid Carcinoma

Most papillary thyroid carcinomas (PTCs) can be diagnosed preoperatively by routine evaluation, such as thyroid ultrasonography and fine-needle aspiration biopsy. Nevertheless, understanding how to differentiate indolent thyroid tumors from aggressive thyroid cancers remains a challenge, which may cause overtreatment. This study aimed to identify papillary thyroid cancer-specific indicators with whole-genome DNA methylation and gene expression profiles utilizing Infinium Methylation EPIC BeadChip (850k) and RNA arrays. In this paper, we report SERINC2 as a potential tumor-driven indicator in PTC. The up-regulated expression levels of SERINC2 were verified in PTC cell lines via qPCR. Then, cell counting kit 8 (CCK-8), wound healing, and flow cytometric assays were performed to confirm the influence of SERINC2 on proliferation and apoptosis in PTC cell lines after intervention or overexpression. Moreover, the investigation of data from the Cancer Dependency Map (DepMap) provided a potential pathway targeted by SERINC2. The activation of the tryptophan metabolic pathway may reduce the dependency of SERINC2 in thyroid cancers. In conclusion, our results demonstrate the whole-genome DNA methylation and gene expression profiles of papillary thyroid carcinoma, identify SERINC2 as a potential tumor-driven biomarker, and preliminarily verify its function in PTC.

Development and validation of an RNA-seq-based transcriptomic risk score for asthma

Recent progress in RNA sequencing (RNA-seq) allows us to explore whole-genome gene expression profiles and to develop predictive model for disease risk. The objective of this study was to develop and validate an RNA-seq-based transcriptomic risk score (RSRS) for disease risk prediction that can simultaneously accommodate demographic information. We analyzed RNA-seq gene expression data from 441 asthmatic and 254 non-asthmatic samples. Logistic least absolute shrinkage and selection operator (Lasso) regression analysis in the training set identified 73 differentially expressed genes (DEG) to form a weighted RSRS that discriminated asthmatics from healthy subjects with area under the curve (AUC) of 0.80 in the testing set after adjustment for age and gender. The 73-gene RSRS was validated in three independent RNA-seq datasets and achieved AUCs of 0.70, 0.77 and 0.60, respectively. To explore their biological and molecular functions in asthma phenotype, we examined the 73 genes by enrichment pathway analysis and found that these genes were significantly (p < 0.0001) enriched for DNA replication, recombination, and repair, cell-to-cell signaling and interaction, and eumelanin biosynthesis and developmental disorder. Further in-silico analyses of the 73 genes using Connectivity map shows that drugs (mepacrine, dactolisib) and genetic perturbagens (PAK1, GSR, RBM15 and TNFRSF12A) were identified and could potentially be repurposed for treating asthma. These findings show the promise for RNA-seq risk scores to stratify and predict disease risk.

Preliminary Study on the Sequencing of Whole Genomic Methylation and Transcriptome-Related Genes in Thyroid Carcinoma.

Thyroid carcinoma is the most prevalent endocrine cancer globally and the primary cause of cancer-related mortality. Epigenetic modifications are progressively being linked to metastasis. This study aimed to examine whole-genome DNA methylation patterns and the gene expression profiles in thyroid cancer tissue samples using a MethylationEPIC BeadChip (850K), RNA sequencing, and a targeted bisulfite sequencing assay. The results of the Illumina Infinium human methylation kit (850K) analyses identified differentially methylated CpG locations (DMPs) and differentially methylated CpG regions (DMRs) encompassing nearly the entire genome with high resolution and depth. Gene ontology and KEGG pathway analyses revealed that the genes associated with DMRs belonged to various domain-specific ontologies, including cell adhesion, molecule binding, and proliferation. The RNA-Seq study found 1627 differentially expressed genes, 1174 of which that were up-regulated and 453 of which that were down-regulated. The targeted bisulfite sequencing assay revealed that CHST2, DPP4, DUSP6, ITGA2, SLC1A5, TIAM1, TNIK, and ABTB2 methylation levels were dramatically lowered in thyroid cancer patients when compared to the controls, but GALNTL6, HTR7, SPOCD1, and GRM5 methylation levels were significantly raised. Our study revealed that the whole-genome DNA methylation patterns and gene expression profiles in thyroid cancer shed new light on the tumorigenesis of thyroid cancer.

A Network-Based Methodology to Identify Subnetwork Markers for Diagnosis and Prognosis of Colorectal Cancer.

The development of reliable methods for identification of robust biomarkers for complex diseases is critical for disease diagnosis and prognosis efforts. Integrating multi-omics data with protein-protein interaction (PPI) networks to investigate diseases may help better understand disease characteristics at the molecular level. In this study, we developed and tested a novel network-based method to detect subnetwork markers for patients with colorectal cancer (CRC). We performed an integrated omics analysis using whole-genome gene expression profiling and copy number alterations (CNAs) datasets followed by building a gene interaction network for the significantly altered genes. We then clustered the constructed gene network into subnetworks and assigned a score for each significant subnetwork. We developed a support vector machine (SVM) classifier using these scores as feature values and tested the methodology in independent CRC transcriptomic datasets. The network analysis resulted in 15 subnetwork markers that revealed several hub genes that may play a significant role in colorectal cancer, including PTP4A3, FGFR2, PTX3, AURKA, FEN1, INHBA, and YES1. The 15-subnetwork classifier displayed over 98 percent accuracy in detecting patients with CRC. In comparison to individual gene biomarkers, subnetwork markers based on integrated multi-omics and network analyses may lead to better disease classification, diagnosis, and prognosis.

Stearoyl-CoA Desaturase (SCD) Induces Cardiac Dysfunction with Cardiac Lipid Overload and Angiotensin II AT1 Receptor Protein Up-Regulation.

Heart failure is a major cause of death worldwide with insufficient treatment options. In the search for pathomechanisms, we found up-regulation of an enzyme, stearoyl-CoA desaturase 1 (Scd1), in different experimental models of heart failure induced by advanced atherosclerosis, chronic pressure overload, and/or volume overload. Because the pathophysiological role of Scd1/SCD in heart failure is not clear, we investigated the impact of cardiac SCD upregulation through the generation of C57BL/6-Tg(MHCSCD)Sjaa mice with myocardium-specific expression of SCD. Echocardiographic examination showed that 4.9-fold-increased SCD levels triggered cardiac hypertrophy and symptoms of heart failure at an age of eight months. Tg-SCD mice had a significantly reduced left ventricular cardiac ejection fraction of 25.7 ± 2.9% compared to 54.3 ± 4.5% of non-transgenic B6 control mice. Whole-genome gene expression profiling identified up-regulated heart-failure-related genes such as resistin, adiponectin, and fatty acid synthase, and type 1 and 3 collagens. Tg-SCD mice were characterized by cardiac lipid accumulation with 1.6- and 1.7-fold-increased cardiac contents of saturated lipids, palmitate, and stearate, respectively. In contrast, unsaturated lipids were not changed. Together with saturated lipids, apoptosis-enhancing p53 protein contents were elevated. Imaging by autoradiography revealed that the heart-failure-promoting and membrane-spanning angiotensin II AT1 receptor protein of Tg-SCD hearts was significantly up-regulated. In transfected HEK cells, the expression of SCD increased the number of cell-surface angiotensin II AT1 receptor binding sites. In addition, increased AT1 receptor protein levels were detected by fluorescence spectroscopy of fluorescent protein-labeled AT1 receptor-Cerulean. Taken together, we found that SCD promotes cardiac dysfunction with overload of cardiotoxic saturated lipids and up-regulation of the heart-failure-promoting AT1 receptor protein.

CD24 gene inhibition and TIMP-4 gene upregulation by Imperata cylindrica's root extract prevents metastasis of CaSki cells via inhibiting PI3K/Akt/snail signaling pathway and blocking EMT

Ethnopharmacological relevanceImperata cylindrica (L.) Raeusch (Gramineae) is a medicinal spice traditionally used in the treatment of hypertension and cancer. Aim of the studyTo assess the anti-metastatic potential of the methanol extract of I. cylindrica roots and determined its mechanisms of action. Material and methodsThe growth inhibition activity of I. cylindrica root extract in vitro and in vivo in human cervical cancer. The scratch assay and Boyden Chamber assay were used to determine the anti-migrative and anti-invasion actions of the plant extract. The whole-genome gene expression profiling using RNA-Seq was performed to determine the differentially expressed genes in CaSki cells after exposure to I. cylindrica to identify its targeted genes related to metastasis. Using protein analysis (western blotting) and gene expression analysis (RTqPCR), the targeted pathways of the key genes that were initially identified with RNA-Seq, were evaluated. ResultsI. cylindrica extract showed dose-dependent cytotoxicity in vitro and in vivo in mice bearing tumors. Furthermore, I. cylindrica root extract significantly inhibited cell migration and cell invasion. After the genome-wide transcriptome analysis, we found that important genes involved in cancer progression and metastasis of cervical cancer, that is, CD24 and TIMP-4 were significantly downregulated and upregulated, respectively. Moreover, I. cylindrica root extract significantly inhibited the PI3/AKT/Snail signaling pathway and blocked the EMT of CaSki cells. ConclusionThese findings provide an anti-metastatic mechanism of action of I. cylindrica root extract toward the human cervical cancer suggesting that this plant maybe developed into selective chemotherapy.

P0531CONTINUOUS HEMODIAFILTRATION WITH PMMA HEMOFILTER MODULATED COMPLEMENT ACTIVATION AND RENAL DYSFUNCTION IN A SWINE MODEL OF SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Sepsis-induced acute kidney injury (AKI) is a growing health care problem, refractory to conventional treatments. This disease is characterized by an overwhelmed immune response against a primary insult that become responsible for renal dysfunction and poor outcome. Therapeutic strategies based on blood purification have been developed for the treatment of this disease. The use of polymethyl methacrylate (PMMA) membrane hemofilter in continuous hemodiafiltration (CHDF) modality showed better hemodynamic stability and efficient renal support in chronic dialysis maintenance. Here we investigated the efficacy of Hemofeel PMMA membrane (TORAY, Japan) in interfering with Complement activation and renal damage in a swine model of sepsis-induced AKI. Method After 3 hours from LPS infusion, 7 hours of PMMA-CVVH treatment or 7 hours of polysulfone (PSF)-CVVH were performed. Animals were sacrificed after 24h from LPS infusion. Histologic and renal function parameters were analyzed in all pigs. Pentraxin-3 (PTX3) and C5b-9 deposits were assessed on renal biopsies. Systemic Complement activation was evaluated by Wieslab kit. Gene expression profile was obtained from isolated PBMCs by Agilent SurePrint G3 Porcine Gene Expression Microarrays. Genespring and R software were used for the analysis. Results were validated by Real-time PCR. Results Analysis of renal biopsies from septic pigs presented increased interstitial leucocyte infiltrate, extensive collagen deposition and diffuse glomerular thrombi compared to healthy pigs (p&amp;lt;0.05). Confocal analysis showed extensive PTX-3 and C5-b9 deposits at tubulo-interstitial level associated with significant activation of systemic complement classical and alternative pathways (p&amp;lt;0.05). Interestingly, PMMA-CVVH treatment significantly reduced local and systemic complement activation, leucocyte infiltrate and tubule-interstitial fibrosis (p&amp;lt;0.05). On the contrary, no significant improvement was observed by PSF-CVVH treatment. Then, we compared the whole-genome gene expression profiles of swine PBMC. We identified 711 differentially expressed genes comparing PBMC before LPS infusion (LPS T0) and after 24 hours from LPS infusion (LPS T24) and 913 genes comparing gene expression profiles of LPS T24 group with that of septic pigs treated with PMMA-CVVH (PMMA T24 group) (fold change &amp;gt;2 ; false discovery rate &amp;lt;0.05). The most modulated genes were Granzime B, Complement Factor B, Complement Component 4 Binding Protein Alpha, IL-12, SERPINB-1 and TIMP-2 that were closely related to sepsis-induced immunological process. Finally, quantitative PCR confirmed the microarray data indicating that Granzime B and Complement Factor B upregulation in PBMC was significantly hampered by PMMA treatment. Conclusion Our data suggest that LPS induced AKI is characterized by activation of Classical and alternative Complement pathways resulting in significant renal tissue damage. By interfering with complement activation and inflammatory response, PMMA membrane might prevent dysfunctional activation of resident renal cells with prevention of sepsis-induced AKI.

Regularized selection indices for breeding value prediction using hyper-spectral image data

High-throughput phenotyping (HTP) technologies can produce data on thousands of phenotypes per unit being monitored. These data can be used to breed for economically and environmentally relevant traits (e.g., drought tolerance); however, incorporating high-dimensional phenotypes in genetic analyses and in breeding schemes poses important statistical and computational challenges. To address this problem, we developed regularized selection indices; the methodology integrates techniques commonly used in high-dimensional phenotypic regressions (including penalization and rank-reduction approaches) into the selection index (SI) framework. Using extensive data from CIMMYT’s (International Maize and Wheat Improvement Center) wheat breeding program we show that regularized SIs derived from hyper-spectral data offer consistently higher accuracy for grain yield than those achieved by standard SIs, and by vegetation indices commonly used to predict agronomic traits. Regularized SIs offer an effective approach to leverage HTP data that is routinely generated in agriculture; the methodology can also be used to conduct genetic studies using high-dimensional phenotypes that are often collected in humans and model organisms including body images and whole-genome gene expression profiles.

Abstract B16: Role of RasGRF2 in AnxA6-mediated growth of TNBC cells

Abstract Introduction: The mechanisms by which annexin A6 (AnxA6) inhibits anchorage-independent tumor cell growth but promotes tumor cell motility remain unclear. Using whole-genome gene expression profiling, we identified RasGRF2 (GRF2), a Ca2+-activated RasGEF, as one of the most upregulated genes following downregulation of AnxA6 in breast cancer (BC) cells. Here, we examined the role of GRF2 as a critical RasGEF in AnxA6-meditated BC cell growth. Methods and Results: We show that the cellular levels of GRF2 are relatively low in AnxA6-high mesenchymal-like BC cells but higher in AnxA6-low basal-like BC cells. We also show that loss of AnxA6 in AnxA6-high BC cells is associated with early onset and rapid growth of xenograft tumors, while upregulation of AnxA6 in AnxA6-low BC cells is associated with a decrease in growth of xenograft tumors. To explore the reciprocal expression of GRF2 and AnxA6, we demonstrate that ionomycin-induced surge in intracellular Ca2+ led to GRF2 downregulation and that AnxA6, GRF2 and Ras proteins are within the same complex. We demonstrate that upon activation of EGFR, GRF2 is rapidly degraded and that the basal GRF2 levels were restored as the receptor activity decreased. The activation/degradation of GRF2 was followed by the activation of ERK1/2, was not affected by chelation of intracellular Ca2+ with BAPTA, but GRF2 was stabilized by chelation of extracellular Ca2+ with EGTA. Lapatinib treatment efficiently blocked the activation of EGFR but did not block the degradation of GRF2. Finally, we show that downregulation of GRF2 and inhibition of EGFR led to a greater inhibition of tumor cell growth. Conclusion: Together, these data identify RasGRF2 as a major effector of AnxA6-dependent breast tumor growth and that reduced cellular levels of GRF2 imply active RasGEF. Combined inhibition of GRF2 and EGFR may be a viable alternative treatment for triple-negative BC. Citation Format: Diva S. Whalen, Sarrah E. Widatalla, Olga Y. Korolkova, Stephen D. Williams, Heather K. Beasley, Amos M. Sakwe. Role of RasGRF2 in AnxA6-mediated growth of TNBC cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B16.

Isoborneol Attenuates Low-Density Lipoprotein Accumulation and Foam Cell Formation in Macrophages.

PurposeIsoborneol has been used in the treatment of cardiovascular disease for several years in China. However, the mechanism is still unclear. The aim of this study was to identify the novel mechanism of isoborneol for its application in atherosclerotic disease.Materials and MethodsThe whole-genome gene expression profiles of MCF-7 cells treated with/or without isoborneol were detected by mRNA microarray analysis. The degree of similarity between the gene expression profiles was compared with the Connectivity Map (CMAP) database. An MTT assay was used to assess the toxicity of isoborneol on RAW 264.7 cells. Oil red O staining and a Dil-ox-LDL uptake assay in RAW 264.7 cells were also used to detect the accumulation of lipids in the macrophages and the uptake of oxidized low-density lipoprotein (ox-LDL).ResultsIsoborneol was proved to have mRNA expression profiles similar to that of ikarugamycin which can inhibit the uptake of ox-LDL. This process has proved to be an important cause of foam cell formation and early atherosclerotic lesions. It is speculated, therefore, that isoborneol may show similar activity to that shown by ikarugamycin. Subsequently, it was shown that RAW 264.7 cells reduced the absorption of ox-LDL and the accumulation of intracellular lipids after treatment with different concentrations of isoborneol.ConclusionThe results indicate that isoborneol inhibits macrophage consumption of ox-LDL, thereby preventing the accumulation of lipids in the macrophages. These results provide evidence for the application of isoborneol in atherosclerotic disease.

Hypoxia-induced modulation of glucose transporter expression impacts 18F-fluorodeoxyglucose PET-CT imaging in hepatocellular carcinoma.

To investigate the molecular mechanisms underlying the variable standard uptake value (SUV) of 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) imaging in hepatocellular carcinoma(HCC) and whether hypoxia-induced glucose transporter expression contributes to the progression of HCC and the rate of glycolysis in HCC cells. Sixteen HCC specimens obtained from patients who underwent pre-treatment staging with 18F-FDG PET-CT imaging were divided into high maximum SUV (SUVmax > 8) and low SUVmax (SUVmax < 5) groups and employed for whole-genome gene expression profiling using GeneChip Human Genome U133 Plus 2.0 Arrays. The relationship between SUVmax and the expression of glucose transporters 1 and 3 (GLUT1 and GLUT3) was further validated using immunohistochemical analysis. The expression of GLUT1 and GLUT3 in different HCC cells under hypoxia and normoxia conditions were monitored by quantitative reverse transcription PCR (RT-qPCR). Glycolysis and FDG uptake by HCC cells were measured using the Seahorse XF glycolysis stress test and 18F-FDG PET-CT imaging. The effect of GLUT1 and GLUT3 on glucose uptake in HCC cells was examined using the fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by detection of fluorescence produced by the cells using flow cytometry. Glucose transporters are differentially expressed between samples from HCC patients with high and low SUVmax. In particular, over-expression of GLUT1 and GLUT3 in high SUVmax patients was correlated with high glucose uptake and overall survival. The expression of GLUT1 and GLUT3 was significantly induced by hypoxia in different HCC cells. High expression of GLUT1 and GLUT3 in HCC cells were correlated with high rates of glycolysis and 18F-FDG uptake. Therefore, our data suggested that hypoxia-induced glucose transporters expression could result in the variations of 18F-FDG PET-CT imaging and progression of HCC, contributing to more aggressive disease phenotypes like large tumor size, recurrence, and poor survival. Over-expression of GLUT1 and GLUT3 significantly increase glucose uptake in HCC cells. Hypoxia-induced glucose transporters expression may therefore be a contributing variable in 18F-FDG PET-CT imaging and progression in HCC.

Aberrant DNA methylation profiling affecting the endometrial receptivity in recurrent implantation failure patients undergoing in vitro fertilization.

DNA methylation profile in mid-secretory phase of endometrium is reported to be varied from other phases in natural menstrual cycle. Therefore, we intended to study the impairment in endometrial receptivity by performing whole-genome methylation and gene expression profiling in endometrium of recurrent implantation failure patients (RIF) during IVF under controlled ovarian stimulation (COS). Endometrial biopsies were collected from IVF-RIF patients (cases, n=6) and healthy fertile oocyte donors (controls, n=6) undergoing COS after 6/7th day of human chorionic gonadotropin administration. The whole-genome methylation and gene expression microarray were performed and analysed by GenomeStudio software (P<.05 by Illumina Custom Model), whereas the enrichment analysis was performed using "Database for Annotation, Visualization and Integrated Discovery" (DAVID, V6.8). Significant differentially methylated genes were correlated with dys-regulated genes using Pearson's correlation. Differential methylation in RIF patients revealed 448 CpG sites. The enrichment analysis showed aberrant methylation in genes involved in immunological response and G protein activity. Methylation in NLRP2 gene in inflammatory pathway had significant negative correlation with gene expression (P=.008), whereas SERPINA5 gene that is already known to be involved in endometrial receptivity was observed to be hypomethylated in promoter region with highest delta beta value and up-regulated in gene expression analysis. The aberrant methylation of genes involved in immunological functions and G protein activation was found to be prevalent which might suggest a role in endometrial receptivity. However, the findings need to be further validated on a larger cohort of IVF-RIF patients.

Role of mitochondria and cardiolipins in growth inhibition of breast cancer cells by retinoic acid

BackgroundAll-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied.MethodsWe used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and –resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data.ResultsATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells.ConclusionsThe observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.

Analysis of genetic networks regulating refractive eye development in collaborative cross progenitor strain mice reveals new genes and pathways underlying human myopia

BackgroundPopulation studies suggest that genetic factors play an important role in refractive error development; however, the precise role of genetic background and the composition of the signaling pathways underlying refractive eye development remain poorly understood.MethodsHere, we analyzed normal refractive development and susceptibility to form-deprivation myopia in the eight progenitor mouse strains of the Collaborative Cross (CC). We used RNA-seq to analyze gene expression in the retinae of these mice and reconstruct genetic networks and signaling pathways underlying refractive eye development. We also utilized genome-wide gene-based association analysis to identify mouse genes and pathways associated with myopia in humans.ResultsGenetic background strongly influenced both baseline refractive development and susceptibility to environmentally-induced myopia. Baseline refractive errors ranged from − 21.2 diopters (D) in 129S1/svlmj mice to + 22.0 D in CAST/EiJ mice and represented a continuous distribution typical of a quantitative genetic trait. The extent of induced form-deprivation myopia ranged from − 5.6 D in NZO/HILtJ mice to − 20.0 D in CAST/EiJ mice and also followed a continuous distribution. Whole-genome (RNA-seq) gene expression profiling in retinae from CC progenitor strains identified genes whose expression level correlated with either baseline refractive error or susceptibility to myopia. Expression levels of 2,302 genes correlated with the baseline refractive state of the eye, whereas 1,917 genes correlated with susceptibility to induced myopia. Genome-wide gene-based association analysis in the CREAM and UK Biobank human cohorts revealed that 985 of the above genes were associated with myopia in humans, including 847 genes which were implicated in the development of human myopia for the first time. Although the gene sets controlling baseline refractive development and those regulating susceptibility to myopia overlapped, these two processes appeared to be controlled by largely distinct sets of genes.ConclusionsComparison with data for other animal models of myopia revealed that the genes identified in this study comprise a well-defined set of retinal signaling pathways, which are highly conserved across different vertebrate species. These results identify major signaling pathways involved in refractive eye development and provide attractive targets for the development of anti-myopia drugs.

Lipid metabolism participates in human membranous nephropathy identified by whole-genome gene expression profiling.

A genomics approach is an effective way to understand the possible mechanisms underlying the onset and progression of disease. However, very limited results have been published regarding whole-genome expression analysis of human idiopathic membranous nephropathy (iMN) using renal tissue. In the present study, gene expression profiling using renal cortex tissue from iMN patients and healthy controls was conducted; differentially expressed genes (DEGs) were filtered out, and 167 up- and 291 down-regulated genes were identified as overlapping DEGs (ODEGs). Moreover, enrichment analysis and protein-protein network construction were performed, revealing enrichment of genes mainly in cholesterol metabolism and arachidonic acid metabolism, among others, with 38 hub genes obtained. Furthermore, we found several associations between circulating lipid concentrations and hub gene signal intensities in the renal cortex. Our findings indicate that lipid metabolism, including cholesterol metabolism and arachidonic acid metabolism, may participate in iMN pathogenesis through key genes, including apolipoprotein A1 (APOA1), apolipoprotein B (APOB), apolipoprotein C3 (APOC3), cholesteryl ester transfer protein (CETP), and phospholipase A2 group XIIB (PLA2G12B).

Identification of differentially expressed genes in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease that affects exocrine glands. To study the molecular mechanism and identify crucial genes/pathways in pSS pathogenesis, the microarray-based whole-genome gene expression profiles from salivary glands of patients with pSS and non-sicca controls were retrieved. After normalization and subsequent batch effect adjustment, significance analysis of microarrays method was applied to five available datasets, and 379 differentially expressed genes (DEGs) were identified. The 300 upregulated DEGs were enriched in Gene Ontology terms of immune and inflammatory responses, including antigen processing and presentation, interferon-mediated signaling pathway, and chemotaxis. Previously reported pSS-associated genes, including HLA-DRA, TAP2, PRDM1, and IFI16, were found to be significantly upregulated. The downregulated DEGs were enriched in pathways of salivary secretion, carbohydrate digestion and absorption, and starch and sucrose metabolism, implying dysfunction of salivary glands during pathogenesis. Next, a protein-protein interaction network was constructed, and B2M, an upregulated DEG, was shown to be a hub, suggesting its potential involvement in pSS development. In summary, we found the activation of pSS-associated genes in pathogenesis, and provide clues for salivary glands dysfunction. Experimental investigation on the identified DEGs in this study will deepen our understanding on pSS.