Ingenuity Pathway Analysis System Research Articles

#3084 EXPLORATION OF PROGNOSTIC FACTORS IN DIABETIC NEPHROPATHY WITH NODULAR LESION USING TRANSCRIPTOME ANALYSIS OF HUMAN KIDNEY TISSUES

Abstract Background and Aims Although the speed of eGFR decline is usually rapid in patients with diabetic nephropathy (DMN) and a nodular lesion (NL), some patients with NL experience a slow eGFR decline. The factors associated with the difference in prognosis remain unknown. In this study, we aimed to explore prognostic factors by performing transcriptome analysis on preserved residual renal biopsy specimens from patients with pathologically diagnosed and prognostically known DMN with nodules. Method Patients who had an annual eGFR drop rate > 5 mL/min/1.73 m2 were defined as rapid decliners (RD) and the others as slow decliners (SD). Twelve patients (RD = 6 and SD = 6) who underwent kidney biopsy, had no other renal comorbidities, and had been followed up for at least one year were included. Total RNA was isolated from renal biopsy tissues in formalin-fixed, paraffin-embedded blocks, and microarray analysis was done. Results At the time of renal biopsy, the eGFR, urine protein, and rate of sclerotic glomeruli were not statistically different between the RD and SD groups, at 49.7±10.1 mL/min/1.73 m2 and 57.5±12.4 mL/min/1.73 m2 (p = 0.258), 7.5±4.2 g/gCre and 4.0±2.7 g/gCre (p = 0.123), and 19.2±13.4% and 25.5±14.4% (p = 0.450), respectively. In microarray analyses (total 58,341 gene probes), genes with little expression (raw signals <50) and with little differences between RD and SD (processed signals <0.3) were excluded, leaving 14,227 genes for further analysis. A total of 1,496 differentially expressed genes (DEGs) were selected with p-values <0.05 and fold change ≥2 between the RD and SD group for further bioinformatic analysis. Using the Ingenuity Pathway Analysis system, 15 canonical pathways and 9 diseases or functions annotations were found to be enriched. In addition, upstream regulator analysis identified 73 upstream regulators. Among them, only two regulators, CBX5 and CCN5, showed p <0.05 and absolute z-score > = 2 as activated and inhibited regulators, respectively, in the RD group. We next performed regulator effects analysis, indicating one regulator network which identified the key upstream regulator CCN5. The regulator network also showed a small number of genes involved in the viral infection, which included CDH1, CD24, and ESR1. Additionally, IPA showed 25 interaction networks ranging from 11 to 38. The fifth-ranked network was associated with Cancer, Organismal Injury, and Abnormalities (score = 32), in which CD24 and ESR1 were included. We, therefore, performed qRT-PCR to compare the expression levels of CDH1, CD24, and ESR1 in the RD group with those in the SD group. The expression levels of CD24 and ESR1 in the RD group were downregulated compared with those in the SD group (p = 0.004 and p = 0.022, respectively) although there was little difference in the level of CDH1 between them (p = 0.638). Conclusion Transcriptomic analysis using renal biopsied tissue revealed that ESR1 and CD24 might be candidates for renoprotective factors in DMN with NL.

Age-dependent oral manifestations of neurofibromatosis type 1: a case\u2013control study

IntroductionMost craniofacial manifestations of neurofibromatosis type 1 (NF1) are considered as a result of tumor compression. We sought to determine salivary changes, caries, and periodontal complications in NF1 patients without tumors in the oral cavity.Objective and methodsEleven NF1 patients without tumors in the oral cavity and 29 matched controls without NF1 were enrolled in this case–control study. Demographic information, medical history, and data of intraoral examinations, including the Decayed, Missing, and Filled Teeth (DMFT) scores and Russel’s periodontal index (PI), were recorded. The functional salivary analysis was performed for sialometry, salivary pH values, and amylase activity. Ingenuity Systems Pathway Analysis (IPA) was conducted to identify mutually activated pathways for NF1-associated oral complications.ResultsNF1 patients were associated with periodontitis (OR = 1.40, 95% CI = 1.06–1.73, P = 0.04), gingivitis (OR = 1.55, 95% CI = 1.09–2.01, P = 0.0002), and decreased salivary flow rates (OR = 1.40, 95% CI = 1.05–1.76, P = 0.005). Periodontal destruction, salivary changes, and dental caries in NF1 patients were age-dependent. Subgroup analyses based on age stratification suggested that salivary flow rates and salivary amylase activities were significantly low in NF1 patients aged over 20 years and that salivary pH values, PI and DMFT scores were significantly high among NF1- controls aged over 20. All oral complications were not significantly presented in NF1 patients aged below 20 years. IPA analyses suggested that cellular mechanisms underlying NF1-associated oral complications involved chronic inflammatory pathways and fibrosis signaling pathway.ConclusionNF1 patients without tumors in the oral cavity presented a comparatively high prevalence of age-dependent oral complications, including periodontal destruction and salivary gland dysfunction, which were associated with chronic inflammatory pathogenesis.

Effect of Garlic Organic Sulfides on Gene Expression Profiling in HepG2 Cells and Its Biological Function Analysis by Ingenuity Pathway Analysis System and Bio-Plex-Based Assays

Garlic organic sulfides are dietary bioactive components with multiple biofunctions to prevent chronic diseases/inflammation and promote human health. DADS (diallyl disulfide), DATS (diallyl trisulfide), and DTS (diallyl tetrasulfide) are typical organic sulfides with similar structures from garlic. However, the structure-activity relationship of garlic organic sulfides remained unknown. The aim of the present study was to investigate the effect of DADS, DATS, and DTS on the gene expression profiling of human hepatocellular carcinoma cells (HepG2) by application of microarray and specialized analysis software, GO, Bio-Plex-based cytokines assay and IPA and analyze their structure-activity relationship according to antioxidant, anti-inflammatory, and metabolic-related properties. According to the microarray data, with the increase of S atom in garlic organic sulfides, its biological activity was gradually enhanced. In the general catalog of GO, garlic organic sulfides mainly affect biological process, molecular function, and cellular component. RT-qPCR results indicated that the microarray data is trustworthy, and the structure-activity analysis data found that more sulfur atoms have more powerful properties; thus, microarray data of DTS was preceded to the subsequent IPA analysis. The results of IPA analysis showed that the top 5 signaling pathways and molecular functions were disturbed by DTS; the molecular functions with the highest scores affected by DTS are cancer, cell apoptosis, and cell proliferation, which imply that the occurrence or metabolism of these diseases is related to the differential expression of the above-mentioned related genes and the activation of signaling channels, and the core of the most significant molecular network is inflammation. Finally, the results found that the secretions of 6 cytokines in macrophages were significantly inhibited by DTS treatment. This is the first study that analyzed the structure-activity relationship of garlic organic sulfides, which will provide useful genetic information for its multi-biofunction and promote their clinical application in the near future.

Abstract PO009: Increased DACH1 mutation frequency in a Kentucky hospital uterine cancer population

Abstract Purpose: DACH1 is a transcriptional repressor and tumor suppressor gene frequently mutated in breast, bladder, and prostate cancer. Most studied in breast carcinoma, DACH1 expression was reduced or lost in invasive breast cancer patients, and its expression was inversely related to tumor diameter, stage, and nodal metastasis, and directly associated with increased survival time. While less studied in uterine cancer, nearly all normal endometrial samples show nuclear expression of DACH1, with DACH1 expression lost in more than half of uterine cancers. Loss of DACH1 expression has been associated with higher surgical stage, positive peritoneal cytology, and lymph node positivity in uterine cancer. In this study, the Oncology Research Information Exchange Network (ORIEN) Avatar database was used to determine the frequency of DACH1 mutations in our Kentucky hospital population with uterine cancer. Experimental Design: Clinical and genomic data were collected for 65 patients with uterine cancer from Markey Cancer Center (MCC) through Total Cancer Care (TCC) ®. TCC is a prospective cohort study with whole-exome tumor sequencing, RNA sequencing, germline sequencing, and lifetime follow-up. The clinical attributes, whole-exome sequencing, RNA sequencing, microsatellite instability, and tumor mutational burden were compared by DACH1 status. EdgeR was used to perform normalization, expression modeling, and difference testing. The network analysis was performed using Qiagen’s Ingenuity Pathway Analysis (IPA) system for core analysis of the RNA sequencing data and overlaid with the Global Molecular Network Overlay in the IPA knowledge base. The Cancer Genome Atlas Project (TCGA) PanCancer Atlas dataset was used for comparison through cBioPortal.org, utilizing endometrial cancer and carcinosarcoma subgroups for analysis totaling 586 patients. Results: Uterine cancer patients at MCC had an increased frequency of DACH1 mutations (12/65 patients, 18.5%) compared to the TCGA uterine cancer population (25/586 patients, 3.8%) with p-value = 1.04E-05. DACH1 mutations were associated with increased tumor mutation count in both TCGA (median 65 vs. 8972, p-value =7.35e-09) and MCC population (490 vs. 2160, p-value = 6E-04). DACH1 mutated patients had a higher tumor mutation burden compared to DACH1 wild-type (24 vs. 6.02, p-value = 4.288E-05). DACH1 mutations showed significant gene co-occurrence patterns with POLE, MLH1, and PMS2. Conclusions: DACH1 mutations are prevalent in Kentucky patients with uterine cancer. These mutations are associated with high tumor mutational burden and co-occur with genome destabilizing gene mutations. These findings suggest DACH1 could be a candidate biomarker for future trials with immunotherapy, particularly in uterine cancers, and may help explain the differences seen in uterine cancer incidence and mortality in Kentucky. Citation Format: McKayla Riggs, Nan Lin, Chi Wang, Dava Piecoro, Rachel Miller, Oliver Hampton, Mahadev Rao, Frederick Ueland, Jill Kolesar. Increased DACH1 mutation frequency in a Kentucky hospital uterine cancer population [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO009.

Cancer Signaling Transcriptome Is Upregulated in Type 2 Diabetes Mellitus

We aimed to explore the differences in the whole transcriptome of peripheral blood mononuclear cells between elderly individuals with and without type 2 diabetes (T2D). We conducted a microarray-based transcriptome analysis of 19 individuals with T2D and 15 without. Differentially expressed genes according to linear models were submitted to the Ingenuity Pathway Analysis system to conduct a functional enrichment analysis. We established that diseases, biological functions, and canonical signaling pathways were significantly associated with T2D patients when their logarithms of Benjamini–Hochberg-adjusted p-value were >1.30 and their absolute z-scores were >2.0 (≥2.0 meant “upregulation” and ≤ −2.0 “downregulation”). Cancer signaling pathways were the most upregulated ones in T2D (z-score = 2.63, −log(p-value) = 32.3; 88.5% (n = 906) of the total differentially expressed genes located in these pathways). In particular, integrin (z-score = 2.52, −log(p-value) = 2.03) and paxillin (z-score = 2.33, −log(p-value) = 1.46) signaling pathways were predicted to be upregulated, whereas the Rho guanosine diphosphate (Rho-GDP) dissociation inhibitor signaling pathway was predicted to be downregulated in T2D individuals (z-score = −2.14, −log(p-value) = 2.41). Our results suggest that, at transcriptional expression level, elderly individuals with T2D present an increased activation of signaling pathways related to neoplastic processes, T-cell activation and migration, and inflammation.

Early moderate exercise benefits myocardial infarction healing via improvement of inflammation and ventricular remodelling in rats.

Thus far, the cellular and molecular mechanisms related to early (especially within 24 hours after acute myocardial infarct (MI)) exercise‐mediated beneficial effects on MI have not yet been thoroughly established. In the present study, we demonstrated that acute MI rats that underwent early moderate exercise training beginning one day after MI showed no increase in mortality and displayed significant improvements in MI healing and ventricular remodelling, including an improvement in cardiac function, a decrease in infarct size, cardiomyocyte apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, and an increase in myocardial angiogenesis, left ventricular wall thickness and the number of cardiac telocytes in the border zone. Integrated miRNA‐mRNA profiling analysis performed by the ingenuity pathway analysis system revealed that the inhibition of the TGFB1 regulatory network, activation of leucocytes and migration of leucocytes into the infarct zone comprise the molecular mechanism underlying early moderate exercise‐mediated improvements in cardiac fibrosis and the pathological inflammatory response. The findings of the present study demonstrate that early moderate exercise training beginning one day after MI is safe and leads to significantly enhanced MI healing and ventricular remodelling. Understanding the mechanism behind the positive effects of this early training protocol will help us to further tailor suitable cardiac rehabilitation programmes for humans.

Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues.

Colorectal cancer (CRC) is one of the most common malignancies and one of the leading causes of cancer death worldwide. Establishing early detection methods or markers of CRC is central to improve the survival rate of CRC patients. Nowadays, new molecular tools have been developed to acquire further knowledge on tumor progression. Comparative proteomics analysis of Vietnamese colorectal carcinoma in different stages was performed to gain an insight into the molecular events taking place in CRC and metastasis. In this study, the comparative protein expression analysis of ten paired CRC and its corresponding noncancerous tissue samples was performed using the combination of isobaric tags for relative and absolute quantitation labeling and mass spectrometry (MS). The data obtained were further analyzed with Ingenuity Pathways Analysis (IPA) system. Based on the MS/MS spectra analyzed by ProteinPilot software, 684 proteins were identified, out of which 215 were observed to be differentially expressed in at least 1 sample pair. Individual protein expression and variation have been identified for each patient. IPA system demonstrated cytoskeletal signaling as the top-ranked functional pathway network associated with the oncogenic function. Our study supplemented the understanding about proteome of Vietnamese CRC patients and identified statistically protein expression differences among samples assisting in finding effective biomarkers for CRC diagnostics.

Gene expression profiling of spontaneously occurring canine mammary tumours: Insight into gene networks and pathways linked to cancer pathogenesis.

Spontaneously occurring canine mammary tumours (CMTs) are the most common neoplasms of unspayed female dogs leading to thrice higher mortality rates than human breast cancer. These are also attractive models for human breast cancer studies owing to clinical and molecular similarities. Thus, they are important candidates for biomarker studies and understanding cancer pathobiology. The study was designed to explore underlying molecular networks and pathways in CMTs for deciphering new prognostic factors and therapeutic targets. To gain an insight into various pathways and networks associated with the development and pathogenesis of CMTs, comparative cDNA microarray expression profiling was performed using CMT tissues and healthy mammary gland tissues. Upon analysis, 1700 and 1287 differentially expressed genes (DEGs, P ≤ 0.05) were identified in malignant and benign tissues, respectively. DEGs identified from microarray analysis were further annotated using the Ingenuity Systems Pathway Analysis (IPA) tool for detection of deregulated canonical pathways, upstream regulators, and networks associated with malignant, as well as, benign disease. Top scoring key networks in benign and malignant mammary tumours were having central nodes of VEGF and BUB1B, respectively. Cyclins & cell cycle regulation and TREM1 signalling were amongst the top activated canonical pathways in CMTs. Other cancer related significant pathways like apoptosis signalling, dendritic cell maturation, DNA recombination and repair, Wnt/β-catenin signalling, etc. were also found to be altered. Furthermore, seven proteins (ANXA2, APOCII, CDK6, GATC, GDI2, GNAQ and MYH9) highly up-regulated in malignant tissues were identified by two-dimensional gel electrophoresis (2DE) and MALDI-TOF PMF studies which were in concordance with microarray data. Thus, the study has uncovered ample number of candidate genes associated with CMTs which need to be further validated as therapeutic targets and prognostic markers.

Dan-Lou Prescription Inhibits Foam Cell Formation Induced by ox-LDL via the TLR4/NF-κB and PPARγ Signaling Pathways.

Atherosclerosis is the major worldwide cause of mortality for patients with coronary heart disease. Many traditional Chinese medicine compound prescriptions for atherosclerosis treatment have been tried in patients. Dan-Lou prescription, which is improved from Gualou-Xiebai-Banxia decoction, has been used to treat chest discomfort (coronary atherosclerosis) for approximately 2,000 years in China. Although the anti-inflammatory activities of Dan-Lou prescription have been proposed previously, the mechanism remains to be explored. Based on the interaction between inflammation and atherosclerosis, we further investigated the effect of Dan-Lou prescription on macrophage-derived foam cell formation and disclosed the underlying mechanisms. In the oxidative low-density lipoprotein (ox-LDL) induced foam cells model using murine macrophage RAW 264.7 cells, the ethanol extract from Dan-Lou prescription (EEDL) reduced ox-LDL uptake and lipid deposition by inhibiting the protein and mRNA expression of Toll-like receptor (TLR)4 and scavenger receptor (SR)B1. After stimulation with ox-LDL, the metabolic profile of macrophages was also changed, while the intervention of the EEDL mainly regulated the metabolism of isovalerylcarnitine, arachidonic acid, cholesterol, aspartic acid, arginine, lysine, L-glutamine and phosphatidylethanolamine (36:3), which participated in the regulation of the inflammatory response, lipid accumulation and cell apoptosis. In total, 27 inflammation-related gene targets were screened, and the biological mechanisms, pathways and biological functions of the EEDL on macrophage-derived foam cells were systemically analyzed by Ingenuity Pathway Analysis system (IPA). After verification, we found that EEDL alleviated ox-LDL induced macrophage foam cell formation by antagonizing the mRNA and protein over-expression of PPARγ, blocking the phosphorylation of IKKα/β, IκBα and NF-κB p65 and maintaining the expression balance between Bax and Bcl-2. In conclusion, we provided evidences that Dan-Lou prescription effectively attenuated macrophage foam cell formation via the TLR4/NF-κB and PPARγ signaling pathways.

Network analysis of microRNAs, genes and their regulation in diffuse and follicular B-cell lymphomas

MicroRNAs (miRs) are short non-coding regulatory RNAs that control gene expression at the post-transcriptional level and play an important role in cancer development and progression, acting either as oncogenes or as tumor suppressors. Identification of aberrantly expressed miRs in patients with hematological malignancies as compared to healthy individuals has suggested that these molecules may serve as novel clinical diagnostic and prognostic biomarkers.We conducted a systematic literature review of articles published between 2007 and 2017 and re-analyzed experimentally-validated human miR expression signatures in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) from various biological sources (tumor tissue, peripheral blood, bone marrow and cell lines). A unique miR expression pattern was observed for each disease. Compared to healthy individuals, 61 miRs were aberrantly expressed in DLBCL and 85 in FL; 20-30% of aberrantly expressed miRs overlapped between the two lymphoma subtypes.Analysis of integrative positive and negative miRNA-mRNA relationships using the Ingenuity Pathway Analysis (IPA) system revealed 970 miR-mRNA pairs for DLBCL and 90 for FL. Through gene ontology analysis, we found potential regulatory pathways that are deregulated in DLBCL and FL due to improper expression of miR target genes. By comparing the expression level of the aberrantly expressed miRs in DLBCL to their expression levels in other malignancies, we identified seven miRs that are aberrantly expressed in DLBCL tumor tissues (miR-15a, miR-16, miR-17, miR-106, miR-21, miR-155 and miR-34a-5p). This specific expression pattern may be a potential diagnostic tool for DLBCL.

Transcriptional profile of ethylene glycol monomethyl ether-induced testicular toxicity in rats

To clarify the molecular mechanism of ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, the potential for EGME-related changes in transcript levels of genes including spermatocyte-specific genes was evaluated in the testis of rats given single dosing of EGME at 200, 600, or 2000 mg/kg. Furthermore, the contribution of decreased testicular testosterone on EGME-induced spermatocyte toxicity was investigated by comparing to transcriptional profile due to a testosterone synthesis inhibitor, ketoconazole (KET), at 30 or 300 mg/kg. EGME at 600 mg/kg or more dose-dependently caused testicular toxicity characterized by degeneration and necrosis of spermatocytes at stage VII–XIV seminiferous tubules. The spermatocyte injury was well correlated with decreased spermatocyte-specific gene expression. Analysis of upstream regulators by the Ingenuity Pathways Analysis system suggested that up-regulation of oxidative stress, protein kinase activation, and histone acetylation was involved in EGME-induced spermatocyte toxicity. Interestingly, KET decreased testicular testosterone to a similar extent compared to the EGME treatment, but KET at up to 300 mg/kg did not show any histopathological abnormality or change in the expression of spermatocyte-specific genes. These results suggested that the decreased testicular testosterone have little impact on EGME-induced spermatocyte injury. In contrast, KET showed trends toward increases in Hsd3b2 and Hsd17b2 mRNAs, presumably resulting from inhibition of androgen synthesis. Transcriptome analysis clearly demonstrated the differential effects of EGME and KET on androgen synthesis. In conclusion, EGME caused spermatocyte toxicity correlated with decreased expression of spermatocyte-specific genes. Furthermore, oxidative stress, protein kinase activation, and histone acetylation were suggested to be involved in EGME-induced testicular toxicity.

Epigenetic changes in blood leukocytes following an omega-3 fatty acid supplementation

BackgroundOmega-3 polyunsaturated fatty acids (n-3 FAs) have several beneficial effects on cardiovascular (CV) disease risk factors. These effects on CV risk profile may be mediated by several factors, including epigenetic modifications. Our objective is to investigate, using genome-wide DNA methylation analyses, methylation changes following an n-3 FA supplementation in overweight and obese subjects and to identify specific biological pathways potentially altered by the supplementation.ResultsBlood leukocytes genome-wide DNA methylation profiles of 36 overweight and obese subjects before and after a 6-week supplementation with 3 g of n-3 FAs were compared using GenomeStudio software. After supplementation, 308 CpG sites, assigned to 231 genes, were differentially methylated (FDR-corrected Diffscore ≥│13│~ P ≤ 0.05). Using Ingenuity Pathway Analysis system, a total of 55 pathways were significantly overrepresented following supplementation. Among these pathways, 16 were related to inflammatory and immune response, lipid metabolism, type 2 diabetes, and cardiovascular signaling. Changes in methylation levels of CpG sites within AKT3, ATF1, HDAC4, and IGFBP5 were correlated with changes in plasma triglyceride and glucose levels as well as with changes in the ratio of total cholesterol/HDL-cholesterol following the supplementation.ConclusionsThese data provide key differences in blood leukocytes DNA methylation profiles of subjects following an n-3 FA supplementation, which brings new, potential insights on metabolic pathways underlying the effects of n-3 FAs on CV health.

Analysis of molecular networks and targets mining of Chinese herbal medicines on anti-aging.

BackgroundMany kidney-tonifying Chinese herbal medicines exert effects on anti-aging by comprehensive interactions of multiple targets. However, the interactions of multi-targets targeted by effective ingredients of kidney-tonifying Chinese herbal medicines are unknown. In this study, to explore the systems pharmacology mechanisms of kidney-tonifying Chinese medicines on anti-aging, we establish the molecular networks with the interactions of multi-targets, analyze bio-functions and pathways with IPA, and calculated the mutual interaction pairs of targets (target pairs) with data mining, respectively.MethodsKidney-tonifying Chinese medicines with anti-aging effects were screened from the Chinese Pharmacopoeia and the literatures. Target proteins of these herbal medicines were obtained from bioinformatics databases. Comparisons of molecular networks, bio-functions and pathways given by Ingenuity Pathway Analysis system showed the similarities and the differences between kidney Yin-tonifying herbal medicines and kidney Yang-tonifying herbal medicines. Target pairs with high correlation related to anti-aging were also discovered by data mining algorithm. And regulatory networks of targets were built based on the target pairs.ResultsTwenty-eight kidney-tonifying herbal medicines with anti-aging effects and 717 related target proteins were collected. The main bio-functions that all targets enriched in were “Cell Death and Survival”, “Free Radical Scavenging” and “Cellular Movement”, etc. The results of comparison analysis showed that kidney Yin-tonifying herbal medicines focused more on “Cancer related signaling”, “Apoptosis related signaling” and “Cardiovascular related signaling”. And kidney Yang-tonifying herbal medicines focused more on “Cellular stress and injury related signaling” and “Cellular growth, proliferation and development related signaling”. Moreover, the results of regulatory network showed that the anti-aging related target pairs with high correlated degrees of Kidney Yin-tonifying herbal medicines included TNF-PTGS2, TNF-CASP3, PTGS2-CASP3, CASP3-NOS2 and TNF-NOS2, and that of kidney Yang-tonifying herbal medicines included REAL-TNF, REAL-NFKBIA, REAL-JUN, PTGS2-SOD1 and TNF-IL6.ConclusionsIn this study, we achieved some important targets, target pairs and regulatory networks with bioinformatics and data mining, to discuss the systems pharmacology mechanisms of kidney-tonifying herbal medicines acting on anti-aging. Mutual target pairs related to anti-aging found in this study included TNF-PTGS2, TNF-CASP3, PTGS2-CASP3, CASP3-NOS2, TNF-NOS2, REAL-TNF, REAL-NFKBIA, REAL-JUN, PTGS2-SOD1 and TNF-IL6. Target pairs and regulatory networks of targets could reflect more potential interactions between targets and comprehensive effects on anti-aging. Compared with the existing researches, it was found that the kidney-tonifying herbal medicines may exert anti-aging effects in multiple pathways in this study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1513-2) contains supplementary material, which is available to authorized users.

Expression of F-actin-capping protein subunit beta, CAPZB, is associated with cell growth and motility in epithelioid sarcoma

BackgroundA previous proteomics study demonstrated the overexpression of F-actin capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.MethodsCellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 protein using INI1-overexpressing EpiS cells.ResultsAll 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression.ConclusionsCAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoprotein CAPZB in the pathogenesis of EpiS remains to be clarified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2235-z) contains supplementary material, which is available to authorized users.

P48: Integrated analysis of genomic and transcriptomic data in clear-cell renal cell carcinoma

Renal cell carcinoma (RCC) is the most widespread kidney tumor, which originates mostly from distal kidney tubules. RCC is the major mortality cause in excretory system cancer in adults, and constitutes about 80% of various kidney cancers. The 5-year survival rate is 60–70%, but lowers significantly in case of metastasis formation. The tumor is relatively resistant to chemotherapy and radiation therapy, but responds to immunotherapy. Target drugs (e.g. sunitinib, bevacizumab, α-interferon, sorafenib) are more preferable in RCC therapy. In most cases RCC is caused by obesity, smoking, arterial hypertension and hereditary factors. 4 patients of Voronezh Regional Clinical Oncology Center, aged from 50 to 75 years old were enrolled in this study. All were diagnosed with renal cell carcinoma confirmed by immunohistochemical analysis. Gene expression profiling was performed using Affymetrix GeneAtlas system with Affymetrix Hunam Gene 1.1 ST DNA- microarrays. Data moralization, statistical analysis and differentially expressed genes (DEG) list creation were performed using Partek Genomics Suite v. 6.6. DNA samples were sequenced on Ion Proton sequencer with Comprehensive Cancer Panel primer pool. Subsequent pathway analysis and biological interpretation were conducted using Ingenuity Pathway Analysis system and Ion Reporter Suite. 3528 genes were differentially expressed with expression change more than 2 times, and 351 genes changed their expression more than 5 times (p-value Only mutations causing either amino-acid substitution in corresponding protein, open reading frame shift, or truncated transcript formation were taken into account. Targeted DNA sequencing revealed 99 common mutations in normal tissues of all test subjects. 14 mutations were localized in SDHB, TRIM33, PDE4DIP, PBX1, ABL2, MTR, VHL, ROS1, PRKDC, CSMD3, MLLT10, TRIP11, PER1, genes and were tumor-specific in all patients. 33 mutations were localized in promoter regions of EGFR, PDGFRA and HNF1A genes. The most representative metabolic and signaling pathways according to Ingenuity knowledge base were “FXR/RXR Activation”, “Atherosclerosis Signaling”, “LXR/RXR Activation”, “Production of NO and ROS in Macrophages”, “Cell migration and adhesion”. Five most downregulated genes among all patients were CALB1 (−187), HPD (−133), KNG1 (−126), SLC36A2 (−126) and PAH (−122). Five most upregulated genes were TNFAIP6 (+34), ANGPT2 (+23), SERPINE1 (+22), CP (+20), HILPDA (+20). Genes with mutations and differentially expressed genes were simultaneously included in pathways analysis in order to generate gene networks with possible upstream regulators (mutated genes) included. This allowed observing a number of gene interactions not present in existing reports. This method of genomic and transcriptomic data integration allowed us to determine the sources of mRNA level variation. A number of mutations and specific DEGs discovered in this study are not registered in existing databases of annotated mutations, which allows us to propose population heterogeneity of RCC causes. These DNA mutations and mRNA level changes could be used as predictive biomarkers of renal cell carcinoma.

A genomic profile of the immune response to stroke with implications for stroke recovery.

The objectives of this study were to determine the change in gene expression between two time points following stroke and to identify biomarkers of stroke recovery through gene expression profiling and pathway analysis. Peripheral blood was collected from 34 ischemic stroke patients (confirmed by magnetic resonance imaging) ≥18 years of age, within 24 hr of symptom onset and 24-48 hr later, and from healthy controls. The Modified Rankin Scale (MRS) was used to determine 30-day recovery. Total RNA was extracted from whole blood in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients at both time points and good versus bad outcome using t-test in GeneSpring. Inflation of Type 1 error was corrected by false discovery rate (FDR), and Ingenuity Systems Pathway analysis (IPA) was performed. A secondary validation cohort was recruited from a local hospital. Three genes were significantly downregulated over time (LY96, IL8, and SDPR; FDR corrected p < .05). This finding was confirmed in a validation cohort of stroke patients (n = 8). IPA revealed cytotoxic T-lymphocyte antigen 4 (CTLA4) signaling was the most significant pathway present in the peripheral whole blood of stroke patients 24-48 hr after onset. When controlling for age and National Institutes of Health Stroke Scale score, high baseline expression of TLR2 and TLR4 significantly predicted worse scores on the MRS. CTLA4 signaling is a novel pathway for the study of stroke-induced immune suppression. Markers of immune dysfunction early after stroke may prove useful for identifying patients with increased risk of poor recovery.

Differential methylation in visceral adipose tissue of obese men discordant for metabolic disturbances

Obesity is associated with an increased risk of Type 2 diabetes and cardiovascular diseases (CVD). The severely obese population is heterogeneous regarding CVD risk profile. Our objective was to identify metabolic pathways potentially associated with development of metabolic syndrome (MetS) through an analysis of overrepresented pathways from differentially methylated genes between severely obese men with (MetS+) and without (MetS-) the MetS. Genome-wide quantitative DNA methylation analysis in VAT of severely obese men was carried out using the Infinium HumanMethylation450 BeadChip. Differences in methylation levels between MetS+ (n = 7) and MetS- (n = 7) groups were tested. Overrepresented pathways from the list of differentially methylated genes were identified and visualized with the Ingenuity Pathway Analysis system. Differential methylation analysis between MetS+ and MetS- groups identified 8,578 methylation probes (3,258 annotated genes) with significant differences in methylation levels (false discovery rate-corrected DiffScore ≥ |13| ∼ P ≤ 0.05). Pathway analysis from differentially methylated genes identified 41 overrepresented (P ≤ 0.05) pathways. The most overrepresented pathways were related to structural components of the cell membrane, inflammation and immunity and cell cycle regulation. This study provides potential targets associated with adipose tissue dysfunction and development of the MetS.

Genome-wide analysis of DNA methylation in an APP/PS1 mouse model of Alzheimer’s disease

To investigate aberrant genome-wide CpG methylation patterns in cortex brain tissue of APP/PS1 mice and as compared to controls, which allows for identification of novel disease-associated genes. This study investigates the genome-wide DNA methylation profiles of the cortex from APP/PS1 transgenic mice and control mice using the Roche NimbleGen chip platform. Functional analysis was then conducted by Ingenuity Pathways Analysis system. The methylated DNA fragments in the genome of each sample were enriched by MeDIP and the whole-genome interrogations were hybridized to the Roche NimbleGen Human DNA Methylation 3x720 K CpG Island Plus RefSeq Promoter Array that cover 15,980 CpG islands and 20,404 reference gene promoter regions of the entire human genome. Analysis reveals 2346 CpG sites representing 485 unique genes as potentially associated with AD disease status pending confirmation in additional study. At the same time, these hyper-methylated genes display familial aggregation. An impairment of the transforming growth factor-β1 (TGF-β1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease, supporting a role for epigenetic change of TGF-β1 in AD pathology. In future research, we will focus on TGF-β1, as it appeared to be the most promising candidate for AD.

IL-17 and TNF Synergistically Modulate Cytokine Expression while Suppressing Melanogenesis: Potential Relevance to Psoriasis

Inflammation-associated pigmentation changes are extremely common, but the etiology behind this clinical observation remains elusive. Particularly, it is unclear how the myriad of cytokines known to be involved in inflammatory skin processes affect epidermal melanocytes. We sought to determine how IL-17 and TNF influence normal human melanocytes, as these two cytokines have been implicated in various skin diseases. IL-17 and TNF jointly stimulated broad inductions of cytokines , including melanoma mitogens CXCL1 and IL-8. Moreover, IL-17 and TNF synergistically inhibited pigmentation-related signaling and melanin production, and induced keratinocytes production of β-defensin 3, an antagonist for melanocortin-receptor 1. When analyzing psoriasis lesions that are known to over express IL-17 and TNF, we observed an increase in melanocyte number and a simultaneous decrease in pigmentation signaling. Furthermore, therapeutic neutralization of TNF and IL-17 with mAbs results in a rapid recovery of pigment gene expression in psoriasis lesions. These results demonstrate that IL-17 and TNF can impact both the growth and pigment production of melanocytes, which may contribute to the pigmentation changes associated with psoriasis. These findings may allow the development of novel therapeutics for pigmentary disorders and bring new insights into the immune milieu surrounding melanocytes and related neoplasms.

Grading Breast Cancer Tissues Using Molecular Portraits

Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grades 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of the tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the likelihood of long-term metastasis-free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.