Rohini Mehta

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

BackgroundGiven the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.ResultsCross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes.ConclusionsWe recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

Molecular signature of adipose tissue in patients with both Non-Alcoholic Fatty Liver Disease (NAFLD) and Polycystic Ovarian Syndrome (PCOS)

BackgroundPolycystic ovarian syndrome (PCOS) is one of the most common reproductive disorders with strong association with both insulin resistance and non-alcoholic fatty liver disease (NAFLD). To untangle the complex relationship between PCOS and NAFLD, we analyzed serum biomarkers of apoptosis, some adipokines and mRNA profiles in the visceral adipose tissue of obese patients with NAFLD who were also diagnosed with PCOS and compared to a group with NAFLD only.MethodsWe included patients with biopsy-proven NAFLD and PCOS (N = 12) and BMI-matched biopsy-proven NAFLD patients without PCOS (N = 12). Expression levels of individual mRNAs and soluble serum biomarkers were compared by non-parametric Mann–Whitney test. The analysis also included Spearman rank correlation tests and multiple regression analysis. For co-correlated genes, the factor analysis was performed.ResultsThe total serum levels of apoptotic biomarker M30 were significantly elevated in PCOS patients with liver steatosis as compared to non-PCOS NAFLD controls (P < 0.02), pointing that androgen-dependent proapoptotic PCOS environment that may directly contribute to NAFLD progression in these patients. Similarly, hyperandrogenism may explain the observed PCOS-specific decrease (P < 0.04) in adipose LDLR mRNA expression that may be connected to the proneness of PCOS patients to NAFLD. The levels of mRNA encoding angiogenesis-associated GSK-3B interacting protein ninein were also significantly increased in the adipose tissue of NAFLD patients with PCOS (P < 0.007). Furthermore, the levels of resistin positively correlated with expression levels of LDLR and prothrombin time (PT).ConclusionAn androgen-dependent proapoptotic PCOS environment may directly contribute to NAFLD progression in these patients. Hyperandrogenism may explain an observed decrease in adipose LDLR mRNA expression. An inflammation-associated increase in the release of resistin into circulation might contribute to the prothrombotic state observed under conditions associated with insulin resistance, including PCOS. The studies of larger cohorts of NAFLD with and without PCOS patients are needed to further assess these potential interactions.

Protein pathway activation associated with sustained virologic response in patients with chronic hepatitis C treated with pegylated interferon (PEG-IFN) and ribavirin (RBV).

Only half of chronic hepatitis C (CH-C) patients treated with pegylated interferon and ribavirin (PEG-IFN+RBV) achieve sustained virologic response) SVR. In addition to known factors, we postulated that activation of key protein signaling networks in the peripheral blood mononuclear cells (PBMCs) may contribute to SVR due to inherent patient-specific basal immune cell signaling architecture. In this study, we included 92 patients with CH-C. PBMCs were collected while patients were not receiving treatment and used for phosphoprotein-based network profiling. Patients received a full course of PEG-IFN+RBV with overall SVR of 55%. From PBMC, protein lysates were extracted and then used for Reverse Phase Protein Microarray (RPMA) analysis, which quantitatively measured the levels of cytokines and activation levels of 25 key protein signaling molecules involved in immune cell regulation and interferon alpha signaling. Regression models for predicting SVR were generated by stepwise bidirectional selection. Both clinical-laboratory and RPMA parameters were used as predictor variables. Model accuracies were estimated using 10-fold cross-validation. Our results show that by comparing patients who achieved SVR to those who did not, phosphorylation levels of 6 proteins [AKT(T308), JAK1(Y1022/1023), p70 S6 Kinase (S371), PKC zeta/lambda(T410/403), TYK2(Y1054/1055), ZAP-70(Y319)/Syk(Y352)] and overall levels of 6 unmodified proteins [IL2, IL10, IL4, IL5, TNF-alpha, CD5L] were significantly different (P < 0.05). For SVR, the model based on a combination of clinical and proteome parameters was developed, with an AUC = 0.914, sensitivity of 92.16%, and specificity of 85.0%. This model included the following parameters: viral genotype, previous treatment status, BMI, phosphorylated states of STAT2, AKT, LCK, and TYK2 kinases as well as steady state levels of IL4, IL5, and TNF-alpha. In conclusion, SVR could be predicted by a combination of clinical, cytokine, and protein signaling activation profiles. Signaling events elucidated in the study may shed some light into molecular mechanisms of response to anti-HCV treatment.

Expression of NALPs in adipose and the fibrotic progression of non-alcoholic fatty liver disease in obese subjects

BackgroundVisceral obesity is often accompanied by non-alcoholic fatty liver disease (NAFLD). Activation of NACHT, LRR and PYD domains-containing proteins (NALPs) may contribute to the release of pro-inflammatory cytokines by adipose and the obesity-associated progression of NAFLD to non-alcoholic steatohepatitis (NASH).MethodsWe analyzed visceral adipose expression of various NALPs and its downstream effectors caspase-1, ASC (Apoptosis-associated speck-like protein containing a CARD), IL-18 (Interleukin-18) and IL-1β (Interleukin- 1Beta) in obese subjects (BMI ≥ 35) with biopsy proven NAFLD.ResultsIn adipose samples collected from NASH and pericellular fibrosis patients cohorts, expression levels of NALPs and IL-1β were lower than that in non-NASH patients. In portal fibrosis, the levels of mRNA encoding anti-inflammatory NALP6 were upregulated. The expression levels of all NALPs were significantly co-correlated. Circulating IL-18 levels were associated with increased liver injury markers AST and ALT and portal fibrosis.ConclusionOur observations point at a possible shift in inflammation and fibrotic response from adipose tissue to liver and a possible negative feedback regulation of tissue inflammation that may instigate NAFLD severity.

Integrated computational approach to the analysis of RNA-seq data reveals new transcriptional regulators of psoriasis

Psoriasis is a common inflammatory skin disease with complex etiology and chronic progression. To provide novel insights into the regulatory molecular mechanisms of the disease, we performed RNA sequencing analysis of 14 pairs of skin samples collected from patients with psoriasis. Subsequent pathway analysis and extraction of the transcriptional regulators governing psoriasis-associated pathways was executed using a combination of the MetaCore Interactome enrichment tool and the cisExpress algorithm, followed by comparison to a set of previously described psoriasis response elements. A comparative approach allowed us to identify 42 core transcriptional regulators of the disease associated with inflammation (NFκB, IRF9, JUN, FOS, SRF), the activity of T cells in psoriatic lesions (STAT6, FOXP3, NFATC2, GATA3, TCF7, RUNX1), the hyperproliferation and migration of keratinocytes (JUN, FOS, NFIB, TFAP2A, TFAP2C) and lipid metabolism (TFAP2, RARA, VDR). In addition to the core regulators, we identified 38 transcription factors previously not associated with the disease that can clarify the pathogenesis of psoriasis. To illustrate these findings, we analyzed the regulatory role of one of the identified transcription factors (TFs), FOXA1. Using ChIP-seq and RNA-seq data, we concluded that the atypical expression of the FOXA1 TF is an important player in the disease as it inhibits the maturation of naive T cells into the (CD4+FOXA1+CD47+CD69+PD-L1(hi)FOXP3−) regulatory T cell subpopulation, therefore contributing to the development of psoriatic skin lesions.

Circulating miRNA in patients with non-alcoholic fatty liver disease and coronary artery disease

Background Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and coronary artery disease (CAD) is the cardiac manifestation of metabolic syndrome. NAFLD is strongly linked to CAD and hepatic steatosis is an independent risk factor for CAD and cardiac mortality. The pathogenic mechanism underlying this association remains poorly understood. In this study, we explored expression of circulating microRNAs (miRNAs) in patients with NAFLD and associated CAD. Results When compared to patients with NAFLD without CAD, patients with NAFLD and CAD had lower circulating levels of miR-132 (0.24±0.16 vs 0.30±0.11, p=0.03), while the circulating levels of miR-143 were higher (0.96±0.90 vs 0.64±0.77, p=0.02). The levels in circulation demonstrated trends opposite to previously observed intracellular levels in patients with CAD. In obese patients with NAFLD, lower circulating levels of miR-145 (1.42±1.00 vs 2.41±1.80), miR-211 (41.26±20.40 vs 57.56±25.45), miR-146a (2.13±1.40 vs 2.90±1.36) and miR-30c (6.92±4.99 vs 11.0±6.92) were detected when compared to lean patients with NAFLD. For miR-161 (0.59±1.19 vs 0.15±0.14) and miR-241 (0.28±0.29 vs 0.16±0.13), higher circulatory levels were detected in the obese patients with NAFLD. These observations suggest altered circulating levels of miRNAs that may serve to balance intracellular levels of miRNA in target tissues. Additional studies examining paired samples of target and producing tissues as well as respective plasma samples will help delineate the regulatory circuits governing the secretion and the uptake of miRNA in multitissue diseases.

Expression of energy metabolism related genes in the gastric tissue of obese individuals with non-alcoholic fatty liver disease

BackgroundStomach is an integral part of the energy balance regulating circuit. Studies exploring the effects of cross-system changes in the energy homeostasis in stomach tissue are scarce. The proximity of the stomach to liver - the most common secondary target affected by obesity – suggests that these two organs are exposed to each other’s local secretion. Therefore, we aimed at expression profiling of energy metabolism associated genes in the gastric tissue of obese non-alcoholic fatty liver disease (NAFLD) patients.MethodsA total of 24 patients with histologically-proven NAFLD were included. In the gastric tissue, gene expression profiling of 84 energy metabolism associated genes was carried out.ResultsThe accumulation of the fat in the liver parenchyma is accompanied by downregulation of genes encoding for carboxypeptidase E (CPE) and Interleukin 1B (IL1B) in the gastric mucosa of same patient. In patients with high grade hepatic steatosis, Interleukin 1 beta encoding gene with anorexigenic function, IL1B was downregulated. The levels expression of 21 genes, including ADRA2B, CNR1 and LEP were significantly altered in the gastric tissue of NAFLD patients with hepatic inflammation. There were also indications of an increase in the opioid signaling within gastric mucosa that may results in a shift to proinflammatory environment within this organ and contribute to systemic inflammation and the pathogenic processes in hepatic parenchyma.ConclusionsWe have shown differential expression of energy metabolism associated genes in the gastric tissue of obese NAFLD patients. Importantly, these gene expression profiles are associated with changes in the hepatic parenchyma as reflected in increased scores for hepatic steatosis, inflammation, fibrosis and NASH. This study suggests the complex interplay of multiple organs in the pathogenesis of obesity-related complications such as NAFLD and provides further evidence supporting an important role for gastric tissue in promoting obesity-related complications.

Expression of Inflammation-Related Genes Is Altered in Gastric Tissue of Patients with Advanced Stages of NAFLD

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.

Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue

Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. Conclusion: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples.

Identification of Transcriptional Regulators of Psoriasis from RNA-Seq Experiments

Psoriasis is a common inflammatory skin disease with complex etiology and chronic progression. To provide novel insights into the molecular mechanisms of regulation of the disease we performed RNA sequencing (RNA-Seq) analysis of 14 pairs of skin samples collected from psoriatic patients. Subsequent pathway analysis and an extraction of transcriptional regulators governing psoriasis-associated pathways was executed using a combination of MetaCore Interactome enrichment tool and cisExpress algorithm, and followed by comparison to a set of previously described psoriasis response elements. A comparative approach has allowed us to identify 42 core transcriptional regulators of the disease associated with inflammation (NFkB, IRF9, JUN, FOS, SRF), activity of T-cells in the psoriatic lesions (STAT6, FOXP3, NFATC2, GATA3, TCF7, RUNX1, etc.), hyperproliferation and migration of keratinocytes (JUN, FOS, NFIB, TFAP2A, TFAP2C), and lipid metabolism (TFAP2, RARA, VDR). After merging the ChIP-seq and RNA-seq data, we conclude that the atypical expression of FOXA1 transcriptional factor is an important player in psoriasis, as it inhibits maturation of naive T cells into this Treg subpopulation (CD4+FOXA1+CD47+CD69+PD-L1(hi)FOXP3-), therefore contributing to the development of psoriatic skin lesions.