Browsing by Subject "Messenger RNA"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Bottom-up, integrated -omics analysis identifies broadly dosage-sensitive genes in breast cancer samples from TCGA(PLOS, 2019-01-17) Kechavarzi, Bobak D.; Wu, Huanmei; Doman, Thompson N.; Biohealth Informatics, School of Informatics and ComputingThe massive genomic data from The Cancer Genome Atlas (TCGA), including proteomics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC), provides a unique opportunity to study cancer systematically. While most observations are made from a single type of genomics data, we apply big data analytics and systems biology approaches by simultaneously analyzing DNA amplification, mRNA and protein abundance. Using multiple genomic profiles, we have discovered widespread dosage compensation for the extensive aneuploidy observed in TCGA breast cancer samples. We do identify 11 genes that show strong correlation across all features (DNA/mRNA/protein) analogous to that of the well-known oncogene HER2 (ERBB2). These genes are generally less well-characterized regarding their role in cancer and we advocate their further study. We also discover that shRNA knockdown of these genes has an impact on cancer cell growth, suggesting a vulnerability that could be used for cancer therapy. Our study shows the advantages of systematic big data methodologies and also provides future research directions.Item Differential profiles of soluble and cellular toll like receptor (TLR)-2 and 4 in chronic periodontitis(PLOS, 2018-12-20) AlQallaf, Hawra; Hamada, Yusuke; Blanchard, Steven; Shin, Daniel; Gregory, Richard; Srinivasan, Mythily; Periodontology, School of DentistryChronic periodontitis is a common inflammatory disease initiated by a complex microbial biofilm and mediated by the host response causing destruction of the supporting tissues of the teeth. Host recognition of pathogens is mediated by toll-like receptors (TLRs) that bind conserved molecular patterns shared by large groups of microorganisms. The oral epithelial cells respond to most periodontopathic bacteria via TLR-2 and TLR-4. In addition to the membrane-associated receptors, soluble forms of TLR-2 (sTLR-2) and TLR-4 (sTLR-4) have been identified and are thought to play a regulatory role by binding microbial ligands. sTLR-2 has been shown to arise from ectodomain shedding of the extracellular domain of the membrane receptor and sTLR-4 is thought to be an alternate spliced form. Many studies have previously reported the presence of elevated numbers of viable exfoliated epithelial cells in the saliva of patients with chronic periodontitis. The objective of this study was to investigate the potential value of salivary sTLR-2 and sTLR-4 together with the paired epithelial cell-associated TLR-2/4 mRNA as diagnostic markers for chronic periodontitis. Unstimulated whole saliva was collected after obtaining informed consent from 40 individuals with either periodontitis or gingivitis. The sTLR-2 and sTLR4 in saliva was measured by enzyme-linked immunosorbent assay. The TLR-2 and TLR-4 transcript in the epithelial cells in saliva was measured by real time polymerase chain reaction. While levels of sTLR-2 exhibited an inverse correlation, sTLR-4 positively correlated with clinical parameters in the gingivitis cohort. Interestingly, both correlations were lost in the periodontitis cohort indicating a dysregulated host response. On the other hand, while the sTLR-2 and the paired epithelial cell associated TLR-2 mRNA exhibited a direct correlation (r2 = 0.62), that of sTLR4 and TLR-4 mRNA exhibited an inverse correlation (r2 = 0.53) in the periodontitis cohort. Collectively, assessments of salivary sTLR2 and sTLR4 together with the respective transcripts in the epithelial cells could provide clinically relevant markers of disease progression from gingivitis to periodontitis.Item Expression of histone deacetylase enzymes in murine and chick optic nerve(2013) Tiwari, Sarika; Belecky-Adams, Teri; Meyer, Jason S.; Marrs, James; Atkinson, SimonEpigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.Item Functional Insights Into Oncogenic Protein Tyrosine Phosphatases By Mass Spectrometry(2012-12) Walls, Chad Daniel; Zhang, Zhong-Yin; Wang, Mu; Wells, Clark D.; Zhang, Jian-TingPhosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.Item Integrated Analysis of Global mRNA and Protein Expression Data in HEK293 Cells Overexpressing PRL-1(Public Library of Science, 2013-09-03) Dumaual, Carmen M.; Steere, Boyd A.; Walls, Chad D.; Wang, Mu; Zhang, Zhong-Yin; Randall, Stephen K.; Biology, School of ScienceBackground: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-ranging cellular effects. Overexpression of PRL-1 can promote cell proliferation, survival, migration, invasion, and metastasis, but the underlying mechanisms by which it influences these processes remain poorly understood. Methodology: To increase our comprehension of PRL-1 mediated signaling events, we employed transcriptional profiling (DNA microarray) and proteomics (mass spectrometry) to perform a thorough characterization of the global molecular changes in gene expression that occur in response to stable PRL-1 overexpression in a relevant model system (HEK293). Principal findings: Overexpression of PRL-1 led to several significant changes in the mRNA and protein expression profiles of HEK293 cells. The differentially expressed gene set was highly enriched in genes involved in cytoskeletal remodeling, integrin-mediated cell-matrix adhesion, and RNA recognition and splicing. In particular, members of the Rho signaling pathway and molecules that converge on this pathway were heavily influenced by PRL-1 overexpression, supporting observations from previous studies that link PRL-1 to the Rho GTPase signaling network. In addition, several genes not previously associated with PRL-1 were found to be significantly altered by its expression. Most notable among these were Filamin A, RhoGDIα, SPARC, hnRNPH2, and PRDX2. Conclusions and significance: This systems-level approach sheds new light on the molecular networks underlying PRL-1 action and presents several novel directions for future, hypothesis-based studies.Item Lineage tracing of Ascl1-expressing cells in the maternal liver during pregnancy(2014) Nambiar, Shashank Manohar; Dai, Guoli; Stocum, David L.; Baucum II, AnthonyTo cope with the high metabolic demands of the body during pregnancy, the maternal liver adapts by increasing its mass and size. This increase is proportional to the increase in total body weight during the course of gestation. The pregnancy-induced maternal liver growth is a result of both hepatocyte hypertrophy and hyperplasia. Microarray analysis of pregnant maternal livers shows markedly different gene expression profiles when compared to a non-pregnant state. Most interesting was the 2,500-fold up-regulation in the mRNA expression of Ascl1, a transcription factor responsible for the differentiation of neural progenitor cells into various neuronal types, during the second half of pregnancy. Our investigation aimed at (1) characterizing the identity of maternal hepatic Ascl1-expressing cells and (2) tracing the fate of Ascl1-expressing cells in the maternal liver during pregnancy. Timed pregnancies were generated and non-pregnant (NP) and pregnant maternal livers were harvested and analysed. To identify the maternal hepatic Ascl1-expressing cells we used the Ascl1GFP/+ reporter mouse line. NP and gestation day 15 (D15) maternal livers were immunostained for green fluorescent protein (GFP). The result shows that GFP-positive, Ascl1-expressing cells are hepatocyte-like cells, which are present in D15 maternal livers, but absent in NP livers. The Rosa26floxstopLacZ/ floxstopLacZ;Ascl1CreERT2/+ mouse line was used to trace the fate of Ascl1-expressing cells during pregnancy. LacZ staining of gestation day 13 (D13) and 18 (D18) maternal livers demonstrates that D13 hepatic Ascl1-expressing cells (labeled with LacZ) undergo hyperplasia to repopulate a large portion of D18 maternal livers. Furthermore, LacZ and HNF4α co-staining of D13 and D18 maternal livers shows the presence of two populations of LacZ-expressing cells: HNF4α+ population and HNF4α- population. HNF4α+ LacZ-expressing cells represent hepatocyte lineage cells that are derived from Ascl1-expressing cells. We observe that, towards the end of pregnancy, a considerable portion of the maternal liver is comprised of hepatocytes derived from Ascl1-expressing cells. Taken together, our preliminary study suggests that pregnancy induces maternal liver turnover via Ascl1-expressing cells.Item miR21 deletion in osteocytes has direct and indirect effects on skeletal muscle in a sex-dimorphic manner in mice(BMC, 2022-10-01) Essex, Alyson L.; Deosthale, Padmini; Huot, Joshua R.; Davis, Hannah M.; Momeni, Nicholas; Bonetto, Andrea; Plotkin, Lilian I.; Anatomy, Cell Biology and Physiology, School of MedicineBackground: Osteocytic microRNA21 (miR21) removal alters cytokine production and bone mass by modulating osteoclast and osteoblast differentiation and activity. Removing osteocytic miR21 increases osteoclast/osteoblast numbers and bone mass in male mice, whereas it decreases osteoclasts/osteoblasts without affecting bone mass in female mice. On the other hand, it leads to sex-independent increases in bone mechanical properties. Because changes in bone remodeling and strength affect skeletal muscle through bone-muscle crosstalk, we investigated whether osteocytic miR21 deletion influences skeletal muscle. Methods: miR21fl/fl mice and 8kbDMP1-Cre mice were mated to obtain miR21-deficient mice primarily in the osteocyte (OtmiR21Δ) and littermate controls (miR21fl/fl). Four-month-old male and female mice were analyzed. Body composition was examined by DXA/Piximus and gene expression was assessed by qPCR. Ex vivo cultures of long bones devoid of bone-marrow cells from male and female 4-month-old were maintained for 48 h. Conditioned media were collected and used for the C2C12 assays. Two-way ANOVA analyses were performed to determine the contributions of genotype and sex and their interaction to the effects of miR21 deficiency. Results: Lean body mass was increased only in female OtmiR21Δ mice, although miR21 levels in soleus muscle were similar in miR21fl/fl (0.05 ± 0.02) and OtmiR21Δ (0.09 ± 0.04) mice. Female, but not male, OtmiR21Δ mice exhibited increased soleus (42%) and gastrocnemius (21%) muscle weight compared to miR21fl/fl littermates. However, muscle strength and gastrocnemius muscle fiber cross-sectional area were unaltered for either sex. Kinase phosphorylation (phospho/total protein ratio) in soleus muscle, measured as a surrogate for kinase activity by means of multiplex analysis, was also selectively changed depending on the mouse sex. Thus, female OtmiR21Δ mice had higher T185/Y187-ERK1/2 but lower S473-Akt phosphorylation than miR21fl/fl controls, while male OtmiR21Δ mice had higher S473-Akt phosphorylation, suggesting sex-dimorphic shifts in anabolic vs. catabolic signaling. Consistently, levels of FOXO3 and MuRF-1, known to be regulated by Akt, were only increased in male OtmiR21Δ mice. Atrogin-1 mRNA levels were upregulated in female OtmiR21Δ mice, suggesting a potential shift in protein regulation. Sex-specific effects were also found by exposing myotube cultures to conditioned media from 48-h-cultured marrow-flushed bones. Thus 5-day differentiated C2C12 myotubes treated with conditioned media of female OtmiR21Δ mice exhibit 12% higher average diameter compared to cells exposed to miR21fl/fl bone conditioned media. Yet, conditioned media from male bones had no effect on myotube size. Conclusions: We present a novel aspect of bone-muscle crosstalk in which osteocyte-derived miR21 influences skeletal muscle size, but not strength, in female but not male mice; whereas, intracellular signaling alterations resulting from loss of miR21 seem to alter protein dynamics in a sex-dimorphic fashion.Item mTORC1 contributes to ER stress induced cell death(2012-12) Babcock, Justin Thomas; Quilliam, Lawrence; Atkinson, Simon; Nakshatri, Harikrishna; Wek, Ronald C.Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.Item REGULATION OF CHOP TRANSLATION IN RESPONSE TO eIF2 PHOSPHORYLATION AND ITS ROLE IN CELL FATE(2012-05) Palam, Lakshmi Reddy; Wek, Ronald C.; Herring, Brian P.; Harris, Robert A. (Robert Allison), 1939-; Skalnik, David G.In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.Item Role of post-transcriptional regulation in human liver(2015-02-11) Chaturvedi, Praneet; Janga, Sarath ChandraMy thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.