Browsing by Subject "Small interfering RNA"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Expression and Function of the PRL Family of Protein Tyrosine Phosphatase
    (2012-12) Dumaual, Carmen Michelle; Stauffacher, Cynthia; Randall, Stephen Karl, 1953-; Malkova, Anna; Sandusky, George Earl, 1945-
    The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.
  • Thumbnail Image
    Item
    Identification, kinetic and structural characterization of small molecule inhibitors of aldehyde dehydrogenase 3a1 (Aldh3a1) as an adjuvant therapy for reversing cancer chemo-resistance
    (2013-10) Parajuli, Bibek; Hurley, Thomas D., 1961-; Zhang, Zhong-Yin; Georgiadis, Millie M.; Zhang, Jian-Ting
    ALDH isoenzymes are known to impact the sensitivity of certain neoplastic cells toward cyclophosphamides and its analogs. Despite its bone marrow toxicity, cyclophos-phamide is still used to treat various recalcitrant forms of cancer. When activated, cyclo-phosphamide forms aldophosphamide that can spontaneously form the toxic phospho-ramide mustard, an alkylating agent unless detoxified by ALDH isozymes to the carbox-yphosphamide metabolite. Prior work has demonstrated that the ALDH1A1 and ALDH3A1 isoenzymes can convert aldophosphamide to carboxyphosphamide. This has also been verified by over expression and siRNA knockdown studies. Selective small molecule inhibitors for these ALDH isoenzymes are not currently available. We hypothe-sized that novel and selective small molecule inhibitors of ALDH3A1 would enhance cancer cells’ sensitivity toward cyclophosphamide. If successful, this approach can widen the therapeutic treatment window for cyclophosphamides; permitting lower effective dos-ing regimens with reduced toxicity. An esterase based absorbance assay was optimized in a high throughput setting and 101, 000 compounds were screened and two new selective inhibitors for ALDH3A1, which have IC50 values of 0.2 µM (CB7) and 16 µM (CB29) were discovered. These two compounds compete for aldehyde binding, which was vali-dated both by kinetic and crystallographic studies. Structure activity relationship dataset has helped us determine the basis of potency and selectivity of these compounds towards ALDH3A1 activity. Our data is further supported by mafosfamide (an analog of cyclo-phosphamide) chemosensitivity data, performed on lung adenocarcinoma (A549) and gli-oblastoma (SF767) cell lines. Overall, I have identified two compounds, which inhibit ALDH3A1’s dehydrogenase activity selectively and increases sensitization of ALDH3A1 positive cells to aldophosphamide and its analogs. This may have the potential in improving chemotherapeutic efficacy of cyclophosphamide as well as to help us understand better the role of ALDH3A1 in cells. Future work will focus on testing these compounds on other cancer cell lines that involve ALDH3A1 expression as a mode of chemoresistance.
  • Thumbnail Image
    Item
    Luminescence-Based MicroRNA Detection Methods
    (2012-08-27) Cissell, Kyle A.; Deo, Sapna K.; Long, Eric C. (Eric Charles); Simpson, Garth; Mao, Chengde
    MicroRNAs (miRNA) are short, 18-24 nucleotide long noncoding RNAs. These small RNAs, which are initially transcribed in the nucleus, are transported into the cell cytoplasm where they regulate protein translation either through direct cleavage of mRNA, or indirect inhibition through binding to mRNA and disrupting the protein translation machinery. Recently, miRNAs have gained much attention due to their implication in numerous diseases and cancers. It has been found that heightened or lowered levels of miRNA in diseased cells vs. healthy cells are linked to disease progression. It is therefore immensely important to be able to detect these small molecules. Current detection methods of Northern blotting, microarrays, and qRT-PCR suffer from drawbacks including low sensitivity, a lack of simplicity, being semi-quantitative in nature, time-consuming, and requiring expensive instruments. This work aims to develop novel miRNA technologies which will address these above problems. Bioluminescent labels are promising alternatives to current methods of miRNA detection. Bioluminescent labels are relatively small, similar in size to fluorescent proteins, and they emit very intense signals upon binding to their substrate. Bioluminescent labels are advantageous to fluorescent labels in that they do not require an external excitation source, rather, the excitation energy is supplied through a biochemical reaction. Therefore, background signal due to excitation is eliminated. They also have the advantage of being produced in large amounts through bacterial expression. Four miRNA detection methods are presented which utilize luminescence-based methods. Three employ Renilla luciferase, a bioluminescent protein, and one is based on fluorescence. The presented methods are capable of detecting miRNA from the picomole (nanomolar) level down to the femtomole (picomolar) level. These methods are rapid, sensitive, simple, and quantitative, can be employed in complex matrices, and do not require expensive instruments. All methods are hybridization-based and do not require amplification steps.
  • Thumbnail Image
    Item
    Methods of MicroRNA Promoter Prediction and Transcription Factor Mediated Regulatory Network
    (Hindawi, 2017) Zhao, Yuming; Wang, Fang; Chen, Su; Wan, Jun; Wang, Guohua; Medical and Molecular Genetics, School of Medicine
    MicroRNAs (miRNAs) are short (~22 nucleotides) noncoding RNAs and disseminated throughout the genome, either in the intergenic regions or in the intronic sequences of protein-coding genes. MiRNAs have been proved to play important roles in regulating gene expression. Hence, understanding the transcriptional mechanism of miRNA genes is a very critical step to uncover the whole regulatory network. A number of miRNA promoter prediction models have been proposed in the past decade. This review summarized several most popular miRNA promoter prediction models which used genome sequence features, or other features, for example, histone markers, RNA Pol II binding sites, and nucleosome-free regions, achieved by high-throughput sequencing data. Some databases were described as resources for miRNA promoter information. We then performed comprehensive discussion on prediction and identification of transcription factor mediated microRNA regulatory networks.
  • Thumbnail Image
    Item
    RNA Therapeutics for Retinal Diseases
    (Taylor & Francis, 2021) Gemayel, Michael C.; Bhatwadekar, Ashay D.; Ciulla, Thomas; Ophthalmology, School of Medicine
    Introduction: In the retina, noncoding RNA (ncRNA) plays an integral role in regulating apoptosis, inflammatory responses, visual perception, and photo-transduction, with altered levels reported in diseased states. Areas covered: MicroRNA (miRNA), a class of ncRNA, regulates post-transcription gene expression through the binding of complementary sites of target messenger RNA (mRNA) with resulting translational repression. Small-interfering RNA (siRNA) is a double-stranded RNA (dsRNA) that regulates gene expression, leading to selective silencing of genes through a process called RNA interference (RNAi). Another form of RNAi involves short hairpin RNA (shRNA). In age-related macular degeneration (AMD) and diabetic retinopathy (DR), miRNA has been implicated in the regulation of angiogenesis, oxidative stress, immune response, and inflammation. Expert opinion: Many RNA-based therapies in development are conveniently administered intravitreally, with the potential for pan-retinal effect. The majority of these RNA therapeutics are synthetic ncRNA's and hold promise for the treatment of AMD, DR, and inherited retinal diseases (IRDs). These RNA-based therapies include siRNA therapy with its high specificity, shRNA to 'knock down' autosomal dominant toxic gain of function-mutated genes, antisense oligonucleotides (ASOs), which can restore splicing defects, and translational read-through inducing drugs (TRIDs) to increase expression of full-length protein from genes with premature stop codons.
  • Thumbnail Image
    Item
    Role of post-transcriptional regulation in human liver
    (2015-02-11) Chaturvedi, Praneet; Janga, Sarath Chandra
    My 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.