Browsing by Subject "microRNA"
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Item INHIBITION OF LET-7I AS A MEANS TO ENHANCE THE NEUROPROTECTIVE EFFICACY OF PROGESTERONE IN THE ISCHEMIC BRAIN(2018-05) Nguyen, Trinh V.; Singh, Meharvan; Basu, Alakananda; Ghorpade, Anuja; Cunningham, Rebecca L.; Yang, ShaohuaThe occurrence of ischemic stroke is relatively rare among pre-menopausal women. Strikingly, this risk doubles every 10 years after the menopausal transition; and women are likely to experience worse outcomes and higher mortality post stroke than men. Since both estrogen (E2) and progesterone (P4) levels decline precipitously following the menopause, this hormonal reduction may, at least partially, contribute to the higher risk and worse outcomes. By inference, these hormones could play a critical role in protecting women against ischemic stroke. In this dissertation project, we focus on P4, the relatively understudied of the two hormones. And while P4 has been shown to be a potent neuroprotectant in various experimental models of stroke, the underlying mechanisms remain unclear. One known mediator of P4's protective function is brain-derived-neurotrophic-factor (BDNF), which has an established role in promoting neuronal differentiation, survival, and synaptogenesis. In addition, emerging literature and data from our laboratory strongly support the indispensable role of glia in P4's neuroprotective program and thus, may also play a significant role in post-stroke recovery. We recently reported that P4 induces a significant release of BDNF from primary astrocytes, through a putative membrane-associated progesterone receptor consisting of progesterone-receptor-membrane-component-1 (Pgrmc1). This receptor is abundantly expressed in various regions of brain and mediates such effects of P4 in the central nervous system (CNS) as anti-apoptotic effects, spinogenesis, and BDNF release. What is not known, however, is how the expression of this receptor is regulated. This dissertation was aimed to elucidate what regulates the expression of Pgrmc1 and BDNF in glia and how such regulation influences the neuroprotective function of P4 in the ischemic brain. Based on the observation that Let-7i regulates the expression of Pgrmc1 in a peripheral cell type, and our in silico analysis that revealed that both Pgrmc1 and BDNF are potential targets of let-7i, we hypothesized that let-7i represses P4's neuroprotective effects by down-regulating the expression of both Pgrmc1 and BDNF in glia, leading to: 1) suppression of P4-induced BDNF release from glia, and 2) attenuation of the beneficial effects of P4 on neuronal survival and markers of synaptogenesis in the ischemic brain. Using primary cortical astrocytes as an experimental model, we found that let-7i negatively regulated the expression of Pgrmc1 and BDNF. This was correlated with a reduction in P4-induced BDNF release from these cells. Under such conditions of reduced expression of both Pgrmc1 and BDNF, P4 was unable to protect primary neurons against oxygen-glucose-deprivation (OGD) or regulate markers of synaptogenesis. In our in vivo model of transient ischemic stroke, we found that protective effects of P4 were greatly enhanced in animals that were concomitantly treated with an inhibitor (antagomir) of let-7i. The combined treatment also enhanced synaptogenesis in the peri-infarct region. Collectively, the data presented here suggested that in the ischemic brain, let-7i negatively influences P4-induced neuroprotection via regulation of the Pgrmc1/BDNF axis. As such, inhibition of let-7i maybe an effective means to enhance the efficacy of P4 in treating ischemic stroke.Item miRNA Profiling of Human Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2021-05) Rangan, Rajiv S.; Tovar-Vidales, Tara; Clark, Abbot F.; Liu, YangGlaucoma is a leading cause of irreversible blindness. Vision loss results from the degeneration and death of retinal ganglion cells (RGCs) and their axons. The primary risk factor for glaucoma is increased intraocular pressure (IOP) (2). Elevated IOP results in aberrations in the biomechanical properties of ocular tissues - including the transmission of biomechanical stretch through the reticulated, fibroelastic region of the optic nerve head (ONH) known as the lamina cribrosa (LC) (6). Cells of the LC are sensitive to biomechanical stretch and respond to increased stretch and pressure to promote the excessive synthesis of extracellular matrix (ECM) proteins and ECM remodeling (15,17). These responses promote a fibrotic environment within the LC that can cause mechanical damage to the axons of RGCs. ONH astrocytes represent one of the major cell types of the LC and are believed to contribute significantly to pathological ECM remodeling at the LC during glaucoma (11). ONH astrocytes also demonstrate a dysregulated pattern of protein expression when exposed to stretch (17). The mechanism that underlies this stretch-induced, aberrant dysregulation is unknown. MicroRNA (miRNA) dysregulation may represent one of the mechanisms contributing to the differential protein expression patterns seen in ONH astrocytes exposed to stretch. In this study we examine the miRNA profiles of ONH astrocytes exposed to cyclic stretch.Item Post-transcriptional and Epigenetic Regulation of MIEN1 in Prostate Cancer(2014-08-01) Rajendiran, Smrithi; Vishwanatha, Jamboor K.; Basu, Alakananda; Berg, Rance E.Migration and invasion enhancer 1 (MIEN1), a gene located in the 17q12 region of the human chromosome, enhances migratory and invasive potential of cancer cells via two mechanisms; activating the Akt dependent NF-κB downstream signaling and facilitating filopodia formation; thereby playing an important role in cancer progression. MIEN1 is highly expressed in many cancers including prostate and breast, but its expression is very basal to null in a variety of normal tissues making it a plausible target for cancer therapy. Though the functions of MIEN1 are known, the reasons for its increased expression in cancer is unknown. Determining the molecular gene regulatory mechanisms by which expression of MIEN1 is curtailed in normal cells will help in developing better targeting strategies. Among the different gene regulatory mechanisms including transcriptional regulation, post-transcriptional modifications and histone and DNA alterations, here we focus on post transcriptional and DNA methylation based regulation of MIEN1. We show that MIEN1 is downregulated post-transcriptionally by miRNA-940 which itself is present in low amounts in cancer cells and tissues compared to the normal counterparts. The miR-940 also contributes to inhibition of cancer progression by attenuating the migration, invasion, anchorage-independent growth and epithelial-to-mesenchymal transition, when ectopically re-introduced into cancer cells. The miR-940 can be detected in circulation and its elevated levels in serum from cancer patients than normal subjects, suggest its potential as a biomarker for prostate cancer diagnosis. MIEN1, like urokinase plasminogen activator, is also suppressed by DNA methylation in normal cells. On the contrary, hypomethylation in cancer, results in its overexpression. The current approach of using global demethylating agents to activate the expression of hypermethylated tumor suppressor genes may in the long run activate tumor promoting genes like MIEN1. Thus, our study supports the notion that gene-centric hypomethylating agents may be a better epigenetic targeting approach to treat cancer. In conclusion, our data confirm the role of post-transcriptional and DNA methylation mediated mechanisms in the regulation of MIEN1.Item REGULATION OF MIEN1 IN PROSTATE CANCER(2014-03) Rajendiran, Smrithi; Parwani, Anil; Hare, Richard; Treuren, Timothy Van; Vishwanatha, JamboorMigration and Invasion ENhancer 1 (MIEN1) is a novel gene located in the 17q12 region of the human chromosome. While there is minimal expression of MIEN1 in multiple normal tissues and cells, it is abundantly elevated in many human cancers including the breast, prostate, gastrointestinal and oral. MIEN1 is a membrane bound signaling molecule that triggers downstream signaling through the AKT/NF-κB pathway (common oncogenic pathways) by up-regulating key proteases (thus aiding cancer progression). MIEN1 has also been shown to have a role in migration and invasion (key processes in cancer spread) of cancer cells by enabling filopodia formation (extensions that enable a cell to move). Thus, by various known and unknown mechanisms, MIEN1 promotes prostate cancer progression. While the cellular functions of MIEN1 have been deciphered, the reasons for its aberrant increased expression in cancer cells are still unclear. Understanding the mechanism(s) involved in the regulation of MIEN1 will aid in developing diagnostic marker(s) or in designing effective therapeutic approach(es) to treat prostate cancer patients. Purpose (a): The overall objective of this study is to identify the deregulated mechanisms leading to the differential regulation of MIEN1 between normal and cancer cells. Commonly deregulated mechanisms encompass alterations at DNA (chromosome) to destabilization at protein (translational) levels. Our study focuses on regulation by microRNA (miR) and methylation. Our hypothesis is that MIEN1 is post-transcriptionally regulated by a specific miR and its proximal putative promoter region is hypermethylated in normal cells. Deregulation of these mechanisms together explain the aberrant increased expression of MIEN1 in cancer. Methods (b): To validate the role of miR in MIEN1 regulation, we have performed various in vitro studies. To determine the global role of the miR, we ectopically expressed it in cancer cells. Additionally, we have used human tissue and serum samples to predict the use of miR-MIEN1 as biomarkers. To demonstrate the importance of methylation in the regulation of MIEN1, we performed global methylation inhibition and specific methyltransferase knockdown. Results (c): Our data indicate that MIEN1 is post-transcriptionally regulated by a specific miR which is highly expressed in normal cells compared to various cancer cells, inversely correlating with MIEN1. Ectopic expression of the miR led to decrease in MIEN1 expression, migratory and invasive potential and anchorage dependent growth of cells and impeded mesenchymal transition. Additionally, the miR expression was higher in the normal glands of prostate tissue compared to the tumor; while the secreted/circulating miR was higher in serum from cancer patients, much like PSA expression patterns; but with more significance than PSA. Inhibition of methylation by pharmacological inhibitors or by individually knocking down the methyltransferases increased MIEN1 in normal cells, indicating the role of methylation in the regulation of this gene. Conclusions (d): After proving our results in a larger cohort of patient specimen, this miR could be a useful non-invasive diagnostic biomarker. Additionally, understanding the regulation of MIEN1 by methylation will provide reasons to revisit the current strategies of methylation inhibition for cancer treatment. Overall, since the importance of MIEN1 as a key signaling molecule in cancer is well established, understanding the mechanisms involved in the regulation will aid in designing more effective therapeutic strategies to treat cancer patients.Item The role of exosomes and microRNA in Astrocyte-mediated HIV-1 Tat neurotoxicity(2016-08-01) Rahimian, Pejman; Johnny J. HeHuman immunodeficiency virus invades the central nervous system (CNS) soon after the initial infection, often leading to neurological complications including cognitive and motor dysfunction, which have been collectively termed HIV/neuroAIDS. The introduction of combination antiretroviral therapy in the mid-1990’s led to reduced viral replication, improved immune function and increased life expectancy among HIV-infected individuals. As a result, the incidence of the most severe form of cognitive impairment due to HIV, so called HIV-associated dementia, reduced dramatically. However, the treatment regimen was not successful in protecting the patients from neuroAIDS as more discrete forms of CNS dysfunction, so-called minor cognitive motor disorders, have become more common. HIV-1 Tat protein is an indispensable factor for successful transcription and replication of the viral genome. Aside from nucleus-bound functions, Tat is diffusely and unconventionally secreted outside of infected cells and contributes immensely to the pathology of neuroAIDS as a potent neurotoxin. The presence of Tat in the CNS despite the implementation of combination anti-retroviral therapy and the strong correlation of pathological hallmarks of neuroAIDS with continued Tat expression in CNS cells warrant a thorough understanding of the partially explained unconventional secretion mechanism(s) by Tat. Exosomal secretion of cargo has been established as an extremely efficient pathway of glia-neuron communications and astrocytes have been shown to utilize this delivery mechanism for the provision of neurotrophic factors and danger-associated molecular patterns to neurons. My dissertation research consisted of two parts. In the first part, we investigated the possibility of exosomal association and distribution of Tat protein from astrocytes and its delivery to neurons. We demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, astrocytoma cell lines, and HIV-infected T cells. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells. In the second part, we investigated the mechanism of neurite shortening by Tat. Dendritic pruning and synaptic loss of neurons are the most prominent pathological hallmarks of neuroAIDS in the cART era. Although Tat has been implicated in the synaptodendritic damage to neurons, the exact mechanisms of this injury by Tat have not yet been elucidated. Several important controllers of dendritic plasticity have been shown to be post-transcriptionally regulated by a brain-enriched microRNA, miR-132, which is abundantly expressed in the brains of the HIV-infected individuals with cognitive impairment. We found significant induction of miR-132 in both astrocytic and neuronal cell lines following Tat transfection. Tat expression in primary astrocytes from our doxycycline-inducible Tat transgenic mice (iTat) and HIV-infected primary human astrocytes also led to significant upregulation of this microRNA. We confirmed the repression of miR-132 target genes involved in the regulation of dendritic length following Tat expression. Using a basic-domain-deletion mutant of Tat we further demonstrated that Tat-induced miR-132 expression involved CREB phosphorylation. Lastly, we showed that following Tat expression in astrocytes, exosome-associated miR-132 was significantly increased and caused neurite shortening in primary mouse cortical neurons. Taken together, these results demonstrate for the first time the role of miR-132 in Tat-induced damage of the dendritic arbor.