21,033 research outputs found

    Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells

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    The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimulation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle

    Integrative multi-omics analysis for the effect of genetic alterations in cancer xenograft and organoid models

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    Department of Biomedical EngineeringDNA damage is a well-recognized factor in the development and progression of cancer. Numerous studies on genetic changes associated with cancer or the DNA repair pathway have been conducted, however, there is still a need for additional research on their function. The establishment of patient-derived xenografts or organoids for the purpose of testing functional genomic approaches is the subject of ongoing research. According to model-specific characteristics, it is not fully understood how these attempts to simulate patient cancer differ from original cancer. To comprehend the distinction between genuine patient cancer and these patient-derived disease models in more depth, multi-omics analysis is required to comprehend the overall genotypes, phenotypes, and environmental variables. Depending on the characteristics of each disease model, distinct omics analysis approaches and factors must be considered. In addition, care must be taken to avoid technical errors when integrating omics data generated by different sequencing equipment. There is currently no golden rule for data integration, but several approaches are being developed. It is crucial to determine the function of genes linked with the DNA repair pathway because these genes contribute to the induction or prevention of cancer. In chapter 1, I identified the interaction between MRE11 and TRIP13 through proximity labeling combined with the SILAC method which is quantitative proteomics using metabolic labeling. TRIP13 depletion doesn???t affect the nuclease activity and conformation of the MRN complex but directly inhibits the interaction of MDC1 with MRN complex and MDC1 recruitment on the DNA damage site. TRIP13 degradation with mirin treatment shows additive effects on ATM signaling activation. In conclusion, TRIP13 regulates immediate-early DNA damage sensing through MRE11 and ATM signaling independently of mirin. When assessing the functional genomic approach using patient-derived disease models, it is essential to determine which aspects of the models' correlation to actual cancer should be properly considered. In chapter 2, I found there are a few overlapped deleterious somatic mutations of the PDX model and their original tumor. I suspected novel mutagen exposure during PDX establishment or sample contamination. However, germline mutations of PDX models are well conserved from original tumors, and their mutational signatures of PDX also mimic that of their tumor. Though the number of overlapped mutations between the PDX model and their tumor was few, brain tumor-specific mutations are found in PDX samples. Especially, histone methylation- and cilia-related gene mutations are enriched in PDX samples. While it suggested these mutated genes are needed for maintaining the stemness of brain tumor PDX model or PDX model would be more appropriate for the samples with high heterogeneity, I have presented precautions and considerations in PDX model genome analysis. Multi-omics analysis that takes into consideration genetic, expressive, and clinical aspects can provide important information for the study of diseases with complicated etiologies, such as cancer, and can contribute to the development of diagnosis and treatment. To utilize colorectal cancer organoids for Companion Diagnostics (CDx), in chapter 3, I characterized patient-derived colorectal cancer (CRC) organoids through well-known genomic markers such as Tumor mutation burden (TMB), Microsatellite instability (MSI) and propose a novel grouping method using sharing same mutation site. The classification of CRC patients was more detailed combined with consensus molecular subtype (CMS) classifications. Additionally, I extract the expression features of the patients who experience recurrence or metastasis after first-line chemotherapy treatment with reference to clinical data. Drug response of CRC organoids by patient group and knockdown of the extracted features in the selected organoids would be validated in further study. In summary, with this dissertation, I conducted functional research on the DNA repair pathway of cancer-related genes, as well as the genetic analysis between patient-derived xenograft and original tumors, and introduced a novel perspective on the diagnosis and treatment of colorectal cancer patients using patient-derived organoids through multi-omics analysis.ope

    Norsk rÄ kumelk, en kilde til zoonotiske patogener?

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    The worldwide emerging trend of eating “natural” foods, that has not been processed, also applies for beverages. According to Norwegian legislation, all milk must be pasteurized before commercial sale but drinking milk that has not been heat-treated, is gaining increasing popularity. Scientist are warning against this trend and highlights the risk of contracting disease from milkborne microorganisms. To examine potential risks associated with drinking unpasteurized milk in Norway, milk- and environmental samples were collected from dairy farms located in south-east of Norway. The samples were analyzed for the presence of specific zoonotic pathogens; Listeria monocytogenes, Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC). Cattle are known to be healthy carriers of these pathogens, and Campylobacter spp. and STEC have a low infectious dose, meaning that infection can be established by ingesting a low number of bacterial cells. L. monocytogenes causes one of the most severe foodborne zoonotic diseases, listeriosis, that has a high fatality rate. All three pathogens have caused milk borne disease outbreaks all over the world, also in Norway. During this work, we observed that the prevalence of the three examined bacteria were high in the environment at the examined farms. In addition, 7% of the milk filters were contaminated by STEC, 13% by L. monocytogenes and 4% by Campylobacter spp. Four of the STEC isolates detected were eaepositive, which is associated with the capability to cause severe human disease. One of the eae-positive STEC isolates were collected from a milk filter, which strongly indicate that Norwegian raw milk may contain potential pathogenic STEC. To further assess the possibilities of getting ill by STEC after consuming raw milk, we examined the growth of the four eae-positive STEC isolates in raw milk at different temperatures. All four isolates seemed to have ability to multiply in raw milk at 8°C, and one isolate had significant growth after 72 hours. Incubation at 6°C seemed to reduce the number of bacteria during the first 24 hours before cell death stopped. These findings highlight the importance of stable refrigerator temperatures, preferable < 4°C, for storage of raw milk. The L. monocytogenes isolates collected during this study show genetic similarities to isolates collected from urban and rural environmental locations, but different clones were predominant in agricultural environments compared to clinical and food environments. However, the results indicate that the same clone can persist in a farm over time, and that milk can be contaminated by L. monocytogenes clones present in farm environment. Despite testing small volumes (25 mL) of milk, we were able to isolate both STEC and Campylobacter spp. directly from raw milk. A proportion of 3% of the bulk tank milk and teat milk samples were contaminated by Campylobacter spp. and one STEC was isolated from bulk tank milk. L monocytogenes was not detected in bulk tank milk, nor in teat milk samples. The agricultural evolvement during the past decades have led to larger production units and new food safety challenges. Dairy cattle production in Norway is in a current transition from tie-stall housing with conventional pipeline milking systems, to modern loose housing systems with robotic milking. The occurrence of the three pathogens in this project were higher in samples collected from farms with loose housing compared to those with tiestall housing. Pasteurization of cow’s milk is a risk reducing procedure to protect consumers from microbial pathogens and in most EU countries, commercial distribution of unpasteurized milk is legally restricted. Together, the results presented in this thesis show that the animal housing may influence the level of pathogenic bacteria in the raw milk and that ingestion of Norwegian raw cow’s milk may expose consumers to pathogenic bacteria which can cause severe disease, especially in children, elderly and in persons with underlying diseases. The results also highlight the importance of storing raw milk at low temperatures between milking and consumption.Å spise mat som er mindre prosessert og mer «naturlig» er en pĂ„gĂ„ende trend i Norge og i andre deler av verden. Interessen for Ă„ drikke melk som ikke er varmebehandlet, sĂ„kalt rĂ„ melk, er ogsĂ„ Ăžkende. I Norge er det pĂ„budt Ă„ pasteurisere melk fĂžr kommersielt salg for Ă„ beskytte forbrukeren mot sykdomsfremkallende mikroorganismer. Fagfolk advarer mot Ă„ drikke rĂ„ melk, og pĂ„peker risikoen for Ă„ bli syk av patogene bakterier som kan finnes i melken. I denne avhandlingen undersĂžker vi den potensielle risikoen det medfĂžrer Ă„ drikke upasteurisert melk fra Norge. I tillegg til Ă„ samle inn tankmelk- og speneprĂžver fra melkegĂ„rder i sĂžrĂžst Norge, samlet vi ogsĂ„ miljĂžprĂžver fra de samme gĂ„rdene for Ă„ kartlegge forekomst og for Ă„ identifisere potensielle mattrygghetsrisikoer i melkeproduksjonen. Alle prĂžvene ble analysert for de zoonotiske sykdomsfremkallende bakteriene Listeria monocytogenes, Campylobacter spp., og Shiga toksin-produserende Escherichia coli (STEC). Kyr kan vĂŠre friske smittebĂŠrere av disse bakteriene, som dermed kan etablere et reservoar pĂ„ gĂ„rdene. Bakteriene kan overfĂžres fra gĂ„rdsmiljĂžet til melkekjeden og dermed utfordre mattryggheten. Disse bakteriene har forĂ„rsaket melkebĂ„rne sykdomsutbrudd over hele verden, ogsĂ„ i Norge. Campylobacter spp. og STEC har lav infeksiĂžs dose, som vil si at man kan bli syk selv om man bare inntar et lavt antall bakterieceller. L. monocytogenes kan gi sykdommen listeriose, en av de mest alvorlige matbĂ„rne zoonotiske sykdommene vi har i den vestlige verden. Resultater fra denne oppgaven viser en hĂžy forekomst av de tre patogenene i gĂ„rdsmiljĂžet. I tillegg var 7% av melkefiltrene vi testet positive for STEC, 13% positive for L. monocytogenes og 4% positive for Campylobacter spp.. Fire av STEC isolatene bar genet for Intimin, eae, som er ansett som en viktig virulensfaktor som Ăžker sjansen for alvorlig sykdom. Ett av de eae-positive isolatene ble funnet i et melkefilter, noe som indikerer at norsk rĂ„ melk kan inneholde patogene STEC. For Ă„ videre vurdere risikoen for Ă„ bli syk av STEC fra rĂ„ melk undersĂžkte vi hvordan de fire eae-positive isolatene vokste i rĂ„ melk lagret ved forskjellige temperaturer. For alle isolatene Ăžkte antall bakterier etter lagring ved 8°C, og for et isolat var veksten signifikant. Etter lagring ved 6°C ble antallet bakterier redusert de fĂžrste 24 timene, deretter stoppet reduksjonen i antall bakterier. Disse resultatene viser hvor viktig det er Ă„ ha stabil lav lagringstemperatur for rĂ„ melk, helst < 4°C. L. monocytogenes isolatene som ble samlet inn fra melkegĂ„rdene viste genetiske likheter med isolater samlet inn fra urbane og rurale miljĂžer rundt omkring i Norge. Derimot var kloner som dominerte i landbruksmiljĂžet forskjellige fra kliniske isolater og isolater fra matproduksjonslokaler. Videre sĂ„ man at en klone kan persistere pĂ„ en gĂ„rd over tid og at melk kan kontamineres av L. monocytogenes kloner som er til stede i gĂ„rdsmiljĂžet. Til tross for smĂ„ testvolum av tankmelken (25 mL) fant vi bĂ„de STEC og Campylobacter spp. i melkeprĂžvene. 3% av tankmelkprĂžvene og speneprĂžvene var positive for Campylobacter spp. og ett STEC isolat ble funnet i tankmelk. L. monocytogenes ble ikke funnet direkte i melkeprĂžvene. Landbruket i Norge er i stadig utvikling der besetningene blir stĂžrre, men fĂŠrre. Melkebesetningene er midt i en overgang der tradisjonell oppstalling med melking pĂ„ bĂ„s byttes ut med lĂžsdriftssystemer og melkeroboter. Forekomsten av de tre patogenene funnet i denne studien var hĂžyere i besetningene med lĂžsdrift sammenliknet med besetningene som hadde melkekyrne oppstallet pĂ„ bĂ„s. Pasteurisering er et viktig forebyggende tiltak for Ă„ beskytte konsumenter fra mikrobielle patogener, og i de fleste EU-land er kommersielt salg av rĂ„ melk juridisk begrenset. Denne studien viser at oppstallingstype kan pĂ„virke nivĂ„ene av patogene bakterier i gĂ„rdsmiljĂžet og i rĂ„ melk. Inntak av rĂ„ melk kan eksponere forbruker for patogene bakterier som kan gi alvorlig sykdom, spesielt hos barn, eldre og personer med underliggende sykdommer. Resultatene underbygger viktigheten av Ă„ pasteurisere melk for Ă„ sikre mattryggheten, og at det er avgjĂžrende Ă„ lagre rĂ„ melk ved kontinuerlig lave temperaturer for Ă„ forebygge vekst av zoonotiske patogener

    Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization

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    The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system

    In vitro investigation of the effect of disulfiram on hypoxia induced NFÎșB, epithelial to mesenchymal transition and cancer stem cells in glioblastoma cell lines

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Glioblastoma multiforme (GBM) is one of the most aggressive and lethal cancers with a poor prognosis. Advances in the treatment of GBM are limited due to several resistance mechanisms and limited drug delivery into the central nervous system (CNS) compartment by the blood-brain barrier (BBB) and by actions of the normal brain to counteract tumour-targeting medications. Hypoxia is common in malignant brain tumours such as GBM and plays a significant role in tumour pathobiology. It is widely accepted that hypoxia is a major driver of GBM malignancy. Although it has been confirmed that hypoxia induces GBM stem-like-cells (GSCs), which are highly invasive and resistant to all chemotherapeutic agents, the detailed molecular pathways linking hypoxia, GSC traits and chemoresistance remain obscure. Evidence shows that hypoxia induces cancer stem cell phenotypes via epithelial-to-mesenchymal transition (EMT), promoting therapeutic resistance in most cancers, including GBM. This study demonstrated that spheroid cultured GBM cells consist of a large population of hypoxic cells with CSC and EMT characteristics. GSCs are chemo-resistant and displayed increased levels of HIFs and NFÎșB activity. Similarly, the hypoxia cultured GBM cells manifested GSC traits, chemoresistance and invasiveness. These results suggest that hypoxia is responsible for GBM stemness, chemoresistance and invasiveness. GBM cells transfected with nuclear factor kappa B-p65 (NFÎșB-p65) subunit exhibited CSC and EMT markers indicating the essential role of NFÎșB in maintaining GSC phenotypes. The study also highlighted the significance of NFÎșB in driving chemoresistance, invasiveness, and the potential role of NFÎșB as the central regulator of hypoxia-induced stemness in GBM cells. GSC population has the ability of self-renewal, cancer initiation and development of secondary heterogeneous cancer. The very poor prognosis of GBM could largely be attributed to the existence of GSCs, which promote tumour propagation, maintenance, radio- and chemoresistance and local infiltration. In this study, we used Disulfiram (DS), a drug used for more than 65 years in alcoholism clinics, in combination with copper (Cu) to target the NFÎșB pathway, reverse chemoresistance and block invasion in GSCs. The obtained results showed that DS/Cu is highly cytotoxic to GBM cells and completely eradicated the resistant CSC population at low dose levels in vitro. DS/Cu inhibited the migration and invasion of hypoxia-induced CSC and EMT like GBM cells at low nanomolar concentrations. DS is an FDA approved drug with low toxicity to normal tissues and can pass through the BBB. Further research may lead to the quick translation of DS into cancer clinics and provide new therapeutic options to improve treatment outcomes in GBM patients

    Characterising the role of the Amyloid Precursor Protein and Glucagon-like peptide-1 analogues in Age-Related Macular Degeneration

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    Age-related macular degeneration (AMD) is a progressive retinal neurodegenerative disorder characterised, in some forms of the disease, by the loss of photoreceptors and the underlying retinal pigment epithelium (RPE) in the macula due to the accumulation of extracellular deposits known as “drusen”. A major component of drusen deposits is the Alzheimer’s disease (AD)-related amyloid beta (AÎČ)-peptide, a 4kDa peptide derived from the larger amyloid precursor protein (APP) through sequential cleavage by enzymes known as ÎČ- and Îł-secretases. Alternatively, in the ‘non-amyloidogenic’ pathway, APP can be processed by a third enzyme, α-secretase, which cleaves within the AÎČ region of the protein thereby preventing the production of toxic peptides as well as producing a larger soluble fragment, sAPPα, known for its neuroprotective and neurotrophic properties. The current project aims to characterise the role played by APP and its proteolytic fragments in AMD using human retinal pigment epithelial cells (ARPE-19) and UV-A light (a known AMD risk factor) as the stressor. In addition, a group of diabetes drugs known as Glucagon Like Peptide-1 (GLP-1) analogues that have previously been purported to reduce neuronal death in AD and Parkinson’s Disease (PD) have been tested for their ability to protect ARPE-19 cells against stress-inducing reagents relative to AMD (UV-A light, hydrogen peroxide and AÎČ-peptides). The results of the current study demonstrate that endogenous cell-associated full-length APP expression was depleted in ARPE-19 cells following UV-A irradiation. Furthermore, ÎČ-secretase but not α-secretase processing of the protein was reduced. Small interfering RNA-mediated depletion of endogenous APP or Îł-secretase (but not α- or ÎČ-secretase) inhibition ablated the detrimental effect of UV-A on cell viability. In contrast, α-secretase and, possibly, Îł-secretase but not ÎČ-secretase activity appeared to promote the longer-term proliferation of ARPE-19 cells in the absence of UV-A irradiation. Furthermore, two of the GLP-1 analogues tested, liraglutide and lixisenatide, were able to restore cell viability after UV-A exposure. Collectively, these data indicate clear links between the expression/proteolysis of APP and the proliferation and resistance of ARPE-19 cells to UV-A irradiation. Whilst these effects are clearly differential, the data warrant further investigation of the role played by APP in AMD. Furthermore, the protective effects against UV-A shown by liraglutide and lixisenatide warrant further investigation of the molecular mechanisms involved with a view to identifying new drug targets for the prevention or treatment of retinal neurodegenerative diseases such as AMD

    Quantification of the Tumor immune Stroma (QTiS) and the metabolic checkpoint molecules in pancreatic cancer: comparison between primary and metastatic tumor

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    Pancreatic cancer (PC) remains one of the most lethal cancers in the world. Currently, surgical resection is still the most effective treatment for PC, yet it only works for a few early-stage patients. Although multiple efforts have been ongoing to treat metastatic pancreatic cancer, the outcome remains unsatisfactory. Previous studies have shown that immune imbalance within the tumor microenvironment (TME) promotes tumor progression. Furthermore, glucose metabolism is essential to providing energy for tumor growth, progression, and distant metastasis. There have been studies on tumor-infiltrating leukocytes (TILs) and energy metabolism in the TME of pancreatic cancer, while research on metastatic PC is unavailable because of the surgical treatment of metastatic pancreatic cancer. We were fortunate to have this opportunity to study 26 cases of metastatic PC in our institution. Quantification of the tumor immune stroma (QTiS) algorithm was used to quantify seven markers after immunohistochemical staining, including four markers of leukocytes (CD3+, CD8+, CD20+, and CD66b+) and three markers of metabolic checkpoint molecules (HIF-1α, GLUT1, PDHK1). Afterward, differences in tumor-infiltrating leukocytes (TILs) and metabolic checkpoint molecules (MCMs) between primary and metastatic lesions of metastatic pancreatic cancer were analyzed. Furthermore, the correlation between seven staining markers and clinical data, including overall survival (OS) and disease-free survival (DFS), was also analyzed. The results showed that the infiltration of CD3+, CD8+, and CD20+ in PC primary tumors was higher than that in metastatic tumors. High inïŹltration of CD20+ TILs (p=0.013) in primary tumors of PC correlates with improved overall survival, and high inïŹltration of CD8+ (p=0.023) in metastatic tumors of PC correlates with improved overall survival. Low level of platelets in blood circulation system associated with improved OS. The density of HIF-1α and PDHK1 in tumor cell area was higher than that in tumor stroma areas of primary and metastatic tumors. Low-density GLUT1 in tumor stroma areas of primary tumors (p=0.009) and metastatic tumors (p=0.01) of PC correlates with improved OS. Notably, in multivariate analysis, CD8+TILs (HR 0.196, 95% CI 0.044-0.872, p=0.032) in metastatic tumors of PC is an independent prognostic factor; and GLUT1 in tumor stromal areas of primary (HR 5.816, 95% CI 1.006-33.624, p=0.049) and metastatic (HR 5.056, 95% CI 1.258-20.324, p=0.022) tumors is independent prognostic factor to metastatic pancreatic cancer. Overall, the present study used the QTiS algorithm to quantify stroma tumor-infiltrating leukocytes in metastatic PC. We extended this method to quantify metabolic checkpoint molecules in tumor cell and stromal areas, efficiently analyzing IHC staining images. Furthermore, we depicted the characters and differences of TILs and MCMs between primary and metastatic lesions of metastatic PC and the correlation between TILs and MCMs with OS and DFS. Our work can contribute to a better understanding of the immune subtypes and energy metabolism in metastatic PC, which could be vital in improving the diagnosis, prognosis, and treatment of advanced PC

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
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