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Etude des mécanismes impliqués dans la diminution d’abondance de l’ARN messager de Microphtalmia-associated transcription factor (MITF-M) en présence d’Interleukine 1-β

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Abstract

T cells directed against tumor-associated antigens can be detected in several types of cancers, including melanoma. Cytotoxic T lymphocytes recognize antigens at the surface of tumor cells and therefore can induce the lysis of those cells. The genes coding for some of these antigens have been identified : they are differentiation genes and are expressed also in normal melanocytes. They encode the tyrosinase, the tyrosinase related proteins 1 and 2, Melan-A/MART1 or gp100/PMEL17.
However, some tumor cells reduce the expression of these antigens and then evade the anti-tumoral immunity. In melanoma cells cultivated in the presence of IL-1, several experiments have shown a decrease in the expression of these differentiation genes and of the transcription factor MITF-M (Microphtalmia-associated transcription factor), which regulates them. It can be suggested that this cytokine, present in the tumoral environment, may be responsible for the decrease in the expression of differentiation antigens and, therefore, favour the escape of tumor cells from the immune system. But, the mechanism used by IL-1 to induce this repression is still unknown. The aim of this work was to study this mechanism.
The first step was to test whether IL-1 acts on the MITF-M promoter or on the mRNA stability. We performed plasmid constructions with the MITF-M promoter (15 kb or 4,5 kb) upstream the luciferase gene and we measured the luciferase activity in the presence or not of IL-1. These experiments do seem not to implicate the promoter in the repression induced by this cytokine.
Then we have tried to evaluate whether there was an instability of the MITF-M mRNA and we expressed plasmid constructs in different tumor cell types (melanomas LB 2259 and LAGI and neuroblastoma SKNBE2) to determine the exact region of the mRNA that can be a target for the signal induced by IL-1. We perfomed constructs with the complete cDNA of MITF or only with the 3’UTR because this region is particulary long (3 kb) and can be responsible for transcript instability. However, although we could not identify the exact region implicated in this regulation, we concluded from our data that the 3’UTR alone was not capable to destabilize the transcript in the presence of IL-1ß.
LB 2259 melanoma cells were also incubated with transcription inhibitors and IL-1 to study MITF-M mRNA decay. These experiments allowed us to conclude that IL-1 does not directly influence MITF-M mRNA stability but promotes the transcription of factors responsible for the decrease in MITF mRNA levels Il a été mis en évidence dans certains types de cancer, dont les mélanomes, qu’il pouvait exister une réponse immunitaire anti-tumorale. Cette réponse est principalement médiée par des lymphocytes T cytolytiques (CTL) capables de reconnaître des antigènes à la surface des cellules de mélanome et d’induire la lyse de ces cellules. Les gènes codant certains de ces antigènes ont été identifiés : il s’agit de gènes exprimés également dans les mélanocytes normaux et qui sont impliqués dans la différenciation des mélanocytes. Ils codent la tyrosinase, les « tyrosinase related proteins », Melan-A/MART1 ou gp100/PMEL17.
Cependant, certaines cellules tumorales vont pouvoir échapper à la réponse immunitaire en réprimant l’expression de ces antigènes de différenciation. Des expériences préalables ont permis d’observer une diminution de l’expression de ces antigènes de différenciation ainsi que d’un facteur de transcription qui les régule, MITF-M (Microphtalmia-associated transcription factor), dans des mélanomes cultivés en présence d’IL-1. Ceci pourrait suggérer que cette cytokine, présente dans l’environnement de la tumeur, serait responsable de la diminution d’expression des antigènes de différenciation et donc de l’échappement à la réponse immunitaire. Cependant, le mécanisme par lequel l’IL-1ß induit cette répression est encore mal connu. Le projet de ce mémoire est d’étudier ce mécanisme.
Une première étape était de savoir si l’IL-1 agit au niveau du promoteur du gène ou au niveau de la stabilité de l’ARNm. Nous avons donc réalisé des constructions contenant le promoteur du gène MITF-M (15 kb ou 4,5 kb) en amont du gène de la luciférase et mesuré l’activité luciférase en présence ou non d’IL-1. Ces expériences ne semblent pas indiquer que le promoteur soit impliqué dans la répression induite par cette cytokine.
Ensuite, pour mettre en évidence une instabilité du transcrit MITF, nous avons réalisé différentes constructions dans plusieurs lignées cellulaires (mélanomes LB 2259 et LAGI et neuroblastome SKNBE2) afin de déterminer la région exacte du transcrit ciblée par l’IL-1ß. Nous avons réalisé des constructions contenant l’ADNc complet de MITF ou uniquement la région 3’UTR étant donné que celle-ci est particulièrement longue (3 kb) et pourrait donc réguler la stabilité du transcrit. Cependant, nous n’avons pas encore pu identifier la région précise impliquée dans cette régulation, la région 3’UTR seule n’étant pas capable d’induire une déstabilisation en présence d’IL-1.
De plus, la lignée de mélanome LB2259 a été cultivée en présence d’inhibiteurs de transcription et d’IL-1, afin d’étudier la vitesse de dégradation de l’ARNm de MITF-M dans différentes conditions. Ceci a permis de conclure que l’IL-1 n’influence pas directement la stabilité de l’ARNm de MITF-M mais favoriserait la transcription d’intermédiaires responsables de la diminution des taux d’ARNm


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Natural and Synthetic Bioactives for Skin Health, Disease and Management
Authors: --- ---
ISBN: 3036560688 303656067X Year: 2022 Publisher: Basel MDPI - Multidisciplinary Digital Publishing Institute

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Abstract

In recent years, researchers have shown a special interest in natural products as alternative methods for the prevention, treatment, and/or management of various skin illnesses and disorders, primarily due to their natural availability, efficacity, reduced cost, and minimal toxicity. In a similar vein, synthetic bioactives, as well as small molecules, have been studied in vitro, in vivo, and in clinical studies as potential drugs or supplements. This Special Issue of Nutrients comprises an array of literature reviews, original communications, and studies on the roles of natural products, synthetic bioactives, and small molecules in a variety of skin disorders and diseases, such as aging, atopic dermatitis, cutaneous T-cell lymphomas, melanoma, allergies, hyperpigmentation, inflammation, and more, which were investigated through in vitro and ex vivo approaches, preclinical trials, cell-based assays, and animal models. The mechanisms of action of certain compounds are proposed, while others are currently under investigation. The authors of the papers also explore technologies designed to improve the bioavailability and delivery of certain compounds. In lieu of ongoing research on a variety of skin disorders, the wealth of information published in this Special Issue can act as a springboard for the development of new preventive and therapeutic modalities for the management of skin diseases.

Keywords

Research & information: general --- Biology, life sciences --- keratinocytes --- rutin --- ascorbic acid --- UV radiation --- proteomics --- 3D cell culture --- borage oil --- triacylglycerol metabolism --- acyl-ceramide --- corneocyte lipid envelope --- epidermis --- anti-melanogenesis --- B16/F10 melanoma cell --- hydroxyoctadecadienoic acid --- Sorghum bicolor --- 3-isobutyl-1-methylxanthine --- mycosis fungoides --- atopic dermatitis --- cutaneous lymphomas --- cornified envelope proteins --- FLG --- microalgae --- Planktochlorella nurekis --- skin cells --- proliferation --- senescence --- holothuroids --- glycosaminoglycans --- inflammation --- ear-inflammation --- whey --- Lactobacillus helveticus --- melanin --- α-melanocyte-stimulating hormone --- tyrosinase --- tyrosinase-related protein 1 --- dopachrome tautomerase --- microphthalmia-associated transcription factor --- cosmetics --- black cumin --- Nigella sativa --- Thymocid® --- skin aging --- glycation --- collagen --- collagenase --- elastase --- melanogenesis --- cosmeceutical --- konjac glucomannan --- ultraviolet B --- human epidermal primary melanocytes --- human embryonic fibroblasts --- anti-inflammatory activity --- antioxidant activity --- Cornus officinalis --- molecular docking --- human high-affinity IgE receptors --- α-MSH --- UVB irradiation --- lotus seedpod extract --- epigallocatechin --- propolis --- skin --- matrix metalloproteinase-1 --- UV --- phosphoinositide 3-kinase --- skin care --- skin health --- bioactive substances --- phytonutrients --- antioxidants --- nutraceuticals --- Perilla frutescens --- cell proliferation --- ultraviolet radiation --- DNA repair --- Lactobacillus plantarum CJLP55 --- acne vulgaris --- sebum --- hydration --- urine bacterial extracellular vesicles --- fungal infections --- nanohydrogel --- polysaccharide --- essential oils --- eggshell membrane --- keratinocyte differentiation --- TRPV --- skin thickness --- Lithospermum erythrorhizon --- NC/Nga --- Th1 --- Th2 --- Th17 --- Th22 --- immune balance --- skin barrier function --- n/a

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