Identification of miR-141 as a new regulator of epidermal homeostatis, in cooperation with miR-203
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ABSTRACT:
PROVIDER: PRJNA1099215 | ENA |
REPOSITORIES: ENA
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Project description:MicroRNAs, small endogenous non-coding RNAs, are involved in the regulation of epidermal homeostasis. Among them, miR-203 was the most described and expressed in human epidermis, promoting keratinocyte differentiation by repressing genes involved in proliferation. To identify other miRNAs involved in this process, the miRNomes of normal human keratinocytes cultured in monolayer (2D) or in 3D reconstructed skin were compared. Besides miR-203, mR-141 was one of the most expressed miRNAs in 3D culture and was overexpressed in 3D vs 2D condition, i.e. during keratinocyte differentiation. Functional experiments revealed that, mostly expressed in the basal layer, miR-141 decreased keratinocyte proliferation and clonogenicity while promoting their differentiation. Target prediction algorithm coupled with transcriptomic data of keratinocytes overexpressing miR-141, as well as 3’UTR luciferase assays enabled to evidence CCND2 mRNA as a direct target of miR-141, leading to its down regulation by miR-141 overexpression. Finally, CCND2 silencing decreased keratinocyte proliferation and induced their differentiation, revealing that miR-141 action was mediated by CCND2. MiR-141 features were also compared with miR-203 in parallel experiments. Although miR-141 displayed similar functions to the ones of miR-203, it exhibited different localization and targets, suggesting a joint participation of miR-141 and miR-203 to engage and maintain keratinocyte towards differentiation, respectively.
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Project description:Identification of miR-141 as a new regulator of epidermal homeostasis, in cooperation with miR-203
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Project description:To identify putative novel specific targets of miR-141-3p, we overexpressed this miRNAs in primary keratinocytes using a synthetic mimic (pre-miR-141-3p) or a synthetic “negative” control mimic (pre-miR-ctrl). RNA samples were harvested 30 hours post-transfection and 3 independent experiments were carried out.
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Project description:We identify numerous miR-203 in vivo targets that are highly enriched for the promotion of cell cycle and cell division. Importantly, individual targets including p63, Skp2 and Msi2 play distinct roles downstream of miR-203 to regulate the cell cycle and long-term proliferation. Together, our findings reveal rapid and widespread impact of miR-203 on the self-renewal program during the epidermal differentiation and provide mechanistic insights for the potent role of miR-203 where coordinated repression of multiple targets is required for the function of this miRNA. We used microarrays to measure transcriptome changes upon miR-203's induction in mouse skin and identified new targets of miR-203. We use two pairs of biological duplicates to perform the microarray analysis from the epidermal samples harvested from K14-rtTA/TRE-miR-203/K14-H2BGFP (DP) and TRE-miR-203/K14-H2BGFP (SP) littermates at P4, 24h after the Dox injection.
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Project description:We identify numerous miR-203 in vivo targets that are highly enriched for the promotion of cell cycle and cell division. Importantly, individual targets including p63, Skp2 and Msi2 play distinct roles downstream of miR-203 to regulate the cell cycle and long-term proliferation. Together, our findings reveal rapid and widespread impact of miR-203 on the self-renewal program during the epidermal differentiation and provide mechanistic insights for the potent role of miR-203 where coordinated repression of multiple targets is required for the function of this miRNA. We used microarrays to measure transcriptome changes upon miR-203's induction in mouse skin and identified new targets of miR-203.
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Project description:The mouse incisor is a remarkable tooth that grows throughout the animalM-bM-^@M-^Ys lifetime. This continuous renewal is fueled by epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of specific miRNAs in this process. Here we describe the role of a novel Pitx2:miR-200c/141:Noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an up-regulation of miR-200c and chromatin immunoprecipitation (ChIP) assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker, amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:Noggin regulatory pathway that is important in epithelial cell differentiation and tooth development. Lower incisors of 3-5 P0 WT and Pitx2-Cre;Dicer1 cKO mices from same litter were dissected and combined for RNA extraction. Two different litters were analyzed. For mRNA microarray, CodeLink Mouse Whole Genome chips (Applied Microarrays) were used according to manufacturerM-bM-^@M-^Ys instruction (done at Genomics Core of TAMU). miRNA from P0 Lower Incisor ameloblast and cervical loops.
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