MiR-146a effect in IFNg and TNFa induced keratinocytes
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ABSTRACT: The aim of the study was to describe the function of miR-146a in human skin keratinocytes in relation to chronic skin diseases. miR-146a precursor and the control were transfected into human primary keratinocytes treated with IFN-gamma, TNF-alpha or left untreated. mRNA expression profiles of each conditions were detected.
Project description:miR-10a has been shown to regulate proliferation and invasiveness of cancer cells and inflammatory responses of endothelial cells. The function of miR-10a in the skin has not been studied before. Here we examined miR-10a expression, regulation and functions in keratinocytes (KCs) in association with atopic dermatitis (AD).
Project description:MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, the function and molecular mechanism of miR-21 in cervical squamous carcinoma has not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the nodal status and differentiation by ISH. Furthermore, we demonstrated that miR-21 regulates cervical squamous cells proliferation, apoptosis, and migration which are HPV16 positive. In order to identify the candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assay, we confirmed the CCL20 gene is one of its targets, which is relative to the HPV16 oncogenes E6 and E7. Our results suggest that miR-21 may be involved in the cervical squamous cell tumorigenesis. Total RNA of cells transfected with anti-miR-21 or scrambled RNA oligonucleotide was extracted using the TRIZOL Reagent according to the manufacturer's instructions. Gene-expression profiling was performed for each pooling RNA sample separately on the GeneChip_ Porcine Genome Array (Affymetrix) at CapitalBio Corporation (Beijing, China) in which GeneChip microarray service was certificated by Affymetrix.
Project description:Mice with deficient expression of RASSF9 exhibit intriguing phenotypes of skin-related pathology, including abnormal thickening of the epidermis, dysregulated proliferation of keratinocytes, and alopecia. To delineate the underlying mechanism, we profiled gene expression in keratinocytes of RASSF9-mutant mice to identify targets whose expressions were affected by RASSF9 gene deletion. Primary keratinocytes from neonatal ICR mice of wildtype control (WT) or homozygous RASSF9 deletion (RASSF9-/-) were harvested for RNA extraction and hybridization on Affymetrix microarrays. For WT, a single microarray hybridization was performed. For RASSF9-/-, one hybridization was performed for each of two independent samples.
Project description:Transcription profiling of the neuroblastoma cell line Kelly following transient transfection with Pre-miR-335 and negative controls to identify potential miR-335 target genes.
Project description:In the epidermis, keratinocytes are involved in physical and first-line immune protection of the host. In this work, we analyzed molecular responses after the addition of contact sensitizers, 2,4-dinitrochlorobenzene (DNCB) using cultured human keratinocytes from the bulge region of a plucked hair follicle (bulge-derived keratinocytes; BDKs) in comparison with neonatal human epidermal keratinocytes NHEK and human monocytic leukemia THP-1. The changes in gene expression by the treatment of DNCB in BDKs were different from those in THP-1. Many genes orchestrating keratinocyte differentiation, which interact TGF-β and BMP signaling pathway, were significantly up-regulated in response to DNCB. Keywords: Comparison analysis of gene expression changes by the treatment of DNCB three cell strains composed of four samples; two BDKs established from different donors, NHEK, and THP-1. One replicate per array.
Project description:The transcriptome analysis of several cancer cell lines transfected by a panel of miRNA, to reveal perturbation on the transcripts regulated by each miRNA transfection.
Project description:Analysis of keratinocyte differentiation in presence or absence of fibroblast-derived JNK-dependent soluble factors in vitro. The hypothesis tested in the present study was that JNK-dependent fibroblast-derived soluble factors promote an efficient keratinocyte differentiation. Results provide important information about reduced expression of a subset of differentiation associated genes in keratinocytes in the absence of JNK-dependent fibroblast-derived soluble factors. In vitro transwell co-culture experiments were performed using jnk1-/-jnk2-/- or wildtype immortalized mouse embryonic fibroblasts (MEFs) and differentiating primary normal human epidermal keratinocytes (NHEK) over a time course of 6 days. Total RNA was obtained from NHEK prior to cultivation with MEFs (day 0) and every second day (days 2, 4, and 6) during co-culture. MEFs have been described previously in Sabapathy et al. (2004) Mol Cell 15:713-25.
Project description:The interaction between the transmembrane glycoprotein surface receptor CD40 expressed by skin keratinocytes (KCs) and its T cell-expressed ligand CD154 was suggested to exacerbate inflammatory skin diseases. However, the full spectrum of CD40-mediated effects by KCs underlying this observation is unknown. Therefore, changes in gene expression after CD40 ligation of KCs were studied by microarrays. CD40-mediated activation for 2 hours stimulated the expression of a coordinated network of immune-involved genes strongly interconnected by IL8 and TNF, while after 24 hours anti-proliferative and anti-apoptotic genes were upregulated. CD40 ligation stimulated the production of chemokines that attracted lymphocytes and myeloid cells from peripheral blood mononuclear cells (PBMCs). Thus, CD40-mediated activation of KCs resulted in a highly coordinated response of genes required for the local development and sustainment of adaptive immune responses. The importance of this process was confirmed by a study on the effects of human papilloma virus (HPV) infection to the KCâs response to CD40 ligation. HPV infection clearly attenuated the magnitude of the response to CD40 ligation and consequently the KCâs capacity to attract PBMCs. The fact that HPV attenuates CD40 signalling in KCs indicates the importance of the CD40-CD154 immune pathway in boosting cellular immunity within epithelia. Total RNA from eight 72 hours 50 IU/ml IFNgamma pre-stimulated primary undifferentiated keratinocyte cultures, four uninfected and four HPV-positive, that were either left unstimulated, or stimulated for 2 hours with IFNgamma and Control- or CD40L-expressing L-cells (mouse fibroblasts), or stimulated for 24 hours with IFNgamma and Control- or CD40L-expressing L-cells. The stimulated samples were generated in duplo. 72 samples were generated in total.