Project description:Human vein umbilical endothelial cells (HUVEC) were transfected with pre-miR control and pre-miR 125b (Ambion) in order to identify targets (direct and indirect) downregulated by miR-125b in endothelial cells. 317 transcripts were downregulated with a fold change ≥ 1.5, among which 99 are predicted direct targets of mir-125b. Computional approach performed with Ingenuity Pathway Knowledge shows that movement of cell and cell-to-cell signalling are the top two biological functions affected bewteen control and transfected cells. Experiments performed in zebrafish and in HUVEC confirmed that mir-125b mainly affects genes involved in cellular movements and junctions. Total RNA was obtained from cells transfected in HUVEC with pre-miR control and pre-miR 125b. Three independent transfections were performed for each condition.
Project description:Human vein umbilical endothelial cells (HUVEC) were transfected with pre-miR control and pre-miR 125b (Ambion) in order to identify targets (direct and indirect) downregulated by miR-125b in endothelial cells. 317 transcripts were downregulated with a fold change ≥ 1.5, among which 99 are predicted direct targets of mir-125b. Computional approach performed with Ingenuity Pathway Knowledge shows that movement of cell and cell-to-cell signalling are the top two biological functions affected bewteen control and transfected cells. Experiments performed in zebrafish and in HUVEC confirmed that mir-125b mainly affects genes involved in cellular movements and junctions.
Project description:We report here the expression profile of microRNAs in human neuronal differentiation in the neuroblastoma cell line SH-SY5Y. Six microRNAs were significantly upregulated during differentiation induced by all-trans¬-retinoic acid and brain-derived neurotrophic factor. We demonstrated that ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both spontaneous and induced differentiation of SH-SH5Y cells, based on neurite outgrowth and neuronal marker expression. In human neural progenitor ReNcell VM cells, miR-125b is also upregulated during differentiation and miR-125b ectopic expression significantly promotes neurite outgrowth. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the microRNA and/or predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the microRNA response elements of ten selected targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation. Keywords: mir125-OE/mir-scrambled control comparison & mir125-KD/mir-scrambled control comparsion
Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global miRNA expression profile of P4 stage hair follicle ORS cells from DTG (K14-rtTA,TRE-miR-125b) and control littermates. Hair follicle cells were isolated from P4 Backskin (Dox since P3 for 24hrs) of DTG (K14-rtTA/TRE-miR-125b/K14-H2BGFP), TRE (TRE-miR-125b/K14-H2BGFP), KrtA (K14-rtTA/K14-H2BGFP) as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment.The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. During FACS, cells were first gated against CD34 (endothelial cells), CD45 (immune cells), CD114 (melanocytes) and DAPI (dead cells). ORS cells were sorted from the remaining cells as α6HiGFPHi.
Project description:Transcriptional profiling of breast cancer cells comparing pre-control transfected cells with cells transfected with pre-miR-125b. We searched for miR-125b targets by systematic screening of mRNA profiling of pre-miR-125b transfected MCF-7 cells and MDA-MB-435 cells. Two-condition experiment, pre-miR-125b Transfected vs. pre-control Transfected MCF-7 cells. One replicate per array.
Project description:We demonstrate that miR-125b, a key node in this microRNA regulatory network, is upregulated in gastric cancer (GC) and associated with poor overall survival through an integrated analysis of microRNA and mRNA profiling of GC revealed a mRNA-regulatory network.So we have employed whole genome microarray expression profiling as a discovery platform to compare the transcriptome profiling of human gastric cells (MKN-45) after 48 hours post-transfection of miR-125b mimic (50nM) and mimic control.Pathway analysis shows that the predicted targets of miR-125b are highly involved in apoptosis/program death pathway,and the robust apoptosis genes, BIK and CASP6 are validated as the directed targets of miR-125b.
Project description:Transcriptional profiling of breast cancer cells comparing pre-control transfected cells with cells transfected with pre-miR-125b. We searched for miR-125b targets by systematic screening of mRNA profiling of pre-miR-125b transfected MCF-7 cells and MDA-MB-435 cells.
Project description:The goal of this study is to define miR-125b-2 target genes in the hematopoietic system by genetic alteration of miR-125b expression levels. Here we report the identification of miR-125b-2 targets in the hematopoietic system by repressing miR125b in megakaryoblastic leukemia (AMKL) cell lines and overexpressing miR-125b-2 in hematopoietic stem and progenitor cells (CD34+-HSPCs)