Differential Ago-RIP-Seq for the identification of miR-375 targets in prostate cancer cells
ABSTRACT: The prostate cancer cell line PC-3 was transfected with pre-miR-375 or control. After 48 hours the cells were lysed and immunoprecipitation was performed using anti-panAgo (...) or isotype anti-IgG antibodies overnight. RNA of total lysates and immunoprecipitation fractions was extracted using miRNeasy kit (Qiagen) and libraries were generated using the Stranded Total RNA Sample Prep Kit (Takara Clontech). The experiment was performed in order to identify potential targets of miR-375 in prostate cancer.
Project description:miR-375 is not expression in human macrophages, however there is an enhanced accumulation of miR-375 in macrophages upon coculture with breast cancer cells. The target landscape of this miR in macrophages is not known. Ago-RIP-Seq was aimed to identify miR-375 targets in human macrophages.
Project description:Nine microRNAs (miRs) previously identified as increased in the sperm of stressed sires were microinjected into C57/Bl6:129S6/SvEvTac mouse single cell zygotes to examine the hypothesis that specific sperm miRs function postfertilization to alter offspring stress responsivity. Derived mice were exmined for hypothalamic-pituitary-adrenal axis stress response in adulthood, and the paraventricular nucleus of the hypothalamus was subsequently collected for gene expression analysis by next gen sequencing. Similar to what we reported previously in our paternal stress model, the majority of diffferenitally expressed genes in the PVN exhibited decreased expression, supporting that an increase of specific sperm miRs in the zygote can elicit long-term genetic reprogramming. Futher, marked changes in the expression of extracellular matrix and collagen gene sets suggested altered blood-brain barrier permeability with potential consequences for neuroendocrine function. mRNA profiling of paraventricular nucleus from adult male mice derived from zygote microinjection of nine miRs (multi-miR; miR-29, miR-30a, miR-30c, miR-32, miR-193-5p, miR-204, miR-375, miR-532-3p, miR-698) or PBS. Six biological replicates per group (12 total cDNA libraries) were multiplexed and sequenced on two identical HiSeq2000 lanes (Illumina).
Project description:We performed Ago HITS-CLIP to identify targets of viral and human miRNAs in latently KSHV-infected PEL cells Ago HITS-CLIP was performed in two latently infected PEL cell lines, BCBL-1 and BC-3; Argonaute-immunoprecipitation of UV cross-linked Ago-miRNA-mRNA complexes, followed by RNA isolation, library construction, and high-throughput sequencing (Illumina GAxII); we performed 3 biological replicates for each cell line, two technical (sequencing) replicates of BCBL-1 biological replicate 1
Project description:In humans, there are four Ago proteins (Ago1–4) and AGO1- and 2 were previously implicated in TGS induced by exogenous siRNAs and microRNAs (miRs) directed against gene promoter transcripts via promotion of changes in histone covalent modifications and DNA methylation. Not-with-standing, many mechanistic details of this process remain poorly defined in human cells, and very little is known about the identity of possible endogenous signals, which may drive this process in human cells. Given the evolutionary conserved role of siRNAs and AGO proteins in TGS and heterochromatin formation, we set out to analyse their possible involvement in senesence-associated repression of E2F target genes. To obtain a detailed picture of AGO-immunoprecipitating miRs (RIP) in senescent cells, we used next-generation sequencing (NGS)(RIP-Seq). We also included histone H3 dimethylated on lysine 9 (H3K9me2) in this analysis to assign potential AGO2-interacting miRs to a repressive chromatin state and unfractionated, cellular RNA from senescent cells for normalisation. Determination of AGO AGO-immunoprecipitating miRs in WI-38 senescent human fibroblast
Project description:To examine the genome-wide gene regulation due to the common seed sequence of the members of miR-302/3272/3273/520 family miRNAs and dsEcad640, we performed microarray profiling of gene expression by the transfection of chemically synthesized dsEcad640, miR-302a,miR-372, miR-373, miR-520c,and miR-520f duplexes into PC-3 cells. Chemically synthesized dsEcad640, miR-302a, miR-372, miR-373, miR-520c,and miR-520f duplexes were transfected into PC-3 cells. At 24 hours after transfection,the total RNA was extracted using the RNeasy Mini Kit (Qiagen). As a control, mock transfected cells treated with transfection reagent without siRNA and miRNA were used.Transfection was carried out four times independently, and the RNA quality was examined using Bioanalyzer (Agilent). Mixed RNA samples (150 ng total) of four experiments were used for the microarray analysis. After cDNA was synthesized with the Agilent One Color Spike Mix Kit, cRNA was prepared using the Quick Amp Labeling Kit (Agilent), labeled with Cy-3, and purified with the RNeasy Mini Kit (Qiagen).The cRNA was hybridized against the Agilent Whole Human Genome Microarray (4 × 44 K multipack format) at 65°C for 17 hours, then washed with Gene Expression Wash Buffer 1 and Wash Buffer 2 (Agilent) in 0.005% Triton X-102. The microarray slide was scanned using the DNA Microarray Scanner (Agilent) and quantified using Feature Extraction Software (Agilent).
Project description:miRNA-mediated gene expression silencing has previously been shown to be important for a variety of physiological and pathological processes. Here, we have explored the role of one bona fide human-specific miRNA (miR-941) in evolution of the human-specific expression and function. Using combination of high-throughput sequencing (GSE26545), miRNA transfection and large-scale PCR of various human populations, we have shown that emergence and rapid expansion of miR-941 might take place on the human evolutionary linage between six and one million years ago. Functionally, miR-941 could be associated with hedgehog and insulin signaling pathways, and thus might potentially play a role in evolution of human longevity. Human-specific effects of miR-941 regulation are detectable in human brain and affect genes involved in neurotransmitter signaling. Furthermore, emergence of miR-941 on the human evolutionary linage was accompanied by the accelerated loss of its binding sites. Taken together, these results strongly implicate the contribution of miR-941 in evolution of the human-specific phenotype. Ago2 Immunoprecipitation (Ago2-IP) experiments after miR-941 overexpression were conducted in 293T cell line. Briefly, All transfections were performed using human 293T cells cultured in 6-well tissue culture plates. Lipofectamine 2000 (Invitrogen) was used for a Synthetic miR-941 or a scrambled oligo transfection, at 30 nmol/l each (final concentration) per 1x106 cells/well of a 6-well plate using DharmaFECT (GE Healthcare). Total 5x106 cells were collected and subjected to Ago2 immunoprecipitation (Ago2-IP) using the RNA isolation kit Mouse Ago2 (Wako Chemicals) according to the manufacturer's instructions. For a negative control, immunoprecipitation was performed using nonimmune IgG beads prepared with the antibody immobilization bead kit (Wako Chemicals). The IP pull down RNA was used as template for an “in vitro” transcription reaction generating biotin-labeled antisense cRNA. The cRNA was analyzed on affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer’s instructions. R RMA package was used to quantify gene expression levels.
Project description:Background: Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we provide initial evidence for potential roles of AKR1C3 in PCa progression. Methods: Spatial distribution of AKR1C3 was analyzed using immunohistochemical staining in prostate adenocarcinoma tissue array. Human PCa PC-3 cells were stably transfected with AKR1C3 cDNA to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analyses were performed to identify pathways that are activated by elevated AKR1C3 expression in PCa cells. Functional confirmation of microarray and bioinformatics results was performed by immunoblot analysis and an in vitro Matrigel angiogenesis assay. Results: Elevated AKR1C3 expression was specifically limited to human prostate adenocarcinoma. Microarray and bioinformatics analysis suggested that elevated AKR1C3 expression in PC-3 cells modulates estradiol and androgen metabolism and activates insulin growth factor (IGF)-1 and Akt signaling pathways. Immunoblots confirmed that phosphorylated levels of IGF-1 receptor (IGF-1R) and Akt are significantly up-regulated in PC3-AKR1C3 as compared to mock transfectants. PC3-AKR1C3 transfectants promoted endothelial cell tube formation in Matrigel as compared to parental PC-3 cells and mock transfectants. Conclusion: Microarray and bioinformatics data followed by biological analyses suggest that elevated AKR1C3 expression in PC-3 cells promotes PCa angiogenesis and aggressiveness. These results suggest AKR1C3 can promote the aggressiveness of PCa through modulating estrogen and androgen metabolism with subsequent activation of growth factor IGF-1 and cytoplasmic Akt signaling pathways. Total RNA from mock- and ACR1C3 transfected PC-3 cells was isolated, with 2 or 3 biological replicates each. Gene expression data from AKR1C3 transfected PC-3 cells were compared with mock-transfected data.