Differential Ago-RIP-Seq for the identification of miR-375 targets in prostate cancer cells
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR950205_1.fastq.gz | Fastqsanger.gz | |||
| ERR950205_2.fastq.gz | Fastqsanger.gz | |||
| ERR950206_1.fastq.gz | Fastqsanger.gz | |||
| ERR950206_2.fastq.gz | Fastqsanger.gz | |||
| ERR950207_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
2017-01-31 | E-MTAB-3691 | biostudies-arrayexpress
Project description:Purpose: to detect expression profile of differentially expressed mRNAs during bovine mammary epithelial cells (MAC-T) transfected with miR-375 inhibitor or negative control (NC) inhibitor in vitro. Methods: bovine mammary epithelial cells were transfected with miR-375 inhibitor or negative control (NC) inhibitor to assess the expression profiles of mRNAs using RNA-seq. Results: silencing miR-375 down-regulated and upregulated the expression of 48 and 15 mRNAs, respectively, in bovine mammary epithelial cells. Conclusion: miR-375 silencing dysregulated the expression of 63 mRNAs in bMECs. Also, miR-375 silencing increased the expression of NR4A1 and PTPN5 genes, all anti-inflammatory genes, via the MAPK signaling pathway. Given the negative correlation between miR-375 expression and NR4A1 and PTPN5 genes, miR-375 potentially promotes inflammation in the mammary gland through the MAPK signaling pathway. The findings of this study provide a new perspective of treating mastitis in cows.
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Project description:We report RNA sequencing data for miR-375 knockout and YAP overexpression lung carcinoid cells (H727). Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated targets indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.
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Project description:Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, ‘RIP-Chip’ experiments enable direct analyses of miRNA targets. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3′ portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3′-untranslated region targeting, and stable AGO association versus mRNA knockdown. For detailed protocol and for full discussion of the results please see Nelson PT et al, Nucleic Acids Res. 2011 Oct;39(18):8163-72. Affymetrix microarrays which were performed 2 days following miRNA transfections: total lysate following miRNA-like transfections; and RNA from anti-Argonaute (2A8 monoclonal antibody) co-Ips
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Project description:Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, ‘RIP-Chip’ experiments enable direct analyses of miRNA targets. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3′ portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3′-untranslated region targeting, and stable AGO association versus mRNA knockdown. For detailed protocol and for full discussion of the results please see Nelson PT et al, Nucleic Acids Res. 2011 Oct;39(18):8163-72. Affymetrix microarrays which were performed 2 days following miRNA transfections: total lysate following miRNA-like transfections; and RNA from anti-Argonaute (2A8 monoclonal antibody) co-Ips RIP-ChIP studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), another physiological miRNA (miR-320), miR-15b*, and three non-physiological additional miRNA-like molecules (miR-107-mut1, mir-107-mut2, and negative control miRNA from Ambion). For details on transfected miRNA sequences, see Nelson PT et al, Nucleic Acids Res. 2011 Oct;39(18):8163-72. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. See Wang WX et al, RNA (2010) 16:394-404 for detailed protocol of RIP-ChIP.
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Project description:We attempted to identify the miR-375-3p target genes using a microarray analysis to evaluate the function and transfection efficiency of miR-375-3p mimic.
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