Development of miRNA expression signatures for induction of histone deacetylase inhibitor (part 2)
ABSTRACT: To clarify the relationship between histone acetylation and miRNA expression, we performed miRNA arrays to detect histone deacetylase inhibitor (OBP-801) induced miRNA regulating AR expression in 22Rv1. Agilent-031181 Unrestricted Human miRNA Microarray (Agilent Technologies, Santa Clara, California) was used according to the manufacturer’s protocols.
Project description:To clarify the relationship between histone acetylation and miRNA expression, we performed miRNA arrays to detect histone deacetylase inhibitor (OBP-801) induced miRNA regulating AR expression in LNCaP and VCaP. Agilent-041686 Unrestricted Human miRNA Microarray (Agilent Technologies, Santa Clara, California) was used according to the manufacturer’s protocols.
Project description:ETS gene fusions have been characterized in a majority of prostate cancers, however key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers. Keywords: Genetic Modification 22RV1 cells were infected with non-targeting siRNA or siRNA against SPINK1. For reported hybridizations, the reference channel is 22RV1 cells infected with non-targeting siRNA. Duplicate hybridizations were performed with duplicate dye flips, for a total of four arrays. Over and under-expressed signatures were generated by filtering to include only features with significant differential expression (PValueLogRatio < 0.01) in all hybridizations and Cy5/Cy3 ratios > or < 1 in all hybridizations.
Project description:FoxA1 has been shown critical for prostate development and prostate-specific gene expression regulation. In addition to its well-established role as an AR pioneering factor,several studies have recently revealed significant AR binding events in prostate cancer cells with FoxA1 knockdown. Furthermore, the role of FoxA1 itself in prostate cancer has not been carefully examined. Thus, it is important to understand the role of FoxA1 in prostate cancer and how it interacts with AR signaling. To address these questions, we generated engineered LNCaP cells with FoxA1 knockdown using shRNA or siRNA, 22RV1 cells with stable FoxA1 knockdown and PC3M cells with FoxA1 stable overexpression. We performed microarray analysis of these cells. We performed microarray analysis on LNCaP cells with FoxA1 knockdown using shRNA or siRNA, 22RV1 cells with stable FoxA1 knockdown and PC3M cells with FoxA1 stable overexpression
Project description:The development and progression of castrate resistant prostate cancer (CRPC), a lethal disease, is thought to be driven by multiple events. A hallmark of CRPC is the ability to evade the cytotoxic effects of anti-androgen therapy. Importantly, persistent androgen receptor (AR) signalling is thought to play a principal role in maintaining CRPC. The precise molecular alterations driving this condition, however, are not clearly understood. Our previous studies identified specific metabolic alterations associated with localized prostate cancer (PCa) and CRPC, implicating metabolic re-programming in disease progression. Building on these findings, using a novel network-based integromics approach, here we show distinct alterations in the Hexosamine Biosynthetic Pathway (HBP) to be critical for sustaining the castrate resistant state. We found expression of the HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) was regulated by androgens and elevated in androgen dependent (AD) PCa while relatively diminished in CRPC possessing either full length AR (AR-FL) or the spliced V7 variant (AR-V7). Genetic loss of function experiments for GNPNAT1 in CRPC-like cells led to increased proliferation and aggressiveness, both, in vitro and in vivo. This was mediated by specific cell cycle genes regulated by the PI3K-AKT pathway activating either AR in cells with AR-FL or SP1-ChREBP (carbohydrate response element binding protein) in cells containing AR-V7. Strikingly, addition of HBP metabolite UDP-N-acetylglucosamine (UDP) to CRPC-like cells reduced the expression of cell cycle genes and attenuated tumor cell proliferation, both in vitro and in vivo. Furthermore, addition of UDP sensitized CRPC-like cells, inclusive of those possessing AR-V7, to enzalutamide, demonstrating the therapeutic value of targeting altered metabolic pathways in lethal PCa. We anticipate that our findings will motivate the development of novel metabolic therapeutic strategies that complement existing treatments for men with lethal prostate cancer We used microarray analysis to determine key molecular alterations associated with inhibition of HBP pathway in CRPC by knocking down GNPNAT1 transcript level using lentiviral particle bearing shRNA in 22Rv1 and LNCaP-ABL cells GNPNAT1 expression was knockdown in two independent prostate cancer cells, 22Rv1 and LNCaP-ABL
Project description:MicroRNA expression profiling was performed in human BMSCs at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 7 days. RNA quantity and quality was assessed spectrophotometrically with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with Agilent RNA 6000 Nano Kit for total RNA. The latest version of Affymetrix platform for miRNA expression analysis (Genechip miRNA 2.0 array) based on mirBase version 12 (http://www.mirbase.org/) was used to obtain miRNA profiles. Normalization and statistical analysis were performed with Partek Genomic Suite 6.6 software by means of ANOVA test. Fold-change (FC) and p-values were applied to generate miRNA differentially expressed lists.
Project description:This SuperSeries is composed of the following subset Series: GSE18588: CpG islands recruit a histone H3 lysine 36 demethylase [Illumina sequencing data] GSE21201: CpG islands recruit a histone H3 lysine 36 demethylase [Agilent data] Refer to individual Series
Project description:miRNA expression was obtained from non-treated or OBP-801 treated TRAP rats which develop prostate cancer. The primary aim was to identify miRNAs which regulate prostate carcinogenesis induced by OBP-801. Overall design: Six week-old male TRAP rats were intravenously injected with 1mg of OBP-801 or saline 2 times a week. After 8 weeks, rats were sacrificed and miRNAs were extracted from ventral and lateral prostate and were run on miRNA arrray.
Project description:Background: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins. Methods: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays. Results: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production Conclusion: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs. MCF7 cells were treated with three different conditions. Treatment-1: MCF7 cells were exposed to hypoxia (0.1% O2) for 48 h and harvested for RNA extraction (n=3). Treatment-2: MCF7 cells were exposed to normoxia for 48 h and harvested for RNA extraction (n=3). Treatment-3: Dicer inhibition in MCF7 cells by transient transfection of siRNAs targeting Dicer. Cells were transfected with 20 nM siRNA duplexes (Shanghai GenePharma Co., Ltd, China), using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. A second transfection was carried out after 24 h following the same protocol. Cells were harvested 24 h after the second transfection and used for RNA extraction (n=3). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Affymetrix miRNA 3.1 Array Strip was used for RNA analysis. This array consisted probe sets unique to human mature and pre-miRNA hairpins. A detailed protocol can be found in the miRNA 3.1 Array Strips technical manual (Affymetrix). In summary, 100-300 ng of total RNA was used to synthesise double stranded cDNA using random hexamers. The cDNA was then amplified to produce antisense cRNA, which was then reverse transcribed in a second cycle of cDNA synthesis. The second cycle incorporates dUTP into the cDNA sequence, which allows it to be fragmented using uracil DNA glycosylase and apurinic/apyrimidic endonuclease I. Following biotinylation, these fragments were hybridised overnight to a Affymetrix miRNA 3.1 array. The arrays were then washed, stained using a fluorescently-labelled antibody, and scanned using a high-resolution scanner. Intensity data were analysed using Partek® software (Partek Inc.). Data were normalised by quantile normalisation and log 2 transformed. Differential expression was determined by ANOVA and corrected for false discovery.
Project description:To screen for epigenetically silenced miRNAs, wecarried out miRNA microarray analysis in three colorectal cancer (CRC) cell lines (HCT116, DLD-1 and RKO) treated with or without 5-aza-2'-deoxycytidine (aza). HCT116 and RKO cells were also treated with aza plus 4-phenylbutyric acid (PBA). In addition, we analyzed HCT116 cells in which the DNA methyltransferase genes DNMT1 and DNMT3B were genetically disrupted (double knockout; DKO cells), thereby abrogating DNA methylation. Expression of a majority of miRNAs was downregulated in all three CRC cell lines tested, as compared to normal colonic mucosa. DAC treatment upregulated expression of a large number of miRNAs in all three CRC cell lines, and combination treatment with DAC plus PBA induced even greater numbers of miRNAs in CRC cells. The most profound effect on the miRNA expression profile was induced by genetic disruption of DNMT1 and DNMT3B in HCT116 cells. CRC cells were treated with 5-aza-2’-deoxycytidine (aza) or aza plus 4-phenylbutyrate (PBA). Nomal colon RNA was purchased from BioChain. Expression of 470 miRNAs was analyzed using Human miRNA Microarray V1 (G4470A; Agilent technologies, Santa Clara, CA, USA).