Comment[ArrayExpressAccession] E-GEOD-44089 MAGE-TAB Version 1.1 Public Release Date 2014-02-05 Investigation Title MicroRNA expression changes associated with specific STAT3 activation Comment[Submitted Name] MicroRNA expression changes associated with specific STAT3 activation Experiment Description Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor in cancer. However, while the protein-coding target genes of STAT3 have been extensively studied, the microRNA target genes of STAT3 are less understood. MicroRNAs are short, non-coding RNAs that regulate messenger RNAs through translational inhibition and transcript degradation. They have been found to be involved in all aspects of cancer biology. Given the roles of both STAT3 and miRNAs in cancer, the function of STAT3 as a transcription factor, and the dearth of known STAT3 miRNA targets, our goal was to identify novel STAT3 miRNA targets relevant to cancer. To do so, we engineered MCF-10A cells with doxycycline-inducible expression of STAT3C. STAT3C is a constitutively-active mutant version of STAT3. Although STAT3 can be activated by various growth factors and kinases, other pathways can be activated as well, which would confound analysis of the results. Thus, the advantage of STAT3C is that it allowed specific and focused activation of STAT3 alone, and this screen represents the first genome-wide survey of miRNA expression changes associated specifically with STAT3 activity. MCF-10A, a non-transformed breast epithelial cell line, was chosen because STAT3C has been reported to be sufficient to cause their neoplastic transformation. Therefore, we reasoned that analysis of STAT3C’s effects in MCF-10A cells would be especially informative for STAT3-regulated miRNAs relevant to cancer. As a result of our study, we identified previously-known as well as novel miRNA targets of STAT3. Doxycycline-inducible MCF-10A cells were seeded, and then untreated (grown in standard growth media alone) or treated with 2μg/ml doxycycline for 48hr. Each condition was performed in biological triplicate (labeled A, B, and C), for a total of 6 samples. Total RNA was harvested and submitted to the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory. MicroRNA expression profiling was performed using TaqMan Low-Density Arrays (TLDA), human miRNA version 2.0A and version 3.0B cards (Applied Biosystems). Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Xiang Xiang Frank Person First Name Michael Michael David Person Mid Initials A Person Email geo@ncbi.nlm.nih.gov Person Affiliation Dana-Farber Cancer Institute Person Address Medical Oncology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA, USA Person Roles submitter Protocol Name P-GSE44089-2 P-GSE44089-1 P-GSE44089-4 P-GSE44089-5 P-GSE44089-3 P-GSE44089-6 Protocol Description Non-normalized data consist of raw Ct values that were reported for each miRNA assayed. Ct values above 30 are considered unreliable, according to the experience of the Molecular Diagnostics Laboratory, due to high intra-sample variability in this regime. Samples that did not show any amplification up to 40 cycles were labeled “Undetermined.” Normalized data were generated by subtracting the average Ct values of the housekeeping genes RNU44 and RNU48 in the sample from the Ct value of each miRNA. Fold-change data were calculated using the delta-delta Ct value method and are shown relative to untreated sample replicate A. Normalized data report miRNA Ct values normalized against housekeeping genes (average Ct values of RNU44 and RNU48 for a particular sample). Fold-change data are relative to sample 1 (untreated condition, replicate A). ID_REF = VALUE = Normalized Non-normalized data consist of raw Ct values that were reported for each miRNA assayed. Ct values above 30 are considered unreliable, according to the experience of the Molecular Diagnostics Laboratory, due to high intra-sample variability in this regime. Samples that did not show any amplification up to 40 cycles were labeled 'Undetermined'. Normalized data were generated by subtracting the average Ct values of the housekeeping genes RNU44 and RNU48 in the sample from the Ct value of each miRNA. Fold-change data were calculated using the delta-delta Ct value method and are shown relative to untreated sample replicate A. Normalized data report miRNA Ct values normalized against housekeeping genes (average Ct values of RNU44 and RNU48 for a particular sample). Fold-change data are relative to sample 1 (untreated condition, replicate A). ID_REF = VALUE = normalized Generation of cDNA and TaqMan qRT-PCR reactions were performed by the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory as specified by the manufacturer. Briefly, 60ng of input RNA was reverse-transcribed using miRNA-specific stem-loop primers. Next, 12 cycles of pre-amplification reactions were performed using miRNA-specific forward primers and a universal reverse primer. Finally, the pre-amplification reactions were diluted, transferred to 384-well plates via microfluidic channels, and subjected to TaqMan miRNA amplification reactions using miRNA-specific FAM-labeled TaqMan probes. n/a Total RNA was harvested using the MicroRNeasy Mini Kit (Qiagen). n/a Protocol Type normalization data transformation protocol normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATED WITH Experimental Factor Type treated with PubMed ID 24473196 Comment[SecondaryAccession] GSE44089 Comment[GEOReleaseDate] 2014-02-05 Comment[ArrayExpressSubmissionDate] 2013-02-05 Comment[GEOLastUpdateDate] 2014-02-05 Comment[AEExperimentType] other Comment[AdditionalFile:Data1] GSE44089_fold_change_cardA.txt Comment[AdditionalFile:Data2] GSE44089_fold_change_cardB.txt Comment[AdditionalFile:Data3] GSE44089_non_normalized_cardA.txt Comment[AdditionalFile:Data4] GSE44089_non_normalized_cardB.txt SDRF File E-GEOD-44089.hyb.sdrf.txt E-GEOD-44089.seq.sdrf.txt