GSE134375
Multi-omics profiling reveals key signaling pathways in ovarian cancer controlled by STAT3
Inhibiting STAT3 signaling reduces tumor progression, metastases and chemoresistance, however the precise molecular mechanism has not been fully delineated in ovarian cancer.  Methods: In this study, we generated STAT3 knockout (KO) ovarian cancer cell lines. Effect of STAT3 KO on cell proliferation, migration and spheroid formation was assessed in vitro and effect on in vivo tumor growth was tested using several tumor xenograft models. We used multi-omic genome-wide profiling to identify multi-level (Bru-Seq, RNA-Seq, and MS Proteomic) expression signatures of STAT3 KO ovarian cancer cells. Overall design: Cells were lysed with TRIzol® Reagent (ThermoFisher Scientific) at room temperature. RNA was further purified with DirectZol kit (Zymo Research, Irvine, CA). RNA quality was assessed using the TapeStation (Agilent Technologies, Santa Clara, CA). Samples with RINs (RNA Integrity Numbers) of 8 or greater were prepared with TruSeq Stranded mRNA Library Prep (Illumina) per the supplier's protocol with 1?g of RNA and 12 cycles of PCR amplification. Libraries were checked for size on the TapeStation and quantified using the Kapa Biosystems library quantification kit (Illumina). The libraries were barcoded, pooled and sequenced on the HiSeq 4000 at the University of Michigan DNA Sequencing Core using 50bp single-end 50bp (OVCAR3 and OVCAR8) and paired-end 50bp (SKOV3) sequencing. Reads were mapped to GRCh38 using STAR v2.5.2 [69] and gene quantifications were calculated using Cufflinks v2.2.1 [70] to quantify refGene annotations. Gene read counts calculated using featureCounts [71] v1.6.1 were used to evaluate differential expression using DESeq2 v1.18.1 [72]. For OVCAR3 and OVCAR8, genes were considered significantly differentially expressed with a mean FPKM > 0.5 and absolute fold change > 1.5 and FDR adjusted p-value < 0.05. For SKOV3, genes were considered significantly differentially expressed with mean FPKM > 0.5 and absolute log2 fold change > 1.5 and FDR adjusted p-value < 0.05. All gene readouts where required to be mappable to both an HGNC and Entrez identifier to be considered for gene set enrichment analyses. Please note: Majority of paper figures are based on OVCAR3=OV3,OVCAR8=OV8, and SKOV3 with CAS9 control.