Gene expression analysis of cancer-associated fibroblasts (CAFs) from the murine breast cancer model MMTV-PyMT after perturbing the expression of Dickkopf-3 (DKK3)

Immortalized (hpv-E6) murine fibroblasts isolated from normal mammary glands and carcinoma stages (CAFs) from the MMTV-PyMT model (FVB/N background) were used in this analysis. CAFs were transfected with two independent RNAi targeting Dickkopf-3 (DKK3) or control RNAi and changes in whole-genome gene expression were evaluated by microarray. In addition, the effect of stable ectopic expression of DKK3 in normal fibroblasts (which normally do not express DKK3) was also evaluated. Analysis demonstrated the role of DKK3 in controlling specific transcriptional programs in CAFs. CAFs were seeded in subconfluency and transfected with control (siCtr) or two independent RNAi targeting DKK3 (siDKK3#2 and siDKK3#3) on duplicate. Normal fibroblasts (NFs) ectopically expressing empty vector (null) or DKK3 were also seeded in subconfluency (duplicates). After 48 h, total RNA was isolated and processed for gene expression analyses as suggested by the manufacturer. Standard expression analysis data (SST-RMA) was used for the analysis.