![]() ![]() One Shot TM TOP10 chemically competent E. As a negative control, the same reaction was performed without insert addition. The digested products were purified with Gel/PCR DNA Fragments Extraction kit, dephosphorylated with rAPid Alkaline Phosphatase kit (Roche, Basel, Switzerland) through incubation at 37 ☌ for 10 min followed by 2 min at 75 ☌ and then ligated using Rapid DNA Ligation kit (Roche, Basel, Switzerland), with samples incubated for 5 min at 20 ☌. Plasmid vector and insert were first digested with NheI and XbaI restriction enzymes (New England Biolabs, Ipswich, MA, USA) through incubation for 1h at 37 ☌. Primer sequences are reported in Table 1. EFEMP1 3′UTR sequence to be cloned was amplified by PCR using ThermoScientific Phusion Hot Start High-Fidelity DNA polymerase kit (Thermo Fisher Scientific, Waltham, MA, USA). However, since only EFEMP1 downmodulation was validated in public datasets comparing tumor vs normal stroma of breast cancer patients, we decided to focus our efforts on studying EFEMP1 contribution to the observed phenotype.ĮFEMP1 3′UTR was cloned into pmirGLO vector plasmid (Promega, Medison, WI, USA), designed to perform luciferase reporter assay and carrying β-lactamase coding region (Ampicillin resistance). These molecules were selected for further analyses since they are known to be involved in the crucial pathways of ECM synthesis and remodelling. Gene expression profile of miR-9 overexpressing NFs revealed EFEMP1, collagen type1 alpha1 (COL1A1) and matrix metalloproteinase-1 (MMP1), as the most significantly modulated genes, being the first two transcripts predicted miR-9 targets. We also showed that these CAF-like cells can increase, in turn, tumor cell aggressiveness. Accordingly, in our previous work by Baroni S et al., we demonstrated that TNBC cells are able to induce the acquisition of CAF-like properties in NFs by releasing the known breast metastamiR miR-9, packaged into exosomes. Indeed miRNAs, small non-coding RNAs involved in post-transcriptional gene regulation, have been proven to act as “messages” to induce the acquisition of malignant traits in stromal cells. Overall, our results demonstrate that miR-9/ EFEMP1 axis is crucial for the conversion of NFs to CAF-like cells under TNBC signaling.Īn increasing body of evidence supports the involvement of microRNAs (miRNAs) in the interaction between tumor and stroma. Moreover, TNBC cells conditioned with the supernatant of NFs transfected with miR-9 or si- EFEMP1 became more resistant to cisplatin. In particular, EFEMP1 siRNA-transfected (si- EFEMP1) fibroblasts have an increased ability to migrate and invade. The luciferase reporter assay showed that miR-9 directly targets EFEMP1 and its silencing recapitulates miR-9-induced pro-tumoral phenotype in fibroblasts. Indeed, upon miR-9 overexpression in NFs, EFEMP1 resulted downmodulated, both at RNA and protein levels. Here we explored the impact of EFEMP1 downmodulation on fibroblast’s acquisition of CAF-like features, and how this phenotype influences neoplastic cells to gain chemoresistance. EGF containing fibulin extracellular matrix protein 1 ( EFEMP1), which encodes for the ECM glycoprotein fibulin-3, emerged as a miR-9 putative target upon miRNA’s exogenous upmodulation in NFs. We previously demonstrated that miR-9, a known metastamiR, is released by triple negative breast cancer (TNBC) cells to enhance the transition of normal fibroblasts (NFs) into cancer-associated fibroblast (CAF)-like cells. MicroRNAs are thought to act as tumor messengers to “corrupt” stromal cells. Tumor growth and invasion occurs through a dynamic interaction between cancer and stromal cells, which support an aggressive niche. ![]()
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