ABSTRACT
Objective: To investigate the effects of Yes-associated protein 1 (YAP 1) gene knockdown in cancer-associated fibroblast (CAF) on the migration and invasion of breast cancer MDAMB- 231 cells. Methods: The expression of YAP1 in breast primary CAF and paired normal fibroblast (NF) in mRNA microarray was analyzed by bioinformatics methods. To verify the authenticity and accuracy of the microarray results, the expressions of YAP1 mRNA and protein in primary and immortalized NF and CAF were detected by real-time fluorescent quantitative PCR and Western blotting. A co-cultured system including breast cancer MDA-MB-231 cells and the conditioned medium of NF or CAF was used, and the effect of fibroblasts on the invasion ability of breast cancer cells was detected by Transwell chamber assay. CAF cells were transfected with the plasmids carrying the specific shRNA targeting YAP 1 gene, then the expressions of YAP1 mRNA and protein in CAF were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The effects of YAP 1 gene interference in CAF on the migration and invasion of MDA-MB-231 cells cultured with CAF conditioned medium were detected by scratch wound healing and Transwell chamber assay, respectively. CAF cells were treated with Hippo pathway inhibitor XMU-MP-1, then the expressions of total YAP1 protein and phosphorylated YAP1 protein in CAF were detected by Western blotting, as well as the invasion ability of MDA-MB-231 cells co-cultured with the CAF conditioned medium was measured by Transwell chamber assay. The expressions of transforming growth factor-beta 1 (TGF -β 1) and interleukin 6 (IL 6), two target genes of YAP1 and the cytokines associated with cell migration and invasion, in CAF with YAP 1 gene interference were detected by real-time fluorescent quantitative PCR. The effect of YAP1-knocked CAF on the extracellular matrix remodeling was tested by Collagen gel contraction assay. Results: Compared with primary NF, the expression of YAP 1 gene was significantly upregulated in primary CAF of breast cancer according mRNA microarray (P < 0.05). The higher expressions of YAP1 mRNA and protein in primary and immortalized CAF were confirmed by real-time fluorescent quantitative PCR (P < 0.05) and Western blotting (P < 0.01). The invasion ability of breast cancer MDA-MB-231 cells was significantly promoted in the coculture system with the conditioned medium of CAF as compared with NF (P < 0.01). The CAF cell line transfected with YAP1 shRNA was successfully constructed, and the expressions of YAP1 mRNA and protein were successfully stably knocked down in CAF (both P < 0.01). Compared with the YAP1-unknocked CAF control group, the migration (P < 0.05) and invasion (P < 0.01) abilities of breast cancer MDA-MB-231 cells were significantly weakened after culture with the conditioned medium of YAP1-knocked CAF. The phosphorylation level of YAP1 protein was decreased in CAF treated with Hippo pathway inhibitor XMU-MP-1 (P < 0.01), while the invasion ability of MDA-MB-231 cells was significantly enhanced after coculture with XMU-MP-1-treated CAF (P < 0.05). The expression levels of TGF-β1 and IL6 mRNAs were down-regulated in YAP1-knocked CAF (both P < 0.05), and the collagen gel shrinking ability of extracellular matrix was significantly decreased in YAP1-knocked CAF group (P < 0.05). Conclusion: The stable knockdown of YAP1 expression in CAF can inhibit the migration and invasion of breast cancer MDA-MB-231 cells through down-regulating the expressions of TGF-β1 and IL6 and remodeling the extracellular matrix.