[DDX5-Targeting Fragile X Mental Retardation Protein Regulates the Wnt/β-catenin Signaling Pathway to Promote Epithelial Mesenchymal Transition in Breast Cancer].

OBJECTIVE

To investigate the role of fragile X mental retardation protein (FMRP) in promoting cell migration and epithelial-mesenchymal transition (EMT) in breast cancer (BC) and the potential mechanisms involved.

METHODS

The mRNA and protein expressions of FMRP in MCF-10A, a normal human breast epithelial cell line, and four breast cancer cell lines, including MCF-7, BT474, MDA-MB-231, and HCC1937, were analyzed by RT-PCR and Western blot. The expression of FMRP in BC tissues was measured by immunohistochemistry (IHC). FMRP expression in BC and its relationship with clinical prognosis were analyzed using GEO database. Lentiviral infection and siRNA interference were used to construct overexpression and interference vectors, respectively, and the human breast cancer cell line MCF-7 was subsequently transfected. A Control group, an interference empty vector group (the NC group), a knockdown vector group (the si group), an overexpression empty vector group (the Lv-NC group), and an overexpression vector group (the Lv- group) were set up. The migration and invasion abilities of the cells were assessed by scratch assay and Transwell assay. The expression of EMT markers, including E-cadherin, an epithelial marker, N-cadherin, an mesenchymal markers, vimentin, zinc finger E-box binding homeobox 1 (ZEB1), and snail family zinc finger 2 (Slug), in the cells of each group was determined by Western blot. The interaction between FMRP and DEAD-box RNA helicase-5 (DDX5) protein was analyzed by immunocoprecipitation combined with mass spectrometry (IP-MS). The regulatory effect of FMRP on DDX5 protein expression was assessed using the protein synthesis inhibitor cycloheximide (CHX) and proteasome inhibitor MG132. In addition, transfection with si vector was conducted to observe whether DDX5 could reverse the effects of overexpression on cell migration and EMT. The localization and expression of β-catenin were determined by immunofluorescence staining, and the expression of core markers of Wnt/β-catenin signaling pathway was examined by Western blot.

RESULTS

FMRP was highly expressed in BC tissues and cells (<0.05), and overall survival (OS) and recurrence-free survival (RFS) of the high expression group were significantly lower than those of the low expression group (<0.05). The migration ability of MCF-7 cells was weakened after knockdown, while overexpression of promoted cell migration (<0.05). After knockdown, the expression of E-cadherin was increased, while the expression levels of N-cadherin, vimentin, ZEB1, and Slug were decreased, which inhibited the occurrence of EMT. In contrast, the overexpression of promoted the EMT process (<0.05). FMRP interacted with DDX5 protein and promoted DDX5 protein stability by blocking the ubiquitin-proteasome pathway. knockdown reversed the effect of overexpression to promote cell migration and EMT (<0.05), effectively inhibited β-catenin nuclear translocation, and decreased β-catenin nuclear distribution. Furthermore, it was found that the expression of p-β-catenin, GSK3β and Axin2 protein was increased and the expression of C-myc protein was decreased after downregulation (<0.05). On the other hand, the expression of these proteins was reversed by combined overexpression (<0.05).

CONCLUSION

FMRP targets DDX5 and promotes BC cell migration and EMT via the activation of the Wnt/β-catenin signaling pathway.