Restin belongs to MAGE superfamily and is known as MAGE H1.
Restin was firstly cloned from HL-60 cells treated with
all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of
Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if
Restin plays a role in
breast cancer cells in vitro and in vivo.
METHODS AND RESULTS: Real-time PCR and western blot were conducted to detect
Restin expression in multiple
breast cancer cell lines and
Restin level was negatively related with cell motility.
Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology,
wound closure assay, transwell migration and invasion assays were performed to detect if
Restin inhibited EMT. Our data showed that
Restin overexpressed cells exhibited classical epithelial cell morphology, and
Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion.
Tumor xenograft model was used to characterize the
biological functions of
Restin in vivo. We found that
Restin overexpression led to reduced lung
metastasis. Real-time PCR, western blot,
luciferase assay and ChIP assay were performed to identify the potential targets of
Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by
Restin. Unexpectedly,
Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by
Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated
Restin interacted with p73, one of p53 family members, which contributed to
Restin-mediated activation of mir-200a/b and suppression of ZEB1/2.
CONCLUSIONS: Taken together, our results suggest that
Restin inhibits EMT and
tumor metastasis by controlling the expression of the
tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between
Restin, EMT and
tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II
proteins may exert a
tumor suppressive effect in vivo.