Smad7 Affects Human Epidermoid Carcinoma Cells Proliferation and Invasion via TGF-β Signaling Pathway

Abstract

Background: This study aimed to explore Smad7 function and possible mechanism in human epidermoid cancer cells A431. Methods: A431 cells were divided into Control group, NC group (transfected with empty vetor) and miR-Smad7 group (transfect with Smad7 recombinant plasmids). Transwell and WST-1 assay were performed to determine A431 cells’ cell migration, invasion and growth. ELISA (enzyme-linked immunosorbent assay) was used to assess secreted TGF-β1 (transforming growth factor beta1) level, and immunofluorescence assay and confocal microscopy was used to assess Smad4 subcellular distribution in A431 cells. TGF-βRI (transforming growth factor beta receptors), Smad, PCNA (proliferating cell nuclear antigen), C-myc, Bcl-2 (B-cell lymphoma-2), Bax and EGFR (epidermal growth factor receptor) protein levels of A431 cells were determined by Western blot. A431 cell cycle and apoptosis was determined using flow cytometry. A431 cells morphology was determined by transmission electron microscope (TEM) micrographs and scanning electron microscope (SEM). Results: The results showed that three Smad7-targeting miR-Smad7s were successfully constructed. A431 cells Smad7 silencing reduced cell invasion, migration and proliferation (p < 0.01) and contributed to enhance PCNA, C-myc, and EGFR protein expression levels (p < 0.01). In addition, miR-Smad7 induced G1 A431 cells arrest and apoptosis (p < 0.01). Specifically, morphology apoptosis phenotype was observed in miR-Smad7 group. Smad4 subcellular distribution showed the presence of Smad4 in the nucleus in miR-Smad7 group. Western blot demonstrated higher TGF-βRI and Smad4 expression levels in the miR-Smad7-2 group compared to the Control group (p < 0.01). Conclusions: In summary, Smad7 affected A431 cells proliferation, migration, invasion and apoptosis, most probably by interfering with the TGF-β signaling pathway.