Figure 6.

Overexpression of miR-200 family genes partially reverses the phenotypic changes caused by GRHL2 knockdown in OSCC cells. (A) SCC4/ShGRHL2 cells were infected with the retroviral vectors expressing miR-200 cluster 1 (miR-200b, -200a, -429) named MSCV-miR-200C1 or miR-200 cluster 2 (miR-200c and -141) named MSCV-miR-200C2. After selection with puromycin (1 μg/ml), the cells were maintained in culture for 7 days to assess altered cell proliferation rate; cell numbers were plotted against time in culture. (B) Western blotting was performed for GRHL2 and epithelial/mesenchymal markers, e.g. E-Cad, N-Cad and p63, in SCC4/EGFP, SCC4/ShGRHL2/MSCV (empty vector), SCC4/ShGRHL2/miR-200C1 and SCC4/ShGRHL2/miR-200C2. (C) Four days after seeding the same cell numbers across the groups, photomicrographs were taken to reveal the altered cell confluence and changes in intercellular adhesion. (D) Tumor spheroid assay was performed with SCC4/EGFP, SCC4/ShGRHL2/MSCV and SCC4/ShGRHL2/miR-200C1. In addition, colony formation assay (E) and Matrigel invasion assay (F) were performed among the indicated cell groups to test the effects of miR-200 gene overexpression on reversing the phenotypic changes induced by GRHL2 knockdown in SCC4 cells.