Fig. 8.

Sp1 positively regulates miRNAs to silence EMT-related gene expression. The 3′UTRs of various EMT-related genes, CD44s-3′UTR, CD44st-3′UTR, β-catenin-3′UTR and ALDH1-3′UTR, were fused to the 3′ end of GFP to study the GFP level in A549 and E2-A549 cells (A, a). After three independent experiments were finished, the levels of GFP were quantitated (A, b). The levels of various Sp1 regulated miRNAs, miR-3194-5p, miR-218-5p, miR-200-5p, miR-193a-5p, miR-182-5p and miR-135-5p, in A549, A549-T24 (B) and E2-A549 (C) cells with or without Sp1 overexpression were studied by q-PCR. After three independent experiments were completed, the levels of miRNAs were quantitated (B, C, b). The level of CD44 in A549 cells with or without overexpression of Sp1 and expression of the indicated miR sponges were studied by Western blotting (D, a). After three independent experiments were finished, the levels of CD44 were quantitated, and statistical analysis was performed with a t test; *p < 0.05 and **p < 0.01. The recruitment of Sp1 (E, a), ERβ (E, b) and acetyl histone 3 (E, c) to the promoter of miR-3194-5p was studied by ChIP assay. After three independent experiments were finished, the results were quantitated, and statistical analysis was performed with a t test; *p < 0.05, **p < 0.01, ***p < 0.005. The level of miR-3194-5p in A549 cells with or without E2, SAHA and TSA treatment was studied by q-PCR (F, a), and the morphology of E2-A549 cells with or without SAHA treatment was evaluated by ×20 microscopy (F, b). After three independent experiments were completed, the results were quantitated