E2F1 controlled MMP 16 expression via E2F1 binding sites in SCLC cells Since E2F1 could regulate expressions of MMP 9, 14 and 15 in NSCLC, together with the observations mentioned above, we performed ChIP to sequence to identify E2F1 target genes in H1688 cell line. sellckchem As summa rized in Additional file 5, Table S3 and Additional file 6, Table S4, many MMP genes including MMP 1, 14, 16, 17, 19, 24, and 25 were found to be regulated by E2F1. MMP 16 was selected for further study due to its higher expression in SCLC tumor. IHC results re vealed that E2F1 was strongly positive in SCLC tumor where MMP 16 was highly expressed, indi cating that E2F1 was associated with the expression of MMP 16. We used MatInspector analysis to identify two putative E2F1 binding sites in the MMP 16 promoter.
H1688, H446, and A549 cells were transfected with luciferase constructs driven by the wild type MMP 16 promoter or the MMP 16 promoter with mutated E2F1 binding sites and an E2F1 expression plasmid. As shown in Figure 5C, overexpression of E2F1 increased the activity of the MMP 16 promoter. Inhibitors,Modulators,Libraries Furthermore, E2F1 could still activate MMP 16 promoter containing the mutated E2F1 binding site, but not mutant 2, indicating that E2F1 could enhance the expression of MMP 16 and that the sequence was required for E2F1 mediated stimula tion of MMP 16 promoter activity. These results indicated that E2F1 stimulated the expression of MMP 16 by bind ing the binding site 2 sequence in the MMP 16 promoter. Sp1 and p65 regulated Inhibitors,Modulators,Libraries MMP 9 expression in SCLC cells Previous studies and our IHC results showed that MMP 9 expression was higher in SCLC and was significantly affected by E2F1 knockdown.
Johnson et al. reported that one E2F1 binding site was predicted in the MMP 9 promoter. Then, we first tested the activity of MMP 9 promoter containing the Inhibitors,Modulators,Libraries E2F1 binding site mutant by a luciferase reporter experiment, and found that this site was not functional. However, our results showed that MMP 9 promoter activity was significantly enhanced when E2F1 was co expressed in H1688, H446, and A549 cells, indicating that E2F1 regulated MMP 9 expression via an indirect pathway. Previous studies suggested that Sp1 and or p65 might be involved in the regulation of MMP transcription. MatInspector analysis identified four putative Sp1 and two putative NF kappa B binding motifs in the MMP 9 promoter.
Activa tion of the wild type MMP 9 promoter Inhibitors,Modulators,Libraries was significantly increased when the cells were cotransfected with a Sp1 expression plasmid. The activity Inhibitors,Modulators,Libraries of the MMP 9 promoter Sp1 binding site mutant 1 and 2 constructs was un changed, but the MMP 9 promoter Sp1 binding site mu tant 3 and 4 constructs showed significantly decreased activity. Together this data indicated that Sp1 upregulated the selleck chemical Regorafenib expression of MMP 9 by binding to sites 3 and 4, but not 1 or 2 in the MMP 9 promoter.