Figure 3. TLR7 is a downstream target of TGF-β/Smad3 and VD signaling.

(a) Total mRNA was isolated from liver tumors of both wild type and Smad3^+/− mice which were fed either low VD or high VD regimen (n = 3 for each group). Gene expression levels of TLR7, TLR9, OAZ1, and P-Para were measured by Q-PCR. Each result shown is representative of three independent experiments. Error bars are shown as standard deviations. (*p < 0.05 [WT Low VD vs. Smad3^+/− Low VD], **p < 0.05 [Smad3^+/− Low VD vs. Smad3^+/− High VD], Student’s t-test). (b) A schematic representation of the TLR7 promoter studied by ChIP analysis. Two sets of PCR primers, designated as P1 and P2, were used for amplification of the TLR7 promoter. SBE (Smad-binding element) positions are numbered relative to the major transcription start site (+1). Black arrow indicates transcription start site. (c) HepG2 cells were treated with 200 pM TGF-β for 2 hrs. ChIP experiments were performed as described in Methods. (d) Gaussia luciferase plasmid containing a TLR7 promoter region was transfected into HepG2 shRNA-Control and HepG2 shRNA-SMAD3 cells. The secondary reporter, secreted Alkaline Phosphatase, serves as an internal control. TLR7 transcriptional activity was analyzed after 200 pM TGF-β and 100 nM VD treatment for 24 hrs. Error bars are shown as SD in (c,d). Each result shown is representative of three independent experiments (*p < 0.01, **p < 0.001, Student’s t-test). (e) Knockdown TLR7 suppresses HepG2 cell growth and cell migration. Cell proliferation of HepG2-siCtrl and HepG2-siTLR7 cells were assessed by colorimetric MTS assays. Transwell migration assays of HepG2-siCtrl and HepG2-siTLR7 cells were performed. Cells were treated with TGF-β1 (200 pM) for 24 hours. HepG2 cells were transfected with control-siRNA or TLR7-siRNA. The expression of TLR in HepG2-siCtrl and HepG2-siTLR7 cells were measured by Q-PCR analyses. Results are presented as mean ± SD (*p < 0.05, **p < 0.01, Student’s t-test).