Figure 4.

SOX9 as an important upstream regulator of PTK7 expression. (A) The correlation of the fold change (FC) in PTK7 and SOX9 expression of HCC tumor compared with nontumor tissue, analyzed using data from GSE45114. (B) Same statistical evaluation of GSE14520 data set as described in Figure 1B for the fold change in SOX9 expression of paired tumor/nontumor HCC samples with different predicted risk of metastasis, by paired t test. (C) The distribution of high-, medium-, and low-SOX9 cases in tumor or nontumor samples of TMA, compared by chi-squared test. (D) The overall survival distribution of TMA patients grouped according to their tumor PTK7 and SOX9 level, compared by log-rank test. (E) The frequency matrix of the DNA-binding motif of SOX9 as reported in JASPAR database. A single SOX9 binding site was predicted on the PTK7 promoter between the -450 and -460 region upstream of transcription start site. (F) Two truncations of the proximal PTK7 promoter region were synthesized and integrated into the pGL3-basic vector backbone for luciferase reporter assay. T1 truncation contained the predicted SOX9 binding site while T2 truncation did not. (G) The ability in driving protein expression of the 2 truncated promoter elements of PTK7 was quantified by the Dual-Glo luciferase reporter assay after transient transfection and compared by Student t test. (H) The physical binding of SOX9 to the PTK7 promoter region was examined by ChIP assay. Targeted enrichment of the predicted binding sequence was quantified by quantitative PCR and compared with mouse IgG immunoprecipitation control (IgG). (I) The messenger RNA (mRNA) level of PTK7 and SOX9 was quantified and validated in Hep3B cells after successful knockdown of SOX9 by short hairpin RNA (shRNA). (J) Western blot validation of the suppression of PTK7 level after SOX9 depletion by shRNA knockdown in Hep3B. H, high; L, low; M, medium; NTC, nontarget control. P values (∗P < .05, ∗∗P < .01, ∗∗∗P < .001).