Fig. 1
The expression and clinical significance of PITX2. A Establishment of an in vitro hepatocyte differentiation model, which induced human embryonic stem (ES) cells into definitive endoderm (DE), liver progenitor cells (LP) and premature hepatocytes (PH). Cells in the four stages of in vitro hepatocyte differentiation model together with two HCC and two adjacent non-tumor tissues were used for deep RNA sequencing (B) The relative expression level of PITX2 and PITX2C in each stage of the in vitro hepatocyte differentiation model was confirmed using qRT-PCR. (*P < 0.05, **P < 0.01, independent Student’s t-test) (C) Bar chart of delta Ct (Top: ΔCtPITX2 = CtPITX2-CtGAPDH; Bottom: ΔCtPITX2C = CtPITX2C-CtGAPDH) in two immortalized liver cell lines and seven HCC cell lines. GAPDH was used as a reference gene. The higher the ΔCt, the lower expression level of target gene. D Representatives of IHC staining images with anti-PITX2 in non-tumor, HCC with moderate PITX2 expression, HCC with high expression (400× magnification). Red arrows indicate the PITX2+ cells. E The relative expression level of PITX2 in HCC clinical samples (Cohort-1) compared to the corresponding non-tumor tissue (Top). ΔΔCt PITX2 = (Ct PITX2 in tumor – Ct GAPDH in tumor)-(Ct PITX2 in non-tumor –Ct GAPDH in non-tumor). As 2-ΔΔCt PITX2 indicates the fold change of PITX2 in tumor relative to the corresponding non-tumor, PITX2 was up-regulated in 53 out of 93 HCC samples with ΔΔCt PITX2 < 0. The relative expression level of PITX2C in HCC clinical samples were evaluated by ΔΔCt PITX2C = (Ct PITX2C in tumor – Ct GAPDH in tumor)-20 as the PITX2C expression was almost absent in non-tumor tissue (Bottom). Five HCC cases showing stable expression of PITX2C as ΔΔCt PITX2C < − 5. Two groups with distinct PITX2C expression: group (+) with ΔΔCt PITX2C < 0; group (−) with ΔΔCtPITX2C > 0. F Kaplan–Meier overall (left) and disease-free survival curve (right) of two HCC groups: PITX2 (+), patients with higher PITX2 expression; PITX2 (−), patients with lower PITX2 expression. G Association of PITX2 expression with tumor size in two HCC cohorts. PITX2 expression was determined by qRT-PCR or IHC. H Association of PITX2C expression with AFP and differentiation in HCC cohort-1. PITX2C expression is defined as positive in HCC samples when ΔΔCt PITX2C < 0. G-H The X-axis represents the number of HCC cases in each group. The P value was calculated using Pearson Chi-square test