STAT3 deficiency prevents hepatocarcinogenesis and promotes biliary proliferation in thioacetamide-induced liver injury

Figure 1 Establishment of hepatic-STAT3 deficient (STAT3^Δhep) mice. Hepatic-STAT3 deficiency enhanced thioacetamide-induced liver injury. A: The exon 21 of STAT3 was flanked by two identically oriented loxP sites (STAT3^flox/flox). To disrupt hepatic-STAT3 gene, STAT3^flox/flox mice were crossed with mice carrying albumin promoter-driven Cre transgene (Alb-Cre). Deficiency of hepatic-STAT3 protein was confirmed by immunoblotting analysis. B: Representative hematoxylin and eosin (H&E) staining images of control and STAT3^Δhep livers with TAA treatment for 16 wk. Hepatic-STAT3 deficiency accelerated inflammation cells infiltration and hepatocytes necrosis. C: Quantification of necrotic hepatocytes. Necrotic hepatocyte was identified with the eosinophilic/vacuolated cytoplasm and nuclear destruction such as karyolysis or Pyknosis. Values represent means ± SE of the mean. ^bP < 0.01 (P = 0.000269) vs control. D: Representative Azan staining of control and STAT3Δhep livers with TAA treatment for 16 wk. E: Quantification of the ratio of fibrosis area to the total area in control and STAT3^Δhep livers. Values represent means ± SE of the mean. ^bP < 0.01 (P = 0.00235) vs control. TAA: Thioacetamide.

Figure 2 Hepatic STAT3 deficiency reduced hepatocyte proliferation and inhibited the development of hepatocellular carcinoma. A: Representative immunohistochemical staining for PCNA in livers of control and STAT3^Δhep mice with TAA treatment for 16 wk. PCNA positive hepatocytes were decreased in STAT3^Δhep mice. Original magnification × 200. B: The number of PCNA positive hepatocytes. The ratio of PCNA positive hepatocytes to all hepatocytes was quantified in three different samples per a group. Values represent means ± standard error of the mean. ^bP < 0.01 (P = 0.000232) vs control. C: Gross liver appearance of control and STAT3^Δhep mice with TAA treatment for 30 wk. Tumor formation with TAA treatment for 30 wk (upper panel). HE staining of representative sections of hepatocellular carcinoma. Original magnification × 100 (lower left panel) and × 400 (lower right panel). D: The number of HCC in control and STAT3^Δhep mice. Values represent means ± SE of the mean (n = 6). ^aP < 0.05 (P = 0.0219) vs control. TAA: Thioacetamide.

Figure 3 Increase in the formation of bile ducts and ductular structure in hepatic STAT3 deficient liver. A: Hematoxylin and eosin staining images of representative areas around portal vein (PV) and central vein (CV) of control and STAT3^Δhep livers following treatment with TAA for 16 wk. TAA treatment caused bile ducts/ductular formation (arrow). B: Immunohistochemistry staining for keratin 19 (KRT19) in control and STAT3^Δhep livers following TAA treatment for 16 wk. Original magnification × 200. C and D: The number of KRT19-positive bile ducts/ductules around PV and CV. To quantify the number of bile ducts/ductules around PV and CV, we observed three different samples per group using 20 × objective lens, imaged five randomly selected cross-sectional veins in the center of field, and calculated the average value after counting the bile duct/ductule number in the total area of the image. Values represent means ± SE of the mean. ^bP < 0.01 [(C) P = 0.00272, (D) P = 0.00187] vs control. TAA: Thioacetamide.

Figure 4 Hepatic STAT3 deficiency upregulated SOX9 expression in thioacetamide-induced liver injury. A: Immunohistochemistry staining for SOX9 in control and STAT3^Δhep livers with TAA treatment for 16 wk. SOX9 expression was markedly enhanced in bile ducts/ductular structure (upper panel) and hepatocytes (lower panel) of STAT3Δhep livers. Original magnification × 200 (upper panel) and × 400 (lower panel). B and C: The number of SOX9 positive bile ducts/ductular structure and hepatocytes. For quantification of SOX9 positive bile ducts/ductular structure, three different samples per a group were observed using a 20 × objective lens and SOX9 positive ducts/ductular structure was counted at five PV areas. For quantification of SOX9 positive hepatocytes, three different samples per a group were observed using a 40 × objective lens and SOX9 positive hepatocytes was counted at five PV areas. Values represent means ± standard error of the mean. ^bP < 0.01 [(B) P = 0.0000103, (C) P = 0.00834] vs control. TAA: Thioacetamide.

Figure 5 YAP activation in STAT3-deficient hepatocyte with thioacetamide treatment. A: Quantitative real-time RT-PCR analysis for SOX-9, KRT 19, YAP, AFP, IL-6, IL-33, Osteopontin, MMP2, and MMP9 mRNA expression levels. The relative expression of each gene mRNA was normalized to GAPDH mRNA (n = 6). ^aP < 0.05 (YAP; P = 0.0134, AFP; P = 0.00347, MMP9; P = 0.00271) and ^bP < 0.01 (SOX9; P = 0.00274, KRT19; P = 0.00402, IL-6; P = 0.00147, MMP2; P = 0.00404) vs control (TAA treatment for 16 wk). B: Immunoblotting analysis for SOX-9, YAP, pY-YAP, p-Src and AFP protein expression levels. GAPDH served as loading control. C: Representative immunohistochemical staining for YAP in livers of control and STAT3^Δhep mice with TAA treatment for 16 wk. Nuclear YAP was augmented in STAT3^Δhep hepatocytes with TAA treatment for 16 wk. Original magnification × 200. D: The number of nuclear YAP positive hepatocytes. Ratio of nuclear YAP positive hepatocytes to all hepatocytes was quantified in three different samples per a group. Values represent means ± SE of the mean. ^bP < 0.01 (P = 0.00312) vs control. TAA: Thioacetamide.