Embryonic morphogen nodal is associated with progression and poor prognosis of hepatocellular carcinoma

Abstract

Background: Nodal, a TGF-β-related embryonic morphogen, is involved in multiple biologic processes. However, the expression of Nodal in hepatocellular carcinoma (HCC) and its correlation with tumor angiogenesis, epithelial-mesenchymal transition, and prognosis is unclear.

Methods: We used real-time PCR and Western blotting to investigate Nodal expression in 6 HCC cell lines and 1 normal liver cell line, 16 pairs of tumor and corresponding paracarcinomatous tissues from HCC patients. Immunohistochemistry was performed to examine Nodal expression in HCC and corresponding paracarcinomatous tissues from 96 patients. CD34 and Vimentin were only examined in HCC tissues of patients mentioned above. Nodal gene was silenced by shRNA in MHCC97H and HCCLM3 cell lines, and cell migration and invasion were detected. Statistical analyses were applied to evaluate the prognostic value and associations of Nodal expression with clinical parameters.

Results: Nodal expression was detected in HCC cell lines with high metastatic potential alone. Nodal expression is up-regulated in HCC tissues compared with paracarcinomatous and normal liver tissues. Nodal protein was expressed in 70 of the 96 (72.9%) HCC tumors, and was associated with vascular invasion (P = 0.000), status of metastasis (P = 0.004), AFP (P = 0.049), ICGR15 (indocyanine green retention rate at 15 min) (P = 0.010) and tumor size (P = 0.000). High Nodal expression was positively correlated with high MVD (microvessal density) (P = 0.006), but not with Vimentin expression (P = 0.053). Significantly fewer migrated and invaded cells were seen in shRNA group compared with blank group and negative control group (P<0.05). High Nodal expression was found to be an independent factor for predicting overall survival of HCC.

Conclusions: Our study demonstrated that Nodal expression is associated with aggressive characteristics of HCC. Its aberrant expression may be a predictive factor of unfavorable prognosis for HCC after surgery.

Figure 1. The expression level of Nodal mRNA and protein in liver cell lines.

1, L02; 2, MHCC97H; 3, Hep3B; 4, HepG2; 5, Huh-7; 6, MHCC97L; 7, HCCLM3. (A) Relative Nodal mRNA expression in liver cell lines measured by qPCR. (B) The expression level of Nodal protein in liver cell lines using Western blotting. Data are Mean ± SD of three replicates.

Figure 2. The expression of Nodal protein in liver tissues.

T, HCC tumor tissue; P, paracarcinomatous tissue; N, normal tissue. (A) Relative Nodal mRNA expression in liver tissues measured by qPCR. (B) The expression level of Nodal protein in liver tissues using Western blotting. Data are Mean ± SD of three replicates. * P<0.05.

Figure 3. Immunohistochemical staining of Nodal, CD34 and Vimentin in HCC tumors.

(A) HCC tumor tissue for hematoxylin-eosin staining (×400). (B) Negative Nodal expression (×400). (C) Low Nodal expression (×400). (D) High Nodal expression (×400). (E) Low Vimentin expression (×400). (F) High expression Vimentin (×400). (G) Low MVD expression (×200). (H) High MVD expression (×200).

Figure 4. Immunohistochemical staining of Nodal in HCC tissues and non-HCC tissues.

(A) Representative images from immunohistochemistry analyses of Nodal expression in normal liver tissue (Non-HCC), HCC without invasion tissue (Non-Invasion-HCC) and HCC with invasion tissue (Invasion-HCC). (B) Percentages of high Nodal expression in Non-HCC cases, Non-Invasion-HCC cases and Invasion-HCC cases.

Figure 5. Inhibition of Nodal by Nodal-specific shRNA in MHCC97H and HCCLM3 cells 24, 48, and 72 h after transfection, respectively.

(A) Relative Nodal mRNA expression in MHCC97H cell measured by qPCR. (B) The expression level of Nodal protein in MHCC97H cell using Western blotting. (C) Relative Nodal mRNA expression in HCCLM3 cell measured by qPCR. (D) The expression level of Nodal protein in HCCLM3 cell using Western blotting. 1, control; 2, 24 h after transfection; 3, 48 h after transfection; 4, 72 h after transfection. Data are Mean ± SD of three replicates. * P<0.05.

Figure 6. Nodal suppressed migration and invasion of HCC cells in vitro.

(A, B) Effect of Nodal knockdown on MHCC97H cell migration and invasion in vitro. (C, D) Effect of Nodal knockdown on HCCLM3cell migration and invasion in vitro. Data are Mean ± SD of three replicates. * P<0.05.

Figure 7. Kaplan-Meier analysis of overall survival curves of patients with HCC according to Nodal expression.

The HCC patients with high Nodal expression showed notably poorer overall survival rates than those with low Nodal expression.