Decreased BMP-7 and p-Smad1/5/8 expression, and increased levels of gremlin in hepatocellular carcinoma

Abstract

The aim of the present study was to investigate the expression of bone morphogenetic protein-7 (BMP-7), gremlin, and p-Smad/1/5/8 in carcinomatous and para-carcinoma tissue specimens from patients with hepatocellular carcinoma (HCC). The association of serum BMP-7 levels with clinicopathological parameters was examined to assess its relevance as a clinical biomarker for HCC. A total of 27 patients with HCC and 7 healthy subjects were included. Gene expression levels of BMP-7 and p-Smad1/5/8 were examined by reverse transcription-quantitative polymerase chain reaction. Immunohistochemical and western blot analysis were performed to determine the protein expression of target genes. The serum levels of BMP-7 were assessed by enzyme linked immunosorbent assay. The mRNA and protein expression of BMP-7 and gremlin were significantly down- and upregulated in HCC tumor tissues, respectively, compared with para-carcinoma tissues (P<0.05). The association of BMP-7 and gremlin expression with the differentiation status of HCC was also analyzed. There was a relatively higher level of BMP-7 and a lower level of gremlin expression in tumor tissues from patients with highly differentiated HCC when compared with poorly or moderately differentiated HCC (BMP-7, F=42.29, P<0.01; gremlin, F=37.93, P<0.01). Furthermore, the level of BMP-7 and p-Smad1/5/8 was decreased in patients with advanced stages of HCC, when compared with stage I HCC. The findings from the present study suggest that the BMP-7/p-Smad signaling pathway may be involved in the pathogenesis of HCC. The serum levels of BMP-7 may serve as a potential biomarker for HCC.

Keywords: bone morphogenetic protein-7; gremlin; hepatocellular carcinoma; p-Smad.

Figure 1.

Reverse transcription-quantitative polymerase chain reaction detection of the mRNA levels of BMP-7 and its endogenous antagonist gremlin in carcinomatous and non-carcinomatous tissue specimens from patients with HCC. Non-carcinoma tissues were used as controls. P<0.05 vs. non-carcinoma tissues. BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.

Figure 2.

Protein expression of BMP-7 and gremlin in carcinomatous and non-carcinomatous hepatic tissue specimens from patients with HCC. (A) BMP-7 expression in a HCC tissue specimen. (B) BMP-7 expression in a non-carcinomatous (para-carcinoma tissues, 3 cm away from tumor margin) tissue specimen. (C) Gremlin expression in a HCC tissue specimen. (D) Gremlin expression in a non-carcinomatous tissue specimen. Black arrows indicate representative positive staining. Magnification, −400. (E) The mean integral optical density per area was calculated. Non-carcinoma tissues were used as controls. P<0.01 vs. non-carcinomatous tissues. BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.

Figure 3.

Protein expression of BMP-7 and gremlin in HCC tissue specimens with varied differentiation status. Carcinoma tissue samples were obtained from patients with (A,B) poor, (C,D) moderate or (E,F) high differentiation. Protein expression of (A,C,E) BMP-7 and (B,D,F) gremlin were analyzed by immunohistochemistry. Magnification, −400. (G) Mean integral optical density per area was calculated. Protein expression of BMP-7 and gremlin in carcinoma tissue samples derived from patients at varied differentiation were compared. P<0.01. BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.

Figure 4.

Reverse transcription-quantitative polymerase chain reaction of BMP-7 and gremlin mRNA expression in HCC tissue specimens from patients at different clinical stages (stage I, II, IIIA or IIIB). mRNA levels of BMP-7 and gremlin in HCC tissue samples derived from patients at varied clinical stages were compared. P<0.05 or P<0.01. BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.

Figure 5.

Western blotting of BMP-7 and p-Smad1/5/8 protein expression in carcinomatous and non-carcinomatous hepatic tissue specimens from patients with HCC at different clinical stages (I, II, IIIA, or IIIB). (A) Representative immunoblots of BMP-7 and p-Smad1/5/8. (B) Protein expression in carcinoma and non-carcinoma tissues was quantified. (C) Protein expression in carcinoma tissues at stages I, II, IIIA or IIIB were quantified. Protein expression of BMP-7 and p-Smad1/5/8 in non-carcinomatous tissue samples and HCC tissue samples at various clinical stages were compared. Non-carcinomatous tissue samples were used as controls. P<0.05 or P<0.01. BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.

Figure 6.

ELISA analysis of serum BMP-7 levels in healthy subjects and patients with HCC. Healthy subjects (n=7); HCC stage I (n=3), II (n=12), IIIA (n=9) and IIIB (n=3). Tissues from healthy subjects were used as controls. **P<0.01 vs. Healthy control. ELISA, enzyme linked immunosorbent assay; BMP-7, bone morphogenetic protein-7; HCC, hepatocellular carcinoma.