Growth differentiation factor 7 alleviates the proliferation and metastasis of hepatocellular carcinoma

Abstract

Background and aims: Inflammatory factors play significant roles in the development and occurrence of hepatocellular carcinoma (HCC). However, the tumor-protective functions of growth differentiation factors (GDFs) in HCC are yet to be clarified. In this study, we aimed to evaluate the expression levels of 10 GDFs in tumor and paratumor tissues from patients with HCC and perform in vitro and in vivo experiments to elucidate the role of GDF7 in regulating the proliferation and metastasis of HCC.

Methods: The gene expression of 10 GDFs was compared between HCC and paratumors using The Cancer Genome Atlas dataset and patient-derived tissues. A tumor microarray containing 108 HCC tissue samples was used to explore the prognostic value of GDF7 expression. Loss-of-function experiments were also performed in vitro and in vivo to investigate the role of GDF7 in HCC.

Results: The mRNA and protein levels of GDF7 were significantly lower in HCC tumors than in paratumors (P < 0.001). Kaplan-Meier analysis showed that decreased GDF7 expression in HCC was associated with worse overall survival (5-year rate: 61.8% vs. 27.5%, P < 0.001) and increased recurrence risk (P < 0.001). Multivariate Cox regression analysis demonstrated that low GDF7 expression, the presence of microvascular invasion, and elevated alpha-fetoprotein (AFP) levels were independent risk factors for tumor recurrence and poor survival. Downregulation of GDF7 also increased the tumor growth in HCC cells and in an HCC xenograft model. GDF7 knockdown promoted migration and invasion via epithelial-mesenchymal transition. Meanwhile, a negative correlation between JunB proto-oncogene (JUNB) and GDF7 was observed in HCC tissues. Modulating JUNB levels altered GDF7 protein expression.

Conclusions: GDF7 is a potential biomarker for predicting superior outcomes in patients with HCC. GDF7 amplification is a potential therapeutic option for HCC.

Keywords: Epithelial–mesenchymal transition (EMT); Growth differentiation factor 7 (GDF7); Hepatocellular carcinoma (HCC); JunB proto-oncogene (JUNB); Metastasis; Proliferation.

Fig. 1

Identifying the tumor-protective growth differentiation factor (GDF) genes. (A) The gene expression of 10 GDFs between hepatocellular carcinoma (HCC) and tumor-adjacent tissues in The Cancer Genome Atlas (TCGA) data and (B) comparison of differential expression of every GDF gene in HCC and tumor-adjacent tissues using TCGA data. The red block indicates increased expression in tumor, whereas the blue block indicates decreased expression in tumor. T, tumor; N, nontumor. (C) The mRNA expression levels of GDF2, GDF6, and GDF7 in the paired HCC and tumor-adjacent tissues (n = 30). Compared by paired Student’s t-test. ^∗∗P < 0.01, ^∗∗∗P < 0.001. Kaplan–Meier analysis of the overall survival and recurrence-free survival in terms of differential expression groups based on (D) GDF2, (E) GDF6, and (F) GDF7. Analyzed by GEPIA (http://gepia.cancer-pku.cn/).

Fig. 2

Decreased expression of growth differentiation factor 7 (GDF7) is associated with worse outcomes in patients with hepatocellular carcinoma (HCC). (A) Representative images of GDF7 expression in HCC tissues and corresponding adjacent tissues, as detected by immunohistochemistry (IHC). Scale bars, 400 μm. (B) A scatter diagram of semiquantitative GDF7 score in HCC tissues and corresponding paratumor tissues (n = 108). Compared by paired Student’s t-test. ^∗∗∗P < 0.001. (C) Representative images of low and high GDF7 expression in HCC tissues, as detected by IHC. Scale bars, 400 μm. Kaplan–Meier survival curves showing (D) overall survival and (E) recurrence-free survival in HCC with low (n = 74) and high GDF7 expression (n = 34). Compared by log-rank test.

Fig. 3

GDF7 knockdown promotes HCC tumor proliferation and growth. (A) The protein expression of GDF7 in different cell lines, detected by Western blotting. (B) Immunoblot analysis of protein extracts from Hep3B and SK-Hep-1 cells treated with control (shCtrl) and three shRNA sequences of GDF7. (C) CCK-8 assay and (D) colony formation assay in Hep3B and SK-HEP-1 cells treated with shCtrl and shGDF7. (E) Tumor growth curves and (F) images of tumors from tumor-bearing mice (n = 5 per group). The data are shown as the mean ± standard deviation. Compared by Student’s t-test. ^∗∗P < 0.01, ^∗∗∗P < 0.001. Abbreviations: CCK-8, cell counting kit-8; GDF7, growth differentiation factor 7; HCC, hepatocellular carcinoma; OD, optical density.

Fig. 4

Growth differentiation factor 7 (GDF7) knockdown increases migration and invasion in hepatocellular carcinoma (HCC) cells. Representative images and data of (A) migration and (B) invasion in Hep3B and SK-Hep-1 cells treated with shCtrl and shGDF7. (C) The expression of epithelial–mesenchymal transition (EMT)-related proteins in shCtrl and shGDF7 of Hep3B and SK-Hep-1 cells, detected by Western blotting. (D) The mRNA levels of EMT-related markers in Hep3B and SK-Hep-1 cells with GDF7 knockdown. (E) Representative images of EMT-related markers and Ki-67 in tumors from tumor-bearing mice, detected by immunohistochemistry. Scale bars, 40 μm. The data are shown as the mean ± standard deviation. Compared by Student’s t-test. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Fig. 5

GDF7 is downregulated by JUNB overexpression. (A) The potential binding site of JUNB to the GDF7 promoter, predicted by JASPAR analysis (https://jaspar.genereg.net/). (B) Detection of GDF7 promoter activity after the overexpression of JUNB in Hep3B and SK-Hep-1 cells by dual-luciferase reporter assay. The data are shown as mean ± standard deviation. Compared by Student’s t-test. ^∗∗∗P < 0.001. (C) The GDF7 protein expression in Ctrl and JUNB overexpression of Hep3B and SK-Hep-1 cells, detected by Western blotting. (D) The GDF7 protein expression in Ctrl and JUNB-knockdown Hep3B and SK-Hep-1 cells, detected by Western blotting. (E) Representative images of GDF7 and JUNB expression in clinical HCC tissues. Scale bars, 100 μm. (F) The correlation between GDF7 levels and JUNB expression in HCC tumors was evaluated in the tumor microarray cohort (n = 108) using Pearson’s correlation analysis. Abbreviations: GDF7, growth differentiation factor 7; HCC, hepatocellular carcinoma; JUNB, JunB proto-oncogene.