GIT1 is a novel prognostic biomarker and facilitates tumor progression via activating ERK/MMP9 signaling in hepatocellular carcinoma

Abstract

Aim: Multiple studies have revealed that G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is overexpressed in many cancers and facilitates tumor progression. However, the role of GIT1 in hepatocellular carcinoma (HCC) remains unclear.

Methods: GIT1 expression was detected in cell lines and 130 pairs of HCC and matched adjacent noncancerous samples. Transwell assay, flow cytometry, caspase 3/7 activity assay, 5-bromodeoxyuridine cell proliferation assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to assess invasion, migration, apoptosis, and proliferation of HCC cells. Furthermore, GIT1 expression was detected by immunohistochemistry to evaluate its correlation with phospho-extracellular signal-regulated kinase (p-ERK)1/2. The regulatory effect of GIT1 on ERK1/2, p-ERK1/2, and matrix metalloproteinase-9 (MMP9) in HCC cells was confirmed by immunoblotting.

Results: In this study, we demonstrated that GIT1 was more highly expressed in HCC samples than that in non-HCC samples, and overexpression of GIT1 was correlated with clinicopathological features of poor prognosis. Clinical analysis demonstrated that GIT1 is an independent prognostic biomarker for predicting overall survival and disease-free survival of patients with HCC. In vitro studies showed that downregulation of GIT1 facilitated HCC cell apoptosis and repressed HCC cell invasion, migration, and proliferation. Overexpression of GIT1 is associated with p-ERK1/2 amplification in HCC tissues. Moreover, downregulation of GIT1 resulted in inactivation of ERK signaling and downregulation of MMP9.

Conclusion: Our findings indicate that GIT1 is an independent prognostic biomarker and facilitates HCC progression via activating ERK/MMP9 signaling.

Keywords: ERK signaling; GIT1; HCC; prognosis.

Figure 1

(A–D) The expression of GIT1 in cell lines and HCC tissues. Notes: (A) GIT1 mRNA expression in the immortalized normal human liver cell line L02 and five hepatoma cell lines (Huh-7, HepG2, SMMC-7721, PLC/PRF5, and MHCC-97H) using qRT-PCR (n=3, *P<0.01, vs L02 group, respectively). (B, i) GIT1 protein expression in the immortalized normal human liver cell line L02 and five hepatoma cell lines (Huh-7, HepG2, SMMC-7721, PLC/PRF5, and MHCC-97H) using immunoblotting (ii) (n=3, *P<0.01, vs L02 group, respectively). (C) GIT1 mRNA expression in HCC tissues (T) and corresponding adjacent no tumorous tissues (N) using qRT-PCR (n=130, P<0.01). (D, i) GIT1 protein expression in HCC tissues (T) and corresponding adjacent no tumorous tissues (N) using immunoblotting (ii) (n=130, *P<0.01). Abbreviations: GIT1, G-protein-coupled receptor kinase-interacting protein 1; HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; vs, versus.

Figure 2

(A–B) Kaplan–Meier survival curves according to GIT1 protein expression in HCC patients. Notes: (A) Overall survival for GIT1 protein expression (log-rank, P=0.000). (B) Disease-free survival for GIT1 protein expression (log-rank, P=0.000). Abbreviations: GIT1, G-protein-coupled receptor kinase-interacting protein 1; HCC, hepatocellular carcinoma.

Figure 3

(A–D) GIT1 regulates apoptosis and proliferation in MHCC-97H cells. Notes: (A) Cell proliferation as measured by BrdU incorporation was inhibited by GIT1-siRNA in MHCC-97H cells (n=3, *P=0.001). (B) As assessed by MTT assay, GIT1 knockdown was found to reduce the viability of MHCC-97H cells (n=3, *P=0.005). (C) The activity of the pro-apoptotic caspases 3 and 7 was upregulated after GIT1 knockdown in MHCC-97H cells (n=3, *P=0.002). (D) Quantification of the apoptotic cell population by flow cytometry. GIT1 knockdown MHCC-97H cells were composed of a larger subset of apoptotic cells compared with the control group (n=3, *P=0.007). Abbreviations: BrdU, 5-bromodeoxyuridine; GIT1, G-protein-coupled receptor kinase-interacting protein 1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; PI, Propidium Iodide; FITC, fluorescein isothiocyanate; h, hours; RLU, relative light unit.

Figure 4

(A–B) GIT1 regulates invasion and migration in MHCC-97H cells. Notes: (A) Representative images show the invasion and migration ability of MHCC-97H cells transfected with control-siRNA or GIT1-siRNA (scale bar: 100 μm). (B) Data are presented as mean relative numbers of invaded or migrated cells from five fields (*P=0.000, **P=0.000). Abbreviations: GIT1, G-protein-coupled receptor kinase-interacting protein 1; siRNA, small interfering RNA.

Figure 5

(A–D) Immunohistochemical staining of GIT1 and p-ERK1/2 in HCC tissues and the effect of GIT1 knockdown on MHCC-97H cells. Notes: (A) The IHC scores of GIT1 and p-ERK1/2 in HCC tissues was notably higher than that in the corresponding adjacent no tumorous tissues (NT), respectively (n=130, *P=0.000, **P=0.000). (B) In cases of high GIT1 expression (a), there was strong p-ERK1/2 expression in the same tissue section (b). Similarly, in the case of low GIT1 expression (c, e), there was no detectable p-ERK1/2 expression in the same tissue section (d, f) (scale bar: 100 μm). (C) Linear regression analysis and Spearman’s correlation test showed a significant positive correlation between GIT1 and p-ERK1/2 expression. (D) HCC cells that had been transfected with indicated siRNAs were treated with ERK inhibitor SCH772984 for 1 hour and Western blotting were performed (n=3, *P=0.001, **P=0.004). Abbreviations: GIT1, G-protein-coupled receptor kinase-interacting protein 1; HCC, hepatocellular carcinoma; IHC, immunohistochemical; MMP9, matrix metalloproteinase-9; p-ERK1/2, phospho-extracellular signal-regulated kinase 1/2.

Figure 5

(A–D) Immunohistochemical staining of GIT1 and p-ERK1/2 in HCC tissues and the effect of GIT1 knockdown on MHCC-97H cells. Notes: (A) The IHC scores of GIT1 and p-ERK1/2 in HCC tissues was notably higher than that in the corresponding adjacent no tumorous tissues (NT), respectively (n=130, *P=0.000, **P=0.000). (B) In cases of high GIT1 expression (a), there was strong p-ERK1/2 expression in the same tissue section (b). Similarly, in the case of low GIT1 expression (c, e), there was no detectable p-ERK1/2 expression in the same tissue section (d, f) (scale bar: 100 μm). (C) Linear regression analysis and Spearman’s correlation test showed a significant positive correlation between GIT1 and p-ERK1/2 expression. (D) HCC cells that had been transfected with indicated siRNAs were treated with ERK inhibitor SCH772984 for 1 hour and Western blotting were performed (n=3, *P=0.001, **P=0.004). Abbreviations: GIT1, G-protein-coupled receptor kinase-interacting protein 1; HCC, hepatocellular carcinoma; IHC, immunohistochemical; MMP9, matrix metalloproteinase-9; p-ERK1/2, phospho-extracellular signal-regulated kinase 1/2.