Research paperTGF-β1 promotes cell migration in hepatocellular carcinoma by suppressing reelin expression
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer and one of the world's top five causes of cancer-related deaths, resulting in approximately 600,000 to 1,000,000 deaths each year in the world (Parkin et al., 2005; Arii et al., 1996; Mazzanti et al., 2008). Even though transplantation and surgical resection are effective treatment options for hepatocellular carcinoma currently (Olsen et al., 2010), uncontrolled tumor metastasis remains the main cause for the poor prognosis of HCC (Liu et al., 2013). Therefore, deepening understanding of the molecular mechanisms of HCC metastasis is essential to develop new therapeutic strategies and improve patients' survival.
It has been well established that initiation of metastasis requires invasion, which is enabled by epithelial–mesenchymal transition (EMT) (Hanahan and Weinberg, 2011). EMT is a process by which epithelial cells loses the cell-cell adhesion and gains migratory and invasive abilities. Decreased expression of epithelial proteins including E-cadherin, and increased expression of mesenchymal proteins such as N-cadherin, fibronectin and vimentin were observed during this process (Chaffer and Weinberg, 2011). The repressed E-cadherin help cancer cells break through the basement membrane and increase invasive properties. It was reported that transforming growth factor-β (TGF-β) is a potent inducer of EMT in cancer, which promotes inactivation of genes encoding E-cadherin and activation of genes encoding N-cadherin and vimentin (Massague et al., 2000; Kasai et al., 2005; Vincent et al., 2009; Xu et al., 2009). Elevated TGF-β1 levels have been observed in the plasma of breast cancer patients and at the invasive fronts of human breast cancer tissues, which correlates well with the observation of lymph node metastasis (Dalal et al., 1993; Chod et al., 2008).
Reelin (RELN) is an extracellular matrix glycoprotein that plays a pivotal role during brain development via manipulating cell-cell interactions (Tissir and Goffinet, 2003). It was reported that RELN is involved in cell migration in neurons. RELN binds to its transmembrane receptors ApoER2 and VLDLR, leading to phosphorylation of Dab1 and src-family kinases (i.e., Src and Fyn) and, through poorly understood intracellular signaling mechanisms, regulates neuronal migration (Gaiano, 2008). In recent years, accumulating studies indicated the important role of RELN in cancer. Suppressed expression of RELN have been observed in several cancer types, including pancreatic cancer, gastric cancer and breast cancer, which is associated with a poor prognosis (Stein et al., 2010; Dohi et al., 2010; Sato et al., 2006). The negative effect of RELN seems to be linked with its ability promoting cell migration. As people shown the inhibition of RELN pathway in cancer cells with normal RELN expression increases cell motility and invasiveness (Sato et al., 2006). In HCC patients' tissues, the hypermethylation of RELN promoter and decreased expression of RELN was also observed (Okamura et al., 2011). Interestingly, it was shown TGF-b1 negatively regulates Reelin expression through Snail in KYSE-30 cells (Yuan et al., 2012). Snail is a zinc finger transcriptional repressor, TGF-b1 treatment increases Snail expression and binding to RELN promoter. The information suggests that RELN may play a critical role in regulation of HCC cell migration.
In this study, we explored the effect of TGF-β1 and RELN on cell migration in HCC cells. We also primarily investigated the relationship between TGF-β1 and RELN. Our data showed that TGF-β1 promotes cell migration while RELN suppresses cell migration, which is consistent with the opposite expression pattern of these two genes observed in HCC cells. Moreover, we also revealed that TGF-β1 enhanced cell migration ability is through inhibiting RELN expression. Overexpression of RELN impaired TGF-β1 induced cell migration. Taken together, our data uncovered that TGF-β1 could promote cell migration by suppressing RELN expression.
Section snippets
Cell culture
All the cell lines (HepG2, Hep3B, SMMC-7721, Bel-7402, Huh7 and L02) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated newborn calf serum, 5100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in 5% carbon dioxide (Gibco Life Technologies, Grand Island, NY, USA). The recombinant human SHH peptide (rhSHH) (R&D Systems, Inc., Minneapolis, MN,
Comparing the expression of TGF-β1 and RELN between normal liver cell and HCC cells
As TGF-β1 was suggested to be a potential candidate as a predictor for HCC, and HCC patients were reported to have significantly higher serum TGF-β1 (Watanabe et al., 2016; Ma et al., 2015), we first examined the expression of TGF-β1 in both normal liver cell and tumor cells. As shown in Fig. 1A and B, liver tumor cells have a significant higher expression of TGF-β1 compared with normal liver cell. Interestingly, an opposite expression pattern of RELN was observed. Both the mRNA and protein
Discussion
Metastasis is key factor in malignant growth of cancer and is associated with poor prognosis. Cancer cells detach from the tumor mass and invade the extracellular matrix and basement membrane. EMT is an essential step for cancer cells to gain these abilities. During this process, gene expression profile alterations, such as decreased expression of epithelial protein E-cadherin and increased expression of mesenchymal proteins including N-cadherin, fibronectin and vimentin are important to
Competing interests
The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article.
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