| Background:Hepatocellular Carcinoma (HCC) is the sixth most common human malignancy in the world, with unfavourable prognosis and a high mortality. HCC ranks as the third leading cause of cancer-related death worldwide, and is a serious threat to the life and health of Chinese. Overall, the long-term survival of HCC remains poor because of the high rates of recurrence and metastasis after hepatectomy. Therefore, studies on the invasion and metastasis of HCC will contribute to explore new therapeutic interventions for HCC and then further improve the treatment level of HCC.Endothelial cell-specific chemotaxis regulator (ECSCR) is a recently identified type I transmembrane protein, preferentially expressed in endothelial cells and vessels of mammalians. It has been reported ECSCR has lots of functions such as promotion of apoptosis, reinforcement of cell-cell adhesion and inhibition of cell migration. Therefore, we speculate ECSCR may play an important role in invasion and metastasis of HCC by regulating apoptosis and cell migration.Aims:To measure ECSCR expression profiles in human HCC tissues and cell lines, and subsequently investigate the correlation of ECSCR expression with prognosis and clinical pathological features; to further validate the biological function and mechanism of ECSCR in HCC by experiments in vivo and vitro.Methods and Results:1. ECSCR is significantly reduced in HCC tissues and cell lines. Firstly, Expression of ECSCR was measured by Real-time PCR, RT-PCR and Western-blot in HCC tissues and adjacent nontumorous liver tissues (ANLT). We discovered that both the mRNA (0.271±0.064vs.1.271±0.153, P<0.05) and protein (0.234±0.047vs.1.097±0.196, P<0.05) expression levels of ECSCR in HCC tissues were significantly lower than those in the corresponding adjacent nontumorous liver tissues. Meanwhile, ECSCR expression profiles were measured by Real-time PCR and Western-blot in normal liver cell line L02and four HCC cell lines HepG2, SMMC-7721, MHCC97-L and HCCLM3. The results indicated that the ECSCR expression was significantly higher in L02cell line than other HCC cell lines (P<0.05).2. The low expression of ECSCR is closely related to clinic pathologic features and prognosis of HCC. We investigated the ECSCR expression in97cases of HCC by immunohistochemistry (IHC). The results showed that the positive expression rate of ECSCR in HCC was significantly lower than that in ANLT (59.8%vs.100.0%, P<0.001). The ECSCR expression was related to tumor size (P=0.036), number of nodules (P<0.001), capsule formation (P=0.001), vein invasion (P=0.001), BCLC staging (P=0.002), TNM classification (P<0.001) and clinical classification of HCC (P=0.001). We found that the patients with low expression of ECSCR had poorer disease-free survival (22.5±3.4vs.41.2±3.6Months,P=0.002) and overall survival (31.1±4.6vs.49.0±3.2Mon, P=0.005) than those with high expression of ECSCR. Multivariate Cox regression analysis revealed that low expression of ECSCR (HR=2.183, P=0.040), vein invasion (HR=3.054, P=0.015), without capsule formation (HR=4.239, P=0.004), BCLC staging (HR=2.469, P=0.046), TNM classification (HR=2.768, P=0.014) and clinical classification of HCC (HR=2.368, P=0.038) were independent risk factors for HCC, suggesting that low expression of ECSCR indicated poor prognosis of HCC.3. ECSCR inhibits the proliferation and migration of HCC cells in vitro. We used lentivirus transfection approach to upregulate the expression of ECSCR in SMMC-7721and HCCLM3cells (SMMC-7721ECSCR, HCCLM3ECSCR) and corresponding vector control cell lines (SMMC-7721Control and HCCLM3Control). The results showed that the growth rates and colony forming numbers of ECSCR overexpression cells had decreased when compared with the vector control ones (P<0.01). The Wound-Healing assay showed that the migration of ECSCR overexpression cells had significantly slower than that of control cells (47%vs.96%, P<0.001;27%vs.86%, P<0.001); and Transwell assay showed a decreased invasion ability of ECSCR overexpression cells (33±8vs.134±11, P<0.001;12±4vs.56±7, P<0.001), comparing to the control ones.4. ECSCR regulates the growth and invasion of HCC by inhibiting TGFβ1signaling. We firstly established a cancer-10-pathway reporter assay in ECSCR overexpression and control cells. The luciferase activity of TGFβ1signal pathway in ECSCR overexpression cells showed a significantly downregulation (P<0.01). We further detected the expression profiles of corresponding proteins by Western-blot. The results showed that the expression levels of TGFβ1(0.154±0.024vs.1.014±0.127, P<0.05), p-ERK (0.479±0.093vs.2.118±0.406, P<0.05) and p-Smad2/3(0.249±0.048vs.1.471±0.215, P<0.05) proteins in ECSCR overexpression cells were down-regulated, while there was no significant change of ERK (0.977±0.208vs.1.012±0.241, P=0.836) or Smad2/3(1.053±0.312vs.0.979±0.283, P=0.903) expression.5. ECSCR suppresses cell proliferation and metastasis of HCC in vivo. We further constructed HCC orthotropic xenograft tumor model in nude mice. Our results showed that the nude mice in HCCLM3ECSCR group had smaller tumor sizes (0.24±0.15vs.0.72±0.39cm3, P<0.05) and a dramatically lower rate of the intrahepatic (33.3%vs.83.3%, P<0.05) and pulmonary (16.7%vs.66.7%, P<0.05) metastasis than those in HCCLM3Control group.Conclusions:In this study, we firstly identified ECSCR was down-regulated in HCC tissues and cell lines, and the expression level of ECSCR was closely correlated to clinic pathological characteristics as well as prognosis of HCC. Upregulation of ECSCR can significantly suppress the cell proliferation and invasion of HCC in vivo and vitro, through regulating TGFβ1signal pathway. Our study suggests ECSCR could be a novel prognostic marker and a therapeutic target for HCC. |
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