Pei Tiemin1, Meng Fanzheng1, Xiao Peng1, Han Jihua1, Song Ruipeng1, Lan Yaliang1, Wang Yan1, Xue Junlin1, Lang Qingfu1, He Zhefeng1, Li Jian1, Guo Zihao1, Liu Guoxing1, Sun Boshi1, Zhao Ming1, Meng Qinghui2, Liang Desen3, Liu Lianxin4. 1. Department of General Surgery, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China. 2. Department of General Surgery, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China. Electronic address: hui-90@163.com. 3. Department of General Surgery, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China. Electronic address: tiemin2008@163.com. 4. Department of General Surgery, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China. Electronic address: liulianxin@ems.hrbmu.edu.cn.
Abstract
BACKGROUND & AIMS: Mucin 13 (MUC13) is reportedly overexpressed in human malignancies. However, the clinicopathological and biological significance of MUC13 in human intrahepatic cholangiocarcinoma (iCCA) remain unclear. The aim of this study was to define the role of MUC13 in the progression of iCCA. METHODS: Expression levels of MUC13 in human iCCA samples were evaluated by immunohistochemistry, western blot, and real-time PCR. In vitro and in vivo experiments were used to assess the effect of MUC13 on iCCA cell growth and metastasis. Crosstalk between MUC13 and EGFR/PI3K/AKT signaling was analyzed by molecular methods. The upstream regulatory effects of MUC13 were evaluated by Luciferase and DNA methylation assays. RESULTS: MUC13 was overexpressed in human iCCA specimens and iCCA cells. MUC13 overexpression positively correlated with clinicopathological characteristics of iCCA, such as vascular invasion and lymph node metastasis, and was independently associated with poor survival. Results from loss-of-function and gain-of-function experiments suggested that knockdown of MUC13 attenuated, while overexpression of MUC13 enhanced, the proliferation, motility, and invasiveness of iCCA cells in vitro and in vivo. Mechanistically, we found that the phosphatidylinositol 3-kinase-AKT signal pathway and its downstream effectors, such as tissue inhibitor of metalloproteinases 1 and matrix metallopeptidase 9, were required for MUC13-mediated tumor metastasis of iCCA. MUC13 interacted with epidermal growth factor receptor (EGFR) and subsequently activated the EGFR/PI3K/AKT signaling pathway by promoting EGFR dimerization and preventing EGFR internalization. We also found that MUC13 was directly regulated by miR-212-3p, whose downregulation was related to aberrant CpG hypermethylation in the promoter area. CONCLUSIONS: These findings suggest that aberrant hypermethylation-induced downregulation of miR-212-3p results in overexpression of MUC13 in iCCA, leading to metastasis via activation of the EGFR/PI3K/AKT signaling pathway. LAY SUMMARY: Mucin 13 overexpression has been implicated in the development of malignancies, although its role in intrahepatic cholangiocarcinoma has not been studied. Herein, we show that mucin 13 plays a critical role in intrahepatic cholangiocarcinoma. Mucin 13 could have therapeutic value both as a prognostic marker and as a treatment target.
BACKGROUND & AIMS: Mucin 13 (MUC13) is reportedly overexpressed in humanmalignancies. However, the clinicopathological and biological significance of MUC13 in humanintrahepatic cholangiocarcinoma (iCCA) remain unclear. The aim of this study was to define the role of MUC13 in the progression of iCCA. METHODS: Expression levels of MUC13 in humaniCCA samples were evaluated by immunohistochemistry, western blot, and real-time PCR. In vitro and in vivo experiments were used to assess the effect of MUC13 on iCCA cell growth and metastasis. Crosstalk between MUC13 and EGFR/PI3K/AKT signaling was analyzed by molecular methods. The upstream regulatory effects of MUC13 were evaluated by Luciferase and DNA methylation assays. RESULTS:MUC13 was overexpressed in humaniCCA specimens and iCCA cells. MUC13 overexpression positively correlated with clinicopathological characteristics of iCCA, such as vascular invasion and lymph node metastasis, and was independently associated with poor survival. Results from loss-of-function and gain-of-function experiments suggested that knockdown of MUC13 attenuated, while overexpression of MUC13 enhanced, the proliferation, motility, and invasiveness of iCCA cells in vitro and in vivo. Mechanistically, we found that the phosphatidylinositol 3-kinase-AKT signal pathway and its downstream effectors, such as tissue inhibitor of metalloproteinases 1 and matrix metallopeptidase 9, were required for MUC13-mediated tumor metastasis of iCCA. MUC13 interacted with epidermal growth factor receptor (EGFR) and subsequently activated the EGFR/PI3K/AKT signaling pathway by promoting EGFR dimerization and preventing EGFR internalization. We also found that MUC13 was directly regulated by miR-212-3p, whose downregulation was related to aberrant CpG hypermethylation in the promoter area. CONCLUSIONS: These findings suggest that aberrant hypermethylation-induced downregulation of miR-212-3p results in overexpression of MUC13 in iCCA, leading to metastasis via activation of the EGFR/PI3K/AKT signaling pathway. LAY SUMMARY: Mucin 13 overexpression has been implicated in the development of malignancies, although its role in intrahepatic cholangiocarcinoma has not been studied. Herein, we show that mucin 13 plays a critical role in intrahepatic cholangiocarcinoma. Mucin 13 could have therapeutic value both as a prognostic marker and as a treatment target.