| Intrahepatic cholangiocarcinoma (ICC) is a primary malignant lethal primary epithelial cancer of the hepatobiliary tract that exhibits characteristics of cholangiocyte differentiation. It represents the secondary liver neoplasm after hepatocellular carcinoma and accounting for 5% to 10% of primary liver cancers[1]. Although considered to be an orphan cancer, the incidence of ICC has increased widely worldwide in recent decades, especially in parts of Eastern Asia[2,3]. At the time of diagnosis, patients generally have disease beyond the limits of resection because of an advanced tumor stage and the resectability rate has been quite low and variable (18-70%) [3]. Unfortunately, the great mass of ICC patients have a poor prognosis even after surgical resection. Thus, an improved understanding of the molecular mechanisms associated with ICC progression is needed and would be beneficial in developing effective therapeutic strategies.The calpain system is an important group of over 12 known mammalian intracellular limited proteases that share a similar catalytic structure [4]. The calpain system has been implicated in tumorigenesis through altered expression and activity in a wide variety of tumor types [5], including breast cancer [6,7], ovarian cancer and pancreatic cancer [8]. Among them, the two ubiquitous isoforms, Capnl and Capn2 are the most widely studied family members and defined by their calcium requirements for in vitro activation [5]. As a subunit of calpains, calpain large subunit 1 (Capn1) plays an important role in calpain catalytic activity However, only few sporadic studies have focused on the role of Capnl in ICC. In our study, we detected the expression of Capn1 in 140 human ICC tissues and matched non-tumor tissues. Then, we inquired into the correlation between Capnl expression and clinicopathological modulus and identified whether Capnl could be a crucial factor for clinical diagnosis in ICC patients. Using siRNA-mediated knockdown expression of Capnl, we also assessed that if Capn1 was involved in ICC cells invasion, migration and proliferation in vitro. Additionally, we further investigated the relationship between the expression Capnl and the expression of the EMT-related markers snail and E-cadherin in ICC samples and cells.Part 1 Expression of Capnl, E-cadherin and Snail in intrahepatic cholangiocarcinoma and the correlation between themObjective:To investigate the expression of Capnl, E-cadherin and Snail in ICC tissues and matched non-tumor tissues and its correlation between them.Methods:Immunohistochemistry was applied in the TMAs of 140 ICC patients’samples to detect the expression of Capn1, E-cadherin and Snail. Western bolt was also used to evaluated the Capnl expression in 4 pair of ICC tumor and peritumor tissues. The correlation between Capnl, E-cadherin and Snail were analyzed by SPSS 19.0Result:Capnl overexpression was correlated with correlated with serum CA19-9 level and tumor number in ICC tissues (p<0.05). Compared with the low expression of Capnl patients, Capnl overexpression patients had a lower OS and higher cumulative recurrence rate. Capnl was an independent prognosis factor in OS and cumulative recurrence rate in univariate and multivariate analysis.Western blot and immunohistochemistry results reveled that Capnl and Snail was elevated and E-cadherin was decreased in ICC tissues. The expression of Capn1 was positive correlated with the expression of Snail and negative with the expression of Snail (p<0.05). Patients with an early recurrence ICC had a higher expression of Capn1 (p<0.05).Conclusions:Capnl and Snail was highly expressed in ICC samples and E-cadherin was low expression in ICC samples. The expression of Capnl was positive correlated with the expression of Snail and negative with the expression of Snail.Part 2 Clinical significance of Capnl and the prognostic value of Capnl, E-cadherin and snail in intrahepatic cholangiocarcinomaObjective:To investigate the Clinical significance of Capnl and the prognostic value of Capnl, E-cadherin and snail in intrahepatic cholangiocarcinoma.Methods:In this part, we applied immunohistochemistry to investigate the expression of Capnl, E-cadherin and Snail in the TMAs of 140 ICC patients’samples to detect the expression of Capnl, E-cadherin and Snail. The clinical significance of Capnl and the prognostic value of Capnl, E-cadherin and snail was analyzed by SPSS 19.0Result:Capn 1 overexpression was correlated with correlated with serum CA19-9 level and tumor number in ICC tissues (p<0.05). Compared to the patients with high expression of Capnl, Snail and low expression of E-cadherin, patients with low expression of Capnl, Snail and high expression of E-cadherin had a lower overall and higher cumulative recurrence rate. Capnl was an independent prognosis factor in OS and cumulative recurrence rate in univariate and multivariate analysis.Conclusions:High Capn1 expression was associated with serum CA19-9 level, tumor number and TNM stage. High expression of Capn1 and Snail and low expression of E-cadherin indicated a poor prognosis in ICC.Part 3 Correlation of Capn1 with the invasion, migration, proliferation and epithelial-mesenchymal transition in Intrahepatic CholangiocarcinomaObjective:To investigate the relationship between Capnl expression and the invasion, migration, proliferation and epithelial-mesenchymal transition in ICC cells.Methods:Expression of Capnl was knocked down in HCCC-9810 and QBC939 cells using RNA interference. Decreased Capnl expression was validated by qRT-PCR and western blot. The invasion and migration ability of ICC cells were measured by transwell and wound healing assays respectively. The MTT assays were used to determine the cells’proliferation. The expression of EMT (epithelial-mesenchymal transition) related markers snail and E-cadherin were evaluated in Capnl down-regulated ICC cells lines.Result:Capn4 expression was reduced by siRNA successfully. Decreased Capnl expression was accompanied by impaired invasiveness, migration and proliferation in ICC cells (p<0.05). The expression of EMT related markers snail and E-cadherin were significantly correlated with Capnl expression in ICC cell lines.Conclusions:Capnl inhibition attenuated the invasion, migration and proliferation of ICC cells in vitro and negatively correlated with the epithelial-mesenchymal transition. |
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