钙网蛋白在胰腺癌上皮细胞间质转化中的作用及机制研究

Pancreatic cancer is one of the most malignant digestive tumor with a 5-year survival rate less than 5%. The poor prognosis is mainly due to its strong local invasion, distant metastasis and chemoresistance in early stage. The above process are also promoted by the effect of epithelial mesenchymal transition (EMT). Thus, it is significant for detecting EMT related genes. Nowadays，there is still controversial for the pro/anti-tumor role of Calreticulin(CRT). Recent reports show CRT is closely related with EMT. However, only two studies report the relationship of CRT with EMT in non-cancerous cells. The corresponding mechanism is poorly understood. Our previous study showed CRT was overexpressed in pancreatic cancer, which had positively associated with tumor lymph node metastasis, UICC stage and poor prognosis of patients. Meanwhile, CRT promoted cell invasion, migration,and chemoresistance in pancreatic cancer,and had a specific regulation on ERK/MAPK signal pathway which was also a main regulator of EMT. Therefore, we tried to investigate whether CRT regulated EMT and concomitant cell biological behaviour of pancreatic cancer via EGFR-ERK/MAPK signal pathway in vitro and vivo. This study is meanful for clarifying the definite role of CRT in cancer, revealing mechanism of pancreatic cancer invasion, metastasis and chemoresistance, inhibiting tumor malignant progression by gene targeted therapy, and improving the prognosis of patients with pancreatic cancer.

胰腺癌是恶性度最高的消化道肿瘤，5年生存率低于5%。预后差归因于其早期易发生胰周侵袭和远处转移，以及对化疗药物的耐药。而上皮细胞间质转化（EMT）正是胰腺癌早期侵袭、转移及耐药的助推器。因此探寻EMT相关分子标记物至关重要。钙网蛋白（CRT）是促癌还是抑癌基因尚存在争议，近年发现参与EMT发生，但至今仅有2篇在非肿瘤细胞中的研究，分子机制还不清楚。我们前期研究发现CRT在胰腺癌中高表达与肿瘤UICC分期、淋巴结转移及不良预后呈正相关。同时CRT促进胰腺癌细胞的侵袭、转移和耐药，并特异性调节ERK表达。ERK/MAPK通路同样是EMT的主要调控机制。因此我们拟体外和体内实验揭示CRT是否依赖EGFR-ERK/MAPK通路参与EMT的发生，最终影响胰腺癌的生物学行为。这对阐明CRT在肿瘤中真正作用；胰腺癌发生侵袭、转移及耐药的机制；应用基因靶向治疗抑制肿瘤恶性进展；改善患者预后具有重要意义。

我们以前的研究表明，Calreticulin（CRT）通过调控ERK/MAPK通路促进胰腺癌的发生发展。接下来我们探讨CRT通过整合素Integrin/EGFR-ERK信号MAPK信号通路促进表皮生长因子（EGF）诱导的胰腺癌细胞上皮间质转化（EMT），这还没有相关研究报道。在胰腺癌Capan-2和AsPC-1细胞中，EGF同时诱导EMT和激活Integrin-EGFR/ERK/MAPK信号，表现在EGF促进Vimentin，MMP9，a-SMA，Integrina5，Integrinβ1，纤连蛋白Fibronectin， c-Myc和磷酸化ERK表达，而E-钙粘蛋白E-cad，ZO-1及β-catenin表达明显减弱 。然而，CRT沉默显著抑制EGF的作用，包括抑制EGF诱导EMT样细胞形态，EGF促进细胞侵袭和迁移，和EGF诱导的E-cad的降低和整合素/EGFR-ERK/MAPK信号通路的关键蛋白（Fibronectin、Integrinβ1，c-Myc和pERK）。此外，CRT可以与Fibronectin、Integrinβ1和c-myc在两株胰腺癌细胞中形成免疫共沉淀，同时通过共聚焦发现以上蛋白存在共免疫荧光共定位。以上结果意味着CRT和整合素/ EGFR-ERK信号MAPK信号通路存在密切相互调控作用。在体内，CRT沉默抑制胰腺癌皮下肿瘤生长和肝转移，并下调Fibronectin、Integrinβ1、c-myc和pERK蛋白水平。在组织中，上述蛋白的过度表达与胰腺癌患者恶性临床病理特征和预后不良密切相关。同时与体内和体外实验结果一致，通过相关性分析观察到了以上蛋白存在正相关性。CRT通过Integrin/ EGFR信号通路促进EGF诱导的胰腺癌EMT发生。CRT将是胰腺癌治疗中很有价值的靶点基因。