The Effect Of Laminin Receptor Overexpression In The Cholangiocarcinoma Cell Immune Escape

Purpose: Tumor invasion and metastasis is a multistage process, involving adherence, movement and proliferation of tumor cells, degradation of extracellular matrix and tumor angiogenesis. In addition, immune system plays an important role in this process. In most cases, escaping from immunologic surveillance and aggression, tumor cells can grow unrestrictedly and metastasize to distant place. Fas and its ligand FasL, which were originally discovered as glucoprotein, by mediating apoptosis, take part in the immunological homeostasis and immune amnesty and are important for the maintenance of physiologic functions of immune privilege organs, such as eyes and testes. Meanwhile, the significance of Fas and FasL in the development of tumors has attached great importance. It was found that the expression of Fas decreased and the expression of FasL increased. The changes of their expression were considered to facilitate the tumor invasion and metastasis by inducing apoptosis of anti-tumor lymphocytes and promoting immune escape of tumor cells.With the facts that the overexpression of Laminin receptor (LNR) was a common manifestation in tumor invasion and metastasis and that Fas/FasL may play a great role in tumor invasion and metastasis by mediating immune escape, we hypothesized that LNR may participate in the process of immune escaping by regulating Fas/FasL signal pathway. Intercellular adhesion molecule-1 (ICAM-1), the primary ligand of lymphocyte function associated antigen-1(LFA-1), is an important molecule mediating cell's adherence and identification. The decrease or even loss of the expression of ICAM-1 would facilitate the tumor cells escaping from the recognization and attack of immune competent cells such as cytotoxic T lymphocytes, NK cells, etc. With the recognition of the intimate relationship between LNR and ICAM-1, we presumed that the LNR overexpression might interfere with recognizing and combining of immune competent cells by impacting the expression of some important molecules, such as ICAM-1, and then promoting the immune escaping of tumor cells mediated by Fas/FasL signal path. On the other hand, down regulation of the expression of LNR on tumor cells could cripple the cholangiocarcinoma cells'resistance of immune response, and thus inhibit the invasion and metastasis of tumor cells.Experimental Design: 1. Investigating the effect of LNR overexpression on the biological behavior of cholangiocarcinoma. The expression of LNR and the cholangiocarcinoma cell strain with high expression of LNR was separated by flow cytometry (FCM). H&E staining, SEM and TEM were used to observe the morphological changes of the cells before and after separation. The expression of skeleton protein was checked by cytoskeleton staining and the invasion and metastasis capability of cholangiocarcinoma cells was observed. 2. Investigating the effect of LNR overexpression on the proteins associated with immune escape of cholangiocarcinoma cells. Down regulation of LNR expression on high metastatic cholangiocarcinoma cell was induced by transfection with LNRshRNA, and the changes of genes expression (FasL, MMP-2, MMP-9, ICAM-1, etc) were detected by real-time PCR, western blot and gelatinase zymography. 3. Approaching the effect of LNR overexpression on the expression of ICAM-1 in cholangiocarcinoma and the molecular mechanism. Using specific inhibitor of metalloproteinase (GM6001) on cholangiocarcinoma cells which were transfected by LNR shRNA, the expression of intercellular adhesion molecule-1 was detected. 4. To investigate the effect of LNR overexpression on the immune competent cells. We established the direct contact and Transwell? co-culture system of Jurkat and cholangiocarcinoma cell, the apoptosis of Jurkat was observed by TUNEL.Results:1. The expression of LNR in cholangiocarcinoma cells with higher metastatic potential was significantly higher than that in cells with lower metastatic potential. The high expression of LNR was related with high metastatic potential in cholangiocarcinoma cells.2. The cytoskeleton staining showed that the cytoskeleton structure of cholangiocarcinoma cells with overexpression of LNR was disordered, and the numbers of the actin body, cell ecphyma and filament were increased. 3. According to the observation by SEM and TEM, the cell surface projection in cholangiocarcinoma with LNR overexpression was more, the lamellar pseudopodia was thicker and the filopodia was more intensive that in cells with lower expression of LNR.4. The kinetic and invasive experiment showed that the moving and invasive capability of cholangiocarcinoma cells with higher expression of LNR enhanced compared with the cells with lover expression of LNR.5. The LNR RNAi plasmid was constructed successfully, and its quality was ensured by sequencing and identification.6. LNR expression in cholangiocarcinoma cells with high metastatic potential was down-regulated after transfected with LNR shRNA, the mRNA and protein expression level of FasL was degraded accordingly, meanwhile the mRNA and protein expression of MMP-2,MMP-9,uPA were degraded, but ICAM-1 which express on cell surface was increased. After using specific inhibitor of metalloproteinase on cholangiocarcinoma cell which was transfected by LNR shRNA, ICAM-1 which express on cell surface was increased compareed with the cell without inhibitor. It suggested that the overexpression of LNR induce the degradation of ICAM-1 of tumor cells by activating the MMP-2 and MMP-9, and lead to the immune escaping of tumor cells.7. LNR expression in cholangiocarcinoma cells with high metastatic potential was down-regulated after transfected with LNR shRNA, and at the same time the mRNA and protein expression level of FasL was degraded accordingly. It would be concluded that there was inherent relationship between the overexpression of LNR and the expression of FasL.8.The apoptotic index of Jurkat cells co-cultured with cholangiocarcinoma cell transfected with LNR shRNA was obviously smaller than that co-cultured with cholangiocarcinoma cell without transfection. It was suggested that the overexpression of LNR would play an important role in the process of immune escape of cholangiocarcinoma.Conclusions:1.The higher expression of LNR may be related with higher metastatic potential of cholangiocarcinoma cells.2.The overexpression of LNR may induce the degradation of ICAM-1 of tumor cells by activating the MMP-2 and MMP-9, and lead to the immune escaping of tumor cells and then facilitate the invasion and metastasis of cholangiocarcinoma cells.3 . Overexpression of LNR may up regulate the transcription of FasL in cholangiocarcinoma cells, and induce the immune escaping and promote the invasion and metastasis of tumor cells through the Fas/FasL signal path.