| Background：A number of studies have suggested that67kDa Laminin receptor (67LR)is associated with tumor invasion and metastasis. Our previous studies demonstrated that67LR is highly expressed in cholangiocarcinoma (CCA) tissues and improved theinvasiveness of CCA cell line QBC939in vitro.67LR is a cell surface receptor has a highaffinity for its primary ligand laminin (LN). Binding of67LR to LN leads to cell adhesionto the basement membrane and activation of intracellular signal transduction, which mayinitiate the invasion and metastasis cascades within tumor cells. Though67LR wassuggested to induce the expression of MMP-2in tumor procession, however, themechanism by which this occurs and how67LR improve the motility of tumor cells is notcompletely understood at present. A recent research showed that67LR was probablyinvolved in epithelial–mesenchymal transition (EMT), modulation of b-catenin expression,and migratory activity of adenoid cystic carcinoma cells CAC2. Our previous studiesshowed that lysyl oxidase-like2(Loxl2) protein was highly expressed in CC, andassociated with differentiation, metastasis and EMT of CCA. Loxl2enhances theaggressiveness of CCA cell line QBC939by inducing EMT in vitro and in vivo. Also,several researchers have observed upregulated levels of Loxl2in breast, colon, esophageal,pancreatic and prostate cancer. Loxl2is key promoter of EMT that mediates the tumor cellsfrom stable epithelial cells to kinetic mesenchymal cells; this change is accompanied bydecreased expression of E-cadherin and upregulation of vimentin and fibronectin. Loxl2contains four repeated copies of scavenger receptor cysteine-rich (SRCR) domains in itsN-termini. The SRCR domains which were found among cell surface and secreted proteinsare known to mediate protein-protein interaction, cell adhesion and cell signaling. All thesestudies enlightened us to study the contribution of Loxl2to67LR-promoted invasive andmetastatic potentials in CC.MATERIALS AND METHODS1.67LR and LOXL2were detected in73cases of Cholangiocarcinoma via immunochemistry staining. The correlations of the proteins and the correlation betweenexpression of proteins and clinical pathological properties were analyzed. Then overallsurvival (OS) and progression-free survival (PFS) were analyzed correlated to pathologicalstaging of CCA and67LR/LOXL2protein expression.2. We cloned67LR and LOXL2into Lentivirus to knockdown or upregulation67LRand LOXL2expression in CCA cell lines QBC939and RBE.3. We measured the metastatic abilitity in vivo of QBC9393and RBE after knockdownor upregulation of LOXL2.4. ex-67LR were transfected into QBC939cells with or without knockdown of LOXL2,and the expression of67LR, LOXL2and the invasive migratory properties of CCA cellswere investigated.5. CCA animal model was established, injecting QBC939or QBC939/LOXL2-/-cellsinto CCA animal model, taking specimens for pathological examination,immunohistochemistry and EMT.RESULTS1.67LR and LOXL2expressed in35.62%and32.88%in73CCA tissues, respectively.Expression levels of67LR and LOXL2were correlated with differentiation, lymph nodemetastasis and TMN stage, respectively (P<0.05).67LR and LOXL2negatively correlatedwith the positive survival time of CCA patients (P<0.05). Positive correlation betweenexpression of67LR and LOXL2were found in73CCA tissues.2. shRNA-67LR and ex-67LR lentivirus were successfully constructed and subcolonesof QBC939and RBE cells with knockdown or overexpression of67LR were selected. InQBC939and RBE and subclones, knockdown of67LR induced decreased expression ofLOXL2while overexpression of67LR induced increased expression of LOXL2. Also,shRNA-LOXL2and ex-LOXL2lentivirus were succesfully constructed and transfected intoQBC939and RBE cells, but no effect to67LR.3. In vitro, migrative ability of QBC939and RBE cells decreased in subclones withknockdown of LOXL2while increased in cells with up-regulation of LOXL2.4. Knockdown of LOXL2reduced migrative ability in QBC939and RBE withup-regulated67LR expression.5. In vivo, result of animal experiments verify the results of in vitro experiment, confirmed the LOXL2play the key role in the67LR-mediated signal pathway. Meanwhile,we detected EMT. RT-qPCR and Western blot have confirmed the occurrence of EMT.Conclusion1.The expression of67LR and LOXL2was closely correlated withdifferentiation,lymph node metastasis and TNM stage in CCA tissues. Expression of67LRand LXOL2negatively with postoperative survival time in CCA patients.2. Expression of67LR correlated positively with expression of LOXL2in CCA tissuesand CCA cell line. And67LR regulated expression of LOXL2in QBC939and RBE cells invitro.3.67LR-LOXL2signal pathway promoted invasive and metastasive ability of QBC93and RBE cells in vitro. Knockdown of LOXL2could decrease invasive and metastasiveability of QBC939and RBE both in vitro and in vivo. LOXL2play the key role in67LR-LOXL2signal pathway.4.67LR-LOXL2signal pathway may be correlated with EMT. |
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