| Background:Pancreatic cancer,known as“the King of cancer",is one of the most malignant human tumors.As a result of the difficulty of the early diagnosis and the limitation of the current treatments of pancreatic cancer,the prognosis of patients with pancreatic cancer is still poor now.90%of pancreatic cancer is pancreatic ductal adenocarcinoma(PDAC).Therefore,it is very urgent and necessary to search for new tumor biomarkers with clinical significance to predict the occurrence and development of pancreatic cancer and to design and develop targeted drugs.Objective:1.To investigate the expression and localization of Asporin and CD44 in PDAC and their clinical significance.2.To explore the effects of Asporin/CD44 signaling axis on the invasion and migration of pancreatic cancer and the underlying mechanisms.Methods:Part 1:Expression and localization of Asporin and CD44 in PDAC and their clinical significanceThe clinical,pathological and prognostic data and paraffin tissues specimens of 132 cases of PDAC and 50 cases of peri-cancerous pancrea were collected.Immunohistochemistry(IHC)and tissue immunofluorescence were used to evaluate the expression and location of Asporin and CD44 in PDAC tissues and peri-cancerous normal tissues.All the results were analyzed by SPSS16.0 statistical software.Part 2:In vitro experimentsEffects of Asporin/CD44 signaling axis on the invasion and migration of PDAC and the underlying mechanism1.Expression and location of Asporin in human activated and deactivated PSCs:Activated human PSCs were induced into the deactivated condition by all-trans retinoic acid(ARTA)after isolated by the outgrowth method and identified by oil red staining and a-Smooth Muscle Actin(a-SMA)immunofluorescence staining.The expression and localization of Asporin in activated and deactivated PSCs were detected by gene chip,RNA sequencing,reverse transcriotion-polymerase chain reaction(RT-PCR),Western Blot and immunofluorescence staining.2.Effects of paracrine Asporin on the capacity of invasion and migration of pancreatic cancer cell lines(Mia PaCa-2 and PANC-1):After knocking down Asporin in PSCs via siRNA,observe and compare the invasion and migration ability of pancreatic cancer cells which were treated by the supernatants of Asporin-knockdown PSCs and control PSCs.3.The expression of Asporin and CD44 in pancreatic cancer cell lines(AsPc-1,BxPC-3,Mia PaCa-2 and PANC-1)and the interaction between Asporin and CD44:The expression of Asporin and CD44 in pancreatic cancer cell lines was detected by RT-PCR,Western Blot and immunofluorescence.And the interaction between Asporin and CD44 was verified by CO-IP.4.The underlying mechanism of paracrine Asporin in the invasion and migration of pancreatic cancer:1)After pretreating Mia PaCa-2 and PANC-1 with rhAsporin,CD44 neutralizing antibody or NF-kappaB transcription factor inhibitor,observe and compare the change of invasion and migration ability,NF-KB transcriptional activity,EMT-related protein,AKT and ERK signaling pathways;2)After pretreating Mia PaCa-2 and PANC-1 by rhAsporin,CD44 neutralizing antibody,AKT inhibitor or ERK inhibitor,detect the change of NF-kappaB transcription activity.5.The effects and underlying mechanism of autocrine Asporin on the invasion and migration of pancreatic cancer:After Asporin was knocked down by siRNA in Mia PaCa-2 and PANC-1,observe the change of the invasion and migration ablility,EMT-related protein,TIMP/MMP family protein,AKT signaling pathway,ERK signaling pathway and NF-kappaB transcription activity in Asporin knockdown group and control group.Part 3:In vivo ExperimentsThe effect of Asporin on the invasion and migration of PDACConstruct pancreatic cancer nude mice orthotopic tumor model by using Asporin-knockdown PANC-1 cell line and control PANC-1 cell line.Then observe the invasion and migration of the pancreatic cancer in two groups,respectively.Results:Part 1:Expression and localization of Asporin and CD44 in PDAC and their clinical significanceAsporin was predominately expressed in the cytoplasm of stromal cells adjacent to pancreatic cancer epithelium and the positive rate was 81.06%(107/132).Asporin could also be expressed in cytoplasm of pancreatic cancer epithelium and the positive expression rate was 24.2%(32/132).However,Asporin was not expressed or weakly expressed in ductal epithelium and stromal cells of normal pancreatic tissues.CD44 was mainly expressed on the membrane of pancreatic tumor epithelial cells and the positive rate was 31.06%(41/132).CD44 could also be expressed in the stromal cells of pancreatic cancer in a fraction of pancreatic cancer tissues.In the normal pancreatic stroma and ductal epithelium,CD44 was weakly expressed or not expressed.Univariate and multivariate analysis suggested that the positive expression of Asporin in tumor stromal cells indicated poor prognosis(P=0.012).Although the positive expression of Asporin in tumor cells was associated with tumor differentiation(P<0.000),there was no correlation between the positive expression of Asporin in tumor cells and poor clinical outcomes(P=0.772).In addition,the positive expression of CD44 was correlated with the prognosis of the pancreatic cancer patients(P=0.004).Part 2:In vitro experimentsEffects of Asporin/CD44 biological axis on the invasion and migration of PDAC and the underlying mechanism1.The expression of Asporin in activated PSCs was significantly higher than that in deactivated PSCs;2.The promotion of the invasion and migration of pancreatic cancer cells by activated PSCs supernatant was significantly attenuated after Asporin knockdown;3.There is a physical interaction between Asporin and CD44 in pancreatic cancer;4.Asporin/CD44 biological axis could promote the nuclear accumulation of NF-?B in Mia PaCa-2 and PANC-1 by activating AKT and ERK signaling pathways.Then the nuclear accumulation of NF-?B promotes the invasion and migration of Mia PaCa-2 and PANC-1 by regulating the EMT biological process;5.The invasion and migration ability of Mia PaCa-2 and PANC-1 were significantly reduced after Asporin knockdown.Asporin knockdown could partially reverse the biological process of EMTand partially inhibited the activation of AKTsignaling pathway,ERK signaling pathway and its downstream the nuclear accumulationof NF-?B.And the expression of TIMP/MMP family protein was not significantly affected by Asporin knockdown.Part 3:In vivo ExperimentsThe effect of Asporin on the invasion and migration of PDACIn order to further confirm the promotion effects of Asporin on pancreatic cancer,we construct pancreatic cancer nude mice orthotopic tumor model by using PANC-1-Asporin-knockdown cell line and PANC-1-NC cell line.We observed that although no local invasion of adjacent organs or distant metastasis were observed in either group by both PET and gross/microscopic examination,Asporin knockdown could significantly reduced the malignant degree of PANC-1 cells.For example,at low magnification,we found that the median diameters were 4.4mm and 3.7mm in PANC-1-NC and PANC-1-Asporin-knockdown groups.And tumors typically infiltrated adjacent pancreas parenchyma diffusely in PANC-1-NC group,whereas an expansive growth with relatively clear border with the neighboring pancreas parenchyma was observed in PANC-1-Asporin-knockdown groups.At high magnification,the mitotic cells in PANC-1-Asporin-knockdown group were significantly less than those in the control group.Conclusion:1.PSC-induced EMT,invasion and migration of PCCs were mediated by the Asporin/CD44 axis.2.The underlying mechanisms involved the AKT and ERK signaling pathways and their downstream transcription factor NF-?B/p65.3.The Asporin/CD44 axis may also exert its effects via an autocrine mechanism in PCCs.4.Asporin may play a critical role in the PSC-PCC interaction and might be a promising therapeutic target and novel prognostic biomarker in pancreatic cancer. |
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