The Effect And Preliminary Mechanism Of ASPM In Angiogenesis, Invasion And Metastasis Of Pancreatic Ductal Adenocarcinoma

Background:Pancreatic cancer,one of the most malignant tumors has worse prognosis,which morbility is increasing in recent years.Exploring the molecular mechanisms of invasion and metastasis of pancreatic ductal adenocarcinoma(PDAC),it would be helpful to understand tumorigenesis and progress,and provide more clinical and cancer therapy evidence.A considerable number of literatures reported that angiogenesis,extracellular matrix(ECM)degradation and epithelial-mesenchymal transition(EMT)correlated with invasion and metastasis.Anti-angiogenesis,ECM degradation and EMT inhibition might play roles in cancer therapy.ASPM,a gene resulted in microcephaly,was down-regulated during tubulogenesis of PDAC,but up-regulated in PD AC cell lines,which promoted aggressiveness of PD AC by maintaining the stabilization of Wnt signal pathway and cancer stem cell characteristics.However,it was unclear whether or not ASPM might participate in angiogenesis,ECM or EMT,and it was also unclear whether or not ASPM might regulate angiogenesis,ECM or EMT through Wnt signal pathway.LRP16,leukemia related protein,a response gene to ERa signaling,which might involve the progress of many tumors,was not reported in PDAC,and it was unclear whether LRP16 might participate in tumorigenesis,invasion and metastasis of PD AC,and its relationship with ASPM need to be investigated in detail.Objective:To evaluate the role of ASPM in PDAC and to identify the possibility as prognostic risk factors,the expression of ASPM,combined with VEGF,MVD,MMP7,and LRP16 were detected and analyzed.The effect and mechanism of ASPM in angiogenesis,EMT,ECM degradation and relationship with Wnt signal pathway were evaluated through RNA and protein level by immunohistochemistry(IHC),RNAscope and RNAi silencing.Methods:1.One hundred and eighteen cases of PDAC and 34 cases of distal normal pancreatic tissue were obtained from PLA General Hospital,to detect the protein and RNA expression by IHC and RNAscope.IHC was carried to analyze the expression of ASPM,LRP16,?-catenin,VEGF and MMP7 in PD AC and normal pancreatic tissues,and clinicopathologic factors were also analyzed.CD34 was carried to label the vascular of PD AC.The relationship between ASPM and LRP16,VEGF,MMP7,?-catenin and MVD was also evaluated respectively.Sixteen cases of lymph node metastasis and 9 cases of liver metastasis were evaluated by RNAscope and IHC to detect the expression of ASPM in primary and metastatic counterpart.2.siRNA technique was carried to silence ASPM gene in PANC-1 and AsPC-1 cell lines.CCK8 and transwell technique were used to detect the growth,proliferation and invasion ability.3.qRT-PCR and Western blot were detected mRNA and protein levels of DVL,?-catenin,VEGF,a-SMA,MMP7 in PANC-1 and AsPC-1 cell lines.The relationship between ASPM and DVL,?-catenin,VEGF,MMP7 and a-SMA,and the probable function of ASPM were also analyzed.Results:1.ASPM expression was correlated with invasion,metasatsis,TNM stage and prognosis in PDAC(P<0.05).ASPM protein and mRNA expression in metastatic tissues was higher than that of primary counterpart.2.Knockdown of ASPM significantly decreased the growth,proliferation and invasion ability of PANC-1 and AsPC-1 cell lines(P<0.05).3.Silencing ASPM of PANC-1 and AsPC-1 cell lines,the mRNA level and protein expression of ASPM,?-catenin,DVL,MMP7 and VEGF in silenced group was lower than those of negative group by qRT-PCR and Western blot(P<0.05).Conclusions:1.ASPM and LRP16 were correlated with prognosis of PDAC,and ASPM might predict the invasion,metastasis,angiogenesis and prognosis of PDAC.2.Knockdown of ASPM significantly decreased the growth,proliferation and invasion ability of PANC-1 and AsPC-1 cells.3.Silencing ASPM of PANC-1 and AsPC-1 cells might down regulate the protein expression and mRNA level of(3-catenin,DVL of Wnt signal pathway,and also down regulate the protein expression and mRNA level of MMP7,a-SMA and VEGF.ASPM might regulate the protein expression and mRNA level of MMP7,a-SMA and VEGF through Wnt-DVL-?-catenin signal pathway.4.ASPM might have influence on invasion and metastasis of PD AC by many ways.ASPM associated with LRP16 might influence tumorigenesis and progress of PD AC,and could be useful cancer therapy targets.