The Effects And Regulatory Mechanisms Of PAFAH1B2 On PDAC

Part one The expression and clinical significances of PAFAH1B2 inPDACObjective: To observe the difference in expression levels of mRNA and protein in PAFAH1B2 form PDAC tissues and corresponding adjacent normal tissues and investigate correlation of expression of PAFAH1B2 protein in clinical,pathological parameters and prognosis of PDAC patients.Methods: Immunohistochemistry,Western blot and qRT-PCR were used to examine expression levels of mRNA and protein of PAFAH1B2 in PDAC tissues and corresponding adjacent normal tissues.The relationship between expression of PAFAH1B2 and clinicopathological parameters and prognosis of PDAC patient was analysized.Results: PAFAH1B2 protein levels in PDAC tissues were significantly increased(P<0.001).One hundred and seventy-nine cases of PDAC were selected from TCGA database and 171 cases of normal pancreatic tissue were selected from GTEx database,analysis mRNA expression level of PAFAH1B2 in PDAC tissue.The results showed that compared with normal tissue acquired from GTEx database,PAFAH1B2 mRNA expression levels of PDAC tissue were significantly increased.Expression of mRNA were tested with real-time quantitative PCR method to detect PDAC and corresponding adjacent normal tissues,the results show that,compared with adjacent normal tissues,mRNA expression levels in PAFAH1B2 PDAC were significant increased(P<0.001).The results of Western blot showed that,compared with corresponding adjacent normal tissues,PAFAH1B2 protein expression in PDAC tissue were significantly increased,the difference was statistically significant(P<0.001).Regional lymph node metastasis and TNM staging between PAFAH1B2 high expression group and low expression group were significantly different(P<0.05).Overall survival and disease progression-free survival of PAFAH1B2 protein low expression group of PDAC patients were significantly higher(P<0.0001)than in patients with PDAC high expression group.Prognosis of PDAC patients with high expression of mRNA PAFAH1B2 were proved to be worse(P<0.0001).Conclusion: The expression level of PAFAH1B2 mRNA and protein in PDAC is significantly increased.The expression of PAFAH1B2 is positively correlated with both regional lymph node metastasis and TNM staging.Overall survival and disease progression-free survival of patients with PDAC of high expression group of PAFAH1B2 protein and mRNA were significantly lower than those of PDAC low expression group.Part two PAFAH1B2 regulates the migration,invasion and metastasis ofPDAC cells.Objective: To investigate the role of PAFAH1B2 in epithelialmesenchymal transition,migration,invasion and metastasis of PDAC cells.Establish an animal model of PDAC liver metastasis on nude mice in order to verify the effect of PAFAH1B2 on hepatic metastasis of PDAC in vivo in animals.Methods: The relationship between tumor samples with high expression of PAFAH1B2 and epithelial-mesenchymal transition of tumor cells was determined by gene enrichment analysis.Apply PAFAH1B2 specific siRNA was transfected into PDAC cell line CFPAC-1 and L3.7-2 to knockdown PAFAH1B2 expression.Western blot and immunofluorescence were used to detect the expression of E-Cadherin,N-Cadherin,Vimentin.The nude mice model of pancreatic cancer with liver metastasis was established.Results: Through GSEA analysis software analyzed 20 cases of raw data samples downloaded from TCGA database of high and low expression PDAC,we found that gene set related to interstitial transdifferentiation of tumor cells enriches in PAFAH1B2 high expression samples.Therefore,we speculate that PAFAH1B2 might be closely related to migration,invasion and metastasis of tumor cells.Western blot analysis showed that silencing the expression of PAFAH1B2 significantly amplified the expression of the characteristic marker E-Cadherin in epithelial cells compared with CFPAC-1 cells transfected with blank siRNA,while expression of N-Cadherin,Vimentin,Snail and Slug were inhibited.Compared with cell L3.7-2 transfected with blank siRNA,overexpression PAFAH1B2 inhibited the expression of E-Cadherin on L3.7-2 cells,amplified the expression of N-Cadherin,Vimentin,Snail and Slug(P <0.05).The expression of E-Cadherin and N-Cadherin by immunofluorescence staining remains the same with the results of Western blot.By counting the number of cells which invaded in the lower surface of Transwell chamber,the expression of the silencing PAFAH1B2 significantly inhibited cell migration and invasion of CFPAC-1(P<0.05),compared to CFPAC-1 cells transfected with blank siRNA.Compared to CFPAC-1 cells transfected with blank siRNA,overexpression of PAFAH1B2 significantly promoted migration and invasion of L3.7-2 cells(P<0.05).Six weeks after implantation,nude mice were executed,and no significant differences were found between the two groups on the size of tumors in situ;counting the number of metastases we found increased number of liver metastases in overexpressed PAFAH1B2 group,which suggests increased ability of metastasis of PDAC cells;we further confirmed the metastasis of the PDAC cells in the liver through HE staining.Conclusion: Silencing PAFAH1B2 expression decreases the ability of invasion and metastasis of PDAC cells,inhibits the occurrence of EMT;overexpressing PAFAH1B2 increases the ability of invasion and metastasis of PDAC cells,amplifies the occurrence of EMT.Overexpression of PAFAH1B2 increased the number of liver metastases in nude mice.Therefore,biological characteristics such as high malignancy and easy metastasis of PDAC in clinic,may be related to the increased expression of PAFAH1B2 gene.Part Three The molecular mechanism of regulating PAFAH1B2 expre-ssion in PDAC cellsObjective: To investigate the role and mechanism of HIF1? in trans-criptional activation of PAFAH1B2 gene of PDAC cell lines.Methods: ChIP assay was performed to detect the interaction of HIF1? with the promoter of PAFAH1B2 gene.The other methods used in the present study were same as above.Results: Immunohistochemistry results indicated: protein expression of PAFAH1B2 and HIF1? in PDAC tissues were positively correlated(R=0.51).Spearman's correlation analysis showed that,mRNA expression of PAFAH1B2 in PDAC tissue was positively correlated with HIF1?(R=0.45),mRNA expression of PAFAH1B2 in HIF1? mRNA high expression group was significantly higher than HIF1? mRNA low expression group.Results of Western blot showed that under hypoxic conditions,protein expression of PAFAH1B2 significantly increased,while expression of HIF1? also significantly increased.The results of real-time fluorescence quantitative PCR remains the same with those of Western blot(P<0.05).We constructed overexpressed plasmid of HIF1?(HIF1A)and small interfering RNA(siHIF1A)to transfect CFPAC-1 cells and L3.7-2 cells,results of Western blot showed that after transfection of HIF1 A plasmid,HIF1? protein expression in both tumor cells significantly increased;while protein expression of PAFAH1B2 significantly amplified.After transfection of siHIF1 A protein,protein expression of HIF1? were significantly inhibited in both tumor cells,and expression of PAFAH1B2 were also inhibited.The results of real-time fluorescence quantitative PCR remains the same with those of Western blot(P<0.05).Chromatin immunoprecipitation showed that HIF1? can bind to the promoter region of PAFAH1B2,and the condition of hypoxia also promotes the binding of HIF1? to the PAFAH1B2 promoter region.Gene analysis of luciferase report revealed that overexpression of HIF1 A significantly increased the activity of promoter of PAFAH1B2 gene.When we modified the HRE sequence of promoter of PAFAH1B2 genefrom GCCTG to GCATG,overexpression of HIF1 A no longer increase the activity of promoter of PAFAH1B2 gene.Conclusion: HIF1? increases activity of promoter of PAFAH1B2 gene by binding to the HRE gene sequence promoter of PAFAH1B2,thereby increasing the expression of PAFAH1B2.PAFAH1B2 is a target gene of HIF1? and promotes the metastasis of PDAC cells.Conclusion:1.Protein and mRNA expression of PAFAH1B2 increases in PDAC tissues.High-expression of PAFAH1B2 is positively correlated with regional lymph node metastasis and TNM staging.Overall survival and progression-free survival of patients with PDAC with high protein and mRNA expression of PAFAH1B2 were significantly lower than those of low protein and mRNA expression of PAFAH1B2.2.Silencing expression of PAFAH1B2 gene,weakens invasion and metastasis of PDAC cells,and inhibits occurrence of EMT;overexpression of PAFAH1B2 gene,amplifies invasion and metastasis of PDAC cells,and increases occurrence of EMT.PAFAH1B2 gene overexpressed in nude mice model of pancreatic cancer with liver metastases,the number of liver metastases significantly increased,animal experiments also verified the above conclusions.3.HIF1? increases activity of promoter of PAFAH1B2 gene by binding to the HRE gene sequence promoter of PAFAH1B2,thereby increasing the expression of PAFAH1B2.PAFAH1B2 is a target gene of HIF1? and promotes the metastasis of PDAC cells.