The Role And Mechanism Of P22^phox In Pancreatic Ductal Adenocarcinoma By Regulating RAS/ERK/HIF-1? Pathway

Part1.Identification and Phenotype of P22^phox Knockout Transgenic MiceObjective: P22^phox is the subunit of NAPDH oxidase,which is involved in the production of ROS and metabolism change.Our earlier study showed that the expression level of P22^phox in PDAC tissue was obviously increased.To investigate the role of P22^phox in the development of PDAC,we specific knockout P22^phox in pancreatic acinar cell and obtain the mice with genotype f ElasCre ERT;KrasG12D/+;P22phox-/-.Identification the genotype that mice carried.Breeding f ElasCre ERT mice and f ElasCre ERT;p22phox-/-mice and identification their genotype and phenotype to figure out whether P22^phox gene knockout would affect the mice health or the development of pancreas in these mice.Methods: Hybridizing f ElasCre ERT mice with KrasG12D/+ mice to obtain mice with genotype of f ElasCre ERT;KrasG12D/+.Mice with the genotype of f ElasCre ERT;KrasG12D/+ are inducible pancreatic ductal adenocarcinoma model.Then hybridizing P22phox-/-mice with f ElasCre ERT;KrasG12D/+ mice to obtain the mice with genotype of f ElasCre ERT;KrasG12D/+;P22phox-/-.The tails of the mice offspring were cut at the age of 2-3 weeks and the DNA was extracted.After the PCR amplification,the electrophoresis process was used to detect the genotype.To our known,we are the first to specific knockout P22^phox gene in pancreatic acinar cell.In order to ensure that P22^phox gene is completely knocked out in pancreatic acinar cell,we extract pancreatic acinar cell from f ElasCre ERT mice and P22phox-/-mice.Protein in pancreatic acinar cell was extracted for Westen Blot to detect the expression of P22^phox.f ElasCre ERT mice and f ElasCre ERT;P22phox-/-mice were sacrificed at the age of 150 days after induction to obtain pancreatic tissue,and histopathology was used to detect their phenotype.Results: After identification the genotype,we obtained the mice with genotype of f ElasCre ERT;KrasG12D/+;P22phox-/-.The p22 protein of pancreas acinar cells was highly expressed in f ElasCre ERT mice,and was not expressed in pancreatic acini of f ElasCre ERT;P22phox-/-mice.The pancreatic tissue phenotypes of f ElasCre ERT mice and f ElasCre ERT;p22phox-/-mice were compared and no significant difference between these groups.None of them had pancreatic acinar lesions,inflammation or tissue fibrosis.Conclusion: We successfully obtained the mice with genotype of f ElasCre ERT;KrasG12D/+;P22phox-/-.Specific knockout P22^phox gene in pancreatic acinar cell will not affect the mice health and the development of pancreas.Part2.The Effect of P22^phox Knockout on Pancreatic Ductal Adenocarcinoma Caused by KrasG12D/+ and High Fat DietObjective: Comparing the pathologic change in the pancreas of f ElasCre ERT;KrasG12D/+ mice under normal diet?ND?and high fat diet?HFD?.To investigate the effect of P22^phox gene knockout on pancreatic ductal adenocarcinoma caused by KrasG12D/+ and HFD.Methods: f ElasCre ERT;KrasG12D/+ mice were induced with Tamoxifen at the age of 70 days.A part of the mice were fed with ND and the other part of the mice were fed with HFD.At the age of 150 days,all mice were sacrificed and pancreatic tissue were harvested for H&E staining to evaluated the histopathological status.f ElasCre ERT mice,f ElasCre ERT;KrasG12D/+ mice,and f ElasCre ERT;KrasG12D/+;P22phox-/-mice were induced with Tamoxifen at the age of 70 days,HFD were given after inducing.A part of the mice were sacrificed at age of 150 days and pancreatic tissue were harvested for evaluating the histopathological status.The other part of the mice were monitored until death to compare the survival time between these groups.Results: The H&E staining of f ElasCre ERT;KrasG12D/+ mice under ND and HFD showed that f ElasCre ERT;KrasG12D/+ mice with HFD had more severe pancreatic lesion than f ElasCre ERT;KrasG12D/+ mice with ND.f ElasCre ERT;KrasG12D/+ mice with ND had Pan IN-1 or Pan IN-2 lesions in pancreas but no high grade Pan IN lesion or tumor.While pancreas of f ElasCre ERT;KrasG12D/+ mice with HFD had Pan IN-3 or even tumor?3 of 10 mice had pancreatic ductal adenocarcinoma?.Compared with f ElasCre ERT;KrasG12D/+ mice with HFD,f ElasCre ERT;KrasG12D/+;P22phox-/-mice with HFD had mild pancreatic lesion.Compare with f ElasCre ERT;KrasG12D/+ mice,the expression level of Amylase and MIST1 were higher in f ElasCre ERT;KrasG12D/+;P22phox-/-mice while the expression level of Alcian Blue,MUC5,?-SMA,Ck19,SOX-9,Sirus Red were decreased in f ElasCre ERT;KrasG12D/+;P22phox-/-mice.In terms of proliferation,f ElasCre ERT;KrasG12D/+ mice with HFD had strong positive Ki67 stain while the Ki67 expression in f ElasCre ERT;KrasG12D/+;P22phox-/-mice were weak.In regard of inflammation,the expression level of CD45 and F4/80 were high in f ElasCre ERT;KrasG12D/+ mice with HFD but low in f ElasCre ERT;KrasG12D/+;P22phox-/-mice with HFD.Conclusion: Oncogenic KrasG12D/+ alone is not sufficient to lead to pancreatic ductal adenocarcinoma.HFD can accelerate the carcinogenic effects of KrasG12D/+.P22^phox gene specific knockout in pancreatic acinar cell could suppress the carcinogenic effects caused by KrasG12D/+ with HFD.Part3.The Mechanism of P22^phox Knockout in Suppressing the Carcinogenic Effects caused by KrasG12D/+ and High Fat Diet.Objective: To investigate the mechanism of P22^phox gene knockout in suppressing the carcinogenic effects caused by KrasG12D/+ and high fat diet.Methods: f ElasCre ERT mice,f ElasCre ERT;KrasG12D/+ mice and f ElasCre ERT;KrasG12D/+;P22phox-/-mice were induced with Tamoxifen at the age of 70 days and then fed with high fat diet.All these mice were sacrificed at the age of 150 days and pancreatic tissue were harvested for protein extraction.Western Blotting was used to detect the expression of HKII,LDHA,HIF-1? and the activation of ERK pathway.Ras Activation Assay Kit was used to detect RAS protein activity.Results: Under the high fat diet stimuli,f ElasCre ERT;KrasG12D/+ mice had significant higher expression level of HKII and LDHA compared with f ElasCre ERT mice.While the expression level of HKII and LDHA in f ElasCre ERT;KrasG12D/+;P22phox-/-mice were much lower than f ElasCre ERT;KrasG12D/+ mice.With high fat diet,f ElasCre ERT;KrasG12D/+ mice had significantly higher percentage of active RAS,stronger activation of ERK signaling pathway and increased expression of HIF-1? than f ElasCre ERT mice.Compared with f ElasCre ERT;KrasG12D/+ mice,f ElasCre ERT;KrasG12D/+;P22phox-/-mice had lower percentage of active RAS,weaker activation of ERK signaling pathway and decreased expression of HIF-1?.Conclusion: The elevated expression of HKII and LDHA in f ElasCre ERT;KrasG12D/+ mice with high fat diet indicated that cells in pancreas had metabolic change,which inverted the metabolic pattern from oxidation phosphorylation to aerobic glycolysis.The change of metabolic network is the feature of tumor cells.The metabolic change in f ElasCre ERT;KrasG12D/+ mice can be regulated by RAS/ERK/HIF-1? pathway.When P22^phox was knocked out in acinar cell,the activity of RAS may have suppressed through decreased intracellular ROS level.And the pathway of RAS/ERK/HIF-1? was inactivated which can impede the reconstruction of metabolic network in cells and delaying the development of cancer.