Effects Of GSH2 Silencing On Pancreatic Cancer Cell SW1990

Pancreatic ductal adenocarcinoma,one of the most lethal human cancers,is the fourth leading cause of cancer deaths and its five-years survival rate remains dismal and below 5%.In U.S.,it makes more than 30000 death per year,and is predicted to become the second leading cause of cancer-related death by 2020.Due to disease presentation at a late stage when PDA is already metastatic,all the therapy have not shown significant benefit,including surgeons,chemotherapy,radiation and other adjuvant therapies.Therefore,extensive research about newer molecular targeted agents is the key of PDAC prevention,early diagnosis and treatment.GS homeobox 2,also known as GSX2,is located on chromosome 4q12.As a member of homeobox gene family,GSH2 encodes transcription factors,regulates cell proliferation,differentiation,apoptosis,migration,and invasion.GSH2 associated SHH participate in the embryonic forebrain development and astrocytomas.SHH signal pathway paly an important role in in the initiation and progression of pancreatic cancer,while GSH2 effect on pancreatic cancer remains unclear.In this study,we detected GSH2 expression in the pancreatic cancer tissues.Using RNA interference to silence GSH2 in pancreatic cancer SW1990 cell,we investigate its effect on cell proliferation,migration,invasion and gemcitabine sensitivity,then to investigate the effect on gene SHH and GLI1 expression,which is the key factor of Hedgehog(HH)pathway.Part? GSH2 gene expression in pancreatic cancer tissueObjective: To detect GSH2 gene expression in pancreatic cancer tissue.Methods and materials: The difference of GSH2 expression between human pancreatic cancer tissues and adjacent normal pancreas(n=3)was detected by immunohistochemistry(IHC).The difference of GSH2 mRNA expression were quantitated by qRT-PCR and GSH2 protein expression were detected by Western blot.Results: Human pancreatic cancer tissues showed strong positive staining for GSH2,while adjacent normal pancreas showed slightly stained.qRT-PCR results revealed that GSH2 mRNA was highly expressed in pancreatic adeaocarcinoma tissues(8.40±0.32)compared to adjacent normal pancreatic(0.95±0.04,t=23.27,p< 0.001).Western blot showed GSH2 protein was highly expressed in pancreatic adeaocarcinoma tissues.Conclusions: GSH2 gene is high-expressed in pancreatic cancer tissues.Part ? Effects of GSH2 silencing on pancreatic cancer cell SW1990 proliferation,migration,invasion,sensitivity to gemcitabine and Hedgehog pathwayObjective: To investigate the effects of GSH2 silencing on pancreatic cancer cell SW1990 proliferation,migration,invasion,sensitivity to gemcitabine and Hedgehog pathway key factors SHH,GLI1.Methods and materials: Silencing GSH2 by siRNA in pancreatic cancer cell SW1990,analysis cell proliferation,migration,invasion,using transfection NC-siRNA cells as control.Combination with gemcitabine treatment,analysis cell proliferation and apoptosis.Constructed plasmid that expresses shRNA of GSH2 correctly and selected one with the best silencing effect.Using transfection NC-siRNA and pLKO.1 puro plasmid cells as control respectively,Silencing GSH2 by siRNA and shRNA,to assess the silencing effect by qRT-PCR and Western blot in the level of mRNA and protein expression,then to investigate the expression of mRNA and protein of Hedgehog pathway key factors SHH,GLI1.Results: Silencing GSH2 by siRNA in SW1990,the absorbtion at 450 nm were 2.89±0.02,lower than control(3.39±0.02;t=20.74,p<0.001);Cell Migration were 157.2±6.71,less than control(349.3±8.33;t=17.95,p<0.001);Cell Invasion were 63.21±1.16,less than control(95.71±3.51;t=8.79,p<0.001).Silencing GSH2 by siRNA and combination with gemcitabine treatment,the absorbtion at 450 nm were 2.48±0.02,lower than control(2.89±0.02;t=17.24,p <0.001);Apoptosis percentage were 39.70%±0.52%,higher than control(26.38%±1.17%;t=10.39,p<0.001).Constructed plasmid that expresses shRNA of GSH2 correctly and selected one with the best silencing effect.Silencing GSH2 by siRNA in SW1990,the expression level of GLI1,SHH mRNA is 0.21±0.01,1.07±0.05 respectively,compared to control,GLI1 mRNA expression decrease significantly(1.00±0.05;t=16.69,p<0.001);SHH mRNA had no significantly change(1.00±0.02;t=1.13,p=0.32).Western blot showed the protein expression of GLI1 decrease,while SHH had no significantly change.Silencing GSH2 by shRNA in SW1990,the expression level of GLI1,SHH mRNA is 0.18±0.004,0.97±0.07 respectively,compared to control,GLI1 mRNA expression decrease significantly(0.98±0.03;t=25.72,p<0.001);SHH mRNA had no significantly change(1.00±0.02;t=0.42,p=0.70).Western blot showed the protein expression of GLI1 decrease,while SHH had no significantly change.Conclusions: Silencing GSH2 gene repress pancreatic cancer cell line SW1990 proliferation,migration,invasion and increase the sensitivity to gemcitabine.Silencing GSH2 gene repress the expression of GLI1 mRNA and protein in HH pathway.Part ? The relationship of GHS2,SHH and GLI1 in pancreatic cancer cell SW1990Objective:To detect the relationship of GHS2,SHH and GLI1 in pancreatic cancer cell SW1990.Methods and materials: Gather SW1990 cell lysis,then get pull-down protein of SHH and GLI1 by Crosslink Magnetic Co-IP kit,and using western blot to detect the protein expression of GSH2 in the pull-down protein of SHH and GLI1 respectively,using normal rabbite IgG as negative control while SW1990 cell lysis as postive control.Results: Postive control expressed GLI1,GSH2 and SHH;negative control showed nothing;detected GSH2 in both SHH and GLI1 pull-down protein.Conclusions: GHS2,SHH and GLI1 has some link in pancreatic cancer cell SW1990.Part ? Effect of GSH2-siRNA in subcutaneous tumors in vivoObjective:To detect the effect of GSH2-siRNA in subcutaneous tumors in vivo.Methods and materials:Transplante pancreatic cancer SW1990 cell tansfected GSH2-siRNA(1x107/100 ul cell suspension per mice)to nude mice in the right arm back subcutaneous.Nude mice were randomly divided into four groups(A group:NC-siRNA;B group : GSH2-siRNA;C group : NC-siRNA+GEM;D group :GSH2-siRNA+GEM).After two weeks,treated group C and D with chemotherapeutic drugs gemcitabine,(100mg/kg body weight,once every three days).The mice were executed after four weeks.Record tumor volume and evaluate the peritoneal and pleural metastases.Results:All mice developed subcutaneous tumor after two weeks and had no peritoneal and pleural metastases until sacrificed.Tumor volume between group A and B showed no signigicant difference(260.1±61.25,216.6±26.64;t=0.59,p=0.572),while tumor volume of group D signigicantly decreased(74.4±16.19)compared to group C(100.5±23.36;t=3.16,p=0.016).Conclusions: Silencing GSH2 gene can repress pancreatic cancer cell line SW1990 proliferation and increase the sensitivity to gemcitabine.Based on the experiments above,this study draw conclusions as follows:1.GSH2 gene is high-expressed in pancreatic cancer tissues.2.Silencing GSH2 gene repress pancreatic cancer cell line SW1990 proliferation,migration,invasion and increase the sensitivity to gemcitabine.3.GHS2,SHH and GLI1 has some link in pancreatic cancer cell SW1990.