| Objective: Pancreatic cancer is one of the most malignant gastrointestinal malignancies.The disease progresses rapidly and has a high degree of malignancy.The 5-year survival rate is less than 6% and the long-term survival rate is less than 15%.It is the eighth leading cause of cancer-related death in the world.Due to the lack of early clinical manifestations,diagnosis of pancreatic cancer is seldom in the advanced stage,surgery is currently the only chance for cure of pancreatic cancer,however,the effect is not very satisfactory.About 80-90% of cases received the surgical treatment can not survive more than 5 years due to early metastasis either preoperative and postoperative.In addition,because the pancreas is not rich in blood supply,so the infiltration of chemotherapy drugs is very limited,this feature leads to the resistence of pancreatic cancer on radiotherapy and chemotherapy.In general,pancreatic cancer is characterized by late onset,early metastasis,frenquent recurrence and poor prognosis,of which the most crucial feature is the early metastasis of pancreatic cancer.Therefore,in view of the characteristics of metastasis of pancreatic cancer,research and development of drugs and treatment methods to improve the prognosis of patients with pancreatic cancer is very necessary.At present,the research on the invasion and metastasis of pancreatic cancer has been a hot topic for many years.However,the specific regulatory mechanisms and key mediating proteins have not yet been completely elucidated.In order to clarify the molecular mechanism of pancreatic cancer invasion and metastasis,in the previous study,we established a non-dissociative pancreatic cancer cell line(PC-1)by using BOP-induced Syrian hamster.After that,PC-1 cells were planted into another Syrian hamster and a dissociated pancreatic cancer cell line(PC-1.0)was established from the liver metastases.The pair of hamster pancreatic cancer cell lines are a pair of homologous cell lines with different invasion and metastasis potential.The main difference is that PC-1.0 is highly invasive and metastatic,while PC-1 is weakly invasive and metastatic.This pair of homologous pancreatic cancer cell line is a good cell model to study the invasion and metastasis of pancreatic cancer.Therefore,the differential proteomic analysis of this pair of cell lines may bring us the proteins more targeting on the invasion and metastasis of pancreatic cancer.The in-depth study of the differentially expressed proteins can be more targeted understanding of the invasion and metastasis of pancreatic cancer related mechanisms.Methods: In this study,we first performed differential proteomic analysis of PC-1.0 and PC-1 cells using two proteomic approaches,2D-DIGE and SILAC.Phosphoglycerate mutase 1(PGAM1)which was significantly upregulated in PC-1.0 in both proteomics was selected were prompted was to do the further research to clarify its impact on pancreatic cancer invasion and metastasis and the involved specific mechanism.In the next study,clinical tissue sections in 54 cases of clinical tissue were used to verify the expression level of PGAM1 between pancreatic cancer tissues and adjacent tissues,between different degree of differentiation of pancreatic cancer and between pancreatic cancer in situ tissue and metastatic tissues by immunohistochemistry.At the cellular level,pancreatic cancer cell lines Aspc-1 and Panc-1 were transiently transfected with PGAM1-siRNA and the silencing efficiency was verified by Western-blot.After successfully silencing PGAM1,the changes of Aspc-1 and Panc-1 cell proliferation were detected by CCK-8;the changes of early apoptosis and cell cycle of Aspc-1 and Panc-1 cells were detected by flow cytometry;The changes of migration and invasion ability of Aspc-1 and Panc-1 cells were detected by Transwell analysis.To further clarify the specific mechanism of PGAM1 in regulating the invasion and metastasis of pancreatic cancer,we used Western-blot and immunofluorescence to confirm the relationship between PGAM1 and PI3K/Akt/mTOR/HIF-1? signal transduction pathway.At the same time,the effect of PGAM1 on epithelial-mesenchymal transition(EMT)on pancreatic cancer cells and the relationship with Wnt/?-catenin signal transduction pathway were also clarified by Western-blot.Results: 1.The results of proteomics showed that there were 42 differentially expressed proteins(>1.5-fold,p<0.05)by DIGE,in which 26 proteins were up-regulated in PC-1.0 cells and 16 proteins were up-regulated in PC-1 cells.A total of 141 differentially expressed proteins(>1.5-fold,p<0.05)were obtained by SILAC,of which 82 proteins were up-regulated in PC-1.0 cells and 59 proteins were up-regulated in PC-1 cells.In order to obtain more accurate results,we overlapping the two groups of data.PGAM1 and HSPE1 were found up-regulated in PC-1.0 cells in both DIGE and SILAC;PDIA3 and CALR were found up-regulated in PC-1 cells in both DIGE and SILAC.Among them,phosphoglycerate mutase 1(PGAM1)was significantly up-regulated in PC-1.0 cells in both proteomics analysis results,and as a glycolysis protein,PGAM1 has a great research space in the regulation of cancer invasion and metastasis.Therefore,PGAM1 was selected for further study.Immunohistochemistry showed that the expression level of PGAM1 in pancreatic cancer tissues was significantly higher than that in adjacent tissues(p<0.05);The expression of PGAM1 was significantly higher in poorly differentiated pancreatic cancer tissues than in moderately and highly differentiated tissues(p<0.05).The expression level of PGAM1 in pancreatic cancer metastasis site was significantly higher than that in primary pancreatic cancer site(p<0.05).The results of CCK8 and flow cytometry showed that the proliferation of pancreatic cancer cells after silencing PGAM1 by siRNA for 48h-72 h was significantly lower than that of the control group(p<0.05),and there was a block in the S phase of the cell cycle(p<0.05),but early apoptosis did not change(p>0.05).Transwell analysis confirmed that silencing PGAM1 decreased the migration and invasion of pancreatic cancer cell lines than the control group significantly(p<0.05).The expression of pho-Akt(ser473)and pho-mTOR(ser4884)in the treated group was significantly higher than that in the untreated group(p<0.05)after treated with PI3K/Akt signal transduction pathway inhibitor Wortmannin and mTOR signal transduction pathway inhibitor Rapamycin for both Aspc-1 and Panc-1 cells(p<0.05).However,after treating Aspc-1 and Panc-1 cells with PGAM1-siRNA,the expression level of PGAM1 in the treatment group was significantly higher than that in the control group(p<0.05).However,the phosphorylation levels of pho-Akt(ser473)and pho-mTOR(ser4884)did not change(p<0.05).During studying the relationship between PGAM1 and HIF-1?,we found that the expression levels of HIF-1? and PGAM1 in HIF-1?-siRNA treated group were significantly lower than those in control group(p<0.05).The expression levels of HIF-1? and PGAM1 in the treatment group were also significantly lower after treating with PGAM1-siRNA than those in the control group(p<0.05).In the study of the effect of PGAM1 on epithelial-mesenchymal transition(EMT)in pancreatic cancer cells,we found that the expression of E-cadherin in the treated group was significantly up-regulated compared with the control group(p<0.05).N-cadherin and Vimenin expression levels were significantly lower than the control group(p<0.05).At the same time,we found that the expression level of pho-?-catenin in PGAM1-siRNA treated group decreased significantly compared with that in control group,whereas the treated group treated with Wnt/?-catenin signal transduction inhibitor XAV-939.In turn,the expression of pho-?-catenin was significantly higher than that of the control group(p<0.05),but the expression level of PGAM1 did not change between the two groups(p>0.05).Conclusions: 1.PGAM1 has an important regulatory role on invasion and metastasis of pancreatic cancer.Clinical analysis showed that the expression of PGAM1 and the degree of pancreatic cancer differentiation and metastasis are closely related.2.PGAM1 can promote the proliferation of pancreatic cancer cells but does not affect the early apoptosis of pancreatic cancer cells.3.PGAM1 can promote the migration and invasion of pancreatic cancer,and this change occurs before PGAM1 promotes cell proliferation and is independent of cell proliferation.4.PGAM1 is regulated by PI3K/Akt/mTOR/HIF-1? signal transduction pathway and located in the downstream of the pathway and has positive interactions with HIF-1?.5.PGAM1 regulates Wnt/?-catenin signaling transduction pathway promotes epithelial-mesenchymal transition(EMT)in pancreatic cancer cells. |
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