The Roles And Mechanisms Of SAV1 In Invasion And Metastasis Of Pancreatic Cancer

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The expression and clinical significance of SAV1 in pancreatic cancerObjective: This section mainly explores the expression level of SAV1 m RNA and protein in pancreatic cancer cell lines,pancreatic cancer tissues and paracancerous tissues;analyzes the correlation between SAV1 expression and clinicopathological factors in pancreatic cancer tissues,and further investigates the effect of SAV1 expression on the prognosis of pancreatic cancer patients.Methods: Pancreatic cancer tissue and paired adjacent normal pancreatic tissue were collected from the affiliated Shanghai First People's Hospital of Nanjing Medical University.The correlation between SAV1 expression and the prognosis of pancreatic cancer patients was analyzed using pancreatic cancer tissue microarray.1.Expression of SAV1 m RNA and protein in pancreatic cancer cell lines and normal pancreatic acinar cells: Difference of SAV1 expression in pancreatic cancer cell lines PANC-1,ASPC-1,CFPAC-1,SW1990 and normal pancreatic adenocarcinoma vesicular cell line HPDE were detected by q RT-PCR and Western blot.2.SAV1 m RNA and protein expression in pancreatic cancer and paracancerous tissue detection: SAV1 m RNA and protein expression levels of fresh frozen pancreatic cancer specimens were detected using q RT-PCR and Western blot.3.Pancreatic cancer SAV1 expression and clinicopathological factors in patients with correlation analysis: SAV1 expression was detected using pancreatic cancer tissue CHIP and immunohistochemical,the differences of the SAV1 expression in pancreatic cancer tissue and adjacent tissues and the correlation between SAV1 expression and the clinicopathological factors in pancreatic adenocarcinoma were analyzed using c2 test.4.Correlation between SAV1 expression and prognosis in pancreatic cancer: Survival curves were analyzed by Kaplan-Meier and the relationship between SAV1 expression and survival of patients with pancreatic cancer was analyzed.Results: 1.SAV1 was low-expressed in four pancreatic cancer cells(PANC-1,ASPC-1,CFPAC-1,SW1990)and high-expressed in normal pancreatic acinus cell line HPDE.2.The m RNA and protein of SAV1 in pancreatic cancer are highly expressed in paracancerous tissues and low in cancer tissues.3.In pancreatic cancer pathology tissue microarray,we found that SAV1 was mainly expressed in the cytoplasm.The SAV1 expression was highly expressed in 58 of 83 pancreatic cancer adjacent tissues cases and was significantly difference with the expression in pancreatic cancer tissues(P <0.05);the expression of SAV1 in cytoplasm was related to the degree of differentiation of pancreatic cancer and lymph node metastasis,however,there is no relationship with gender,age,tumor size,T,M,TNM,tumor location and nerve invasion.4.The expression level of SAV1 is positively correlated with the overall survival rate.Conclusion: 1.SAV1 was low expression in pancreatic cancer cells and tissues,normal pancreatic acinar cells but overexpression in adjacent tissues;the expression of cytoplasmic SAV1 was related to pancreatic cancer differentiation,lymph node metastasis.Part ? The role of SAV1 in the migration,invasion and EMT in PDACObjective: In this section,we wants to explore the malignant phenotype,including the migration,invasion,apoptosis of tumor cells and EMT after SAV1 gene was silenced or overexpressed.Methods: In the first part,the expression of PANC-1,ASPC-1,CFPAC-1,SW1990 and SAV1 were higher expressed in PANC-1 cells and lower expressed in ASPC-1 cells.We transfected human SAV1 si RNA into pancreatic cancer cell line PANC-1 and transfected lentiviral vector p Lenti-EF1a-EGFP-P2A-Puro-CMV-SAV1-3 Flag into As PC-1 cells to down-regulate the expression of SAV1.Cell scratch assay and transwell assay were used to detect the effect of SAV1 silencing and overexpression on the migration and invasion ability of human pancreatic cancer cells.Flow cytometry was used to detect the effect of SAV1 silencing and overexpression on the apoptosis of human pancreatic cancer cells.Finally,q RT-PCR Western blot was used to detect the expression of EMT-related proteins E-cadherin and Vimentin after SAV1 silencing and overexpression.Results: Cell scratch assay and transwell assay showed that the SAV1 si RNA treatment group significantly enhanced the migration and invasion ability.Flow cytometry revealed that silencing SAV1 can inhibit the apoptosis of pancreatic cancer cells.SAV1 overexpression significantly reduced the migration and invasion ability.Overexpression of SAV1 can promote apoptosis in pancreatic cancer cells.Western blot results showed that the expression of EMT marker Vimentin increased and the expression of E-cadherin decreased after SAV1 down-regulation;while the expression of Vimentin decreased and the expression of E-cadherin increased after SAV1 over-expression.Conclusion: Down-regulation of SAV1 expression enhances the ability of invasion,inhibits apoptosis and promotes EMT,while up-regulation of SAV1 attenuates the ability of invasion,promotes apoptosis and reverses EMT of human pancreatic cancer cells.Part ? Effect of 5-Aza-Cd R on pancreatic carcinoma proliferation?apoptosis and SAV1 gene expressionObjective: The role of upstream regulatory genes of SAV1 in pancreatic cancer is still not clear,this section want to explore the impact of methylation on the expression of SAV1,and study the impact of methylation inhibitor 5-aza-2'-deoxycytidine(5aza-Cd R)on proliferation,clone formation and apoptosis of human pancreatic cancer cells.Methods: SAV1 Cp G island was present in the promoter region of SAV1 detected by bioinformatics method;pancreatic cancer As PC-1was then treated with 5aza-Cd R,the changes of SAV1 expression were detected by q RT-PCR and Western blot.CCK8 experiment and cell cloning formation assay were used to detect the cell proliferation and cloning ablity of As PC-1 cells in human pancreatic cancer with methylation inhibitor 5aza-Cd R.The changes of apoptosis in As PC-1 cells were detected by flow cytometry after treated with methylation inhibitor 5aza-CdR.Results: SAV1 Cp G island was present in the promoter region of SAV1 detected by bioinformatics method.Western blot showed that the expression of SAV1 was up-regulated after 5-a-methylation inhibitor 5aza-Cd R treated in human pancreatic cancer cells As PC-1(P<0.05),and the results of q RT-PCR were the same as Western blot.The clone formation rate of As PC-1 cells in 5aza-Cd R(5u M,10 u M,30u M)decreased by 52%,75% and 84% respectively(P<0.001).Flow cytometry showed that the methylation inhibitor 5 aza-Cd R treatment of human pancreatic cancer AsPC-1cells,increased tumor cell apoptosis.Conclusion: SAV1 expression is related to the promoter methylation level.Methylation inhibitor 5-aza-Cd R can effectively reverse the abnormal methylation of SAV1 gene in As PC-1 cells cultured in vitro and induce the expression of SAV1.Methylation inhibitor 5-aza-Cd R inhibits the cloning ability of As PC-1 cells in pancreatic cancer,inhibits tumor cell proliferation and promotes apoptosis of tumor cells.