Effect Of ASAP1 Gene On Malignant Biological Behavior Of Gastric Cancer And Its Mechanism

Objective To detect the effect of adenosine diphosphate ribosylation factor guanylate kinase 1(ASAP1)on malignant biological behavior of gastric cancer and its mechanism,and to explore its possible molecular mechanism in the progress of gastric cancer.Methods1.We detected the expression of ASAP1 in gastric cancer tissues and adjacent tissues by immunohistochemistry and analyzed the relativity between the ASAP1 expression and clinical pathological characteristics.2.We tested the ASAP1 expression in normal gastric epithelial cells GES-1 and three gastric cancer cell line by RT-q PCR and Western blot in protein and m RNA level.Then we obtained the target gastric cancer cells for subsequent experiments.3.We got the ASAP1 overexpression cell BGC823 and knockdown cells BGC823 and MKN45 through the molecular biology techniques,such as lentiviral packaging,infection and screening.4.CCK8 assay,clone formation assay,Annexin/PI dual staining assay,Transwell invasion assay and scratch assay were applied to detect the effect of oe ASAP1 or si ASAP1 on gastric cancer malignant biological behavior.5.WB was used to detect the changes in proliferation,apoptosis,invasion,metastasis,angiogenesis and epithelial-mesenchymal protein level after silencing ASAP1.6.ASAP1 knockdown out nude mice model was successfully built.Mouse xenograft study was used to detect the change of the cells’ prolifetation in vivo.Results1.The expression levels of ASAP1 in GC tissues were significantly higher than those in normal gastric mucosal tissues(P=0.012).The expression levels of ASAP1 was associated with depth of invasion,lymph node metastasis and pathological stage(P<0.05).In addition,ASAP1 expression level was negatively associated with 5-year disease-free survival rate and overall survival rate(P<0.05).2.The m RNA and protein levels of ASAP1 in three gastric cancer(BGC823,MKN45,MGC803)are higher than gastric epithelial cells GES-1(P<0.05).BGC823 and MKN45 were selected to the subsequent experiments.3.We successfully screened a gastric cancer cell line BGC823 stably overexpressing ASAP1 and BGC823 and MKN45 stably silencing ASAP1.4.CCK8 and clone formation assays showed that the proliferation activity was remarkably higher in os ASAP1 group,which was lower in si ASAP1 group.Annexin/PI dual staining assay revealed that the apoptosis rate in os ASAP1 group was decreased significantly,while it was rised in si ASAP1 group.Transwell and scratch assays found that the numbers of cell permeating septum and the scratch healing rate in oe ASAP1 group were greatly increased,wheras they were decreased in si ASAP1 group(P<0.01).5.WB showed the protein expressions of Cleaved-Caspase3,Cleaved-PARP,E-cadherin were upregulated and of MMP2,MMP9,VEGFA,N-cadherin and Vimentin were downregulated in si ASAP1 group(P<0.01).6.Mouse xenograft study showed that the tumor volume and weight were significantly lower in si ASAP1 group(P<0.01).The tumor weight inhibition rate was 78.67%.Conclusions ASAP1 is highly expressed in gastric cancer tissues.The expression of ASASP1 is closely related to clinicopathological factors and prognosis ASAP1 may represent novel molecular markers for the pathophysiology and prognosis of GC.ASAP1 is also highly expressed in gastric cancer cells.Overexpression of ASAP1 obviously promote gastric cancer cell proliferation,invasion,migration and reduce apoptosis,while its knockdown exerted an oppoiste effect.ASAP1 may inhibit the malignant biological behavior of gastric cancer by downregulating VEGFA,MMP2,MMP9,suppressing cancer cell EMT and upregulating Cleaved-Caspase3,Cleaved-PARP.