Study On The Effect Of Astrocyte Elevated Gene-1 On Pancreatic Cancer Cells

Objective: Pancreatic cancer is a highly malignant gastrointestinal tumor,in contrast to the general trend of most malignancies,and its morbidity and mortality continue to rise.About 90% of pancreatic cancers are ductal adenocarcinomas that are derived from ductal epithelium.Due to a lack of typical symptoms in the early stage,pancreatic cancer is easily misdiagnosed.The median survival of this cancer is less than 6 months and the prognosis is poor.Astrocytes elevated gene 1(AEG-1)gene encoding a 582 amino acid proteins was initially identified in primary fetal brain astrocytes.Protein AEG-1 predominantly localizes in the perinuclear region and endoplasmic reticulum and contains a single pass transmembrane and three putative domains responsible for lysine-rich nuclear localization signals.Elevation of AEG-1 is frequently observed in human cancers,including endometrial cancer,hepatocellular carcinoma,glioma and links to poor clinical outcomes.Previous gain-and loss-of-function studies have shown that the over expression of AEG-1 promotes the growth,angiogenesis,and metastasis of various malignancies.Although the m RNA and protein expression levels of AEG-1gene were higher in pancreatic cancer cell lines and tissues,there is no further investigation about the role of AEG-1 in the tumorigenesis of pancreatic cancer.Interfering with the cell cycle has proven highly successful in cancer treatment in the clinics,and the cell cycle inhibitors are under development for the treatment of pancreatic cancer.Knockdown of AEG-1 is often reported to promote the G0/G1 phase arrest in cancer cells in vitro,but the role of AEG-1 in the cell cycle in different types of cancer cells differs.More effort is needed to elucidate its role in regulating the cell-cycle progress of pancreatic cancer cells.The invasion and metastasis of a tumor require the degradation of extracellular matrix(ECM),in which the matrix metalloproteinases(MMPs)play crucial regulatory roles.Researches showed that the forced over expression of AEG-1 induced migration and invasion of osteosarcoma cells by augmenting the MMP-2 transcription activity.The over expression of MMP-2 has been observed in pancreatic cancer tissues.Therefore,in this study,the role of AEG-1 in cell migration and invasion of pancreatic cancer cells was explored,with a focus on MMPs.In the present study,a specific short-hairpin RNA(sh RNA)was used to target the m RNA of AEG-1 gene and inhibited its expression in two pancreatic cancer cell lines.The proliferation,cell cycle distribution,apoptosis and mobility of normal and AEG-1-silenced pancreatic cancer cells were evaluated.Methods: Obtaining the stable AEG-1 silencing transfected cells.Quantitative Real-time PCR(q RT-PCR)and Western blot to detect the expression of Bax?Bcl-2?MMP-2?MMP-9??-catenin?p-?-catenin?cleaved caspase 3?p-AKT S473?AKT and ?-actin.CCK-8 was used to assess cell proliferation.Colony formation assay and sphere formation assay.The cell apoptosis and cycles were analyzed by flow cytometry.Wound healing assay and transwell assay were used to detect the abilities of migration and invasion.Xenograft tumor model and evaluation the apoptosis of xenograft tumor tissues.Results: 1.We found that AEG-1 expressed at a higher level in As PC-1 and PANC-1cells.Therefore,these two cell lines were then transfected with AEG-1 specific sh RNA or NC sh RNA,and the m RNA and protein expression levels of AEG-1 were examined by q RT-PCR and Western blot.Results of q RT-PCR and Western blot showed that the AEG-1 m RNA and protein levels were downregulated about 70% in both As PC-1 and PANC-1 cells.2.AEG-1 silencing inhibits the proliferation of pancreatic cancer cells in vitro.The effect of AEG-1 silencing on the growth of pancreatic cancer cells in vitro was detected by the CCK-8 assay.The proliferation of As PC-1 and PANC-1 cells with stable low expression of AEG-1 was significantly suppressed.Colony formation assay further confirmed that knockdown AEG-1 significative reduction in the colony forming ability of As PC-1 and PANC-1 cells,colony formation rates of both AEG-1silencing cells were lower than NC sh RNA cells.Furthermore,the stemness of both cell lines was examined with spheres formation assay experiments.The size and number of tumor spheres were inhibited after cells transfected with AEG-1 sh RNA.In addition,by analyzing the cell cycle,we found that the silencing of AEG-1 induced a G0/G1 arrest in both pancreatic cancer cell lines.3.AEG-1 silencing promotes apoptosis in PC cells.Cell apoptosis was detected on a flow cytometer.The apoptotic percentage was increased.Moreover,the levels of apoptosis-related proteins were determined by Western blot compared with the cells transfected with NC sh RNA,the expression level of anti-apoptotic protein Bcl-2 was downregulated and that of the pro-apoptotic proteins Bax and cleaved Caspase-3 were upregulated in cells transfected with AEG-1 sh RNA.4.AEG-1 knockdown inhibits the migration and invasion of PC cells.Wound healing assay was performed to analyze the effect of AEG-1 knockdown on the migration of pancreatic cancer cells.Silencing of AEG-1 in As PC-1 and PANC-1 cells significantly delayed wound closure and inhibited the migration ability in AEG-1 silenced cells.In addition,the results of transwell assay showed that the numbers of invasive cells were significantly reduced in AEG-1-silenced cells.Moreover,the expression levels of invasion-related protein MMP-2 and MMP-9 were detected by Western blot.5.AEG-1 affected the AKT/?-catenin signal pathway The expression of AKT and p-AKT,p-?-catenin and nuclear ?-catenin were determined by Western blots.The p-AKT protein level was decreased when AEG-1 silencing in both As PC-1 and PANC-1 cells.Besides,the level of p-?-catenin was upregulated but ?-catenin in nuclear was significantly downregulated in cells which AEG-1 was knockdown.6.AEG-1 knockdown suppresses the growth of xenograft tumor in vivo.Control As PC-1 and PANC-1 cells and those expressing a stable lower AEG-1 were injected into nude mice.The tumors formed by AEG-1-silenced PC cells were much smaller than that formed by the control cells.Furthermore,downregulation of AEG-1 by sh RNA significantly reduced the expression of MMP-2 and MMP-9 as well as the expression of AEG-1.The apoptotic cells in the xenografts were detected by TUNEL staining assay,the results demonstrated that AEG-1 silencing increased apoptotic cells in xenograft tumor tissues.Conclusion: That cells with low AEG-1 expression exhibited weak proliferation,migration,and invasion,and were arrested at G0/G1 phase.The growth of xenograft tumors formed by AEG-1 silenced pancreatic cancer cells was slower than those generated by control cancer cells.The acceleration of cell cycle progression is a hallmark of many malignant tumors,including pancreatic cancer.By knocking down the expression of endogenous AEG-1,we found that both As PC-1 and PANC-1 cells were arrested at the G0/G1 stage.Although the effects of AEG-1 knockdown on the cell cycle progress of cancer cells have been reported before,our study was the first to show this in pancreatic cancer cells.The escape of apoptosis of a tumor cell is a major cause of tumorigenesis.The downregulation of AEG-1 mediated by anti-cancer drugs,micro RNAs that target its m RNA or small interference RNA has been reported to accelerate the apoptosis in cancer cells.By analyzing the phosphatidylserine,we observed that AEG-1 sh RNA promoted apoptosis in both As PC-1 and PANC-1 cells.Pro-and anti-apoptotic proteins that control the permeabilization of the mitochondrial outer membrane constitute the Bcl-2 family.We found that AEG-1 silencing could induce a downregulation in anti-apoptotic Bcl-2 and an up-regulation in pro-apoptotic Bax,leading to an accelerated apoptosis in pancreatic cancer cells.The balance between the pro-and anti-apoptotic Bcl-2 members determines whether a cell undergoes apoptosis.Besides Bcl-2 and Bax analyzed in this study,AEG-1 has also been observed to interact with other Bcl-2 family members.For instance,in primary human fetal astrocytes,the over expression of AEG-1 could phosphorylate the Bcl-2agonist of cell death(Bad).Bad is a BH-3-only protein that is known as an apoptosis "sensitizer",and its phosphorylation is linked to cancer cell survival.The phosphorylation of Bad leads to its sequestration by 14-3-3 proteins and causes the translocation to the cytosol.For AEG-1 overexpression can induce Bad phosphorylation,we deduce that AEG-1 knockdown can promote its dephosphorylation,hence leading to apoptosis in pancreatic cancer cells.In addition,downregulation of AEG-1 could affect the chemosensitive of tumor cells via the Bax/bcl-2 pathway.There is the Bax/bcl-2 pathway,in addition to AEG-1 was also reported to inhibit the chemosensitive of tumor cells by promoting autophagy,or directly upregulating multidrug resistance gene 1(MDR1),transcription factor LSF and thymidylate synthase(TS).More efforts will be devoted to study the mechanisms underlying the role of AEG-1 in cancer apoptosis in the near future.As zinc endopeptidases,MMPs can cleave nearly all components in ECM.In pancreatic cancer,they can be produced by both tumor and stromal cells,and their high expression is associated with the increased rate of tumor metastasis and invasion.Several previous studies have linked the migration and invasion of cancer cells induced by AEG-1 with the up-regulation in MMPs,such as MMP-2,MMP-9,MMP-7,etc.In this study,the mobility reduction and the MMP-2 and MMP-9 down regulation simultaneously occurred in pancreatic cancer cells if AEG-1 was silenced.The tissue inhibitors of metalloproteinases(TIMPs)are biological MMP regulators.Their N-terminal domain is recognized as the "inhibitory domain" that is responsible for MMP inhibition.Although the MMP-mediated ECM degradation is associated with AEG-1-mediated cancer cell migration and invasion,whether AEG-1 affects TIMPs is unknown.The overexpression of AEG-1 can activate pathways that are responsible for cancer cell survival,metastasis and chemoresistance,such as NF-?B,Akt and Wnt/?-catenin signaling pathways.Discovery of molecules that can interfere with the above-mentioned pathways holds the potential to restrain the growth and metastasis of pancreatic cancer.Studies have found that depletion of AEG-1 suppressed Akt activity and inhibited Bcl-2 expression,and FGF18 following activation of the Akt/GSK3?/?-catenin pathway to up-regulated the MMP-2 and MMP-9.AKT is a key central effector protein of multiple signal transduction pathways.Phosphorylated AKT activates its downstream molecules and is closely related to tumor invasion,metastasis,proliferation and apoptosis.The ?-catenin can interact with a variety of proteins,such as E-cadherin,Lymphoid enhancer factor 1(LEF1)and transcription factor 4(TCF4),involved in Wnt signaling pathway and intercellular adhesion,tumor cell metastasis and other processes.Activated AKT can increase the concentration of?-catenin in tumor cells,promote ?-catenin translocation into the nucleus,thereby accelerating the proliferation of tumor cells.Additionally,AEG-1 silencing demonstrated inhibition of the PI3K/AKT pathway and increased cell apoptosis has been reported by researchers.To further explore the molecular mechanism by which AEG-1 inhibits the proliferation of pancreatic cancer cells,we detected the AKT/?-catenin signaling pathway associated protein.The results showed that p-AKT protein level was decreased when AEG-1 silencing in both As PC-1 and PANC-1 cells.The level of p-?-catenin was upregulated,but ?-catenin in nuclear was significantly downregulated in cells which AEG-1 was knockdown.Consistent with MMP-2,the expression of MMP-9 was also decreased in PDAC cells when the AEG-1 was knocked down.Thus,knock-down of AEG-1 may affect the expression of MMP-2,MMP-9 and Bcl-2,and further inhibit cell migration by the suppression of AKT/?-catenin pathway.On the basis of the present work,further experiments will be performed to investigate by the role of AEG-1 in pathways involved in the tumorigenesis of pancreatic cancer.In conclusion,our study demonstrates that the knockdown of AEG-1 inhibits the proliferation,migration and invasion,and promoted apoptosis in pancreatic cancer cells in vitro.The cell cycle of AEG-1-silenced cells is arrested at G0/G1 stage.These results suggest that pancreatic cancer cells require AEG-1 to maintain their survival and metastasis,suggesting AEG-1 as a potential target in the treatment of pancreatic cancers.