The Effects And Mechanisms Of UBA2 On Proliferation,Migration And Invasion Of Gastric Cancer

Background and Objective:Gastric cancer is the fourth most common malignancy and the second leading cause of cancer-related death globally.In China,the incidence and cancer-related mortality of gastric cancer is second only to lung cancer.At present,surgery is still the main treatment for patients with early stage gastric cancer.Nevertheless,due to high heterogeneity,occult onset and rapid development of the disease,most patients with gastric cancer are diagnosed at middle and late stages,and the treatment often requires of combination with other methods such as chemotherapy.With new insights into etiology,pathology and molecular mechanisms of the disease,there has been some progress in the selection of chemotherapy regimens and the development of targeted drugs at molecular level for gastric cancer,but few significant effects have been achieved.In addition,the limitation of curative effect may be related to the complex process of gastric cancer development,which often involves changes in multiple genes and signaling pathways.Therefore,further elucidating the pathogenesis of gastric cancer,exploring new molecular targeted drugs and benefiting the survival of patients are still the key issues to be solved in the treatment of gastric cancer.Small ubiquitin-like modifiers(SUMOs)are newly discovered ubiquitin-like molecules that can reversibly modify post-translated proteins and regulate the stability,localization and interaction of target proteins with other proteins.Several studies have indicated that SUMOylation is involved in the tumorigenesis and development of gastric cancer.However,ubiquitin-like modifier activating enzyme 2(UBA2),as an important component of E1 enzyme in SUMOylation,has few research on its role in the tumorigenesis and development of gastric cancer.The effects and mechanisms of UBA2 on proliferation,migration and invasion of gastric cancer were not fully elucidated.In order to investigate the role of UBA2 in the tumorigenesis and development of gastric cancer and its underlying molecular mechanisms,firstly,we detected the levels of UBA2 mRNA and UBA2 protein in human gastric cancer tissues by The Cancer Genome Atlas(TCGA)database,immunohistochemistry(IHC)and Western blotting,and analyzed the correlation between UBA2 protein level and clinicopathological indicators as well as overall survival rate.Secondly,BGC-823 cell line with high expression level of UBA2 protein and SGC-7901 cell line with low expression level of UBA2 protein were used to construct gastric cancer cell lines with knock-down and over-expression of UBA2 by lentivirus infection respectively.Finally,the effects of UBA2 on proliferation,migration and invasion of gastric cancer cells were observed in vivo and in vitro.The expression levels of cell cycle-related proteins,epithelial-mesenchymal transition(EMT)-related proteins and invasion-related proteins were detected and the molecular mechanisms were investigated.Through the above studies,we revealed the roles and molecular mechanisms of UBA2 in the tumorigenesis and development of gastric cancer,and provided research basis for UBA2 as a therapeutic target for gastric cancer.Methods:1.Gastric cancer and paracancerous tissue specimens were collected from the First Hospital of Jilin University from August 2013 to August 2018.The expression of UBA2 protein was examined in 132 patients.Among 132 cases,100 cases(100 gastric cancer tissues and 22 paracancerous tissues)were examined by IHC,32 paired fresh tissues were examined by Western blotting.The difference of UBA2 expression level between gastric cancer tissues and paracancerous tissues was compared.The clinical data of 100 patients were collected and the correlation between UBA2 expression and clinicopathological parameters as well as overall survival rate of gastric cancer patients was analyzed.StarBase website was used to retrieve UBA2 mRNA level of patients with gastric cancer in TCGA database,and the results of UBA2 mRNA in gastric cancer and normal gastric tissues were obtained and analyzed.2.Experiments in vitro:(1)Using lentivirus infection technology,BGC-823 and SGC-7901 gastric cancer cells were infected.Gastric cancer cell lines with knock-down and over-expression of UBA2 were constructed respectively.BGC-823 cells were divided into 3 groups: normal cell group(Normal group),control group(Control group)and siRNA-UBA2 infected group(siUBA2 group);SGC-7901 cells were also divided into 3 groups: normal cell group(Normal group),empty vector group(EV group)and UBA2 infected group(UBA2 group).Western blotting assay was used to detect the expression level of UBA2 protein in BGC-823 and SGC-7901 gastric cancer cells after infection;(2)MTT,colony formation assay and flow cytometry were used to detect the effects of UBA2 on proliferation and cell cycle of gastric cancer cells.The changes of cell cycle-related proteins(p21,p27,Cyclin D1 and CDK4)were detected by Western blotting assay;(3)The effects of UBA2 on migration and invasion of gastric cancer cells were detected by wound healing assay,transwell migration and invasion assay.The expression levels of invasion-related proteins(MMP2,MMP7 and c-Myc)and EMT-related proteins(E-cadherin and Vimentin)were detected by Western blotting;(4)MTT assay was used to evaluate the effect of UBA2 on chemosensitivity of gastric cancer cells after adding cisplatin;(5)Western blotting was used to detect the expression levels of key proteins in Wnt/?-catenin signaling pathway(total ?-catenin,p-?-catenin and nuclear ?-catenin).T-cell factor(TCF)/lymphoid enhancer-binding factor 1(LEF1)double luciferase reporter gene assay was used to analyze the effect of UBA2 expression on the activity of Wnt signaling pathway;(6)Western blotting was also used to detect the expression levels of key proteins(Akt and p-Akt)in Akt signaling pathway.The effects of UBA2 expression on Akt signaling pathway-related proteins(Akt,p-Akt,p27,Cyclin D1 and CDK4)were further examined after adding Akt activator(SC79)and PI3 K inhibitor(LY294002).3.Experiments in vivo: (1)Nude mice were subcutaneously injected with BGC-823 cells with knock-down of UBA2(siUBA2 group)and Control group cells,SGC-7901 cells with over-expression of UBA2(UBA2 group)and EV group cells to construct models of nude mice xenograft tumors.The volume and weight of xenograft tumors were measured and analyzed statistically;(2)Western blotting was used to detect the expression levels of cell cycle-related proteins(p21,p27 and Cyclin D1)in xenograft tumors.Results:1.The level of UBA2 mRNA in gastric cancer tissues were significantly higher than that in normal gastric tissues,and the expression level of UBA2 protein in gastric cancer tissues was significantly higher than that in paracancerous tissues;the expression level of UBA2 protein was related to the degree of differentiation(P<0.001),Lauren classification(P=0.003),vascular invasion(P=0.045),TNM stage(P=0.048),T classification(P=0.042)and N classification(P=0.026)of gastric cancer.There was no correlation with age(P=0.514),gender(P=0.508),tumor size(P=0.381)or perineural invasion(P=0.061).The overall survival rate of patients with low expression of UBA2 in gastric cancer tissues was significantly better than that of patients with high expression of UBA2(P<0.05).2.Experiments in vitro:(1)BGC-823 gastric cancer cell line with UBA2 knock-down and SGC-7901 gastric cancer cell line with UBA2 over-expression were successfully constructed by lentiviral infection;(2)Compared with Control group cells,BGC-823 cells with UBA2 kncok-dwon showed marked decrease in cell proliferation and proportion of S phase in cell cycle,down-regulation of cell cycle-related proteins Cyclin D1 and CDK4,while up-regulation of p21 and p27.The ability of proliferation in SGC-7901 cells with UBA2 over-expression was significantly enhanced compared with EV group cells,the proportion of S phase in cell cycle was increased,the expression levels of Cyclin D1 and CDK4 were up-regulated,while the expression levels of p21 and p27 were down-regulated;(3)Compared with Control group cells,the abilities of migration and invasion in siUBA2 group cells were decreased,and the expression levels of MMP2,MMP7,c-Myc and Vimentin were down-regulated,while the expression level of E-cadherin was up-regulated.The abilities of migration and invasion in SGC-7901 cells with UBA2 over-expression were significantly enhanced in comparison with EV group cells,the expression levels of MMP2,MMP7,c-Myc and Vimentin were up-regulated,and the expression level of E-cadherin protein was down-regulated;(4)The chemosensitivity of BGC-823 cells with UBA2 knock-down was higher than that of Control group cells,the chemosensitivity of SGC-7901 cells with UBA2 over-expression was lower than that of EV group cells;(5)In BGC-823 cells,the ratio of TOP Flash/FOP Flash in siUBA2 group was significantly lower than that in Control group.The expression levels of total and nuclear ?-catenin were down-regulated,and the expression level of p-?-catenin was up-regulated.In SGC-7901 cells,the ratio of TOP Flash/FOP Flash in UBA2 group was significantly higher than that in EV group,the expression levels of total ?-catenin and nuclear ?-catenin were up-regulated,while the expression level of p-?-catenin was down-regulated;(6)In BGC-823 cells,the expression level of p-Akt in siUBA2 group was significantly lower than that in Control group,while the expression level of p-Akt in SGC-7901 cells with UBA2 over-expression was significantly higher than that in EV group,and there was no statistical difference in the expression of Akt.After adding LY294002 in UBA2 group of SGC-7901 cells,the expression levels of p-Akt,Cyclin D1 and CDK4 were significantly down-regulated,the expression level of p27 was significantly up-regulated,while the expression level of Akt was not statistically different.After adding Akt activator SC79 in siUBA2 group of BGC-823 cells,the expression levels of p-Akt,Cyclin D1 and CDK4 were significantly up-regulated,the expression level of p27 was significantly down-regulated,and the expression level of Akt was not statistically different.3.Experiments in vivo:(1)The nude mice model of subcutaneous xenograft tumors was successfully constructed.Compared with Control group,the weight and volume of xenograft tumors were significantly decreased in siUBA2 group;compared with EV group,the weight and volume of xenograft tumors were significantly increased in UBA2 group;(2)Compared with Control group,the expression level of Cyclin D1 was downregulated in siUBA2 group,while the expression levels of p21 and p27 were up-regulated.Compared with EV group,the expression level of Cyclin D1 was up-regulated in UBA2 group,while the expression levels of p21 and p27 were down-regulated,which were consistent with the results in vitro.Conclusions:1.The levels of UBA2 mRNA and UBA2 protein in gastric cancer tissues were significantly up-regulated.The expression of UBA2 protein was positively correlated with worse differentiation and high TNM stage of gastric cancer.UBA2 was also a risk factor for clinical prognosis.2.Knock-down and over-expression of UBA2 can inhibit and promote the proliferation,migration and invasion of gastric cancer cells,respectively.3.UBA2 can regulate the expression levels of cell cycle-related proteins,invasion-related proteins,and EMT-related proteins,which are related to Wnt/?-catenin and Akt signaling pathways in gastric cancer.4.UBA2 might be involved in the tumorigenesis and development of human gastric cancer and act as a potential target for the treatment of gastric cancer.