The Role Of Ubiquitin-conjugating Enzyme UBE2C In Gastric Cancer

Objective: The ubiquitin-conjugating enzyme 2C(UBE2C)is well known to interact with the anaphase-promoting complex/cyclosome(APC/C)E3 ligase to promote cell cycle through mitosis.As an APC/C specific E2,UBE2 C has a direct role in regulation of the spindle assembly checkpoint by catalyzing ubiquitination of one or more components of the APC–Cdc20–checkpoint protein complex.Cells over-expressing UBE2 C are failed to maintain spindle checkpoint activity after entering mitosis due to sustained activation of APC/C and excessive degradation of CyclinB,resulting in chromosome missegregation and aneuploidy,which may eventually lead to tumor formation.Amplification and/or overexpression of ube2 c have been reported in many malignancies,and high expression of UBE2 C is associated with poor clinical outcomes.However,the role of UBE2 C in gastric cancer(GC)still remains largely elusive.In this study,we set about to determine the expression status of UBE2 C in GC tissues and serum,following with the analysis of the correlation between UBE2 C level and the clinicopathological characteristics as well as the prognosis of GC patients.The potential of UBE2 C as a serum diagnostic marker for early GC is also evaluated.Cellular functional assays are carried out to further elucidate the underlying mechanisms of UBE2 C involving in proliferation,apoptosis and cell cycle regulation of GC cells,thus providing novel insights into the early diagnosis and precise treatment of GC.Methods: 1.Collecting GC tissue samples and performing the following assays and analysis:1)Quatitive Real-time PCR(qRT-PCR)and Western Blotting(WB)were performed to determine the expression of UBE2 C in 30 pairs gastric cancer samples and adjacent normal tissues.2)Immunohistochemical staining was used to assess the expression of UBE2C in 216 paraffin-embedded gastric cancer tissues.To analyze the relationship between UBE2 C expression and clinicopathological parameters and Ki-67 and Her-2.3)Kaplan-Meier method was used to study the relationship between UBE2C expression and 5-year survival rate of gastric cancer patients;multivariate COX regression analysis was used to analyze the prognostic factors.2.Perform the following experiments in GC cell lines:1)The expressions of UBE2 C protein in normal gastric epithelial cells and GC cell lines were determined by WB.2)In UBE2 C high-expressing GC SNU-1 cells,UBE2 C knockdown plasmid was constructed and verified,and co-transfected into the 293 T cells with the packaging plasmids.Mature lentiviral particles were harvested from the culture supernatant.Twenty-four hours after the infection of SNU-1 cells,Puromycin was used to select the SNU-1 cells with silenced UBE2 C.WB was performed to confirm the knockdown of UBE2 C.3)The cell proliferation was analyzed by WST-1 assay,the cell cycle and apoptosis analysis were performed by flow cytometry and the expressions of PARP,p-ERK,p-AKT,p-Wee1,p53 and Bcl-2/Bax were determined by WB after knockdown of UBE2 C.3.Performing the following assays and analyses using serum samples:1)To explore the potential of UBE2 C as a serum marker for early diagnosis of GC,ELISA assays were used to determine the levels of UBE2 C in serum samples from 50 healthy volunteers,30 cases of chronic gastritis and 30 cases of GC,respectively.2)The difference of serum UBE2 C levels between preoperative and postoperative plasma is explored.The correlation between serum UBE2C level and clinicopathological parameters was also analyzed.Results: 1.The expression status of UBE2 C in GC:1)The expression level of UBE2 C was higher in gastric cancer tissues than that in normal gastric mucosa tissues,and the difference was statistically significant.2)The results of immunohistochemistry showed that UBE2 C was mainly accumulated in the cytoplasm of tumor cells.The positive rate of UBE2C staining was 67.6% in gastric cancer tissues,while the positive rate only of UBE2C staining was 19.4% in the adjacent normal tissues..The expression levels of Ki67 and Her-2 were associated with the expression level of UBE2 C.UBE2C expression levels were statistically correlated to lymph node metastasis,serosal invasion,and TNM staging in gastric cancer patients,but not correlated to age,gender,tumor differentiation,Lauren classification,and tumor location.3)Kaplan-Meier analysis showed that the high expression of UBE2 C was significantly correlated with the poor prognosis of GC patients.Multivariate COX regression analysis showed that UBE2 C was an independent prognostic factor for GC.2.The functional and mechanical assays in GC cells:1)The expression level of UBE2 C in normal gastric epithelial cells GES-1 was significantly lower than that in GC cells.The expression level was high in SNU-1 cell lines.2)Knockdown of UBE2 C inhibited the proliferation of SNU-1 cells.There were significant differences between the two groups of cells at each time point of detection.After 48 hours silencing of UBE2 C,the apoptosis rate of the cells was increased from 3.02±0.36% to 18.01±2.15%(P<0.01)determined by flow cytometry.After knockdown of UBE2 C,SNU-1 cells were arrested in the G2/M phase.3)The levels of some key proteins involving in cell proliferation and apoptosis were determined by WB after knockdown of UBE2 C,and our results showed that the levels of cleaved-PARP and p-p53 were increased,while p-Wee1 protein level and the ratio of Bcl-2/Bax was reduced.3.The serum UBE2 C level in GC patients:1)In serum,the level of UBE2 C protein in gastric cancer patients was significantly higher than that in normal healthy people and that in chronic gastritis patients.The differences were statistically significant.2)The UBE2 C level in serum was significantly correlated to tumor serosal invasion,Lauren classification,and TNM staging,but not correlated to age,gender,tumor size,degree of differentiation,and location of the tumor.There was no significant difference of serum UBE2 C levels between preoperative and postoperative patients.Conclusions: 1.The expression level of UBE2 C was significant higher in gastric cancer tissues than that in normal gastric mucosa tissues,which was correlated to lymph node metastasis,serosal infiltration and TNM staging.UBE2 C could serve as an independent prognostic factor for GC.2.Knockdown of UBE2 C in GC cells resulted in G2/M phase arrest,promoted apoptosis and slower proliferation rate of SNU-1 cells,suggesting UBE2 C may represent a potential therapeutic target of GC,which provides novel insights into the precise treatment strategy for GC patients.The wee1 and p53 and Bcl-2 pathways may have involved in the above fuctional impairments in UBE2C-deficint GC cells,however,the underlying molecular mechanism warrants further investigation.3.In the serum of gastric cancer patients,the expression level of UBE2 C was significantly higher than those of normal people and chronic gastritis patients.The serum UBE2 C level was closely related to the key clinicopathalogical characteristics of gastric cancer,including tumor serosal invasion,Lauren classification,and TNM staging.These results suggested that UBE2 C may serve as an potential serological marker for early diagnosis of GC.