Molecular Regulation Of UBE2T On Gastric Cancer Cell

Ubiquitin-conjugating enzymes?E2 enzymes?such as UBE2T target proteins for ubiquitination and degradation via the proteasome.Here,we examined the effects of UBE2T on the progression of gastric cancer.UBE2T is highly expressed in gastric tumors and gastric cancer cells.siRNA-mediated suppression of UBE2T inhibited gastric cancer cell proliferation and colony formation,promoted cell cycle arrest at G2/M phase and cell apoptosis and death.Suppression of UBE2T also attenuated the invasive and metastatic abilities of gastric cancer cells by altering expression of epithelial-mesenchymal transition?EMT?-related factors.A xenograft model in which nude mice were injected with UBE2T knockdown human gastric cancer cells confirmed that suppression of UBE2T also decreased tumor formation and growth in vivo.Expression levels of CCND1,Phospho-GSK3?,WNT family members,and MYC were all affected by UBE2T knockdown.These results suggest that UBE2T plays a critical role in gastric cancer,and that it may serve as a useful prognostic biomarker and therapeutic target in gastric cancer patient.The paper consists of five Chapters.Chapter 1:UBE2T expression was increased in gastric tumors:To investigate the effects of UBE2T on gastric cancer progression,UBE2T expression was examined in130 gastric tumor samples and in 39 para-carcinoma tissues using IHC.As shown in this study,UBE2T expression was obviously increased in gastric tumors compared to para-carcinoma tissues?A:40×,B:100×,C:400×?.The number of samples with high expression of UBE2T was also higher in gastric tumor samples than para-carcinoma samples?P<0.05?.UBE2T expression was then examined in samples with different degrees of differentiation;However,no differentiation-dependent differences were observed?P>0.05?.Chapter 2:siRNA-mediated knockdown of UBE2T in gastric cancer cell:Given that increased UBE2T expression is associated with carcinogenesis.We next examined whether suppression of UBE2T could inhibit and attenuate proliferation,invasion,and metastatic abilities in gastric tumors or not.To that end,siRNA was used to knock down UBE2T expression in three common gastric cancer cell lines:SGC-7901,BGC-823,and AGS.We find that puromycin resistant screening?A?and green fluorescent protein?GFP?detection?B?revealed that siRNA lentivirus infection was successful in SGC-7901,BGC-823,and AGS cells.UBE2T expression was then measured using real-time PCR.GAPDH was used as a native control and relative UBE2T expression?UBE2T/GAPDH?was evaluated.siRNA infection dramatically decreased UBE2T gene expression in SGC-7901,BGC-823,and AGS cells?P<0.01?.Western blots revealed that UBE2T protein expression also dramatically decreased in SGC-7901,BGC-823,and AGS cells after siRNA infection.Cells in which was inhibited UBE2T expression were named shUBE2T,while the negative control cells transfected with empty vector were named shCtrl.Chapter 3:Suppression of UBE2T inhibited growth and colony formation,increased G2/M phase cell cycle arrest,and promoted apoptosis in gastric cancer cells:To examine the effects of UBE2T suppression on cancer cell proliferation,cellomics detection and MTT assays were conducted.Cellomics is the discipline of quantitative cell analysis using bioimaging methods.After siRNA lentivirus infections,proliferation clearly decreased in SGC-7901 cells.This indicated that UBE2T expression was associated with cell proliferation and that suppression of UBE2T inhibited the proliferation of SGC-7901 cells.MTT detection in SGC-7901cells produced similar results.The OD₄₉₀90 value,which is indicative of the number of living SGC-7901 cells,also decreased in a time-dependent manner after siRNA lentivirus infections.MTT detection also indicated that suppression of UBE2T inhibited the proliferation of SGC-7901 cells in a time-dependent manner,reaching maximum inhibition on the 5th day.We then conducted a tumor colony formation assay.in both SGC-7901 and BGC-823 cells,the number of colonies was much lower on the 13th day after plating in shUBE2T groups compared to the shCtrl groups?P<0.01?,indicating that suppression of UBE2T inhibits colony formation in gastric cancer cells.Flow cytometry?FCM?was then performed to examine the effects of suppression of UBE2T on cell cycle progression.The percentages of shUBE2T cells in the G1 and S phases of the cell cycle were decreased,while the percentage in the G2/M phase was obviously increased,compared to shCtrl cells?P<0.01?.This indicated that down-regulation of UBE2T increased cell cycle arrest at the G2/M phase.Because abnormal cell cycle progression can result in cellular apoptosis,we examined apoptosis in gastric cancer cells using FCM to detect Annexin V staining.The percentage of apoptotic cells greatly increased after the siRNA lentivirus infection in both SGC-7901 and BGC-823 cells?P<0.01?,Chapter 4:Suppression of UBE2T attenuated tumor formation and growth:we performed in vivo studies to confirm the effects of UBE2T on tumor formation and growth observed in the in vitro experiments.We injected shRNA lentivirus-infected BGC-823 gastric cancer cells in which UBE2T expression was greatly inhibited into nude mice to generate the KD group.Ten mice each were assigned to the KD and NC groups.Tumors grew rapidly in the NC group,indicating that the gastric cancer cells proliferated quickly.In contrast,tumors grew much more slowly in the KD group.Mice were sacrificed on the 18th day after injection and the tumors were collected.Tumors from the NC group were larger and heavier than those from the KD group(P<0.01.These results indicate that suppression of UBET2 inhibited the growth of human gastric carcinoma cells,and consequently tumor formation and proliferation,in vivo.Chapter 5 Path-array analysis for UBE2T Analysis of UBE2T-related signaling factors and networks.To explore the regulation of UBE2T in gastric cancer,path-array analysis was performed on 3 samples each from the NC and KD groups using gene chips.Sample quality was confirmed prior to the analysis.Correlations between samples in each group were analyzed;A clustering dendrogram was used to identify differences in gene expression between the two groups.Compared to the control were up-regulated,while a total of 282 genes were down-regulated,in the KD group.Genes for which no obvious changes.Shorter distances indicate a more similar relationship.Meta-analysis was performed to detect pathways that might be regulated byUBE2T using IPA online software.The results suggested that many cellular events,such as growth and proliferation,movement,and cell death and survival,are involved in the regulation of UBE2T.A possible regulation network for UBE2T based on our results and the results of previous studies.The expression of UBE2T-related factors identified in the path-array analysis was then analyzed at the gene level using real-time PCR and at the protein level using Western blots in tumor cells.CDH1,Phospho-GSK3B,WNT7B,and WNT5A mRNA expression were increased in the KD group compared to the NC group.In contrast,CCND1 and MYC mRNA expression were decreased in the KD group.The Western blot results were similar to those obtained using RT-PCR.WNT7B,MYC,and WNT5A protein expressions were decreased and almost undetectable in the NC group.Phospho-GSK3B protein expression also increased by 42.1%,in the KD group.In contrast,CDH1 protein expression,which was very weakly expressed in the NC group,increased in KD group.CCND1 protein expression also decreased by 85.2%in the KD group.Except for WNT7B and WNT5A,for which protein expression decreased although mRNA expression was unchanged,similar changes in expression were observed at both the mRNA and protein levels for all factors examined.Taken together,these results and previous research indicates that UBE2T might play a critical role in gastric cancer and might serve as useful potential biomarker and therapeutic target in gastric cancer patients.