Effect Of UBR5 On The Survival Of Gallbladder Carcinoma Patients And Its Biological Function In Gallbladder Cancer Cells

Background:As one of common biliary malignant tumor?BMT?,the incidence of gallbladder cancer ranks the 7^th place in the malignant tumors of digestive tract,it is high malignant and progresses rapidly with poor prognosis,the 5-year survival rate is only about 10%.For now,radical resection is still the optimal treatment of patients with gallbladder cancer,however the 5-year survival rate after radical resection is only about 16.5%,and easy to relapse and metastasis after surgery.Because the pathogenesis of gallbladder cancer is concealed and it is lack of corresponding specific clinical symptoms and early diagnosis indexes,most of the patients are always diagnosed at an advanced stage,when local invasion and distant metastasis has occurred,in this case the patients has already lost the chance of a radical surgical treatment.Besides,the effects of radiotherapy,chemotherapy or other adjuvant therapy for the treatment of intermediate and late gallbladder cancer are also very limited,even the high sensitivity of gemcitabine,the effectiveness of its treatment is only 36%.So by studying the relevant mechanisms to optimize present therapy and find new targets is extremely urgent.UBR5 is a Drosophila tumor suppressor gene.Mammal UBR5 is a HECT domain family?homologous E6 related protein at the carboxy terminus?and E3 ubiquitin ligase recognition positive determinants.The human UBR5 gene has 59 exons encoding about 10 kb of mRNA and>300 kDa of protein,and is widely expressed in various cell types.UBR5 is highly conserved in multicellular animals,has unique structural features,and is involved in the regulation of DNA damage response,metabolism,transcription and apoptosis.Therefore,UBR5is a key regulator of cell signaling related to the field of tumor biology.In recent years,a large number of studies have found that UBR5 plays an important role in the development of many tumors,and UBR5 expression may be closely related to the proliferation and metastasis of malignant tumors.In addition,some researches have shown that UBR5 may also be associated with the resistance or the sensitivity of some cancer cells on chemotherapeutics,but the specific mechanism is not yet clear.However,the study of UBR5 is still relatively rare in gallbladder cancer.Therefore,it is necessary to ascertain its impact on gallbladder cancer and related mechanisms to fill gaps in this research area.Objective:Observing the expression of UBR5 gene in gallbladder tumor and combine the clinical information of cases to analyze the risk factors affecting UBR5 protein expression and explore the risk factors affecting the prognosis of patients.UBR5 gene in human gallbladder cancer cell line GBC-SD was silenced by liposome method to study its effects on cell proliferation,cell cycle and apoptosis.Methods:1.39 gallbladder tumor specimens were collected from Tianjin Nankai Hospital from January 2014 to October 2017.In addition,the gallbladder cancer tissue chip of clinical data were purchased,so a total of 79 cases were analyzed.All patients did not receive any treatment before surgery and had complete case data and follow-up results.All specimens were paraffin-embedded sections,and the expression of UBR5protein was detected by immunohistochemistry.Chi-square test was used to statistically analyze the correlation between the case data and the UBR5 protein expression level.2.Lentivirus was used to transfect shRNA into human gallbladder cancer GBC-SD cells to silence the UBR5 gene,thereby constructing a gallbladder cancer GBC-SD cell line that stably expresses UBR5 protein.3.The mRNA and protein levels of UBR5 gene in gallbladder cancer cells after gene silencing were detected by semi-quantitative PCR and Western blotting,respectively.Flow cytometry was used to detect the effect of UBR5 gene silencing on the cell cycle and apoptosis of gallbladder cancer GBC-SD cells.Results:1.The level of UBR5 expression affected the size of tumor growth,tumor histological type and differentiation?p<0.05?,but there was no significant difference with age,gender,distant metastasis,lymph node metastasis,clinical stage and Nevin stage?p>0.05?.The survival analysis of the patients was also performed.The level of UBR5 expression,distant metastasis and differentiation were closely related to the prognosis of the patients?p<0.05?.2.Stably knockdown sh-UBR5-GBC-SD cell line was constructed,and the expression of green fluorescent protein was over 90%.The lowest expression of UBR5 protein was determined in sh1-UBR5-GBC-SD cell line by semi-quantitative PCR and Western blotting.3.Compared with the scrambled control group SH0,the growth rate of gallbladder cancer cells was significantly reduced,cell cycle was arrested in the G2/M phase,the G1/G0 phase ratio was decreased,the proportion of S phase was not changed significantly,and the cell apoptosis rate was increased significantly,after UBR5 gene knockdown.These results indicated that UBR5 knockout inhibitd the growth of GBC-SD cells in vitro.Conclusion:1.When UBR5 was highly expressed in gallbladder cancer,the patients had larger tumors,more adenocarcinomas,and higher degree of malignancy;patients with higher UBR5 expression had a shorter survival period than patients with low UBR5expression.2.Our group has constructed GBC-SD cell line that stably expresses UBR5 protein,and the gallbladder cancer GBC-SD-SH1 cells were screened for the highest knockdown efficiency.3.In human gallbladder cancer GBC-SD cells,low expression of UBR5 inhibited cell growth,induced cell apoptosis,and regulated cell cycle.The specific mechanisms need to be further studied.This experiment provided the most basic and necessary evidences for the effects of UBR5 gene on human gallbladder cancer cells.