Ubiquitin Conjugating Enzyme E2T Facilitates Proliferation Of Hepatocellular Carcinoma Via Activiation Of AKT Pathway

Background and objectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors.At present,the incidence of HCC is increasing every year,exceeding 750000 new cases each year according to the latest data,ranking fifth in clinical morbidity of all the malignant tumors worldwide;and the mortality takes the second among the tumor related death.HCC has the characteristics of unconspicious sympotoms,fast progression and poor prognosis,till now,the best treatment of HCC is operation and liver transplantation.Although the other treatment options including Percutaneous Ethanol Injection(PEI),Transcatheter Arterial Chemoembolization(TACE),radiation therapy,immunotherapy and molecular targeted therapy have already had some effects,but the 5-year incidence doesn't improved significantly.One of the important reasons for that is that so little mechanism we have known about HCC that we haven't found a molecular to guide us to make early diagnosis,prognosis evaluation and also find treatment target.So it is critical to explore the molecular regulatory net insides HCC and investigate the mechanism underlying development and progression of HCC.Ubiquitin-proteasome system extensively exits in eukaryocytes,playing a role in cell cycle regulation,cell signal transduction and cell apoptosis.This system contains ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2 and ubiquitin ligase E3.More and more studies showed that ubiquitin-conjugating enzymes(E2)have a close relationship with oncogenesis in varied types of cancers.ubiquitin-conjugating enzymes UBE2C which is upregulated in lung cancer,esophagus cancer,breast cancer and many other cancers can serve as an early diagnostic and prognostic molecular of esophagus cancer;UBC13 can facilitate metastasis breast cancer via activating TAK1-P38 signaling pathway.As a member of ubiquitin-conjugating enzyme E2,ubiquitin-conjugating enzyme E2T(UBE2T)plays a regulatory role in many kinds of cancers.In the early study,Takata et al.firstly found UBE2T was upregulated in the specimens of bladder cancer,lung cancer and prostate cancer.Then Yuichi et al.found UBE2T expression can regulate the level of single ubiquitin FANCD2,thereby regulating FA pathway,blocking cell DNA damage repair pathways,cause the occurrence of tumor.Some recent studies have shown the increased expression of UBE2T in lung cancer tissue compared with non-tumor tissue,and the lung cancer cells with overexpressing UBE2T possessed significantly higher colony-forming ability,indicating UBE2T expression level is closely related to the initiation of lung cancer.Study of Ueki et al.suggested that suppressing UBE2T expression or inhibiting its activity can restrain the degradation of BRCA1,inhibiting the proliferation of breast cancer cells.Ramaekers et al.found that in the condition of hypoxia,the downregulation of UBE2T can increase the chemotherapy sensitivity of cancer cells.These results suggest that UBE2T involves in the development of oncogenesis as an oncogene.Cao Chuanhui master in our research group tested the expression of UBE2T in digest system tumors by tissue microarray and immunohistology.He found that UBE2T was upregulated in HCC,esophagus cancer,gastric cancer and colon cancer.Then he focused on the expression of UBE2T in HCC,he confirmed the expression of UBE2T in HCC tissues using western blot and qRT-PCR.He also found that there were 60%positive UBE2T expression in 133 paraffin embedded cancer tissues while there was only 15%in the paired non-cancer tissues using immunohistochemistry.Survival analysis showed that UBE2T can act as an independent prognostic risk factor for HCC.This is the further study of Cao's research which focuses on the biological function of UBE2T and the mechanism underlying.In this study,first of all,we extanded the HCC patients' sample size to 171 and used immunohistochemistry to explore the expression level of UBE2T in these 171 HCC specimens.Then we analyze UBE2T's effect on the prognosis of HCC patients by the method of Kaplan-Meier and COX regression model.Then,by the method of overexpression and knockdown,we explored its role in the proliferation and metastasis in HCC and the underlying mechanism,thus illustrating its influence on the initiation and development in HCC,finally laying the theoretical basis for HCC new prognostic molecular markers.Methods1.The expression of UBE2T in HCC.We use immunohistochemistry to detect the expression of UBE2T in the paraffin sections of primary HCC(n=171),then grading its expression level.We apply Kaplan-Meier method to analyse the influence of UBE2T on prognosis of HCC,Spearman rank correlation method to analyse the relationship between the UBE2T expression and clinical pathological parameters,and COX proportional hazards regression model to do univarible and multivariable factor analysis on the relationship between the clinical pathological parameters and the prognosis of primary HCC to make clear UBE2T whether can be an independent risk factor for the prognosis of primary HCC.2.The biological effect of UBE2T in HCC.The overexpressing and knockdown HCC cell lines are established by transducing overexpressing lentivirus construct Lv-UBE2T and knockdown lentivirus construct Lv-shRNA-UBE2T to HCC cells respectively.The transducer efficiency was confirmed by western blot.We used CCK-8 to test the change of cell proliferation,we used flow cytometry to test the change of cell cycle and apoptosis,and we also used western blot to determine the expression level of the protein markers of proliferation,cell cycle and apoptosis before and after transduction.The in vivo model was established by injecting overexpressing and knockdown HCC cell lines subcutaneously into nude mice.3.The mechanism of UBE2T in initiation and development in HCC.We used western blot to detect the phosphorylation of the key component in cell signaling pathways in HCC cells before and after overexpression and knockdown of UBE2T,by using pathway inhibitors,we used CCK-8 to test the change of proliferative ability in HCC cells.Results1.Analysis of UBE2T expression in HCC.The UBE2T expression in 171 cases of paraffin-embedded liver cancer specimen was tested by immunohistochemical method.Result showed the higher expression of UBE2T in HCC-tissue compared to non-tumor tissues.Spearman rank correlation was used to analyze the relationship between the expression of UBE2T and HCC clinicopathological parameters.We found that the expression was significantly related to the number of the tumors(P<0.001),BCLC staging(P<0.000),portal vein tumor thrombus(P<0.001),distant metastasis(P=0.017).On contrast,there was no significant relationship between the expression and age,gender,pathological grade,tumor size,recurrence.We performed the Kaplan-Meier to analyze UBE2T and primary liver cancer prognosis.By using COX proportional hazards regression model single factor and multi-factor to analyze the relationship between various clinicopathological parameters and primary liver cancer prognosis,it was found that UBE2T(HR=1.837,P=0.012),recurrence(HR=1.516,P=0.048)and Portal vein tumor thrombus(HR=2.089,P=0.024)were both the independent risk factors of HCC prognosis.2.UBE2T induces cell cycle progression,inhibits apoptosis,facilitates proliferative ability in HCC cells.We explored the biological function of UBE2T by overexpressing and knockdown in HCC cells.Cell proliferative ability was tested by CCK-8,we found that cells gained a higher ability after stable expression of UBE2T,whereas knockdown of UBE2T can make cells grow more slowly.The in vivo nude mice model was established by injecting stable overexpressing cells and knockdown cells subcutaneously.The mice carried the overexpressing cells had a shorter expectancy of tumors than the ones with vehicle cells,on the contrary,the tumors in mice with the knockdown cells had lower formation ability.Also,we used flow cytometry to examine the change in cell cycle.A transition repression of cells from G2/M phase was found in Huh7 cells after the knockdown of UBE2T.3.UBE2T promote development of HCC by activating Akt/GSK-3?/?-catenin pathway.Next,we discuss the mechanism involved in the regulatory function of UBE2T in HCC.We found that after overexpressing UBE2T in HCC cells,the phosphorylated Akt was significantly upregulated,and downregulated after knockdown.Then we tested the expression level of the downstream factors of Akt including GSK-3? and ?-catenin.In UBE2T overexpressing cells,p-GSK-3?(deactivating form)and ?-catenin increased while decreased in knockdown cells,which indicated that UBE2T may fuction via Akt/GSK-3?/?-catenin signaling pathway.In order to confirm the result,we used Akt pathway inhibitor MK-2206 2HCL to block the pathway,the proliferative ability of HCC cells was impaired by CCK-8 assay.All these results showed that UBE2T modulate HCC proliferation via Akt/GSK-3?/?-catenin signaling pathway.Conclusions1.UBE2T was upregulated in HCC,and was closely related to the prognosis,and it can be used as an independent prognostic risk factor of HCC.2.UBE2T induces cell cycle progression,inhibits apoptosis,facilitates proliferative ability in HCC cells.3.UBE2T promotes development of HCC by activating Akt/GSK-3?/?-catenin pathway.