Roles Of UBE2V1 And UBE2V2 On Growth And Metastases Of Liver Cancer Cells And Its Mechanism

Introduction:Primary liver cancer (PLC) is one of the most common malignancies worldwide with increasing incidence and accounts for the third leading cause of cancer-related mortality. This disease tends to occur in livers damaged by alcohol abuse,chronic infection with hepatitis B and C and cirrhosis. An important contributing factor to the development of a tumor is the balance between the activation and inactivation of the proto-oncogenes and anti-oncogenes.Thus, to discover and clarify the reasons for these disorders will be benefit for illuminating the mechanism on pathogenesis of HCC and for developing effective treatment.UBE2V1, also named CROC 1,locates in chromosome 20q13.2 and is expressed as at least four different splice variants. Constitutive expression of exogenous UBE2V1 proteins in HT-29-M6 cells inhibits their capacity to differentiate upon confluence and induces changes in their cell cycle behavior, associated with an inhibition of the mitotic kinase cdk1.UBE2V1 participates in intracellular signalling pathways regulating diverse cellular processes involved in induction of the human FOS promoter.Consistent with a role in immortalization, we have also observed elevated expression of UBE2V1 in several tumor-derived cell lines.Some studies have found that the expression of UBE2V1 is elevated in almost all cancer cell lines,and UBE2V2 gene expression is also elevated in some but not all cancer cell lines. These results suggest that, UBE2V1 and UBE2V2 expression may be involved in cell differentiation and tumorigenesis in a subset of tissues.UBE2V1 and UBE2V2 are kinds of the E2 ubiquitin-conjugating enzymes,but lacke their enzymatic activity.TRAF6, a RING domain protein, functions together with Ubc13/UBE2V1 to catalyze the synthesis of unique polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. IKK is activated through the assembly of K63-linked polyubiquitin chains and then active the NF-kappaB pathway.Recently a study reported that one of the identified compounds, NSC697923, specifically inhibits the activity of Ubc13- UBE2Vl,and showed that this compound impedes NF-kappaB activation in ABC-DLBCL cells and demonstrate that the compound inhibits the proliferation and survival of both ABC-DLBCL and GCB-DLBCL cell lines and survival of primary DLBCL cells.Some study showed that in MDA-MB-231 breast cancer cells, the UBE2V1 transcript level is moderately elevated compared to normal breast cells. Overexpression of UBE2V1 alone in MDA-MB-231 cells is sufficient to activate NF-kB, which in turn upregulates the MMP1 expression to enhance breast cancer cell metastasis. More importantly, experimental depletion of UBE2V1 in MDA-MB-231 cells reduces MMP1 expression and reduces their ability to grow tumors and metastasize in a xenograft mouse model.UBE2V2,also named hMMS2,is a human homologs of MMS2 in the yeast. The yeast MMS2 gene is involved in protection of cells from a variety of DNA damage, since disruption of the MMS2 gene not only results in an increased killing by MMS and UV irradiation, but also dramatically increases the spontaneous mutation rate. Genetic analyses indicate that MMS2 functions in the error-free post-replication repair (PRR) branch of the RAD6 pathway.UBC13 and MMS2 can form a heterodimeric ubiquitin-conjugating enzyme. And two other members of the RAD6 group, UBC13 and MMS2, form a heterodimeric ubiquitin-conjugating enzyme, which catalyses the formation of non-canonical multi-ubiquitin chains linked via K63 of ubiquitin Both enzymes, RAD6 and UBC13-MMS2, are recruited to chromatin by interaction with the RING-finger-containing, DNA-binding proteins RAD 18 and RAD5, respectively for DNA repair.Recently,a study pointed out that in ER-positive/HER2-negative tumors, the expression of UBE2V2 was associated with poor prognosis.So many studies have showen that UBE2V1 and UBE2V2 could participate in a various of cellular processes,including the activation of NF-kappaB pathway and DNA repair,these processes,which are related with tumorigenesis and carcinogenesis,involve in the proliferation and differentiation of cells,the regulations of cell cycles and the overcome of cell senescence.But until now,there is no studies about UBE2V1 and UBE2V2 in liver cancers.Therefore, the aim of the research was to discover the physiological function of UBE2V1 and UBE2V2 and its role in the growth of liver cancer, using modern experimental methods such as cloning technology, western blotting, establishment of an subcutaneous xenotransplanted tumor model of human liver cancer cells in nude mice,in vivo imaging of mouse, Immunological Histological Chemistry and so on.Objective:1. Detect the expression of UBE2V1 and UBE2V2 in normal liver cells and liver cancer cells.2. Build the recombination liver cancer cells expressing UBE2V1 and UBE2V2.3. Explore the effect on proliferation and metastases of liver cancer cells of UBE2V1 and UBE2V2.4. Detect the machanism of the influence on liver cancer cells of UBE2V1 and UBE2V2.Methods:1. The UBE2V1 and UBE2V2 gene were amplified by PCR,using specific primers. After PCR, restriction, and then connected to the lenti-virus vector LV5, LV5-UBE2V1 and LV5-UBE2V2 lenti-virus were constructed by company. Then the LV5-UBE2V1 and LV5-UBE2V2 lenti-virus infected the liver cancer cells. Infected cells screened by puro for one weeks, carried out to expand the training, for subsequent experiments.2. Recombinant cells and negective control cells were seeded with equal number of cells into 24 well-plate,cck8 was added into cells every 24h,after 3h,OD450 were detected, then drawed the growth curves.3.Recombinant cells and negetive control cells were seeded with equal number of cells into 6 well-plate, analysis on colony formation of recombinant cells by plate clone assay was performed.4.Recombinant cells and negetive control cells were seeded with equal number of cells into 6 well-plate,a wound should be did,and DMEM without serum was added into the cells,and then detect the distance the cells crawling at the same time to detect the ability of migration of cells.5.Recombinant cells and negetive control cells were seeded with equal number of cells into transwell chambers,and then the cell numbers of going through the membrane were detected at the same time to investigate the ability of migration of cells.6. Recombinant cells and negetive control cells were seeded with equal number of cells into transwell chambers with ECM matrigel and then the cell numbers of going through the membrane were detected at the same time to investigate the ability of invasion of cells.7.Groups of recombinant cell lines were injected the nude mice into armpit of the ministry by subcutaneously and successfully built xenograflt tumors in nude mice. Tumor growth curve was observed and the expression of ki67 and actived-Caspase 3 Were detected in the tumors with IHC.8. Groups of recombinant cell lines were injected into the tail veins of nude mice to establish the model of liver cancer transfection,which,along with the in vivo imaging assay,were to detect the influence of UBE2V1 and UBE2V2 on the liver cancer cells.9. The expression of c-myc、UBE2V1、UBE2、p-AKT and p-ERK were detected by western blotting to investigate the mechanism of promotion of liver cancer cells transfection by UBE2V1 and UBE2V2.10. The expression of c-myc、UBE2V1、UBE2V、p-AKT and p-ERK of tumors were detected by IHC to investigate the mechanism of promotion of liver cancer cells transfection in vivo by UBE2V1 and UBE2V2.Results:1. Successfully constructed a lenti-virus vector of high expression of UBE2V1 and UBE2V2, and the hepatoma cell lines were infected with lenti-virus.The Cells were screened with puro, and Western Blot was performed to detect the expression of UBE2V1 and UBE2V2. Results showed that the cells infected by LV5-UBE2V1 and LV5-UBE2V2 have significantly increased the expression level of UBE2V1 and UBE2V2, Which suggested to have already built a stable expression UBE2V1 and UBE2V2 hepatoma cell successfully.2. CCK8 assay was used to detect the growth curve of recombinant cells with ectopic expression of UBE2V1 and UBE2V2, and the corresponding control cells and results show that over expression of UBE2V1 and UBE2V2 in recombinant cells have no significant influence on cell growth (P>0.05)3. Plate colony formation experiments showed that, with highly expression of UBE2V1 and UBE2V2, colony formation ability was no significantly weaker or stronger than the control group (P>0.05). This confirmed that UBE2V1 and UBE2V2 can nor promote or inhibit the colony formation ability of liver cancer cells4.The wound healing assay showed that UBE2V1 and UBE2V2 could promote the ability of migration of the liver cancer cells in vitro (P<0.05)5. Transwell migration experiments showed that both UBE2V1 and UBE2V2 increase the migration of liver cancer cells in vitro (P<0.05)6. Transwell invasion experiments showed that both UBE2V1 and UBE2V2 increase the migration of liver cancer cells in vitro (P<0.05)7. Groups of recombinant cell lines were injected into the nude mice armpit of the ministry by subcutaneously and successfully built xenograft tumors in nude mice. Tumor growth curve was observed and found that the tumors with injection of liver cancer cells highly expressing UBE2V1 and UBE2V2 in nude mice,compared with the control nude mice, growed faster and larger.The IHC assay showed that the expression of ki67 was higher in the recombinant cell lines highly expressing UBE2V1 and UBE2V2 (P<0.05),while the expression of actived-Caspase 3 was lower in the recombinant cell lines (P<0.05)8. Groups of recombinant cell lines were injected into the tail veins of nude mice,and then were detected the transfection of the liver cancer cells,which,along with the in vivo imaging assay,showed that UBE2V1 and UBE2V2 can promote the transfection of liver cancer cells (P<0.05).9. Western blot detection of the phosphorylation level of ERK and AKT,found that after over expression of UBE2V1, the expression of phosphorylation of AKT and ERK has not significant change, while over expression of UBE2V2 was able to promote the expression of the phosphorylation of AKT and phosphorylation of ERK.10. In nude mice tumor, immunohistochemical analysis showed that on xenograft tumor sections highly expressed UBE2V1 and UBE2V2, for UBE2V1, the expression level of phosphorylated AKT was higher than the control group (P<0.05),while for UBE2V2, the expression level of phosphorylated AKT and ERK was both higher(p>0.05).Conclusions:UBE2V1 and UBE2V2 could promote the migration and invasion of liver cancer cells in vitro,rather than growth. And in vivo,all the abilities of growth and migration of liver cancer cells can be promoted by UBE2V1 and UBE2V2. The experiments show that for UBE2V1,the activation of PI3K/Akt/mTOR pathways may involve in these processes, but for UBE2V2, PI3K/Akt/mTOR and MAPK pathways may both involve in these processes.