The Role Of Uba2 And Its Regulatory Mechanism In Colorectal Cancer

Background and Objective:Colorectal cancer(CRC)is a malignant tumor with high invasion and metastasis.CRC is the third most common malignant tumor in the world,ranking fourth in China.Because CRC has a higher death rate,it is a serious threat to human health.Surgical treatment and chemoradiotherapy are the main treatments for CRC.Although targeted therapy has been used in the treatment of CRC in recent years,its 5-year survival rate and 10-year survival rate are still not high.This may be due to the reason that CRC is caused by multiple pathways and multiple genetic changes.Therefore,it is an urgent problem to clarify the pathogenesis of CRC and to find the optimal target for molecular target therapy.Uba2 is one of the components of E1 activation enzyme in the process of SUMOylation,which plays an important role in the process of SUMOylation.SUMOylation can regulate some signal transduction pathways,such as cytokines,Wnt,growth factors and hormone signaling pathways.At the same time,it was found that SUMOylation participate in a variety of pathological processes.Therefore,it is considered that Uba2 is important to the physiological and pathological processes of the body.The recent studies found that Uba2 was closely associated with some diseases,including certain tumors.Uba2 expression was significantly higher than that of adjacent tissues,and was related to the pathological grading,proliferation,invasion,metastasis and drug resistance in lung cancer,liver cancer and gastric cancer.However,the role of Uba2 in the occurrence and development of CRC has not been studied.Does Uba2 participate in the formation of colorectal cancer and how it plays a role? Can inhibition of Uba2 expression be effective in inhibiting tumor progression and effective targeting of CRC?Therefore,this study firstly detected the expression level of Uba2 mRNA in human CRC tissue.Secondly we choose HT-29 and DLD-1 cell lines high-expressed Uba2 and HCT-15 cell line low-expressed Uba2 as the research object,and use lentivirus technology to change the level of Uba2 expression.In addition,HT-29 colorectal cancer cell with normal and down-regulated expression of Uba2 were used to construct themodel of subcutaneous xenografted tumor of nude mice.The effect of Uba2 on CRC was studied in vivo and in vitro.Therefore,this study provides a basis for the pathogenesis of CRC,and provides new ideas for the effective targe of targeted therapy.Methods:(1)To analyse the level of the Uba2 mRNA expression in human normal colorectal tissue and cancer tissue,the Oncomine database website(https://www.oncomine.org)was used.(2)In vitro: HT-29,DLD-1 and HCT-15 cells were infected with lentivirus,and the CRC cell lines infected was stabilized by ampicillin resistance.The protein expression levels of Uba2 in HT-29,DLD-1 and HCT-15 cells were detected by Western blot(WB)method.The effects of Uba2 expression on cell proliferation,cell invasion and metastasis were detected by MTT,wound healing,Transwell assay,WB experiment and gelatin zymography assay.The changes of various genes and signaling pathways in CRC cell lines after down-regulation of Uba2 expression were detected by using gene chip technology.The effects of Uba2 expression on the key proteins in Wnt signaling pathway(GSK3β,total β-catenin,phosphorylation β-catenin,TCF-1,MMP7)were detected by using luciferase reporter assay and WB.(3)In vivo: HT-29 colorectal cancer cells down-regulated Uba2 expression and normal HT-29 colorectal cancer cells were used to construct the model of xenografted tumor of nude mice.The volume and quality of tumor were evaluated and analyzed.Results:(1)The expression level of Uba2 mRNA in CRC tissues increased significantly compared with the adjacent tissues.(2)In vitro: HT29 and DLD-1 cell lines down-regulated Uba2 expression and HCT-15 cell line up-regulated Uba2 expression were successfully constructed.Compared with the control group,the proliferation had significantly decreased in HT-29 and DLD-1cells and decreased as well as the ability of migration and invasion.The proliferation,migration and invasion of HCT-15 cells up-regulated Uba2 expression were significantly increased compared with the control group.In HT-29 and DLD-1 cells,MMP2 and MMP9 protein and Vimnetin protein related to EMT were significantly reduced after down-regulation of Uba2 expression,and up-regulated E-cadherin protein expression.In HCT-15 cells,MMP2 and MMP9 protein and Vimnetin protein related to EMT of Uba2 group were significantly higher than those in the control group,and the E-caherin proteinwas lower than the control group.It is shown that inhibition of Uba2 expression can inhibit the metastasis of CRC.Gene chip results show that 650 genes changed in total,and 492 genes were up-regulated and 158 genes were down-regulated.Furthermore,the expression of multiple signaling pathways changed after inhibition of Uba2 expression compared with the control group.In addition,the chip results showed that Uba2 was related to a variety of biological functions,such as molecular adhesion,motion and migration.The level of luciferase reporter after down-regulating the expression of Uba2 was significantly lower than control group in the HT-29 and DLD-1 cells,and the proteins including total β-catenin,nuclearβ-catenin,TCF-1,MMP7 protein expression were decreased,GSK3β,phosphorylationβ-catenin were up-regulated in the Wnt signaling pathway;The opposite results were observed in HCT-15 cell.(3)In vivo : The model of xenografted tumor of nude mice was successfully constructed.Compared with the control group,the mass and volume of the tumor were reduced after down-regulating Uba2 expression.Conclusions:(1)Uba2 expression was significantly higher in CRC than adjacent tissue.(2)In vitro experiments showed that down-regulation of Uba2 protein expression could inhibit the proliferation,invasion and metastasis of colorectal cancer cells.Up-regulation of Uba2 protein expression can promote the proliferation,invasion and metastasis of colorectal cancer cells.It indicates that Uba2 plays a positive regulatory role in the occurrence and development of colorectal cancer.The mechanism may be that activating the expression of WNT signaling pathway after up-regulating Uba2 expression by increasing the nuclear transposition of β-catenin,thereby enhancing the interaction between β-catenin and TCF.(3)In vivo experiments suggested that down-regulation of Uba2 expression could significantly reduce the incidence of colorectal cancer.(4)Uba2 may be a potential target for CRC treatment and this study can provide new ideas for molecular targeted therapy of CRC.