The Role And Mechanism Of Ube2v1 In Colorectal Cancer Metastasis

Background and ObjectEconomic development is accompanied by an improvement in the standard of living,followed by changes in the dietary structure.From a worldwide perspective,colorectal cancer has developed into one of the most common digestive system tumors.In recent years,with the progress of medical and technological progress,colorectal cancer diagnosis and treatment has made great progress;but its morbidity and mortality is still at a high level,and colorectal cancer metastasis and poor prognosis is leading to death main reason.Therefore,the study of the occurrence and metastasis of colorectal cancer mechanism,for patients with early diagnosis,targeted therapy and survival prognosis are of great significance.Ubiquitination is a fundamental translational modification of intracellular homeostasis,and the family of binding enzymes(E2)is a key component of the ubiquitin protease complex system.Ube2v1 is a variant of the E2 family of proteins.At present,it has been found that Ube2v1 regulates inflammation by activating the NF-kappa-B pathway,but its role in colorectal cancer is unclear.Autophagy is closely related to the occurrence and development of tumors.The role of autophagy in tumors is more complicated,especially in colorectal cancer.In our study,we found that Ube2v1 inhibited autophagy.In the colorectal cancer cells,Ube2v1 inhibited the expression of LC3,Beclin1,ATG3,ATG5,ATG7,ATG4,ATG4 a,ATG4b,ATG4 c,ATG4d,ATG16L1 and other gene transcription levels by promoting the ubiquitination degradation of deacetylase Sirt1 and reduced the deacetylation of histone H4K16 by the direct interact with Ubc13.Subsequently,we based on the construction of Ube2v1 overexpression and knockdown of colorectal cancer cell lines,through cell biology experiments and nude mice in vivo experiments,found Ube2v1 promoted colorectal cancer metastasis by EMT.We present the hypothesis that Ube2v1 promotes colorectal cancer metastasis by inhibiting autophagy.To verify this hypothesis,we first constructed a lung metastatic model of Ube2v1 colorectal cells by injection of nude mice tail vein.We then injected rapamycin(Rapamycin)and trehalose(Trehalose)in the mice,Mycobacterium and trehalose are autophagic inducers,compared with autophagic inducer before and after feeding the mouse lungs.This suggests that our autophagic primers are able to attenuate the progression of Ube2v1 to colorectal cancer.Finally,we found that Ube2v1 was highly expressed in clinical colorectal cancer samples,which was closely related to the poor prognosis of patients with colorectal cancer.Our study linked Ube2v1 in the ubiquitin protease complex system to the transfer of autophagy and colorectal cancer.In this study,we found that Ube2v1 promoted the metastasis of colorectal cancer and elucidated its mechanism.Ube2v1 combined with Ubc13,through the ubiquitin proteasome pathway degradation Sirt1,weaken Sirt1 deacetylation,which at the transcription level to inhibit the expression of autophagy-related genes to promote the EMT process.And provide clues and basis for the design of targeted and target cell-specific colorectal cancer metastasis interventions based on the important components of the ubiquitin proteasome system.Methods1,Western blot(WB),RT-PCR and immunohistochemistry(IHC)were used to detect the expression of Ube2v1 in human colorectal cancer cells and tissues;2,Immunofluorescence staining(IF)was used to detect the expression of autophagy pathway and epithelial to interstitial metastasis(EMT)in human colorectal cancer cells;3,Observation of autophagic vesicles by transmission electron microscopy;4,TMT screening of human colorectal cancer cells in the Ube2v1 effect of the protein;5,Studying the direct;interaction between the protein by co-immunoprecipitation(CO-IP);6,Cell biology experiments(scratches,migration,invasion);7,Nude mice subcutaneously implanted tumor tumor test,nude mice tail vein injection construction transplantation model;Results?,Ube2v1 inhibits autophagy pathway in colorectal cancer cellsThe expression of Ube2v1 and WB in colorectal cancer cell lines SW480 and DLD-1 showed that the expression of autophagy index LC3 and Beclin1 was decreased and the expression of P62 was increased.The results showed that Ube2v1 was positively regulated by LC3 and Beclin1,and the positive regulation of P62 was still existed.In the SW480 cell,Ube2v1 was overexpressed by transmission electron microscopy,and the autophagy was observed by transmission electron microscope.(P <0.05).The number of autophagic vesicles in the Ube2v1 group was significantly lower than that in the control group(p <0.05),and the number of autophagic vesicles in the Ube2v1 group was significantly lower than that in the control group(p <0.05)).IF results showed that Ube2v1 knocked low after the number of autophagic bodies increased,P62 expression decreased.?,Ube2v1 inhibits Sirt1 enhances the level of histone H4K16 deacetylation and further regulates the expression of downstream autophagy pathway-related genes at the transcriptional level.Ube2v1 overexpression of colorectal cancer cells SW480,screening analysis of Ube2v1 overexpression significantly inhibited histone expression by TMT,so we speculate that Ube2v1 involved in histone modification.We found that Ube2v1 was able to up-regulate the expression of acetylated H4K16 by WB.Since Sirt1 was involved in the regulation of H4K16 deacetylation,we examined whether Ube2v1 was deacetylated by Sirt1.The results showed that Ube2v1 inhibited Sirt1 expression,while H4K16 acetylation level increased.Ube2v1 overexpression at the same time overexpression of Sirt1,detection of H4K16 acetylation level,found that H4K16 acetylation level down.The above results suggest that Ube2v1 increases the acetylation level of H4K16 by inhibiting Sirt1.The results of RT-PCR showed that overexpression / knockdown of Ube2v1 and overexpression / knockdown of Sirt1 both regulated transcriptional autophagy-related genes at the transcriptional level.Ube2v1 overexpression / knockdown at the protein level regulates Sirt1,RT-PCR results show that Ube2v1 can not regulate Sirt1 at the transcriptional level.Combined with Ube2v1 this ubiquitin ligase,we speculate that Ube2v1 regulation of Sirt1 is likely to be through the ubiquitination pathway.?,Ube2v1 promotes ubiquitination degradation of Sirt1 by Ubc13 in colorectal cancer cells.The results of WB assay showed that Ube2v1 could promote the degradation of Sirt1 by adding protein synthesis inhibitor actinone(CHX)in SW480 cells.The results showed that Ube2v1 could not directly bind to Sirt1,but Ube2v1 overexpression could promote the ubiquitination of Sirt1.As a result,Ube2v1 and Ubc13 interacted to form Ube2v1-Ubc13 complex to play ubiquitin ligase,and our results The inhibition of Ube2v1 and Ubc13 can reduce the ubiquitination degradation of Sirt1.?,Ube2v1 promotes colorectal cancer by inhibiting autophagy.WB results showed that the expression of E-cadherin and ?-catenin in the important epithelial cells of EMT was decreased after Ube2v1 expression,and the expression of Vimentin,Fibronectin and N-cadherin were increased in stromal cells.After we knocked down Ube2v1,we further confirmed The above results suggest that Ube2v1 promotes colorectal cancer cell EMT progression.In order to show whether Ube2v1 regulates EMT through autophagy,we selected autophagy-related genes ATG5 and ATG7,and the transcriptional level of these two genes showed a significant change after Ube2v1 overexpression or low expression.In colorectal cancer cells at the same time knock low Ube2v1 and ATG5 / ATG7,we found that Ube2v1 overexpression to promote the process of EMT will be weakened.?,Ube2v1 promotes tumor metastasis by inhibiting autophagy.The lung metastasis model was constructed by injecting Ube2v1 overexpressing colorectal cancer cells by tail vein.The results of HE staining showed that the transfection of overexpression of Ube2v1 was more and more(p <0.05)than that of control group(p <0.05).In addition,the volume and number of metastatic tumors decreased(p <0.05)under the action of autophagy-primamycin-rapamycin.The expression of Beclin1 was decreased and the expression of E-cadherin was decreased in Ube2v1 cells.The autophagic induction of intraperitoneal injection of rapamycin was induced by immunohistochemical staining in the lungs of several groups of mice.,The expression of EMT-related proteins was picked up;by feeding another autophagic-inducer,trehalose,the results were consistent with the treatment of rapamycin.DescriptionUbe2v1 is capable of ubiquitination degradation of Sirt1 at the protein level,thereby regulating the deacetylation of H4K16,which regulates autophagy-related genes at the transcriptional level and affects EMT.So can Sirt1 be able to regulate EMT directly at the transcription level? We were overexpressed and knocked down Sirt1 in colorectal cancer cells,and the expression of EMT important genes was detected by RT-PCR.The results show that Sirt1 can not directly regulate EMT at the transcription level.?,The expression of Ube2v1 in colorectal cancer was higher than that in adjacent tissues and was closely related to poor prognosis.RT-PCR was used to detect the expression of Ube2v1 in 39 pairs of human colorectal cancer specimens.It was found that the transcription level of Ube2v1 in colorectal cancer tissues was generally higher than that in the adjacent tissues.IHC results showed that 86 pairs of paired human colorectal Ube2v1 in colorectal cancer tissues also showed high expression in cancer specimens,especially in distant metastatic specimens of Ube2v1 showed high expression.Using the Public Gene Expression Profile(GSE17537),the prognosis of colorectal cancer patients with high expression of Ube2v1 was poorer.