The Mechanism Of RNF126 In Regulating The Progression Of Colorectal Cancer By Mediating P53 Ubiquitination Degradation

Background:Colorectal cancer(CRC)is one of the most common cancers in the world,with the fourth highest mortality rate,following lung cancer,breast cancer,and prostate cancer.Each year,Colorectal cancer accounts for almost 10% of the diagnosed cancers and related deaths worldwide.Nearly 900,000 people die of colorectal cancer each year.In China,the morbidity and mortality of Colorectal cancer are among the top five in cancer patients.Including the imbalance of tumor suppressor genes and oncogenes,Dysfunction of multiple molecular signaling pathways plays a decisive role in the occurrence and development of colorectal cancer.Ubiquitin proteasome system is a post-translational modification that plays an important role in the regulation of tumor signaling pathways.Ubiquitin is the post-translational modification of proteins,which plays an important role in ensuring cell homeostasis.The covalent binding of ubiquitin and protein substrates can be mediated by the enzymatic cascade of ubiquitin activating enzyme E1,ubiquitin conjugating enzyme E2 and ubiquitination ligase E3.Ubiquitin ligase E3 which largely determines the specificity of the ubiquitination reaction can recruit substrates and promote or directly catalyzes the transfer of ubiquitin to the substrates.Ubiquitin ligase E3.Gene alteration,abnormal expression or dysfunction of ubiquitin ligase E3 that impair the degradation of oncogenes or accelerate the degradation of tumor suppressors,can lead to the occurrence and progression of humans cancer.The pathogenesis of colorectal cancer is complex and diverse,involving somatic mutations,abnormal gene fusion,deletion or amplification,and epigenetic changes,which may lead to abnormal expression or functional changes of ubiquitin ligase E3 in colorectal cancer.A large number of studies have shown that the functional deficiency of ubiquitin ligase E3 is involved in the molecular etiology and pathogenesis of colorectal cancer.The role of abnormally expressed ubiquitinated ligase E3 in colorectal cancer depends on the ubiquitinated substrate protein.In recent years,the potential role of ubiquitination ligase E3-mediated ubiquitination modification in the occurrence and progression of colorectal cancer has been increasingly understood.RNF126 is a novel E3 ubiquitin ligase.Although it plays an oncogenic role in some solid tumors,its potential role in colorectal cancer has been rarely reported.Therefore,in this study,we explored the potential role and molecular mechanism of RNF126 in the development of colorectal cancer.Part?:Differential expression of RNF126 in colorectal cancer tissues and relationship between overexpression of RNF126 and clinicopathological parametersObjective:We observed the expression of RNF126 in colorectal cancer tissues and paired adjacent tissues.We investigated the correlation between RNF126 and p53 expression in colorectal cancer tissues.We investigated the relationship between overexpression of RNF126 and clinicopathological parameters in patients with colorectal cancer.We analyzed the relationship between the overexpression of RNF126 and postoperative survival time in patients with colorectal cancer.Methods:In this study,the expression of RNF126 in 147 cases of colorectal cancer and paired adjacent tissues was detected by immunohistochemical technique.The expression of p53 in colorectal tissue was detected.Correlation analysis was conducted to explore the correlation between RNF126 and p53 expression in colorectal cancer tissues.the protein expression of RNF126 in 24 cases of CRC specimens and paired adjacent normal tissues was detected by western blot.The m RNA expression of RNF126 in 24 cases of CRC specimens and paired adjacent normal tissues was detected by q RT-PCR.The overexpression of RNF126 and the corresponding clinicopathological significance with CRC patients were analyzed via chi-squared test.Kaplan-Meier method was used to estimate the survival of CRC patients with high RNF126 or p53 expression.Results: RNF126 was overexpressed in human colorectal cancer tissues compared with paired adjacent normal tissues(54.4%,80/147 vs 30.6%,45/147,p<0.01).The overexpression rate of mutant p53 in colorectal cancer specimens was 51%(75/147).Correlation analysis indicated that there was no correlation between overexpression of RNF126 and overexpression of p53 in colorectal cancer tissues(r=0.114,p=0.166).WB and q RT-PCR further verified that RNF126 protein and m RNA levels were significantly higher in 24 CRC tissues than in the paired adjacent normal tissues.The relationship of RNF126 overexpression with clinical data is summarized.RNF126 overexpression was tightly associated with tumor size(p=0.021),T stage(p=0.030),lymph node metastasis(p=0.006),and TNM stage(p=0.001)of CRC patients.In Kaplan Meier assay,CRC patients with high RNF126 expression had a worse overall survival(p=0.003).Conclusion:RNF126 was overexpressed in CRC specimens compared with paired adjacent normal tissues,which was tightly associated with tumor size,T stage,lymph node metastasis,TNM stage,and the poor survival of CRC patients.There was no correlation between the overexpression of RNF126 and mutant p53 in colorectal cancer tissues.Part?:The Correlation and molecular mechanism of RNF126,p53 and p21 in CRCObjective:To explore Correlation and molecular mechanism of RNF126 and p53-p21 in CRC cells.Methods:Cell transient transfection technique was used to construct RNF126 overexpressed and silencing cell lines.The interactions between RNF126 and p53-p21 were detected by Western blotting and co-immunoprecipitation,and the protein level and ubiquitination level of p53 were detected.To investigate whether RNF126 mediates the ubiquitination of p53,Combined with proteasome inhibitor,MG132.Results: The silencing efficiency of RNF1 26 was detected by Western blot.the silencing effect is good,suggesting that RNF126 silencing cell line was successfully constructed.RNF126 silencing did not change p53 and its downstream target p21 expression in mtp53Colo-205 and SW620 cells.In RNF126 silencing HCT116 cells,We observed that p53 and p21 expression was significantly increased,but p Rb was decreased.which was reversed by p53 si RNA.In RNF126 overexpression HCT-8 cells,p53 and p21 expression was significantly decreased,but p Rb was increased.However,MG132,as a classic proteasome inhibitor,reversed RNF126 overexpression-inhibited p53 and p21,and RNF126 overexpression-promoted p Rb expression.Both RNF126 silencing and overexpression had no effect in the m RNA levels of p53 and p21.RNF126 was co-immunoprecipitated with p53 and p21 in HCT116 cells.p53 was also co-immunoprecipitated with RNF126 and p21 when using p53-lysates immunocomplex as the immunoprecipitated target.we detected the weakened ubiquitination of p53 from the p53-lysates immunocomplex in RNF126 silencing HCT116 cells following the increase of p53 protein expression.Conversely,the enhanced ubiquitination of p53 was observed in the p53-lysates immunocomplex in RNF126 overexpression HCT-8 cells following the decrease of p53 protein level.Conclusion: 1.RNF126 could not regulate the protein expression of mutant p53.2.RNF126 can regulate the expression of wild-type p53 via Enhancing p53 Ubiquitination and Degradation,and then regulate the expression of downstream p21 and p Rb.Part ?: Regulation of RNF126 on proliferation,migration,invasion and chemotherapeutic drug sensitivity of colorectal cancer cellsObjective:To explore the regulation of RNF126 on proliferation,migration,invasion and chemotherapeutic drug sensitivity of colorectal cancer cells.Methods:The effects of silencing and overexpression of RNF126 on invasion and migration of colorectal cancer cells were investigated by Transwell method.The effect of silencing and overexpression of RNF126 on cell proliferation was observed by MTT assay.The regulation effect of RNF126 silencing or overexpression on the cell cycle of colorectal cancer cells was studied by flow cytometry.MTT assay was used to observe the effect of silencing and overexpression of RNF126 on the sensitivity of colorectal cancer cells to chemotherapy drugs.RNF126 silenced stable transfected cell lines were constructed to observe the effect of RNF126 on colorectal cancer in vivo by tumor formation experiment in nude mice.Silencing RNF126 can inhibit the proliferation,migration and invasion of colorectal cancer cells,which was significantly reversed by p53 si RNA.Overexpression of RNF126 can promote cell proliferation,migration and invasion,which was significantly reversed by p53 si RNA.Silencing RNF126 can reduce the resistance of colorectal cancer cells to chemotherapy drugs,while overexpression of RNF126 can enhance the resistance of colorectal cancer cells to chemotherapy drugs.In vivo,the tumor formation test results of nude mice indicated that the growth of transplanted tumor volume was significantly inhibited in the silenced RNF126 group compared with the control group.The weight of transplanted tumor in nude mice treated with silenced RNF126 was significantly lower than that in control group.Conclusion: 1.In CRC cell lines expressing wild-type p53,RNF126 promotes the proliferation,invasion,migration and drug resistance of CRC by enhancing ubiquitination of p53 degradation.2.Silencing RNF126 has an inhibitory effect on colorectal cancer in vivo.