| Colorectal cancer(CRC)is the most common malignancy in the digestive system and has an insidious onset.Persistent tumor proliferation and metastasis are common causes of death in CRC.Despite numerous studies,the mechanism that simultaneously drives tumor growth and metastasis are still poorly understood.Our previous work found that Myocyte Enhance factor 2A(MEF2A),a member of the MEF2 family,was highly expressed in CRC tissues and was closely related to the poor prognosis of CRC patients.Simultaneously,in vitro and in vivo experiments confirmed that up-regulation of MEF2 A promoted the proliferation and metastasis of CRC.Whether the function of MEF2 A in CRC is limited to metastasis deserves further investigation,and the pathways and small molecule drugs that affect the protein stability of MEF2 A have hardly been reported in tumors.Metformin is a first-line hypoglycemic drug for patients with type 2diabetes mellitus,which is characterized by high efficiency,safety,and low cost.In recent years,a large number of studies have found that metformin plays an important role in anti-aging and anti-tumor in addition to the hypoglycemic effect.Cancer treatment drugs need to go through a long process from development to market,so while developing new drugs,many studies also start to explore new uses of old drugs.Metformin has been widely studied in tumors due to its drug properties and has been proved to have anti-proliferation and increase the sensitivity of tumor cells to chemotherapeutic drugs in many types of cancers.At present,the treatment of CRC mainly includes surgery,chemotherapy,targeted therapy,and immunotherapy.Among them,fluorouracil,oxaliplatin,and calcium folate are considered first-line treatments for CRC.Due to neurotoxicity,hepatotoxicity,drug resistance,and other adverse reactions,seeking new effective treatment drugs is still an important task to ameliorate the prognosis of CRC patients.In the preliminary experiment,we found that MEF2 A protein levels were low in tumor tissues of CRC patients with diabetes who received metformin treatment.We wonder whether metformin can regulate MEF2 A in CRC and whether it can benefit patients by affecting MEF2 A to inhibit the progression of CRC.Objective: This study aims to investigate the role of MEF2 A in the proliferation of CRC and the mechanism of MEF2 A protein stability maintenance,as well as to search for small molecule compounds that affect the stability of MEF2 A protein to intervene in the progression of CRC.(1)Investigate the role and mechanism of MEF2 A in CRC progression.(2)Identify metformin as a small molecule compound to inhibit MEF2 A expression in CRC.(3)Confirm that metformin degraded MEF2 A through the ubiquitinproteasome pathway.(4)Demonstrate that metformin mediated MEF2 A degradation by inducing the expression of STUB1,an E3 ligase.Methods:(1)Real-time quantitative PCR(q PCR),Western blot,immunofluorescence(IF),and immunohistochemical staining(IHC)were used to detect m RNA or protein expression levels of various genes in cells or tissues.(2)CRC cell lines were treated with three autophagy inhibitors,a proteasome inhibitor,a pan caspase inhibitor,respectively,and the protein level of MEF2 A was detected by Western blot.(3)Proteomics was used to evaluate the changes in cellular components,biological processes,and molecular function in metformintreated cells;RNA-seq was used to screen downstream target genes and key signaling pathways of MEF2 A.(4)Ubibrowser online database is used to predict the E3 ubiquitin ligase of MEF2 A,and PROMO and JASPR databases jointly predict gene transcription factors and potential binding sites.(5)The protein interaction between MEF2 A and E3 ubiquitin ligase was determined by immunoprecipitation(CO-IP).(6)The direct transcriptional regulatory relationship between MEF2 A and target genes was verified by the dual-luciferase reporter system and chromatin immunoprecipitation(Ch IP).(7)CCK8 and Ed U assays were performed to detect cell viability and proliferation.(8)CRC xenograft tumor model was established by subcutaneous inoculation of CRC cells in nude mice.Intraperitoneal injection of metformin was used to test its effect on CRC growth.(9)Follow up on the use of hypoglycemic drugs and prognosis of CRC patients with diabetes,and analyze the effect of metformin on the overall survival time of patients.Results:(1)MEF2A promoted the growth of CRC cells and subcutaneous tumors in nude mice.(2)RNA-seq results of MEF2 A overexpression showed that potential downstream target genes of MEF2 A were enriched in the WNT signaling pathway,and MEF2 A overexpression promoted luciferase activity driven by TCF/LEF promoter in CRC.(3)MEF2A bound to the-1428 to-1452 and-929 to-943 regions of the CTNNB1 promoter to initiate the transcription of CTNNB1.(4)Metformin inhibited the proliferation of CRC cells and the growth of CRC xenograft tumors.Among 105 CRC patients with diabetes who received metformin treatment had a much longer overall survival time than those who did not.(5)Metformin down-regulated MEF2 A levels in human CRC tissues,subcutaneous tumor tissues from CRC mouse models,and CRC cell lines by affecting the protein stability of MEF2 A.(6)The inhibitory effect of metformin on the proliferation of CRC could be partially blocked by up-regulating MEF2 A expression.(7)Metformin inhibited WNT/β-catenin activity by reducing the stability of MEF2 A.(8)MEF2A protein in CRC is mainly degraded through the ubiquitinproteasome pathway.(9)Metformin promoted the ubiquitination and degradation of MEF2 A in an indirect binding manner.(10)STUB1 was the E3 ubiquitin ligase that mediated the ubiquitination and degradation of MEF2 A,and the two mainly interacted in the nucleus.STUB1 bound to the Gln/Pro enrichment domain of MEF2 A and promoted ubiquitination of MEF2 A at K64 and K68.(11)Metformin promoted the degradation of MEF2 A depending on the up-regulation of STUB1.Conclusions:(1)MEF2A transcription up-regulated CTNNB1 and activated WNT/β-catenin signaling pathway to promote CRC proliferation.(2)Metformin inhibited the growth of CRC through down-regulated protein level of MEF2 A in vitro and in vivo.(3)Metformin induced degradation of MEF2 A through the ubiquitinproteasome pathway.(4)Metformin promoted the expression of STUB1,an E3 ligase,mediating MEF2 A degradation.Figures: 32,References: 167... |
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