MIER1 Affects Colorectal Cancer Progression By Regulating EMT And Stemness Via Sp1

Objective:Colorectal cancer(CRC)is one of the most common malignancies,and shows a year-to-year increase in incidence and mortality rates.The cancer cells stemness and epithelial-to-mesenchymal transition(EMT)are crucial in the proliferation and metastasis of CRC.Sp1 is a transcription factor that can promote the progression of CRC through various pathways.MIER1 is a member of the mesoderm-induced early response protein family,which plays an important role in tumor progression,but the role of MIER1 in CRC is unclear.The purpose of this paper is to investigate the role and mechanism of MIER1 in CRC progression.Materials and methods:1.The expression of MIER1 in TCGA pan-cancer was analyzed by bioinformatics.Cancer tissue,paracancerous tissue and clinical data of CRC patients were collected,and Western Blot method was applied to detect MIER1 expression in 7 cases of CRC tissues.112 wax blocks of CRC patients were processed for immunohistochemical staining to analyze the correlation between MIER1 and clinicopathological characteristics of CRC.2.The expression of MIER1 in five CRC cell lines was detected by Western Blot,MIER1 overexpressing SW480 cell line and MIER1 knockdown HCT116 cell line was established by lentivirus infection,and further CCK8,Transwell invasion assays and cell scratching assays were performed.3.The correlation between MIER1 and Sp1 as well as the expression of Sp1 in CRC was analyzed using CCLE,TCGA and GEO databases.Coimmunoprecipitation assay and Western Blot were applied to explore the interaction and regulation of MIER1 and Sp1.Downregulate Sp1 expression in MIER1 overexpressing SW480 cells,and then the CCK8 and Transwell invasion assays were performed.4.Analyze the relationship between MIER1 and CRC stemness as well as EMT by bioinformatics.The expression of CRC stemness and EMT markers were examined by Western Blot after MIER1 overexpression and knockdown.Downregulate Sp1 expression in MIER1 overexpressing SW480 cells,and changes in the expression of EMT and stemness markers were detected by Western Blot.5.A subcutaneous tumorigenic model was established in nude mice,the growth volume of the tumor was recorded,and the final volume and mass of the tumor were measured.A spleen-injected hepatic metastatic model was established in nude mice,and HE staining was performed on the liver and spleen.Results:1.MIER1 was highly expressed in 18 malignancies.Expression of MIER1 was significantly higher in CRC tissue than in paracancerous tissues(P < 0.05).Patients with high MIER1 expression in CRC were more likely to have poorer N stage(P = 0.020)and vascular infiltration(P =0.012).A higher level of MIER1 expression was associated with a greater risk of recurrence and metastasis(HR = 2.244)or death(HR = 3.067)risk in CRC patients.2.MIER1 expression was lower in SW480 cells and was higher in HCT116 cells.In MIER1 overexpressing SW480 cells,the cell proliferation ability was significantly enhanced compared with the control group,the number of invasion cells in Transwell invasion assay was significantly increased,and the scratch healing rate was significantly faster in the scratch assay;in HCT116 cells with down-regulated MIER1 expression,the cell proliferation ability was significantly weaker compared with the control group,the number of invasion cells in Transwell invasion assay was significantly reduced,and the scratch healing rate was significantly slower in the scratch assay.3.The expression of MIER1 was positively correlated with Sp1,and Sp1 was highly expressed in CRC.Co-immunoprecipitation showed an interaction between MIER1 and Sp1.Sp1 expression was increased after upregulation of MIER1 expression in SW480 cells and decreased after downregulation of MIER1 expression in HCT116 cells.Downregulation of Sp1 expression in MIER1 overexpressing SW480 cells resulted in significant inhibition of cell proliferation and invasive ability.4.Ferroptosis,cuproptosis,m6 A methylation regulators and immune checkpoint molecules were significantly differentially expressed in MIER1 high and low expression groups.Upregulation of MIER1 resulted in the acquisition of a mesenchymal phenotype in SW480 cells.The stemness score was significantly higher in MIER1 high expression samples.Upregulation of MIER1 expression promoted the expression of mesenchymal and stemness markers,and inhibited the expression of epithelial markers in SW480 cells.Downregulation of MIER1 expression inhibited the expression of mesenchymal and stemness markers,and promoted the expression of epithelial markers in HCT116 cells.Downregulation of Sp1 expression inhibits mesenchymal and stemness markers,and promotes expression of epithelial markers in MIER1 overexpression SW480 cell.5.In the subcutaneous tumorigenic model of nude mice: in the MIER1 high expression group,tumor growth rate was higher,and tumor mass and volume also increased significantly,expression of mesenchymal and stemness markers increased significantly,and expression of epithelial markers decreased significantly in tumor tissue;in the MIER1 low expression group,tumor growth rate was lower,tumor mass and volume decreased significantly,expression of mesenchymal and stemness markers decreased significantly,and expression of epithelial markers increased significantly in tumor tissue.In the spleen-injected hepatic metastatic model of nude mice: in the MIER1 high expression group,the liver of nude mice showed obvious intravascular emboli,the volume of spleen tumor in situ increased significantly,and the number of liver metastases increased significantly;in the MIER1 low expression group,the volume of spleen tumor in situ of nude mice decreased significantly.Conclusion:1.MIER1 is highly expressed in CRC,and MIER1 high expression is an independent poor prognostic factor in CRC;2.MIER1 promotes CRC progression by regulating EMT and stemness via Sp1;3.MIER1 regulates tumorigenic and hepatic metastatic capacity of CRC cells in vivo.