Runt-related Transcription Factor 1(RUNX1) Promotes Colorectal Cancer Metastasis By Upregulating MUC13 Through Wnt/β-catenin Pathway

Background and objectives:Among the leading causes of cancer-related deaths worldwide,colorectal cancer(CRC)is the third most common cancer.In patients with CRC,conventional treatment strategies include endoscopy,surgery,cytotoxic chemotherapy,and targeted therapy.Despite major advances in clinical treatment strategies for CRC,metastases still occur in more than 50%of patients who undergo resection.Accordingly,there is an urgent need to identify novel functional genes and biomarkers involved in CRC pathogenesis,which would aid in developing effective treatment options.The role of runt-related transcription factor 1(RUNX1),also termed as acute myeloid leukemia 1(AML1),is well-established in hematological malignancies.Furthermore,several recent studies have explored the involvement of RUNX1 in promoting solid tumor development.The present study explores the expression,clinicopathological characteristics,and survival prognosis of RUNX1 expression in CRC to elucidate the molecular mechanisms through which RUNX1 promotes colorectal carcinogenesis and metastasis,determine the downstream targets of RUNX1,and identify relevant signaling pathways.Methods:Multiple bioinformatics online databases were searched to predict the expression of RUNX1 and MUC13 in colorectal cancer and its impact on the prognosis of colorectal cancer.Immunohistochemistry(IHC)analysis was conducted on a tissue microarray(TMA)containing 90 cases of colorectal cancer tissues and corresponding adjacent non-tumor tissues.Also,pathological sections of liver tissue from 15 patients with colorectal cancer liver metastases were obtained from the Department of Pathology of Wuhan Tongji Hospital to evaluate the expression of RUNX1 and MUC13 in colorectal cancer and its correlation with the clinicopathological characteristics,survival,and prognosis of colorectal cancer.Colorectal cancer(daughter line F3)cells with enhanced metastatic ability were obtained by continuous in vivo screening of tumor cells(maternal line F0)following intrasplenic injection of Caco-2 human colorectal adenocarcinoma cells into the spleens of nude mice used as a liver metastasis model.In vitro analyses included CCK-8,colony formation,Annexin V-FITC/PI cell apoptosis,cell cycle,EdU,scratch,and Transwell migration and invasion assays.In vivo analyses included intra-splenic injection liver metastasis assay and subcutaneous tumor formation assay,performed using nude mice.These assays were performed to evaluate the effects of RUNX1 overexpression or knockdown on colorectal cancer cell proliferation,apoptosis,migration,and invasion in vitro and growth/metastasis in vivo.RNA sequencing(RNA-Seq)was performed on F0 and F3 cells to identify potential targets associated with highly invasive phenotype.Multiple regulatory targets of RUNX1 were identified by ChIP-Seq.Further analyze the intersection of the results of ChIP-Seq and RNA-Seq,and identify the genes that are not only correlated with highly invasive phenotype of colorectal cancer cells(the gene significantly up-regulated relative to F0 in RNA-seq),but also serve as the direct target of RUNX1(the gene that RUNX1 binds to in ChIP-seq).To determine whether RUNX1 interacts with MUC13,a co-immunoprecipitation(Co-IP)assay was performed.The Spearman’s rank correlation analysis of RUNX1 and MUC13 protein levels was performed based on the IHC results using the same TMA.The online database was used to further verify the relationship between them.RUNX1 and MUC 13 lentivirus were co-transfected for rescue experiments.The effects of interference with RUNX1 and MUC 13 gene expression on malignant biological behavior of colorectal cancer cells were investigated by phenotypic experiments in vitro and in vivo.qPCR and Western Blot were used to verify the effects of interference with the expression of RUNX1 and MUC 13 genes on the activity of molecules related to the signal pathway,thus exploring the potential transcriptional regulation mechanism of RUNX1.Results:The results of IHC analysis of pathological sections in 90 cases of colorectal cancer and 15 cases of liver metastasis of colorectal cancer in TMA and several independent online databases all showed that the expression of RUNX1 and MUC 13 in colorectal cancer tissue and liver metastatic tumor tissue increased,and its high expression was closely associated with the more aggressive and advanced clinicopathological features and poor prognosis.A highly invasive F3 cell line was successfully constructed.In vitro and in vivo experiments confirmed the enhanced proliferation,migration and invasive abilities of F3 compared with F0.The potential target genes for RUNX1 transcriptional regulation from the overlap between the RUNX1 binding downstream target genes in the ChIP-Seq test were identified,along with the significantly higher expressed genes in F3 compared to F0 cells(using RNASeq).Conclusively,MUC13 was identified as the target gene.The results of Co-IP assay confirmed the efficient interaction between RUNX1 and MUC13 protein.A Spearman’s rank correlation test conducted by integrating TCGA and GTEx databases showed a significant positive correlation between RUNX1 and MUC13.The Spearman’s rank correlation on the IHC results using the same TMA show a significant positive correlation between RUNX1 and MUC13 protein levels in colorectal cancer tissues.In rescue experiments and in vitro and in vivo phenotypic assays,co-transfection of RUNX1 and MUC13 lentivirus revealed that MUC13 knockdown could partially reverse the procancer effect of RUNX1 in colorectal cancer.Considering qPCR and western blotting analyses of signaling pathway-related molecules,we found that MUC13 knockdown could partially counteract RUNX1-mediated EMT induction in colorectal cancer cells and further reduce the activity of the Wnt/β-catenin signaling pathway activated by RUNX1.Conclusions:This study revealed that the abnormally high expression of RUNX1 in colorectal cancer can enhance the proliferation,migration and invasion of colorectal cancer cells in vivo and in vitro,thus affecting the progression of colorectal cancer.RUNX1 can regulate the transcription of MUC13,and RUNX1 promotes the activation of EMT and βcatenin/Wnt signal pathway by up-regulating MUC13 in colorectal cancer.Therefore,RUNX1 and MUC13 may be effective therapeutic targets for colorectal cancer,and these findings have potential significance for tumor therapy.Part Ⅰ:Expression of RUNX1 and its impact on clinicopathological features and survival prognosis analysis in colorectal cancerObjective:To study the differential expression of runt-related transcription factor 1(RUNX1)gene in clinical tissue samples of colon cancer and its corresponding Para cancerous tissues;to analyze the relationship between the expression of RUNX1 and various clinicopathological characteristics and the long-term prognosis of colon cancer.Methods:Bioinformatics databases,including TCGA,GEO,GTEx,GEPIA,TIMER,HPA,and CPTAC,were searched to predict the expression of RUNX1 in colorectal cancer and its impact on the prognosis of colorectal cancer.IHC analysis was performed on a TMA containing 90 cases of colorectal cancer tissues and corresponding adjacent non-tumor tissues.Also,pathological sections of liver tissue from 15 patients with colorectal cancer liver metastases were obtained from the Department of Pathology of Wuhan Tongji Hospital to evaluate the expression of RUNX1 in colorectal cancer and its correlation with the clinicopathological characteristics,survival,and prognosis of colorectal cancer.The expression levels of RUNX1 in five human colorectal cancer cell lines(HCT 116,HT29,SW480,Caco-2 and LoVo)and five human hepatocellular carcinoma cell lines(Huh7,HepG2,Hep3B,SK-Hep1 and MHCC97-H)were detected by qPCR and Western blot;the results were compared with normal human colonic epithelial cells(NCM460)and human normal liver cell line(LO-2)respectively.Results:The analysis of TIMER and GEPIA online databases revealed the significantly elevated expression of RUNX1 in most types of digestive system tumors.Five GEO datasets containing colorectal cancer samples(GSE37182,GSE44861,GSE71187,GSE14297 and GSE49355)showed that RUNX1 expression was significantly elevated in colorectal cancer compared to that in normal samples.IHC analysis of 90 colorectal cancer tissues in the TMA showed that RUNX1 was significantly elevated in tumor tissues relative to the corresponding adjacent non-tumor tissues.IHC analysis of the liver pathological sections of metastasized colorectal cancer revealed elevated RUNX1 expression.Examination of the Clinical Proteomics Tumor Analysis Consortium(CPTAC)and Human Protein Atlas(HPA)databases both showed significantly elevated RUNX1 in colorectal cancer tumor tissues.Ninety patients with colorectal cancer were divided into low or high RUNX1 expression groups.The Chi-square test was used to evaluate the relationship between the level of RUNX1 expression and clinicopathological characteristics and clinical outcomes of these colorectal cancer patients.Higher RUNX1 expression was associated with higher grading(χ2=7.283,p=0.007),higher AJCC stage(χ2=11.072,p<0.001),higher T stage(χ2=12.532,p<0.001),and higher N stage(χ2=8.598,p=0.003),as well as clinical outcomes(χ2=5.657,p=0.017).Kaplan-Meier survival analysis based on the clinical data of 90 patients in TMA and the GEPIA database jointly demonstrated that patients with high RUNX1 expression had a shorter survival time.The qPCR and western blot results showed that RUNX1 expression was elevated in various colorectal cancer and hepatocellular carcinoma cell lines compared to that in the NCM460 and LO-2 normal cell lines.Conclusions:RUNX1 expression was significantly higher in colorectal cancer tissues than in adjacent non-tumor tissues,and was elevated in metastatic lesions.High expression of RUNX1 was associated with more aggressive clinical features and poor prognosis.The findings implicate RUNX1 as a biomarker for the prognosis of colorectal cancer.Part Ⅱ:Effect of RUNX1 on malignant biological behavior of colorectal cancerObjective:To investigate the effects of RUNX1 on malignant biological behaviors,such as proliferation,apoptosis,migration,invasion,and metastasis of colorectal cancer cells.Methods:Colorectal cancer(daughter line)cells with enhanced metastatic ability were first obtained by continuous in vivo screening of tumor cells(maternal line)following intrasplenic injection of Caco-2 human colorectal adenocarcinoma cells into the spleens of nude mice used as a liver metastasis model.Liver metastasis appeared approximately three to four weeks following injection.The primary tumor cells were isolated from the liver metastases and injected into the spleen of the next batch of mice.This process was repeated for a total of three rounds of in vivo selection.The third-generation Caco-2 cells were designated F3.The parent Caco-2 was designated F0 for convenience of comparison.In vivo and in vitro experiments verified significant differences in the malignant biological behavior of F0 and F3.A lentiviral vector for the stable overexpression and stable knockdown of RUNX1 was constructed.F0 cells were treated with RUNX1 overexpression(OE-RUNX1)and F3 cells were treated with shRNA for RUNX1 knockdown(sh-RUNX1).Lentiviral transfection was followed by qPCR and western blot experiments to detect the overexpression efficiency of genes and proteins.In vitro analyses included CCK-8,colony formation,Annexin V-FITC/PI cell apoptosis,cell cycle,EdU,scratch,and Transwell migration and invasion assays.In vivo analyses included intra-splenic injection liver metastasis assay and subcutaneous tumor formation assay,performed using nude mice.These assays were performed to evaluate the effects of RUNX1 overexpression or knockdown on colorectal cancer cell proliferation,apoptosis,migration,and invasion in vitro and growth/metastasis in vivo.Immunofluorescence(IF),western blot,qPCR analyses and examination of online databases were used to verify if the tumor-promoting effect of RUNX1 in this study was associated with the occurrence of EMT.Results:A highly invasive F3 cell line was successfully constructed.In vitro and in vivo experiments confirmed the enhanced proliferation,migration and invasive abilities of F3 compared with F0.The highly malignant phenotype of F3 was associated with the occurrence of EMT.RUNX1 overexpression and knockdown lentiviral vectors were successfully constructed and transfected into the F0 and F3 cells,respectively.qPCR and western blot analyses verified that the transfected F0 and F3 cells achieved stable ectopic overexpression or knockdown of RUNX1,respectively.CCK-8,colony formation,and EdU assays confirmed that RUNX1 overexpression enhanced the proliferation of colorectal cancer cells.The Annexin V-FITC/PI cell apoptosis assay confirmed that RUNX1 overexpression hindered cell apoptosis.The cell cycle assay confirmed that RUNX1 overexpression stimulated cell cycle progression from the G2/M phase to S phase.The wound healing assay and Transwell migration and invasion assay confirmed that RUNX1 overexpression enhanced the migration and invasion ability of colorectal cancer cells.The above experiments of malignant biological behaviors showed that RUNX1 knockdown resulted in opposite behaviors.In vivo experiments of intrasplenic injection of liver metastasis in nude mice confirmed that RUNX1 overexpression resulted in an increased incidence of liver metastasis and more extensive areas of liver metastatic lesions,and significantly reduced the overall survival of mice.Subcutaneous tumor formation experiments in nude mice confirmed a significant increase in tumor volume and weight;the staining of tumor Ki67 was enhanced,suggesting a stronger proliferation ability in vivo.The above in vivo experiments with RUNX1 knockdown suggested opposite behaviors.IF,Western blot,qPCR and online database information verified that the tumor-promoting effect of RUNX1 was related to induction of EMT.RUNX1 can lead to the elevated expression of EMT indicators and EMT-related transcription factors.Conclusions:RUNX1 can play a tumor-promoting role in the development of colorectal cancer.RUNX1 can promote malignant biological behaviors,such as proliferation,migration,invasion,and metastasis,of colorectal cancer cells in vitro and in vivo,and induce the development of EMT.Part Ⅲ:Identifying MUC13 as the downstream target gene of RUNX1 transcriptional regulationObjective:Screening and verification confirmed MUC13 as the downstream target gene of RUNX1 transcriptional regulation.This finding further established the correlation between RUNX1 and MUC13 expression;however,further investigations revealed that MUC13 expression in colorectal cancer tissues is in turn closely correlated with clinicopathological characteristics and tumor prognosis.Methods:RNA-Seq was performed on F0 and F3 cells that are significantly different regarding invasion ability.ChIP-Seq was also performed on F0 cells with RUNX1 overexpression and corresponding RUNX1 negative F0 cells,and the overlapping results of the ChIP-Seq and RNA-Seq data were evaluated.Then the downstream-regulation target was screened out.The interaction was further validated by co-immunoprecipitation(Co-IP).MUC13 expression in multiple gastrointestinal tumors was identified using TCGA database.Two independent GEO microarray datasets(GSE14297 and GSE37182)were used to analyze the difference in MUC13 expression levels between normal and colorectal cancer tissues.MUC13 expression was verified at the protein level based on the CPTAC and HPA databases.to the resulting data was compiled and a Spearman’s rank correlation test between RUNX1 and MUC13 expression levels was performed.IHC analysis was performed on a TMA containing 90 cases of colorectal cancer tissues and corresponding adjacent non-tumor tissues.Also,pathological sections of liver tissue from 15 patients with colorectal cancer liver metastases were obtained from the Department of Pathology of Wuhan Tongji Hospital to evaluate the expression of MUC13 in colorectal cancer and its correlation with the clinicopathological characteristics,survival,and prognosis of colorectal cancer.The Spearman’s rank correlation analysis of RUNX1 and MUC13 protein levels was performed based on the IHC results using the same TMA.The expression levels of MUC13 in five human colorectal cancer cell lines(HCT 116,HT29,SW480,Caco-2 and LoVo)and five human hepatocellular carcinoma cell lines(Huh7,HepG2,Hep3B,SK-Hep1 and MHCC97-H)were detected by qPCR and western blot;the results were compared with normal human colonic epithelial cells(NCM460)and human normal liver cell line(LO-2)respectively.Results:The potential target genes for RUNX1 transcriptional regulation from the overlap between the RUNX1 binding downstream target genes in the ChIP-Seq test were identified,along with the significantly higher expressed genes in F3 compared to F0 cells(using RNASeq).Conclusively,MUC13 was identified as the target gene.The results of Co-IP assay confirmed the efficient interaction between RUNX1 and MUC13 protein.The analysis of the TCGA database revealed the significantly elevated expression of MUC13 in most types of digestive system tumors.Two independent GEO microarray datasets containing colorectal cancer samples(GSE14297 and GSE37182)showed that MUC13 expression was significantly elevated in colorectal cancer compared to that in normal samples.A Spearman’s rank correlation test conducted by integrating TCGA and GTEx databases showed a significant positive correlation between RUNX1 and MUC13.IHC analysis of 90 colorectal cancer tissues in the TMA showed that MUC13 was significantly elevated in tumor tissues relative to the corresponding adjacent non-tumor tissues.IHC analysis of the liver pathological sections of metastasized colorectal cancer revealed elevated MUC13 expression.Examination of the CPTAC and HPA databases both showed significantly elevated MUC13 in colorectal cancer tumor tissues.The Spearman’s rank correlation on the IHC results using the same TMA show a significant positive correlation between RUNX1 and MUC13 protein levels in colorectal cancer tissues.Ninety patients with colorectal cancer were divided into low or high MUC13 expression groups.The Chi-square test was used to evaluate the relationship between the level of MUC 13 expression and clinicopathological characteristics and clinical outcomes of these colorectal cancer patients.Higher MUC 13 expression was associated with higher AJCC stage(χ2=13.098,p<0.001),higher T stage(χ2=8.855,p=0.003),and higher N stage(χ2=10.490,p=0.001),as well as clinical outcomes(χ2=6.988,p=0.008).Kaplan-Meier survival analysis based on the clinical data of 90 patients in TMA demonstrated that patients with high MUC 13 expression had a shorter survival time.The qPCR and western blot results showed that MUC 13 expression was elevated in various colorectal cancer and hepatocellular carcinoma cell lines compared to that in the NCM460 and LO-2 normal cell lines.Conclusions:MUC13 is the downstream target gene of RUNX1 transcriptional regulation.MUC 13 expression was significantly higher in colorectal cancer tissues than in adjacent non-tumor tissues.High expression of MUC 13 was associated with more aggressive clinical features and poor prognosis.MUC 13 is positively correlated with RUNX1 in colorectal cancer tissues.Part Ⅳ:Transcriptional regulation of MUC13 by RUNX1 impacts the Wnt/β-catenin signaling pathway activityObjective:To explore the effect of RUNX1-mediated transcriptional regulation of MUC13 on the malignant biological behavior of colorectal cancer cells and potentially related pathways to comprehensively clarify the underlying molecular mechanisms.Methods:Following RUNX1 overexpression and knockdown,qPCR and western blotting were performed to detect expression levels of molecules related to the Wnt/β-catenin signaling pathway and elucidate whether RUNX1 can impact the activity of the classical Wnt/βcatenin pathway.Co-transfection of RUNX1 and MUC13 lentivirus was performed for rescue experiments.To assess the effects of MUC13 and RUNX1 co-transfection on the proliferation,apoptosis,migration,and invasion abilities of colorectal cancer cells,we performed CCK-8,clone formation,EdU,cell cycle,Annexin V-FITC/PI apoptosis,scratch,and Transwell migration/invasion assays.We next determined the combined action of MUC13 and RUNX1 on the metastatic ability of colorectal cancer cells in vivo.Accordingly,nude mice were administered an intrasplenic injection of liver metastasis.IHC staining was performed to analyze the correlation between RUNX1 and MUC13 protein levels in mouse liver metastatic lesions.IHC staining for Ki67 was used to detect the combined effect of RUNX1 and MUC13 on colorectal cancer cell proliferation.qPCR and western blotting were performed to verify the expression of signaling pathwayrelated molecules and determine whether RUNX1 could affect the EMT phenotype and Wnt/β-catenin signaling in colorectal cancer cells via transcriptional regulation of MUC13.Results:Based on qPCR and western blotting analyses,RUNX1 could affect the activity of the classical Wnt/β-catenin signaling pathway.In rescue experiments and in vitro and in vivo phenotypic assays,co-transfection of RUNX1 and MUC13 lentivirus revealed that MUC13 knockdown could partially reverse the pro-cancer effect of RUNX1 in colorectal cancer.Considering qPCR and western blotting analyses of signaling pathway-related molecules,we found that MUC13 knockdown could partially counteract RUNX1-mediated EMT induction in colorectal cancer cells and further reduce the activity of the Wnt/β-catenin signaling pathway activated by RUNX1.Conclusion:The transcription factor RUNX1 positively regulates MUC13 expression by binding to the downstream target MUC13,thereby promoting the development and metastasis of colorectal cancer,inducing the EMT phenotype and enhancing the activity of the Wnt/β-catenin signaling pathway.