Deciphering the miR-200a-3p/RUNX1 Axis: A Novel Oncogene Signature in Colorectal Cancer

doi:10.21203/rs.3.rs-4844859/v1

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Research Article

Deciphering the miR-200a-3p/RUNX1 Axis: A Novel Oncogene Signature in Colorectal Cancer

https://doi.org/10.21203/rs.3.rs-4844859/v1

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Background

The dual role of carcinogenic or tumor suppressor makes Runt related transcription factor 1 (RUNX1) a new diagnostic markers or therapeutic target for colorectal cancer (CRC). In CRC, the relationship between RUNX1 and prognosis, biological function, and potential microRNA directly involved in the regulation of RUNX1 are unclear.

Methods

Gene expression of RUNX1 in colorectal cancer (CRC) was comprehensively analyzed using data from The Cancer Genome Atlas (TCGA) and Oncomine databases. Kaplan-Meier survival curves were constructed to assess the clinical and prognostic status associated with RUNX1 expression in CRC patients. The correlation between clinical features and RUNX1 expression was analyzed in the GSE17536 dataset using the Chi-square test. The relationship between RUNX1 expression and overall survival (OS) in CRC was investigated through both univariate and multivariate Cox regression analyses. Genes co-expressed with RUNX1 were identified using Spearman correlation analysis. The potential functions of RUNX1 in CRC were elucidated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. MiRNAs that negatively regulate RUNX1 expression were identified using TargetScan, ENCORI, and miRDB databases. The relationship between miR-200a-3p expression levels and clinicopathologic characteristics, as well as the prognosis of CRC patients, was analyzed using the Chi-square test. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to determine the expression levels of RUNX1 and miR-200a-3p in CRC cell lines (HCT-116, HT-29, SW480, and SW620). The interaction between RUNX1 and miR-200a-3p was confirmed through a luciferase reporter assay.

Results

Compared with normal tissues, RUNX1 mRNA expression was up-regulated in most cancer tissues, including CRC. RUNX1 expression was closely correlated with TNM stage in CRC patients (P < 0.05). The high expression level of RUNX1 mRNA (HR: 2.198, 95%CI: [1.200, 4.027]) could be used as an independent risk factor for overall survival (OS) in CRC patients. The mRNA level of RUNX1 in CRC patients was significantly correlated with OS (P < 0.01), disease-free survival (DFS) (P < 0.01), and disease-specific survival (DSS) (P < 0.001). RUNX1 co-expressed genes are mainly involved in GO entries such as development and growth, differentiated cell morphogenesis, and KEGG signaling pathways such as adhesion plaques and adhesion junctions. miR-200a-3p may be the miRNAs with direct regulatory role of RUNX1. The expression of miR-200a-3p was significantly correlated with T stage (P = 0.03) and M stage (P = 0.026). Low expression of miR-200a-3p was significantly associated with poor prognosis in CRC patients (P = 0.02). The expression levels of RUNX1 and miR-200a-3p in CRC cell lines were negatively correlated. RUNX1 has specific binding sites with miR-200a-3p. The results of dual luciferase reporter gene detection showed that compared with three groups, Luc-3'UTR + mimic-NC, Luc-NC + miR-200a-3p mimic and Luc-NC + mimic-NC, luciferase activity of Luc-3'UTR + miR-200a-3p mimic group was significantly decreased (P < 0.05), suggesting that miR-200a-3p may be a direct negative regulator of RUNX1.

Conclusion

High expression of RUNX1 might function as an oncogene in CRC. The up-regulated expression of RUNX1 is associated with poor prognosis after CRC, which can be used as a biomarker of prognosis in CRC patients. This study is the first to report that RUNX1 is a direct negative regulatory target of miR-200a-3p in CRC and can be used as a potential therapeutic target for CRC patients.

Colorectal cancer

RUNX1

miR-200a-3p

Prognosis

Oncogene signature

Colorectal cancer (CRC) is one of the common malignant tumors with a high incidence and a sharp increase with age [1]. According to authoritative data statistics, CRC is the tumor with the third highest number of new cases and the second highest number of death cases worldwide [2]. The pathogenesis of CRC is complex, and a large number of risk factors are known to contribute to the development of it, such as age > 60, family history of CRC, inflammatory bowel disease, obesity, and poor dietary habits (processed meat, low dietary fiber diet), etc. [1, 3, 4]. Currently, the genes known to cause CRC recurrence include human epidermal growth factor receptor 2 (HER-2), MET proto-oncogene (MET), KRAS proto-oncogene (KRAS) and epidermal growth factor receptor (EGFR) mutations [5]. At present, it is one of the directions of most cancer treatments to study the tumor from the gene level and find out the targeted therapeutic targets. In recent years, with the in-depth study of RUNX family, more and more relevant studies have been conducted in malignant tumors, and it is expected to become a new therapeutic target.

RUNX family genes are a class of nuclear transcription factors with conserved protein sequences, which play an important regulatory function in cell lineage-specific genes, cell differentiation and development [6]. In the human genome, the RUNX family consists of three members: RUNX1, RUNX2, and RUNX3. RUNX2 is highly expressed in pancreatic cancer cells and is also highly expressed in tumor-associated fibroblasts, while it is less expressed in normal pancreatic tissues. Studies have shown that RUNX2 can be involved in regulating the expression of extracellular matrix regulatory factors such as matrix metalloproteinases 1 (MMP1), thus affecting the tumor microenvironment [7]. RUNX3 plays a dual role in promoting tumor migration but inhibiting tumor cell proliferation in pancreatic cancer [8]. In recent years, studies on the relationship between RUNX1 gene and cell apoptosis and chemotherapy resistance have gradually attracted attention [9, 10]. RUNX1 plays a dual role of carcinogenic or tumor suppressor in hematologic tumors. In addition, RUNX1 activates or inhibits related genes or signaling pathways in different solid tumors, thus playing a role in carcinogenesis or tumor suppression. The role of RUNX1 varies with different tumor types.

MicroRNA (miRNA) has attracted more and more attention due to its inherent timing and spatial characteristics and high regulatory accuracy. miRNA can be involved in the occurrence and development of almost all tumors including colorectal cancer, and they are highly closely related to the initiation of tumor inhibition, tumor proliferation, tumor invasion and metastasis, tumor colonization and heterotopic, tumor angiogenesis [11, 12]. Although there have been many studies on miRNA molecules and colorectal cancer, due to the "one-to-many" characteristics of molecular regulation of miRNA, many different miRNA molecules play roles in the same type of tumor. Even the same miRNA in the same type of tumor may play different roles by targeting and regulating different genes. Therefore, there are still broad research prospects in the field of miRNA.

In this study, the expression level of RUNX1 in colorectal cancer tissues was analyzed based on bioinformatic methods. The effect of RUNX1 RNA expression on the prognosis of colorectal cancer patients was further investigated, and its correlation with the clinical data of CRC patients and its biological function were analyzed. Finally, miRNA that may be directly involved in the negative regulation of RUNX1 were discussed and studied to provide a new therapeutic target for the early intervention and targeted treatment of CRC.

2.1 Profiling of RUNX1 expression data

mRNA expression profiles across various tissues were obtained from the TCGA database and the Genotype-Tissue Expression (GTEx) project, which serve as valuable resources for studying gene expression. The Gene Expression Profiling Interactive Analysis (GEPIA) web server http://gepia.cancer-pku.cn/, a tool for analyzing RNA-Seq data derived from TCGA and GTEx, was utilized to explore the expression patterns and correlations of RUNX1 across different tissues and cancer types.The expression levels of RUNX1 in distinct stages and subtypes of liver cancer were assessed using both GEPIA and the UCSC Xena project, which has recalculated all raw expression data from TCGA. Additionally, GEPIA was employed to analyze the prognostic significance of RUNX1 expression in colorectal cancer (CRC) tissues [13].The relationship between RUNX1 mRNA levels and DNA copy number alterations in liver cancer cell lines was investigated using data from the Cancer Cell Line Encyclopedia (CCLE) project. Information regarding RUNX1 gene alterations was accessed through cBioPortal OncoPrint http://www.cBioPortal.org/index.do, a platform that provides comprehensive genomic data on cancer cell lines[14].

2.2 Prognostic nomogram constructed using RUNX1 expression analysis in patients with CRC

Expression levels of RUNX1 in colorectal cancer (CRC) and adjacent non-cancerous tissues were analyzed using patient data from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) dataset GSE17536 [15]. Variables were examined and transformed for evaluation in a Cox proportional hazards regression model, and survival analysis was conducted to assess clinical outcomes in CRC patients.Overall survival (OS) and relapse-free survival (RFS) were defined consistent with a previously published study [16]. To determine the prognostic role of RUNX1 in CRC, 177 tumor specimens were collected from consecutive CRC tumor resections performed between August 6, 2009, and the final follow-up day on August 3, 2020. Tumor stages were assessed using the 2010 International Union Against Cancer Tumor-Node-Metastasis (TNM) classification system [17], with curative resection defined as previously described [18].Clinicopathological data for the CRC patients were retrieved from hospital medical records. Survival data were obtained from the Social Security Death Index, telephone interviews, and medical records [19].

2.3 GO and KEGG analysis

For the analysis of RUNX1 gene co-expression in colorectal cancer (CRC), the TCGA Provisional dataset was selected and further analyzed using the Co-Expression functions of cBioPortal [14, 20] and LinkedOmics [21]. Genes exhibiting a Spearman correlation coefficient greater than 0.5 with RUNX1 were identified as co-expressed genes.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the identified co-expressed genes using DAVID Bioinformatics Resources v6.7 [22]. A P-value cut-off of less than 0.05 was applied to filter for significant functional and pathway enrichment.Protein-protein interaction (PPI) networks for the targets were constructed using the STRING database [23]. The resulting PPI network was visualized using Cytoscape software.

2.4 Analysis of miRNAs associated with RUNX1 expression in CRC

The LinkedOmics platform [21] was utilized for the discovery, comparison, and understanding of associations within and across omics datasets related to RUNX1 expression in colorectal cancer (CRC). miRNAs associated with RUNX1 expression were identified using databases such as LinkedOmics and TargetScan. Overlapping miRNAs related to RUNX1 expression across different datasets were determined using Venny 2.1.0 software [25] http://bioinfogp.cnb.csic.es/tools/venny/.Kaplan-Meier plotter analysis [24] http://kmplot.com/ was conducted to assess the correlation between miRNA expression and the overall survival (OS) of CRC patients. The optimal cutoff for miRNA expression was automatically selected based on the analysis, which included 379 CRC patients. The relationship between miRNA expression and the clinicopathological features of these patients was analyzed using the LinkedOmics database. The false discovery rate (FDR) was calculated using the Benjamini-Hochberg method.

2.5 Cell culture and transfection

Human colorectal cancer (CRC) cell lines HCT-116, HT-29, SW480, SW620, and SW1116 were obtained from the American Type Culture Collection (ATCC, USA). HCT-116 and HT-29 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Invitrogen). The remaining cell lines were cultured in McCoy's 5A medium (Invitrogen), also supplemented with 10% FBS. All cells were maintained in a humidified incubator with 5% CO2 at 37°C. Transfection of the cells was carried out using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocols. The vectors used for transfection were synthesized by GenePharma (Shanghai, China) and included the miR-200a-3p mimic and a mimic negative control (mimic NC). Cells transfected solely with the transfection reagent were designated as the mock group.

2.6 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from the cells was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and subsequently used to synthesize complementary DNA (cDNA) with the PrimeScript RT reagent Kit (TaKaRa), following the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) was performed to quantify the expression levels of miR-200a-3p and RUNX1 mRNA using the SYBR Green I Master Mix kit (Invitrogen) and the 7500 Real-Time PCR System (Applied Biosystems, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA (snRNA) served as the internal controls for RUNX1 and miR-200a-3p expression, respectively. The relative expression levels were determined using the 2^^−ΔΔCt method and normalized to the respective endogenous control genes.

2.7 Luciferase reporter assay

A luciferase reporter assay was employed to validate the interaction between RUNX1 and miR-200a-3p. The 3'-untranslated regions (3'-UTRs) of the wild type (WT) and mutant type (MT) of RUNX1 were cloned into firefly luciferase reporter vectors containing Renilla luciferase (Promega, Madison, WI, USA). Subsequently, these vectors were co-transfected with either the miR-200a-3p mimic or mimic negative control (mimic NC) into tumor cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity was measured 48 hours post-transfection using the SecrePair Dual-Luciferase Reporter System (Promega Corporation, Mannheim, Germany).

2.8 Statistical analysis

Differences in mRNA expression levels of genes between colorectal cancer (CRC) tissues and adjacent normal tissues were assessed using Student's t-test. The correlation between gene expressions was evaluated with Spearman's correlation coefficient. Kaplan-Meier survival analysis was conducted to estimate survival percentages, and differences in these percentages were compared using the generalized log-rank test. The diagnostic significance of RUNX1 was determined by constructing a receiver operating characteristic (ROC) curve. Stratified analyses of RUNX1 expression levels were performed to assess the relative hazard ratios for the prognosis of CRC patients.All statistical analyses were conducted using two-tailed tests and were executed with SPSS version 21.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered to indicate statistical significance.

3.1 Expression and distribution of RUNX1 in human tumor tissues

By mining the Oncomine data, we found that RUNX1 mRNA expression was up-regulated in multiple types of cancer (blue represents low RUNX1 expression in the corresponding tumor, red represents high RUNX1 expression, Fig. 1A). In addition, based on The Cancer Genome Atlas (TCGA), we found that RUNX1 expression was significantly upregulated in colorectal cancer tissues (n = 286) compared with normal tissues (n = 41) (red for tumor tissue, blue for normal tissue, Fig. 1B).

3.2 Prognostic analysis of RUNX1 gene expression in colorectal cancer

In this study, GEPIA database was used to assess the association between RUNX1 mRNA levels and overall survival (OS) and disease-free survival (DFS) in CRC patients. According to RUNX1 mRNA levels, CRC samples were divided into high expression group and low expression group (> 50% for high expression group and < 50% for low expression group). The results showed that RUNX1 mRNA levels were significantly associated with overall survival (P = 0.029, Fig. 2A) and disease-free survival (P = 0.0082, Fig. 2B) in CRC patients.

3.3 Relationship between RUNX1 expression and clinical pathological characteristics of CRC patients

We searched GEO database and comprehensively analyzed the basic information and clinical features of 177 primary CRC patients with GSE17536 data. The group was grouped according to the median RUNX1 expression value (< 9.52 for low expression group, > 9.52 for high expression group). Among 177 CRC patients, 92 cases had high RUNX1 expression and 85 cases had low RUNX1 expression. Among 177 CRC patients, 92 cases had high RUNX1 expression and 85 cases had low RUNX1 expression. Correlation analysis (Chi-square test) was conducted between RUNX1 expression value and corresponding clinicopathological data of CRC patients (including age, gender, degree of differentiation and TNM stage, etc.). The results showed that RUNX1 expression was closely correlated with TNM pathological stage of CRC patients (P < 0.05). There was no significant correlation with the gender and age of CRC patients (P > 0.05) (Table 1).

Univariate analysis showed that the degree of tumor differentiation, TNM stage and RUNX1 expression level were correlated with the prognosis of patients with colorectal cancer (Table 2). Multivariate Cox regression model uses HR value as risk assessment parameter. The results of multivariate analysis showed that TNM staging and RUNX1 expression were correlated with the prognosis of colorectal cancer patients (Table 3). Cox regression analysis showed that high expression of RUNX1 mRNA (HR: 2.198, 95%CI: [1.200, 4.027]) (Table 3) could be an independent risk factor for survival in patients with colorectal cancer. Kaplan-Meier curve analysis showed that OS, DFS and DSS of colorectal cancer patients in the high RUNX1 RNA expression group were significantly lower than those in the low expression group, with statistical significance (P < 0.05) (Fig. 3).

Table 1

Analysis of the relationship between RUNX1 expression and clinical characteristics of CRC patients (GSE17536)
Characteristics		RUNX1 expression		P-value
		Low (n)	High (n)
Age (y)	≤ 55	14	22	0.26
	>55	71	70
Gender	Male	45	51	0.76
	Female	40	41
Grade	1/2	74	75	0.53
	3	11	16
TNM stage	I/II	46	35	0.03
	III/IV	39	57

Chi-square test analysis (P < 0.05).

Table 2

Univariate Cox regression analysis of the relationship between RUNX1 expression and overall survival of colorectal cancer
Characteristics		Univariate Analysis
Characteristics		HR (95% CI)	P-value
Age	≤ 55 vs.>55	0.758(0.412, 1.395)	0.373
Gender	Male vs. female	0.843(0.491, 1.446)	0.534
RUNX1	Low vs. high	2.654(1.466, 4.806)	0.001
Grade	1/2 vs. 3	2.389(1.277, 4.468)	0.006
TNM stage	I/II vs. III/IV	5.473(2.673, 11.206)	0.001

Cox proportional hazard regression analysis (P < 0.05); HR: hazard ratio; CI: confidence interval.

Table 3

Multivariate Cox regression analysis of the relationship between RUNX1 expression and overall survival of colorectal cancer
Characteristics		Multivariate Analysis
Characteristics		HR (95% CI)	P-value
Age (y)	≤ 55 vs.>55	1.096(0.578, 2.080)	0.779
Gender	Male vs. female	1.053(0.601 1.844)	0.857
RUNX1	Low vs. high	2.198(1.200, 4.027)	0.011
Grade	1/2 vs. 3	1.787(0.941, 3.392)	0.076
TNM stage	I/II vs. III/IV	4.628(2.226, 9.623)	0.001

Cox proportional hazard regression analysis (P < 0.05); HR: hazard ratio; CI: confidence interval.

3.4 Cluster analysis of RUNX1 co-expressed genes and construction of protein interaction network

By mining the LinkedOmics database, 379 samples of correlation omics from CRC were queried and the differential genes were analyzed (Fig. 4A). We obtained the top 50 genes that are significantly positively correlated with RUNX1 expression (Fig. 4B) and the top 50 genes that are negatively correlated with RUNX1 expression (Fig. 4C). Then, we mined the cBioPortal and LinkedOmics database, and selected the overlapped genes with Spearman correlation coefficient greater than 0.5 as the RUNX1 co-expressed genes. The results of Venn diagram showed that 55 co-expressed genes were obtained (Fig. 4D). The genes, which always have similar expression changes in a physiological process or different tissues, can be considered that these genes are functionally related.

In order to preliminarily explore the potential molecular mechanism of RUNX1 in CRC, we performed functional cluster analysis of 55 genes co-expressed with RUNX1 to predict the function of RUNX1. The Metascape database was used for GO and KEGG enrichment analysis (Fig. 4F, Table 4). Through GO analysis, it was found that RUNX1 co-expressed genes were mainly involved in a variety of biological processes, including development and growth, differentiated cell morphogenesis, extracellular matrix tissue construction, and wound healing response. KEGG analysis showed that RUNX1 co-expressed genes were mainly involved in adhesion plaques and adhesion junctions signaling pathways.

Meanwhile, the protein-protein interaction network (PPI network) was constructed by analyzing the 55 protein genes in STRING database. The results showed that RUNX1 directly interacted with TRPS1, ELK3, FGFR1, BICC1, and ZNF521 proteins (Fig. 4E).

Table 4

GO and KEGG pathways enrichment analysis of RUNX1 co-expressed genes
GO	Category	Description	Count	Log10(P)	Log10(q)
GO:0048589	GO BP	developmental growth	15	-10.76	-6.44
GO:0000904	GO BP	cell morphogenesis involved in differentiation	15	-10.12	-6.1
GO:0030198	GO BP	extracellular matrix organization	11	-9.22	-5.38
GO:0001501	GO BP	skeletal system development	12	-8.75	-5.13
GO:0007423	GO BP	sensory organ development	11	-7.44	-4.16
GO:0048729	GO BP	tissue morphogenesis	11	-6.54	-3.34
GO:0001568	GO BP	blood vessel development	11	-5.97	-2.93
GO:0034329	GO BP	cell junction assembly	7	-5.87	-2.89
GO:0009611	GO BP	response to wounding	10	-5.54	-2.73
GO:0001503	GO BP	ossification	8	-5.46	-2.68
hsa04510	KEGG Pathway	Focal adhesion	6	-5.25	-2.5
hsa04520	KEGG Pathway	Adherens junction	4	-4.67	-2.01
GO:0097435	GO BP	supramolecular fiber organization	9	-4.62	-1.98
GO:1905114	GO BP	cell surface receptor signaling pathway involved in cell-cell signaling	8	-4.1	-1.62

3.5 Prediction of miRNA targeting negative regulation of RUNX1

By mining 379 CRC-related RNAseq omics samples from LinkedOmics database that were positively and negatively correlated with RUNX1 (P < 0.001) (Fig. 5A), 193 miRNAs were negatively correlated with RUNX1 expression (Spearman correlation analysis). The gene heat map showed the top 50 miRNAs that were significantly positively correlated with RUNX1 expression (Fig. 5B) and the top 50 miRNA that were negatively correlated with RUNX1 expression for subsequent analysis (Fig. 5C). Then, the upstream miRNA with RUNX1 as the regulatory target genes were predicted by retrieving "TargetScan" databases. Overlapped miRNAs in "TargetScan", "ENCORI" and "miRDB" databases were analyzed by intersection, and 30 overlapped miRNAs were finally obtained (Fig. 5D). Then, the intersection of 30 overlapping miRNAs and the top 50 miRNAs that were significantly negatively correlated with RUNX1 expression was selected to obtain 6 miRNAs, which were miR-200a, miR-18b, miR-192, miR-141, miR-215, and miR-18a (Fig. 5E). By analyzing the information of binding sites and scores in the database, we speculated that miR-200a-3p might be the miRNA gene upstream regulated by RUNX1, which needs further experimental verification.

3.6 Relationship between miR-200a-3p expression and clinical characteristics and prognosis of CRC patients

In this study, 286 CRC-related RNAseq samples from the TCGA database were used to analyze the relationship between miR-200a-3p expression and clinical characteristics and prognosis of CRC patients (Table 5). The results showed that the expression of miR-200a-3p was significantly correlated with T stage (P = 0.03) and M stage (P = 0.026). Meanwhile, prognostic analysis of miR-200a-3p expression in CRC patients suggested that low expression of miR-200a-3p was significantly associated with poor prognosis in CRC patients (P = 0.02) (Fig. 6).

Table 5

Relationship between miR-200a-3p expression and clinical characteristics of patients with CRC
Characteristics		N	miR-200a-3p level
Characteristics		N	Low	High	P-value
Age(y)	≤ 55	66	30	36	0.4
Age(y)	>55	219	113	106	0.4
T stage	T0-2	51	18	33	0.03
T stage	T3-4	234	124	110	0.03
N stage	N0	156	71	85	0.3
	N1	78	41	37
	N2	49	28	21
M stage	M0	201	94	107	0.026
M stage	M1	34	23	11	0.026
Stage	I-II	146	66	80	0.14
Stage	III-IV	124	68	56	0.14
Tumor location	Proximal	210	103	107	0.58
Tumor location	Distal	71	38	33	0.58

Chi-square test analysis (P < 0.05).

3.7 Relationship between miR-200a-3p and RUNX1 in CRC cell lines

In addition, the effect of miR-200a-3p on the expression of RUNX1 was further confirmed in CRC cells. HCT116, HT29, SW480 and SW620 CRC cell lines were used in the current study. As shown in Fig. 7A and 7B, we observed that the expression of RUNX1 was significantly upregulated, while the expression of miR-200a-3p was remarkably downregulated. These results were consistent with the data obtained from bioinformatics analyses. To verify that the RUNX1 was a directly target gene of miR-200a-3p, luciferase reporter assay was performed in the SW1116, which had the significant differential expression of RUNX1 and miR-200a-3p. The overexpression of miR-200a-3p by miR-200a-3p mimic could decreased the luciferase activity of WT 3'-UTR of RUNX1 (P < 0.01), but no effect was observed in the RUNX1-MT group (Fig. 7C-D), indicating that there was a direct interaction between miR-200a-3p and RUNX1. Moreover, the expression of RUNX1 was suppressed in the cells with overexpression of miR-200a-3p (Fig. 7E), which further implied that miR-200a-3p targeted negative regulation of RUNX1.

The existing body of literature has consistently demonstrated that the RUNX1 gene exhibits elevated levels of expression across a spectrum of malignancies, correlating with an unfavorable prognosis in various human cancers [25–27]. Recent scholarly inquiries have discovered that RUNX1 has the capacity to enhance the proliferation of leukemia cells, underscoring its multifaceted roles in diverse hematological malignancies. As the depth of research progresses, it has become increasingly evident that RUNX1's influence extends beyond hematological neoplasms, with significant implications for solid tumors as well. A plethora of contemporary studies have illuminated the dualistic nature of RUNX1, suggesting that it may either stimulate or suppress tumor cell proliferation, survival, and differentiation within the context of various solid tumors, through the modulation of genes pertinent to tumorigenesis [9, 10]. Nonetheless, the extant literature is somewhat sparse in terms of elucidating the diagnostic and prognostic implications of RUNX1 in colorectal cancer (CRC), as well as the underlying molecular mechanisms through which RUNX1 contributes to the etiology of CRC. This investigation marks the pioneering effort to systematically assess the clinical relevance of RUNX1 in CRC, utilizing a comprehensive analysis of the GEPIA database, the GSE17536 dataset, and RNA-seq data sourced from The Cancer Genome Atlas (TCGA). The findings reveal a notably elevated expression of RUNX1 in CRC samples within the aforementioned repositories. These observations collectively imply that RUNX1 could be a pivotal factor in the pathogenesis of CRC.

Emerging research has identified that the RUNX1 transcription factor is capable of binding to coactivators of transcription, which are integral regulatory proteins within signaling pathways, thereby exerting control over the transcriptional regulation of a spectrum of genes [28]. The induction of RUNX1 expression by IL-1β is hypothesized to be orchestrated by the P38 mitogen-activated protein kinase (MAPK) signaling molecule, which in turn orchestrates the expression of a cadre of molecules pivotal for invasion and angiogenesis, including matrix metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-19) and vascular endothelial growth factor A (VEGFA) [29]. To elucidate the functional role of RUNX1 in colorectal cancer (CRC), we undertook a comprehensive bioinformatics analysis, employing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methodologies, to scrutinize genes co-expressed with RUNX1. Our findings underscore the multifaceted involvement of RUNX1 in a plethora of biological processes, such as cell morphogenesis integral to differentiation, the orchestration of the extracellular matrix, the development of the skeletal system, the assembly of cell junctions, and the formation of focal adhesions. Furthermore, RUNX1 has been implicated in the modulation of several signaling pathways, with a notable role in the regulation of adherens junctions.

To delve deeper into the clinical ramifications of RUNX1 in colorectal cancer (CRC), our analysis of datasets from GSE17536 and The Cancer Genome Atlas (TCGA) has uncovered a correlation between RUNX1 expression and the malignancy of CRC phenotypes, encompassing variables such as age, gender, tumor grade, TNM stage, and patient survival. Our findings indicate that elevated RUNX1 expression is significantly and positively associated with the advanced TNM pathological stage of CRC (P < 0.05). Prior research has intimated that RUNX1 could be instrumental in the facilitation of CRC cell metastasis and recurrence. In alignment with our preceding analysis, subsequent Kaplan-Meier survival analysis has illuminated that CRC patients exhibiting RUNX1 expression levels above the median are significantly linked to a diminished overall survival (OS) and relapse-free survival (RFS). Consequently, our data collectively suggest that RUNX1 could function as an oncoprotein, exerting a pivotal influence in the neoplastic transformation of CRC.

MicroRNAs (miRNAs) have emerged as pivotal biomarkers and therapeutic targets in the pathology of various conditions, notably in the realm of oncology [30]. This novel class of short noncoding RNAs exerts post-transcriptional regulatory effects and is intricately involved in a multitude of physiological and pathological processes. Consequently, the identification of miRNAs is deemed essential for the advancement of therapeutic strategies for colorectal cancer (CRC). Accumulating evidence has underscored the functional roles of several miRNAs in CRC, with miR-200a-3p being a prominent example [31–33]. Ubiquitously expressed across various tissues, miR-200a-3p plays a significant role in vital life processes and has been increasingly implicated in the pathogenesis of numerous cancers.Previous studies have reported a marked downregulation of miR-200a-3p in CRC cell lines and clinical tissues [32]. In the present investigation, we observed a diminished expression level of miR-200a-3p in human CRC tissues relative to adjacent non-CRC tissues, suggesting a functional role for miR-200a-3p in the progression of CRC. Furthermore, we discovered an inverse correlation between RUNX1 expression and miR-200a-3p levels. Computational predictions have identified potential binding sites for RUNX1 mRNA on miR-200a-3p, suggesting that RUNX1 may serve as a target of miR-200a-3p. Additionally, elevated miR-200a-3p expression was found to be significantly associated with age at diagnosis, pathological stage, T stage, and overall survival in CRC patients. Ultimately, Kaplan-Meier survival analysis revealed that diminished miR-200a-3p expression is significantly linked to a reduction in overall survival among CRC patients. miR-200a-3p, which is downregulated in CRC, has been shown to exert an anti-proliferative effect on CRC cells both in vitro and is correlated with TNM stage and differentiation grade.In summary, the findings of the current bioinformatics analysis study underscore a potential association between RUNX1 expression and miR-200a-3p, highlighting their interplay in the pathophysiology of CRC.

In colorectal cancer (CRC) cells, the levels of RUNX1 and miR-200a-3p were assessed. Elevated miR-200a-3p was found to diminish RUNX1 expression, suggesting its role as a repressive miRNA in colorectal cancer (CRC). This finding corroborated with bioinformatics data. Furthermore, RUNX1 was validated as a direct target of miR-200a-3p, evidenced by diminished luciferase activity and reduced RUNX1 levels in miR-200a-3p-overexpressing cells.

Analysis of colorectal cancer (CRC) tissues revealed higher RUNX1 expression compared to normal tissue, correlating with poor prognosis and offering potential for personalized CRC management. This study utilized qRT-PCR to measure RUNX1 and miRNA-200a-3p in CRC cell lines, identifying a negative correlation between their levels. The dual-luciferase assay confirmed RUNX1 as a direct target of miRNA-200a-3p's negative regulation. Clinical data analysis linked RUNX1 expression to CRC's TNM staging, with high RNA levels of RUNX1 emerging as an independent risk factor for patient survival. RUNX1 mRNA levels significantly predict overall, disease-free, and disease-specific survival in CRC, suggesting its utility in prognostic evaluation. Elevated RUNX1 expression may also serve as a novel biomarker for rectal cancer, guiding individualized CRC diagnosis and therapy.

Elevated RUNX1 expression in colorectal cancer (CRC) tissues is oncogenic, with its upregulation linked to adverse patient outcomes and serving as a prognostic biomarker. RUNX1 is a direct target of miR-200a-3p's negative regulation, positioning it as a potential therapeutic target for CRC.

CRC, colorectal cancer; mRNA, messenger RNA; RNA, ribonucleic acid; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; DMEM, Dulbecco’s minimum essential medium; miRNA, microRNAs; FBS, fetal bovine serum; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; OS, overall survival; RFS, relapse free survival; RUNX1, Runt-related transcription factor 1; OD, optical density; kD, kilodalton; LB, lysogeny broth; DEPC, diethylpyrocarbonate; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; UTR, untranslated Region.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The data supporting the findings of this study are available within the article.

Competing Interests

The authors declare no competing interests.

Funding

This research was supported by the 2022 &2023 Hebei introduction of foreign expert intelligence projects [NO. YZ202201, YZ202305], Hebei Natural Science Foundation [NO. H2020206374, H2021206306], Hebei clinical medicine excellent talents project of Province [NO. LS202001] and Hebei Provincial Health Commission Medical Science Research Project [NO.20240925]. The funding sources were not involved in the study design, the collection, analysis, and interpretation of data; in the writing of the report, neither in the decision to submit the article for publication.

Authors' contributions

Xingkai Su, Xia Jiang and Fangjian Shang conducted and completed the data analysis and manuscript writing. Yingchao Gao, Jianwei Ma, Mei Wang and Haobo Wang completed the literature retrieval and data mining. Yuanyuan Wang and Zengren Zhao provided some good suggestions and supervision. All authors contributed to the article and approved the submitted version.

Acknowledgements

We wish to extend our thanks to any individual for their assistance in the conduction of this study.

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Deciphering the miR-200a-3p/RUNX1 Axis: A Novel Oncogene Signature in Colorectal Cancer

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Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1 Introduction

2 Materials and methods

2.1 Profiling of RUNX1 expression data

2.2 Prognostic nomogram constructed using RUNX1 expression analysis in patients with CRC

2.3 GO and KEGG analysis

2.4 Analysis of miRNAs associated with RUNX1 expression in CRC

2.5 Cell culture and transfection

2.6 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.7 Luciferase reporter assay

2.8 Statistical analysis

3 Results

3.1 Expression and distribution of RUNX1 in human tumor tissues

3.2 Prognostic analysis of RUNX1 gene expression in colorectal cancer

3.3 Relationship between RUNX1 expression and clinical pathological characteristics of CRC patients

3.4 Cluster analysis of RUNX1 co-expressed genes and construction of protein interaction network

3.5 Prediction of miRNA targeting negative regulation of RUNX1

3.6 Relationship between miR-200a-3p expression and clinical characteristics and prognosis of CRC patients

3.7 Relationship between miR-200a-3p and RUNX1 in CRC cell lines

4 Discussion

5 Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1