Introduction

Colorectal cancer (CRC) is the third most prevalent cancer globally with substantial morbidity and mortality (Sung et al. 2021). Surgical intervention has proven effective in managing early-stage CRC patients, while most patients were diagnosed at advanced stage or with distant metastases (Paczek et al. 2022). Despite achievement in diagnosis and therapeutic approaches for colorectal cancer over the past decade, the 5-year survival rate for advanced patients remains unsatisfactory. Consequently, it is urgent to delve deeper into the underlying pathogenesis of CRC and develop novel therapeutic strategies to address the unmet healthcare challenge.

microRNAs (miRNAs) are short, non-coding RNA molecules consisting of approximately 18–24 nucleotides. They play a significant role in gene expression regulation by binding to the 3′ untranslated regions (3′-UTRs) of target mRNA molecules, leading to mRNA degradation or inhibition of translation. Subsequently, miRNAs exert a negative regulatory effect on target genes. miRNAs have been implicated as crucial regulators of tumor suppressor genes and oncogenes (Chen et al. 2019b; Ji et al. 2014), and are involved in various biological processes, including cell proliferation, apoptosis, and cell cycle regulation (Chen et al. 2019a).

Extensive research has underscored the strong correlation between dysregulated miRNAs and the progression of colorectal cancer. These aberrantly expressed miRNAs hold promise as novel targets for CRC therapies (Ciesla et al. 2011). Notably, miR-140-3p has been observed to display altered expression patterns in various cancer types, including lung cancer, breast cancer, spinal cord chordoma, and hepatocellular carcinoma and so on (Taheri et al. 2023). miR-140-3p functions as a tumor suppressor, playing a crucial role in various cellular processes and influencing signaling pathways. Its impact on tumor development involves regulating downstream target genes. For instance, it targets ATP8A1 in non-small cell lung cancer and tripartite motif 28 in breast cancer (Dong et al. 2016; Zhou et al. 2019). Additionally, studies indicate that miR-140-3p can influence drug resistance by targeting the pregnenolone X receptor in hepatocellular carcinoma cells (Li et al. 2018). In colorectal cancer (CRC), lower plasma exosome miR-140-3p levels are observed in patients compared to healthy individuals. This downregulation is also seen in liver metastasis (Liu et al. 2021). In cell lines, miR-140-3p has been shown to inhibit cell proliferation, migration, and invasion by targeting BCL9 and promoting apoptosis by targeting BCL2 and PD-L1 (Jiang et al. 2019; Liu et al. 2021). Nonetheless, the specific expression profile, clinical implications and signaling networks of miR-140-3p in the context of CRC remain largely unexplored.

The homeobox gene (HOXA) family comprises a group of transcriptional regulators with highly conserved sequences. The homeobox A9 (HOXA9), a member of the HOXA family, has been extensively investigated, mainly in acute myeloid leukemia (AML). HOXA9 plays a critical role in promoting blood cell expansion, altering cellular differentiation, and driving malignancies (Li et al. 2013; Talarmain et al. 2022). In recent years, there has been a growing interest in the role of HOXA9 in solid tumors, and its aberrant expression is closely associated with patient prognosis (Tang et al. 2022). Notably, a study by Watanabe et al. revealed that HOXA9 overexpression in colorectal cancer tissues was linked to lymph node metastasis (Watanabe et al. 2018).

In this study, we have identified a significantly downregulated miRNA, miR-140-3p, in colorectal cancer, and have discovered HOXA9 as a novel target of miR-140-3p. Furthermore, this research provides clinical data supporting the significance of miR-140-3p and HOXA9 expression and their potential applications in the context of CRC.

Materials and methods

Bioinformatics analysis

miRNA expression datasets (GSE122187, GSE110402, GSE39845) were retrieved from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The raw data were analyzed by R. The ratio of miRNA expression in colorectal tumors compared to adjacent normal tissues was calculated as the fold change (Fc), and differentially expressed miRNAs were defined as those with |LogFc|> 1 and p < 0.01. To identify commonly differentially expressed miRNAs among the three datasets, we employed the VENN drawer online tool. To analyze the expression of miR-140-3p and its potential target genes, we utilized the ENCORI database (https://starbase.sysu.edu.cn/), a comprehensive online resource for miRNA analysis. Additionally, the GEPIA2 database (gepia2.cancer-pku.cn) and the TIMER database (https://cistrome.shinyapps.io/timer/) were utilized to investigate the expression of HOXA9 in colorectal cancer and normal tissues, as well as to explore the correlation between miR-140-3p and HOXA9. Finally, we employed the ENCORI database to analyze the prognostic significance of miRNAs and potential regulatory genes.

Clinical data collection

We collected paraffin-embedded tissues and adjacent normal paraffin tissues from patients who underwent surgical resection for pathologically confirmed colorectal adenocarcinoma at Shanxi Cancer Hospital between January 2019 and June 2019. The patient cohort consisted of 109 males and 101 females, with ages ranging from 28 to 89 years and a median age of 62 years. Among these cases, 193 had complete follow-up information. Our inclusion criteria were as follows: (1) no preoperative antitumor treatment administered through any route; (2) complete clinical records available; (3) confirmation of adenocarcinoma through pathological examination of colorectal cancer biopsies and absence of tumor cell infiltration in the adjacent para-cancerous tissue.

Tissue microarray preparation

A tissue microarray block instrument with a 2 mm aperture was used to create the paraffin microarray chip. A total of 210 colorectal cancer samples were re-evaluated based on hematoxylin–eosin stained (HE) sections, and the typical tumor areas were identified and marked on the pathological sections under a microscope. Using a puncture needle, the corresponding tissues marked on the HE sections were carefully picked and punched into the prepared blank tissue chip. Once the chips were prepared, they were placed upside down on the surface of a blank slide. Subsequently, the chips were heated at 55 °C in a drying oven for 7 min, followed by cooling down at room temperature outside the oven for 7 min. This heating and cooling process was repeated until the picked tissue was fully in contact with the paraffin on the chip.

Immunohistochemical staining

All tissue specimens were fixed in 10% neutral formalin, dehydrated in a gradient of alcohol, and then made transparent in xylene. Subsequently, the specimens were embedded in paraffin. The paraffin tissue microarrays used for immunohistochemical (IHC) staining had a thickness of 3 μm. For the automatic immunohistochemical staining, the Roche VENTANA BenchMark XT Immunohistochemical Stainer was employed. The rabbit anti-human HOXA9 monoclonal antibody (Catalog No.: MA5-37888, Thermo Scientific USA) was diluted at a ratio of 1:400 in the antibody dilution. Other mediums and buffers were prepared as working solutions according to the requirements of the platform, with the exception of the primary HOXA9 antibody.

Interpretation of results

The localization of the HOXA9 protein was primarily observed in the cytoplasm, with a lesser presence in the nucleus. Positive HOXA9 staining was characterized by the presence of brownish-yellow granules in the cytoplasm and/or nucleus of cancer cells. Two qualified pathologists conducted the examination of immunohistochemical staining results using a double-blind method. In cases of disagreement, a third pathologist re-evaluated the sections to determine a consensus score. The scoring system for HOXA9 immunostaining was defined as follows according to the method published in the literatures before(Yuan et al. 2018): absence of staining or positive staining in less than 5% of cells was denoted as “−”; positive staining in 5–25% of cells was denoted as “ + ”; positive staining in over 25% but less than 50% of cells was denoted as “++”; and positive staining in over 50% of cells was denoted as “+++”. Tissue sections with scores “++” and “+++” were classified as high HOXA9 expression, while those with “−” or “+” scores were classified as low HOXA9 expression.

qRT-PCR assay

A total of 100 paraffin-embedded CRC tissues and 10 matched adjacent normal tissues, with available follow-up data, were utilized for the analysis of miR-140-3p and HOXA9 mRNA expression. The extraction of miRNA and mRNA was carried out using the respective extraction kits. Subsequently, the RNA was reverse transcribed into cDNA using the MonScriptTM miRNA First Strand cDNA Synthesis Kit (Tailing Reaction) following the provided instructions. The reaction system was prepared using the tailing method with cDNA as a template. PCR amplification was performed using an ABI Step One Plus Fluorescent PCR instrument (Applied Biosystems, USA) as instructed by the MonAmpTM ChemoHS qPCRMix kit. The qRT-PCR program was set as follows: 95 °C for 10 min (pre-denaturation) → 95 °C for 10 s (denaturation) → 60 °C for 10 s (annealing) → 72 °C for 30 s (extension) for 40 cycles. GAPDH and U6 were separately used as reference genes for mRNA and miRNA measurements. The primer sequences are provided in Table 1. The experiments were conducted in triplicates, and the expression levels of miR-140-3p and HOXA9 mRNA in the tested samples were calculated based on the Ct values obtained using the 2^(-ΔΔCt) relative quantification method, respectively.

Table 1 The primer sequences employed in this study
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Follow-up visit

The follow-up period commenced on the day of surgery, and the endpoint for the follow-up visit was either the date of the patient’s death or June 30, 2022. Follow-up data of 193 cases were available for analysis. To assess the overall survival differences, a Kaplan–Meier overall survival curve was constructed.

Statistical analysis

For data analysis, the SPSS 26.0 software was utilized. The qualitative variables were presented as the number of cases, and group comparisons were performed using the χ2 test. Quantitative variables were expressed as mean ± standard deviation, and group comparisons were conducted using the Student’s t test. The correlation between miR-140-3p and HOXA9 expression was evaluated using the Spearman’s rank correlation coefficient. The overall survival difference was analyzed using the log-rank test. Univariate and multivariable prognostic analysis was conducted using the COX proportional hazards regression model. A p value of less than 0.05 was considered statistically significant, indicating a significant difference between groups or associations between variables.

Results

miR-140-3p is downregulated in colorectal carcinoma

Three miRNA expression datasets (GSE122182, GSE110402, and GSE39845) were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs were identified using the following criteria: |LogFc| > 1 and p < 0.01. The overlapping miRNAs that showed differential expression in all three datasets were determined using the VENN drawer online tool (Fig. 1a). 10 miRNAs were found to be differentially expressed, namely miR-497, miR-139-5p, miR-140-3p, miR-30a-star, miR-29b-2-star, miR-30a, miR-143-star, miR-139-3p, miR-195, and miR-133a. While several of these miRNAs have been extensively studied in colorectal cancer (CRC) (Pidikova et al. 2020), the clinical application and regulation networks of miR-140-3p which is one of the most distinct miRNA and has reported having important biological functions in cellular level remain underexplored. Therefore, we focused on miR-140-3p for further investigation. Consistently, analysis of the ENCORI database confirmed that miR-140-3p is significantly downregulated in colon cancer tissues compared to paired normal tissues (Fig. 1b).

Fig. 1
figure 1

Bioinformatic analysis was conducted to investigate the expression of miR-140-3p and HOXA9 in colorectal cancer (CRC) and their correlation. a Differentially expressed miRNAs were identified by analyzing three datasets (GSE122182, GSE110402, and GSE39845) from the GEO database. Differentially expressed miRNAs were identified using the following criteria: |LogFc| > 1 and p < 0.01. Fc = expression of colon cancer tissue/adjacent normal tissue. b miR-140-3p expression was evaluated in colon cancer tissue samples (n = 450) and compared to normal colon tissue samples (n = 8). The data used for this analysis was obtained from ENCORI. c Overall survival analysis was performed on a cohort of 447 colon cancer patients using the ENCORI database. The Kaplan–Meier analysis revealed a significant decrease in cumulative survival among patients with low miR-140-3p expression (represented by the brown line) compared to those with high expression (represented by the green line). The statistical significance was determined using the log-rank test (p = 0.041). d Four miRNA target gene databases (PITA, microT, PicTar, and TargetScan) predicted HOXA9 as a target gene of miR-140-3p. The prediction data were generated via ENCORI. e The binding sites of miR-140-3p with HOXA9 were investigated. f HOXA9 mRNA expression was analyzed in tumor and normal tissue samples from patients with colonic and rectal adenocarcinoma. The dataset included 275 colon adenocarcinoma (COAD) tumor samples, 92 rectal adenocarcinoma (READ) tumor samples, 349 COAD normal tissue samples, and 318 READ normal tissue samples. The analysis was conducted using GEPIA2. g The expression profile of HOXA9 mRNA was examined in a pan-cancer context, specially focusing on its downregulation in tumor sections of COAD and READ. The analysis included 457 COAD tumor samples, 166 READ tumor samples, 41 COAD normal tissue samples, and 10 READ normal tissue samples. The data was obtained through TIMER. h In colon cancer, a negative correlation between miR-140-3p and HOXA9 expression was observed in a sample size of 450, with statistical significance (p < 0.05). This correlation data was generated through ENCORI

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To investigate the clinical significance of dysregulated miR-140-3p expression, we performed survival curve analysis using the ENCORI database. The analysis revealed that patients with lower miR-140-3p expression, whose expression levels below the average, exhibited a significantly poorer prognosis (Fig. 1c). These findings suggest that miR-140-3p likely plays a crucial role in colorectal cancer and may have clinical relevance as a potential prognostic indicator for this disease.

HOXA9 is a potential novel target of miR-140-3p

To elucidate the functional role of miR-140-3p, we employed the comprehensive ENCORI database to identify potential downstream target genes. Among the candidates, HOXA9 emerged as a highly scored and novel target which did not report before (Fig. 1d). Since miRNAs typically exert their functions by binding to target gene mRNA sequences to degrade mRNA or inhibit translation, we performed a sequence comparison and identified a significant binding region between miR-140-3p and HOXA9 mRNA (Fig. 1e), indicating that HOXA9 may be a potential binding target of miR-140-3p.

Additionally, we investigated the expression profiles of HOXA9 using the GEPIA2 database (Fig. 1f) and the TIMER database (Fig. 1g). Interestingly, contrary to miR-140-3p, HOXA9 exhibited upregulation in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) tumor tissues. To further validate the relationship between miR-140-3p and HOXA9, we analyzed their correlation, and the ENCORI data revealed a negative correlation in colon cancer tissues (Fig. 1h).

In summary, our analysis demonstrates that miR-140-3p is downregulated, while HOXA9 is upregulated in tumor tissues compared to normal tissues, indicating an unfavorable prognosis. Importantly, miR-140-3p has the potential to bind to the mRNA sequence of HOXA9. These findings strongly suggest that HOXA9 represents a novel target of miR-140-3p, and miR-140-3p may regulate the development of colorectal cancer by targeting HOXA9.

miR140-3p and HOXA9 axis is correlated with lymph node metastasis

To validate the findings from bioinformatic analysis above, we conducted experimental assays using residual paraffin-embedded tissue samples in our institute. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of miR-140-3p and HOXA9. The results demonstrated that miR-140-3p expression was significantly downregulated in carcinoma tissues (0.87 ± 0.13, n = 100) compared to adjacent normal tissues (n = 10) (Fig. 2a). In contrast, HOXA9 mRNA expression was elevated in tumor tissues compared to matched normal tissues (1.14 ± 0.1, both n = 10) (Fig. 2b).

Fig. 2
figure 2

Expression analysis of miR-140-3p and HOXA9 in clinical samples. a, b The a illustrates the expression levels of miR-140-3p, while the b demonstrates the mRNA expression of HOXA9 in tumor and normal tissues obtained from clinical samples. c Representative images of hematoxylin and eosin (HE) staining and immunohistochemical staining of HOXA9 are provided to showcase the histopathological features and intensity and distribution of HOXA9 of both tumor and matched normal tissue samples captured at a magnification of ×40. d The statistical analysis presents quantitative data derived from the immunohistochemical staining of HOXA9. The results highlight a significant increase in the ratio of highly expressed HOXA9 within the tumor area

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Immunohistochemical staining was performed to examine the protein expression of HOXA9 in colorectal cancer and paired normal tissues. The results revealed that HOXA9 protein was predominantly localized in the cytoplasm, with a smaller fraction detected in the nucleus (Fig. 2c). The high expression rate of HOXA9 protein was observed in colorectal cancer tissues compared to adjacent normal tissues (63.33%, 133 cases vs 45.71%, 96 cases) (Fig. 2d). In summary, our analysis of clinical samples confirmed the downregulation of miR-140-3p and the upregulation of HOXA9 in colorectal tumor tissues.

Furthermore, we investigated the relationship between miR-140-3p or HOXA9 expression and the clinicopathological characteristics of colorectal cancer patients. Based on the mean value of miR-140-3p expression, 100 randomly selected colorectal cancer patients were divided into the high expression group (36 cases) and the low expression group (64 cases). Similarly, based on immunohistochemical results for HOXA9, 210 colorectal cancer patients were divided into the low HOXA9 expression group (77 cases) and the high expression group (133 cases). The relationship between miR-140-3p and HOXA9 expression in colorectal cancer tissues and the clinicopathological characteristics of patients was analyzed. The results (Table 2) indicated that low miR-140-3p expression was associated with lymph node metastasis (p < 0.05), and high HOXA9 expression was associated with lymph node metastasis and lympho-vascular invasion (p < 0.05). However, miR-140-3p and HOXA9 expression levels showed no significant association with age, gender, differentiation, TNM stage, tumor diameter, and tumor type, tumor localization, KRAS mutation, p53 mutation and MSI status (p > 0.05). Ki67 expression correlated to some extent with miR-140-3p and HOXA9 expression, although the associations were not statistically significant (p = 0.078 and p = 0.091, respectively). In summary, the dysregulation of the miR-140-3p and HOXA9 axis in colorectal cancer is closely correlated with lymph node metastasis.

Table 2 Associations between miR-140-3p and HOXA9 expression levels and clinicopa-thological characteristics in patients diagnosed with colorectal cancer
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HOXA9 is an independent prognosis factors for colorectal cancer patients

The Kaplan–Meier survival curves were generated by our cohort to assess the effect of miR-140-3p (Fig. 3a) and HOXA9 (Fig. 3b) expression on the prognosis of colorectal cancer patients. The results revealed that the 3-year overall survival rate was 86.1% (31 case/36 case) in the miR-140-3p high expression group, while the miR-140-3p low expression group exhibited significantly poorer outcomes (3 years OS: 67.2%, 43 case /64 case) (p < 0.05). Similarly, for HOXA9, the 3-year overall survival rate was 73% (89 cases/122 cases) in the high expression group and 87.3% (62 cases/77 cases) in the low expression group, indicating a worse prognosis in the high HOXA9 expression group compared to the low expression group (p < 0.05).

Fig. 3
figure 3

Prognostic analysis of miR-140-3p and HOXA9 expression in colorectal cancer patients. a Among our cohort of colorectal cancer patients, the miR-140-3p low expression group (n = 64) exhibited a significantly poorer prognosis in comparison to the miR-140-3p high expression group (n = 36) (p = 0.048). b Similarly, within our colorectal cancer patient cohort, the HOXA9 high expression group (n = 122) demonstrated a significantly unfavorable prognosis compared to the HOXA9 low expression group (n = 71) (p = 0.033)

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Cox univariate regression analysis (Table 3) demonstrated that, in addition to the known prognostic factors such as differentiation, TNM stage, and lymph node metastasis, miR-140-3p and HOXA9 expression levels also significantly influenced the prognosis of colorectal cancer patients. Further, Cox multivariate regression analysis, after adjusting for potential confounding factors (Table 3), identified TNM stage and HOXA9 as independent risk factors for the prognosis of colorectal cancer patients, emphasizing their clinical significance.

Table 3 Univariate and Multifactorial Cox regression analysis investigating the prognostic factors influencing the outcomes of patients with colorectal cancer
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In conclusion, low miR-140-3p expression and high HOXA9 expression were associated with poor prognosis in colorectal cancer patients. Notably, HOXA9 emerged as an independent risk factor for the prognosis of these patients, highlighting its potential as a prognostic biomarker in colorectal cancer management.

Discussion

Colorectal cancer (CRC) is a significant malignant tumor with a high mortality rate due to recurrence and metastasis, despite advancements in treatment modalities such as surgery, radiotherapy, chemotherapy, and immunotherapy (Dekker et al. 2019; Kennedy et al. 2011). Biomarkers are crucial tools for CRC diagnosis, classification, and prognostic prediction. MiRNAs have emerged as important players in tumor pathogenesis, diagnosis, and treatment, prompting us to screen for miRNAs that can serve as markers and targets for CRC diagnosis and treatment. In this study, we identified miR-140-3p as an important miRNA in CRC development and uncovered HOXA9 as its novel target.

miR-140-3p has been to some extend investigated in other cancer types, often functioning as a tumor suppressor. For instance, low miR-140-3p expression in gastric cancer patients has been associated with poor prognosis and unfavorable clinicopathological features. Overexpression of miR-140-3p inhibits gastric cancer cell migration, invasion, and proliferation (Wang et al. 2021). Similarly, in bladder cancer, miR-140-3p hinders cancer cell proliferation and invasion by directly targeting FOXQ1 (Wang et al. 2020b). However, miR-140-3p can also act as a tumor promoter, as demonstrated in ovarian cancer, where it inhibits NK cell recruitment and diminishes cytotoxic effects, thereby promoting ovarian cancer cell growth through the mediation of MAPK1 (Wang et al. 2020a).

HOXA9, along with other AbdBC-type HOXA genes, plays a crucial role as a transcription factor in hematopoiesis and leukemogenesis (Abramovich et al. 2005; de Bock et al. 2018). Elevated HOXA9 expression has also been observed in breast cancer. In ovarian cancer, HOXA9 expression induces normal peritoneal fibroblasts to adopt a cancer-associated fibroblast (CAF) phenotype, stimulating the growth of ovarian cancer and endothelial cells (Ko et al. 2012). Additionally, HOXA9 has been implicated in chemotherapy resistance to 5-fluorouracil in CRC (Ju et al. 2017). However, studies have shown that HOXA9 hypermethylation promotes hepatocellular carcinoma development (Kuo et al. 2014), suggesting that HOXA9 may have different roles in different cancer types.

In our study, bioinformatic analysis revealed low expression of miR-140-3p and high expression of HOXA9 in colorectal cancer tissues. Furthermore, miR-140-3p was found to regulate HOXA9 by binding to its mRNA, a finding that was confirmed through examination of clinical samples. Our limitation lies in not directly verifying their interaction, which would have provided favorable evidence of their connection. Clinical data demonstrated that low miR-140-3p expression and high HOXA9 expression were associated with lymph node metastasis and poor prognosis in CRC patients. Notably, HOXA9 emerged as an independent risk factor for colorectal cancer patients. It is intriguing that in our multivariate analysis, miR-140-3p did not emerge as an independent risk factor. This absence of significance could be attributed to the robust interaction observed between miR-140-3p and lymph node metastasis, as illustrated in Table 2. In multivariate Cox analysis, the assessment of a variable’s significance takes place while considering other variables concurrently. It is plausible that the significance of miR-140-3p and lymph node metastasis diminished when accounting for their interactions or dependencies with other covariates. Consequently, these factors did not demonstrate statistical significance in the multivariate Cox analysis.

Indeed, miR-140-3p and HOXA9 exhibit substantial functional overlap and share common targets. Both miR-140-3p and HOXA9 play crucial roles in cell stemness, proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). For instance, in breast cancer, the downregulation of miRNA-140 promotes proliferation and inhibits apoptosis by influencing SOX2 expression (Zhang et al. 2012). Intriguingly, HOXA9 synergistically regulates SOX2 and BCL2 to suppress apoptosis and mediate leukemogenesis (Miyamoto et al. 2021). In colorectal cancer (CRC), BCL2 is recognized as a direct target of miR140-3p (Liu et al. 2021). It has been shown that miR-140-3p regulates the PI3K/AKT, JAK/STAT, mTOR, TGFβ/Smad, and Wnt pathways in tumors (Taheri et al. 2023), while HOXA9 influences the expression levels of PI3K/AKT, JAK/STAT, mTOR, NF-kB, HIF1a, and the Wnt pathway (Tang et al. 2022). Therefore, miR-140-3p and HOXA9 may engage in crosstalk at various cellular levels.

Our data showed miR-140-3p and HOXA9 predict the poor prognosis and indicated its expression related to clinical lymph node metastasis. Previous studies have pointed that the Wnt pathway is a shared signaling of both miR-140-3p and HOXA9, playing a crucial role in the EMT process (Xu et al. 2021, Gonzalez and Medici 2014). Bioinformatic analysis in CRC has revealed a positive relationship between HOXA9 and β-catenin (gene: CTNNB1), while miR140-3p exhibits the opposite trend (Supplementary Fig. 1). Consequently, we hypothesize that the miR-140-3p-HOXA9 axis may influence lymph node metastasis and contribute to poor outcomes through the Wnt/β-catenin-mediated EMT process, necessitating further exploration in future studies.