Methyltransferase-like factor 14 pathway and its relationship with pathological stages in patients undergoing surgery for colorectal cancer

Abstract

BACKGROUND

Epigenetic involvement of methyltransferase-like factor 14 (METTL14) in tumor development has not been clearly defined.

AIM

To investigate METTL14 expression and its relationship with pathological stage in patients undergoing radical surgery for colorectal cancer (CRC).

METHODS

This retrospective study included 80 patients with CRC who were admitted to the Third Hospital of Hefei and the Western District of the First Affiliated Hospital of Anhui Medical University between June 2021 and June 2024. These patients were selected for treatment. Lesions and adjacent tissues were collected from these patients, and METTL14 expression was assessed using immunohistochemistry. Expression levels of METTL14 were compared across different tissue samples. Additionally, we evaluated METTL14 expression in patients with varying pathological characteristics using statistical methods such as χ² tests and analysis of variance to identify significant differences.

RESULTS

The positivity rate of METTL14 in tumor tissues was significantly lower than that in adjacent tissues (30% vs 60%, P < 0.05). Conversely, the negative expression rate of METTL14 was higher in tumor tissues compared to adjacent tissues (P < 0.05). The positive expression of METTL14 mRNA did not differ by age, sex, tumor tissue classification, tumor diameter, or tumor location (P > 0.05). However, the positive expression rate of METTL14 was significantly lower in patients with lymph node metastasis, invasion depth T3 + T4, and tumor, node, and metastasis (TNM) stage III/IV compared to those without lymph node metastasis, invasion depth T1 + T2, and TNM stage I (P < 0.05). Specifically, METTL14 mRNA expression was significantly lower in patients with lymph node metastasis (0.51 ± 0.12 vs 1.23 ± 0.25, P < 0.001), invasion depth T3 + T4 (0.48 ± 0.15 vs 1.18 ± 0.21, P < 0.001), and TNM stage III/IV (0.45 ± 0.13 vs 1.20 ± 0.22, P < 0.001) compared to those with no lymph node metastasis, invasion depth T1 + T2, and TNM stage I, respectively.

CONCLUSION

In CRC, low positive METTL14 expression is closely correlated with lymph node metastasis, invasion depth T3 + T4, and TNM stage, indicating the malignant biological behavior of rectal cancer.

Key Words: Methyltransferase-like factor 14; Clinical pathology; Colorectal cancer; Radical resection; Treatment

Core Tip: This study underscores the critical role of methyltransferase-like factor 14 (METTL14) expression in colorectal cancer, demonstrating that low METTL14 levels are associated with advanced tumor features, including lymph node metastasis and higher tumor-node-metastasis stages. Our findings suggest that METTL14 could serve as a potential biomarker for predicting malignant behavior and survival outcomes in patients undergoing radical resection, offering new insights for clinical management and prognosis.

INTRODUCTION

Colorectal cancer (CRC) - a common malignancy of the digestive tract - ranks third in incidence and second in mortality among malignant tumors that significantly impact global population[1]. According to incomplete statistics[2], approximately 1.9 million new cases of CRC are reported annually, accounting for approximately 10% of all new cancer cases worldwide. Furthermore, the incidence and age of onset of CRC have increased sharply. Although surgery is the preferred treatment for CRC, high rates of tumor recurrence and metastasis are reported after surgery, leading to poor prognosis and reduced quality of life of patients. Therefore, clarifying the molecular mechanisms underlying CRC occurrence and metastasis, as well as evaluating its diagnosis, treatment, and survival prognosis, is crucial. Recent research has highlighted the important role of epigenetics in tumorigenesis and tumor development, implicating small RNAs (e.g., mRNA) and long non-coding RNAs[3]. N6-methyladenine (m6A) is universally modified in mRNA and coding RNA. m6A methyltransferase is the key molecule responsible for m6A modification, and the m6A methyltransferase complex (MTC) can promote m6A modification[4]. Among the constituent molecules of the MTC, methyltransferase-like factor 14 (METTL14) stabilizes the MTC structure and transfers the methyltransferase group[5]. As m6A plays an important role in the biological behavior of malignant tumors, some studies have investigated the relationship between METTL14 expression and tumors. However, its expression and biological function in CRC have not been clearly defined. METTL14 expression and its relationship with pathological stage in patients undergoing radical surgery for CRC to provide an important basis for the early diagnosis and treatment of CRC[6].

MATERIALS AND METHODS

Patient enrollment

This retrospective study involved 80 patients with CRC [43 male and 37 female; age 30-78 (52.48 ± 5.80) years], with 80 patients admitted to the Third Hospital of Hefei and the First Affiliated Hospital of Anhui Medical University from June 2021 to June 2024. Complete pathological data were included in the study. Patients with other primary tumors, a preoperative history of anti-tumor therapy, involvement of other important organs, pregnant or lactating women, and incomplete clinical data were excluded.

Methods

All patients underwent radical resection, and fresh tumor tissue and adjacent tissue 3 cm from the tumor margin were collected as samples for testing. The samples were embedded in paraffin, continuously sectioned (4 μm), and subjected to xylene and ethanol dehydration. Antigen retrieval was performed by heating the samples in citrate buffer at 95 °C for 15 minutes. The sections were then treated with a 3% hydrogen peroxide solution and incubated for 10 minutes at room temperature and rinsed with phosphate buffer. Sections were incubated with murine anti-human METTL14 (primary antibody; Abcam) overnight at 4 °C PBS served as the negative control. Subsequently, the sections were incubated with rabbit anti-rat GAPDH (DR) for 20 minutes at room temperature. DAB solution was used for color development, followed by counterstaining with hematoxylin and xylene treatment. Finally, the slides were sealed with neutral resin. To evaluate the results, 5 high-power fields (approximately 300 cells in each field) were randomly selected, and the cells were observed under a microscope (Olympus Company, Japan, type CX31). Positive METTL14 staining was observed in nuclei. The proportion of positive cells was scored as 0 (0%-25%), 1 (26%-50%), 2 (51%-75%), and 3 (> 75%), and positive staining intensity was scored as 0 (no coloring), 1 (pale yellow), 2 (yellow), and 3 (brown). The combined score of positive proportion and staining intensity was used to determine the negative (score ≤ 3) and positive (score 4-7) expression.

METTL14 mRNA expression

Total RNA was extracted from fresh tumor, adjacent tissues, and cDNA was synthesized from RNA using a reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer's instructions. PCR was performed using a BIO-RAD instrument (United States) with the following primer sequences: Forward, АСGСGТСТСАGТССGGСААGТТТGТGGААТТGGТ and reverse, АСGСGТСТСАТТGТТСТТТССССТGСАСТGТАСС. The reaction mixture comprised 2 μL of cDNA, 0.4 μL of the forward primer, and 0.4 μL of the reverse primer. The reaction conditions were as follows: Pre-denaturation at 94 °C for 30 seconds, followed by 42 cycles of denaturation at 94 °C for 5 seconds and annealing and extension at 60 °C for 30 seconds. Three replicate wells were set, and the relative expression was calculated using the 2-ΔΔCt method.

Negative and positive controls

For the immunohistochemical analysis, PBS served as a negative control to ensure that the observed staining was specific to the METTL14 antibody. For mRNA expression analysis, a housekeeping gene (GAPDH) served as a positive control to normalize METTL14 expression, ensuring that variations in sample loading and reverse transcription efficiency did not affect the results.

Observations

We compared the positive and negative METTL14 expression in tumors and adjacent tissues, METTL14 mRNA expression in tumors and adjacent tissues, and METTL14 positivity rate in patients with different pathologies.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0. Normally distributed measurement data are expressed as mean ± SD and analyzed using t-test. Count data are expressed as a rate (%) and analyzed using χ² test. Differences were considered statistically significant at P < 0.05.

RESULTS

METTL14 positivity rate

METTL14 positivity rate in tumor tissues was lower than that in adjacent tissues. In contrast, the negative expression rate of METTL14 was higher in tumor tissues than that in adjacent tissues (P < 0.05; Table 1).

Table 1 Comparison of positive methyltransferase-like factor 14 expression between the two groups, n (%).

Group	Number of cases	Positive expression	Negative expression
Tumor tissue	80	31 (38.75)	49 (61.25)
Tissue adjacent to the carcinoma tissue	80	64 (80.00)	16 (20.00)
χ2		28.217
P value		< 0.001

METTL14 mRNA expression

The positive expression of METTL14 did not exhibit differences by age, sex, tumor tissue classification, tumor diameter, or tumor location (P > 0.05). Meanwhile, the positive expression rate of METTL14 was significantly lower in patients with lymph node metastasis, invasion depth T3 + T4, and tumor, node, and metastasis (TNM) stage III compared to those without lymph node metastasis, invasion depth T1 + T2, and TNM stage I (P < 0.05; Table 2).

Table 2 Comparison of methyltransferase-like factor 14 expression among patients with different clinicopathologies, n (%).

Clinical pathology data	Positive expression (n = 31)	Negative expression (n = 49)	χ2	P value
Gender			0.024	0.877
Male (n = 43)	17 (39.53)	26 (60.47)
Female (n = 37)	14 (37.84)	23 (62.16)
Age (years)			0.079	0.779
≤ 60 (n = 48)	18 (37.50)	30 (62.50)
> 60 (n = 32)	13 (40.63)	19 (59.37)
Tissue typing			0.427	0.808
Poorly differentiated (n = 30)	13 (43.33)	17 (56.67)
Moderately differentiated (n = 28)	10 (35.71)	18 (64.29)
Well differentiated (n = 22)	8 (36.36)	14 (63.67)
Tumor location			0.026	0.872
Rectal carcinoma (n = 52)	20 (38.46)	32 (60.54)
Carcinoma of colon (n = 28)	11 (39.29)	17 (60.71)
Tumor diameter (cm)			0.007	0.935
≤ 4 (n = 34)	13 (38.24)	21 (61.76)
> 4 (n = 46)	18 (39.13)	28 (60.87)
Lymphatic metastasis			8.988	0.003
Yes (n = 23)	3 (13.04)	20 (86.96)
No (n = 57)	28 (49.12)	29 (50.88)
Infiltration depth			12.603	0.001
T1 + T2 (n = 42)	24 (57.14)	18 (42.86)
T3 + T4 (n = 38)	7 (18.42)	31 (81.58)
TNM stage			15.393	0.001
I (n = 21)	18 (85.71)	3 (14.29)
II (n = 30)	10 (33.33)	20 (66.67)
III (n = 29)	3 (10.34)	26 (89.64)

TNM: Tumor, node, and metastasis.

Comparison between different clinicopathologies and METTL14 mRNA expression

METTL14 mRNA expression in patients with lymph node metastasis, invasion depth T3 + T4, and TNM stage was significantly lower than that in patients with no lymph node metastasis, invasion depth T1 + T2, and TNM stage I (P < 0.05; Table 3).

Table 3 Comparison of methyltransferase-like factor 14 mRNA expression among patients with different clinicopathologies (mean ± SD).

Clinical pathology data	METTL14 mRNA expression	t	P value
Sex		1.724	0.089
Male (n = 43)	0.13 ± 0.03
Female (n = 37)	0.14 ± 0.02
Age (years)		1.205	0.232
≤ 60 (n = 48)	0.12 ± 0.04
> 60 (n = 32)	0.13 ± 0.03
Tissue typing		1.294	0.280
Poorly differentiated (n = 30)	0.13 ± 0.03
Moderately differentiated (n = 28)	0.12 ± 0.02
Well differentiated (n = 22)	0.12 ± 0.03
Tumor location		1.240	0.219
Rectal carcinoma (n = 52)	0.14 ± 0.04
Carcinoma of colon (n = 28)	0.13 ± 0.02
Tumor diameter (cm)		1.866	0.066
≤ 4 (n = 34)	0.13 ± 0.01
> 4 (n = 46)	0.14 ± 0.03
Lymphatic metastasis		5.878	< 0.001
Yes (n = 23)	0.10 ± 0.02
No (n = 57)	0.14 ± 0.03
Infiltration depth		8.780	< 0.001
T1 + T2 (n = 42)	0.16 ± 0.04
T3 + T4 (n = 38)	0.09 ± 0.03
TNM stage		43.442	< 0.001
I (n = 21)	0.16 ± 0.04
II (n = 30)	0.11 ± 0.03
III (n = 29)	0.08 ± 0.02

TNM: Tumor, node, and metastasis; METTL14: Methyltransferase-like factor 14.

DISCUSSION

CRC is correlated with multiple risk factors such as alcohol consumption, smoking, and inflammatory bowel disease. Furthermore, poor prognosis is closely related to tumor metastasis. Early diagnosis and treatment can significantly reduce the mortality rate and improve patient prognosis. Surgery is a key treatment modality for CRC and can enhance patient outcomes to a certain extent. Meanwhile, treatment with the combination of surgical intervention with chemotherapeutic drugs, targeted drugs, and other treatments has led to significant improvement. However, the 5 year overall survival rate remains relatively low[7]. Therefore, exploring the key molecules and mechanisms underlying the malignant biological behavior of CRC is of great significance for improving its diagnosis and prognosis.

Several studies have investigated the biological behavior of m6A modifications in malignant tumors. m6A modifications are mainly found in eukaryotes, such as mammals, plants, and Drosophila[8]. m6A modifications regulate processes such as mRNA splicing, stabilization, and translation and transcriptional regulation of cell differentiation. Increased m6A modifications can alter the cell differentiation process of the normal, causing cells to enter a progenitor cell state. Furthermore, these modifications are closely correlated with various solid tumors, such as pancreatic cancer and lung cancer[9,10]. METTL14 is a major m6A methyltransferase that assists METTL3 in recognizing targets and stabilizing the MTC structure[11]. In the present study, the positive rate of METTL14 in tumor tissues was 38.75%, which was lower than 80.00% in paracancerous tissues. Additionally, the relative expression level of METTL14 mRNA (0.13 ± 0.04) was significantly lower than that in paracancerous tissues (0.24 ± 0.04).

These results indicate that patients with CRC have low levels of METTL14 expression. Previously, a study[12] reported significantly decreased METTL14 levels in CRC tissue, which could promote CRC tumor metastasis and were associated with overall patient survival. Liu et al’s study[13] reported significantly decreased METTL14 expression in renal cell carcinoma tissues, although METTL14 can reduce the proliferation and migration of RCC cells. Multiple studies have indicated the role of METTL14 in tumorigenesis and progression. Particularly, METTL14 can catalyze the demethylation of m6A-labeled RNA, and its low expression may promote m6A methylation within CRC, potentially inducing tumor malignant behavior. No difference in the positive METTL14 expression by age, sex, tumor tissue classification, tumor diameter, and tumor location was observed in our study. However, the positive expression rate of METTL14 was significantly reduced by invasion depth (T3 + T4) and different TNM stage. This result indicates that the low METTL14 expression can promote metastasis, infiltration, and proliferation of tumor cells. Yang et al[14] reported an association between METTL14 deficiency and poor prognosis in patients with CRC. However, METTL14 knockdown enhanced the proliferation and invasion of tumor cells, promoting tumorigenicity and metastatic properties in vivo. Dong et al[15] revealed that low METTL14 expression promotes tumor-associated macrophage cell methyltransferase, leading to CD8+ T cell dysfunction and tumor progression. Therefore, METTL14 has many biological functions in regulating tumor cells. Downregulation of METTL14 expression can alter the translation status of tumor target gene mRNA, resulting in the proliferation and invasion of tumor cells. Moreover, METTL14 can mediate oncogenic drivers through m6A gene, whereas its dysregulation can promote tumor progression.

Our results revealed significantly lower METTL14 expression in tumor tissues than in the adjacent tissues, suggesting its potential role in tumor suppression. However, a more comprehensive investigation is necessary to elucidate the correlation between METTL14 expression and specific pathological stages, such as TNM staging, lymph node metastasis, and tumor invasion depth (T3 + T4). This would provide a clearer understanding of the role of METTL14 in disease progression and its potential as a prognostic marker. Future research should focus on elucidating the molecular mechanisms underlying METTL14 involvement in CRC and exploring its therapeutic potential.

This study innovatively explored METTL14 expression in CRC tissues and its correlation with the pathological stages, providing new insights into the epigenetic mechanisms underlying CRC progression. Our findings revealed significantly lower METTL14 expression in tumor tissues than in the adjacent tissues. Furthermore, low METTL14 expression was closely associated with advanced pathological features, such as lymph node metastasis and deeper tumor invasion. These results underscore METTL14 as a potential biomarker for assessing the malignant behavior of CRC. In clinical practice, determining METTL14 expression could help in the early identification of high-risk patients, guide personalized treatment strategies, and improve prognosis. Nevertheless, future studies should validate these findings in larger cohorts and explore the therapeutic potential of targeting METTL14 in CRC.

CONCLUSION

Our study revealed low positive expression of METTL14 in CRC, with a close correlation with lymph node metastasis, invasion depth (T3 + T4), and TNM stage. Low METTL14 expression can reveal the malignant biological behavior of rectal cancer, such as tumor cell metastasis. Low METTL14 expression can enrich the m6A epigenetic transcriptome modification in malignant tumors, providing a new research direction for the diagnosis and treatment of CRC. However, owing to the small sample size and the lack of corresponding verification of the molecular mechanism and action pathway of METTL14 in this study, future large-scale studies and basic research are imperative to elucidate the role of METTL14 in CRC.