Expression significance of biomarker MORC4 in colorectal cancer patients and its relationship with pathological features and prognosis

Abstract

BACKGROUND

Colorectal cancer (CRC) is one of the most common malignant gastrointestinal tumors worldwide, with high incidence and mortality rates.

AIM

To investigate the expression significance of the chromatin-remodeling protein MORC family CW-type zinc finger 4 (MORC4) as a biomarker in CRC patients, and to explore its relationship with pathological features and prognosis.

METHODS

A total of 143 CRC specimens and 57 adjacent tissue specimens, surgically removed from our hospital between January 2020 and January 2021, were collected. MORC4 protein expression was assessed using immunohistochemistry after paraffin embedding. The relationship between MORC4 protein expression and clinicopathological characteristics of patients was analyzed. Kaplan-Meier survival curves were plotted to analyze the relationship between MORC4 protein expression and prognosis in CRC patients.

RESULTS

Compared with adjacent tissues, the expression rate of MORC4 protein in CRC tissues was significantly higher (P < 0.05). No significant difference was observed in the high expression rate of MORC4 protein in CRC tissues among patients of different gender, age, tumor location, tumor diameter, and primary tumor status (P > 0.05). However, significant differences were found in the high expression rate of MORC4 protein in patients with different degrees of differentiation, lymph node metastasis, distant metastasis, tumor-lymph node-metastasis stage, and serum carcinoembryonic antigen levels (P < 0.05). Compared with patients with low MORC4 expression, patients with high MORC4 expression had a worse prognosis (P < 0.05).

CONCLUSION

The upregulation of MORC4 expression in CRC patients is closely related to disease severity and prognosis, suggesting its potential as an evaluation biomarker, which warrants further investigation.

Key Words: Colorectal cancer; Biomarker; MORC family CW-type zinc finger 4; Expression significance; Pathological features; Prognosis; Relationship

Core Tip: To investigate the expression significance of the chromatin-remodeling protein MORC family CW-type zinc finger 4 as a biomarker in colorectal cancer patients, and to explore its relationship with pathological features and prognosis. The upregulation of MORC family CW-type zinc finger 4 expression in colorectal cancer patients is closely related to disease severity and prognosis, suggesting its potential as an evaluation biomarker, which warrants further investigation.

INTRODUCTION

Colorectal cancer (CRC) is one of the most common malignant gastrointestinal tumors worldwide, with high incidence and mortality rates[1]. According to global cancer statistics[2], CRC ranks third in cancer incidence and second in cancer-related deaths, following lung cancer. Particularly in economically developed regions, the incidence of CRC is steadily rising due to factors such as population aging, lifestyle changes (e.g., high-fat diets, lack of exercise), and increasing rates of obesity and diabetes[3]. Although early screening techniques (such as colonoscopy and computed tomography colonography) and advancements in treatment have improved early diagnosis and treatment of CRC in recent years, and some patients’ prognoses have been improved, the overall survival (OS) rate of patients with advanced CRC remains low, with a 5-year survival rate of less than 15%[4]. The development of CRC involves the combined effects of genetic, environmental, and lifestyle factors, leading to abnormal proliferation and malignant transformation of intestinal cells[5]. In recent years, as tumor molecular biology research has deepened, the molecular mechanisms of CRC have gradually been revealed, and the role of biomarkers in tumor development, diagnosis, and prognosis assessment has garnered increasing attention[6]. Biomarkers not only provide new avenues for early diagnosis and molecular subtyping of CRC but also lay the foundation for personalized treatment and prognosis evaluation[7].

The chromatin-remodeling protein MORC family CW-type zinc finger 4 (MORC4) is a protein involved in DNA repair, gene expression regulation, and cell cycle control[8]. Its abnormal expression in various malignant tumors has attracted widespread attention in recent years. MORC4 may influence tumor occurrence and progression by regulating chromatin remodeling, affecting cell proliferation, differentiation, and apoptosis[9]. Previous studies have shown that MORC4 is highly expressed in various tumors, including breast cancer, liver cancer, and gastric cancer, and is closely related to tumor invasiveness and poor patient prognosis[10]. However, there is a lack of systematic research on the expression of MORC4 in CRC and its relationship with pathological features and prognosis. Therefore, this study aims to explore the expression of MORC4 in CRC and its relationship with patients’ clinicopathological characteristics and prognosis. By analyzing the expression levels of MORC4 in CRC tissues and adjacent normal tissues, we aim to reveal its role in tumor progression and evaluate its potential as a prognostic marker for CRC, providing new theoretical support and potential therapeutic targets for CRC diagnosis and treatment.

MATERIALS AND METHODS

Tissue materials

A total of 143 CRC specimens and 57 adjacent non-cancerous tissue specimens resected via surgery at our hospital between January 2020 and January 2021 were collected. After embedding in paraffin, immunohistochemistry was performed to examine the expression of the MORC4 protein. Clinical and pathological data of the 143 CRC patients are shown in Table 1. This study was approved by the Ethics Committee of our hospital.

Table 1 Clinical and pathological data of colorectal cancer patients.

Pathological characteristics	Cases	Proportion (%)
Gender	-	-
Male	81	56.64
Female	62	43.36
Age (years)	-	-
< 60	19	13.29
≥ 60	124	86.71
Tumor location	-	-
Left side	112	78.32
Right side	31	21.68
Tumor diameter (cm)	-	-
≤ 5	94	65.73
> 5	49	34.27
Degree of differentiation	-	-
Low differentiation	21	14.69
Moderate + high differentiation	122	85.31
T stage	-	-
T1 + T2 stage	13	9.09
T3 + T4 stage	130	90.91
N stage	-	-
N0 stage	88	61.54
Nx stage	55	38.46
M stage	-	-
M0 stage	127	88.81
M1 stage	16	11.19
TNM stage	-	-
I + II	85	59.44
III + IV	58	40.56
Serum CEA (μg/L)	-	-
< 5	67	46.85
≥ 5	76	53.15

TNM: Tumor-node-metastasis; CEA: Carcinoembryonic antigen.

Major reagents

The major reagents used in this study were as follows: MORC4 primary antibody (Batch No.: GR256479-6) was purchased from Abcam, MA, United States; secondary antibody (Batch No.: Wp18031101), citrate buffer (Batch No.: 18070601), and hydrogen peroxide (Batch No.: 180105) were provided by Fuzhou Maixin Biotechnology Development Co., Ltd.; DAB (diaminobenzidine) staining solution (Batch No.: Wp18111303), hematoxylin staining reagent (Batch No.: 2018052901), and crystal violet (Batch No.: 20181025) were obtained from Shanghai Beyotime Biotechnology Co., Ltd. All reagents were used in strict accordance with the manufacturer’s instructions during the experiment to ensure the accuracy and reproducibility of the results.

Detection of MORC4 protein expression in tissues

The expression of MORC4 protein in CRC tissues was detected using standard immunohistochemistry techniques. Positive controls were CRC tissue sections with known high MORC4 expression, as confirmed by pre-experiments using western blot and immunohistochemistry. Negative controls included sections incubated without the primary antibody or with an isotype-matched immunoglobulin G antibody at the same concentration and conditions.

Paraffin-embedded sections were prepared from 143 CRC tissues and 57 adjacent normal tissues. During the experiment, the sections were first incubated in 80% methanol for 10 minutes to block endogenous peroxidase activity. The sections were then washed with phosphate buffered saline (PBS), and antigen retrieval was performed using a 0.1 mmol/L citrate buffer solution to ensure full antigen exposure. After another PBS wash, 50 μL of primary anti-MORC4 antibody (dilution ratio 1:100) was added, and the sections were incubated at 37 °C for 1 hour to allow sufficient binding to the target protein. After washing with PBS, horseradish peroxidase-labeled secondary antibody (50 μL) was added, and the sections were incubated again at 37 °C for 1 hour to enhance the antigen-antibody reaction signal. After secondary antibody incubation, the sections were incubated with streptavidin-peroxidase for 1 hour to ensure signal amplification. Finally, the sections were stained using DAB color development and counterstained with hematoxylin. After mounting, the results were observed using a light microscope. Staining results were independently evaluated by two experienced pathologists blinded to the sample identity. MORC4 protein was primarily localized in the nucleus of tumor cells, with positive staining appearing as brown-yellow granules. The percentage of stained cells in each field of view was scored: Less than 5% stained cells = 0 points, 6%-25% = 1 point, 26%-50% = 2 points, 51%-75% = 3 points, and more than 75% = 4 points. The intensity of staining was also scored, with no staining = 0 point, weak staining = 1 point, moderate staining = 2 points, and strong staining = 3 points. The final score was the product of these two scores: 0-1 points were considered negative (-), 2-4 points were weakly positive (+), 6-8 points were moderately positive (++), and 9-12 points were strongly positive (+++). In the analysis, - to + were defined as low expression, while ++ to +++ were defined as high expression.

Comparison of prognosis in patients with different levels of MORC4 protein expression

For the postoperative prognosis follow-up, patient survival data were recorded via telephone or outpatient visits. The follow-up period started from the date of surgery and ended at the patient’s death or the follow-up cutoff date of February 1, 2024. OS was defined as the time from the surgery date to the first occurrence of all-cause death or the follow-up cutoff. Based on the immunohistochemical scoring criteria, patients were divided into the MORC4 high-expression group and the low-expression group, and the survival differences between the two groups were compared.

Statistical analysis

Experimental data were visualized using GraphPad Prism 8, and data analysis was conducted using SPSS 22.0. Count data were expressed in the form of [n (%)], and group comparisons were made using the χ² test. For normally distributed measurement data, and independent samples t-tests were conducted for intergroup comparisons. Survival analysis was performed using the Kaplan-Meier method to plot survival curves, and the Log-rank test was used to assess survival differences between patients with different levels of MORC4 expression. Statistical significance was set at P < 0.05.

RESULTS

Comparison of MORC4 protein expression between two types of tissues

Immunohistochemical staining showed that compared to adjacent non-cancerous tissues, brownish-yellow nuclear staining was concentrated in CRC tissue samples, and the high expression rate of MORC4 protein in CRC tissue samples (62.24%) was significantly higher than in adjacent non-cancerous tissues (12.28%), with a statistically significant difference (P < 0.05), as shown in Table 2.

Table 2 Comparison of MORC family CW-type zinc finger 4 protein expression between two types of tissues, n (%).

Tissue type	Low expression	High expression	χ²	P value
CRC tissue (n = 143)	54 (37.76)	89 (62.24)	40.750	< 0.001
Adjacent tissue (n = 57)	50 (87.72)	7 (12.28)

CRC: Colorectal cancer.

Comparison of high MORC4 expression rate in CRC tissue among patients with different pathological characteristics

There were no statistically significant differences in the comparison of high MORC4 protein expression rates in CRC tissues among patients of different genders, ages, tumor locations, tumor diameters, and T stages (P > 0.05). However, statistically significant differences were observed in patients with different levels of differentiation, N stage, M stage, tumor-nodes-metastasis (TNM) stage, and serum carcinoembryonic antigen (CEA) levels (P < 0.05), as shown in Table 3.

Table 3 Comparison of high MORC family CW-type zinc finger 4 expression rate in colorectal cancer tissue among patients with different pathological characteristics, n (%).

Pathological characteristics	Cases (n = 143)	High MORC4 expression (n = 89)	χ²	P value
Gender	-	-	0.705	0.401
Male	81	48 (59.26)	-	-
Female	62	41 (66.13)	-	-
Age (years)	-	-	0.356	0.550
< 60	19	13 (68.42)	-	-
≥ 60	124	76 (61.29)	-	-
Tumor location	-	-	0.015	0.902
Left side	112	70 (62.50)	-	-
Right side	31	19 (61.29)	-	-
Tumor diameter (cm)	-	-	0.827	0.362
≤ 5	94	56 (59.57)	-	-
> 5	49	33 (67.35)	-	-
Degree of differentiation	-	-	5.772	0.016
Low differentiation	21	18 (85.71)	-	-
Moderate + high differentiation	122	71 (58.20)	-	-
T stage	-	-	0.911	0.339
T1 + T2 stage	13	6 (46.15)	-	-
T3 + T4 stage	130	83 (63.85)	-	-
N stage	-	-	5.760	0.016
N0 stage	88	48 (54.55)	-	-
Nx stage	55	41 (74.55)	-	-
M stage	-	-	7.612	0.005
M0 stage	127	74 (58.27)	-	-
M1 stage	16	15 (93.75)	-	-
TNM stage	-	-	7.706	0.005
I + II	85	45 (52.94)	-	-
III + IV	58	44 (75.86)	-	-
Serum CEA (μg/L)	-	-	19.270	< 0.001
< 5	67	29 (43.28)	-	-
≥ 5	76	60 (78.95)	-	-

MORC4: MORC family CW-type zinc finger 4; TNM: Tumor-nodes-metastasis; CEA: Carcinoembryonic antigen.

Impact of MORC4 protein expression level on CRC patient prognosis

After 3 years of follow-up, of the 143 CRC patients, 112 survived (78.32%), 26 died (18.18%), and 5 were lost to follow-up (3.50%). The postoperative survival time ranged from 15 to 36 months. Survival analysis showed that the 3-year OS rate of patients with high MORC4 expression in CRC tissue was lower than that of patients with low MORC4 expression (P < 0.05), as shown in Figure 1.

Figure 1 Impact of MORC family CW-type zinc finger 4 protein expression level on colorectal cancer patient prognosis. MORC4: MORC family CW-type zinc finger 4.

DISCUSSION

In recent years, the incidence and mortality rates of CRC have been rising globally, especially in countries and regions with rapid economic development, where changes in the environment and lifestyle have significantly increased the risk of the disease[11]. The etiological factors of CRC are highly complex and are usually the result of the combined effects of genetic susceptibility and environmental factors[12]. Recent studies have found that behavioral factors such as unhealthy diets, lack of exercise, obesity, and smoking are closely associated with the occurrence of CRC[13]. However, despite advances in medical technology improving the early detection and treatment of CRC, many patients are still diagnosed at an advanced stage, resulting in a poor prognosis[14]. Therefore, actively exploring biomarkers related to CRC prognosis and using them for early diagnosis and personalized treatment is of great clinical value in improving the survival rate of CRC patients.

The MORC protein family is a highly conserved superfamily of nuclear proteins, with its members broadly involved in critical biological processes such as gene expression regulation and DNA damage repair[15]. Members of the MORC protein family typically contain three characteristic domains: First, the N-terminal MutL-type ATPase domain, which plays a key role in DNA repair and gene transcription regulation[16]; second, the CW-type zinc finger domain, which participates in chromatin regulation by recognizing lysine 4 methylation modification of histone 3[17]; and finally, the C-terminal coiled-coil domain, which mainly regulates cell functions through protein-protein interactions and subcellular localization[18]. Additionally, as a member of this family, the MORC4 protein contains four potential small ubiquitin-related modifier binding sites, allowing it to bind to the C-terminal binding protein corepressor, thereby regulating gene expression suppression processes[19]. In recent years, MORC4 has gradually attracted attention in cancer research. Studies have found that this protein plays a crucial role in oncogenesis, participating in various biological behaviors of tumor cells, such as proliferation, migration, invasion, and apoptosis[20]. Other studies have shown that MORC4 exhibits abnormally high expression in several malignancies, particularly in breast cancer, and its high expression is closely associated with tumor malignancy[21]. Knockdown of MORC4 expression can significantly inhibit the growth of breast cancer cells and promote apoptosis, thereby slowing disease progression.

In this study, through the analysis of tissue samples from 143 CRC patients, we found that MORC4 expression levels in CRC tissues were significantly higher than in adjacent normal tissues, indicating that MORC4 may play an important oncogenic role in the development of CRC. Further analysis revealed that MORC4 expression was closely associated with several clinicopathological characteristics. There was no significant difference in the high expression rate of MORC4 between different patients based on gender, age, tumor location, tumor size, or T stage (P > 0.05), suggesting that these factors may not be directly related to MORC4 expression levels. However, high expression of MORC4 was closely associated with tumor differentiation degree, lymph node metastasis, distant metastasis, TNM stage, and serum CEA levels, with statistically significant differences (P < 0.05). These results suggest that high MORC4 expression may reflect a higher aggressiveness and metastatic tendency of CRC, and is associated with poorer prognosis. Additionally, the survival analysis results further support the prognostic value of MORC4. In this study, the OS of CRC patients with high MORC4 expression was significantly lower than that of patients with low expression (P < 0.05), indicating that MORC4 may not only be an oncogenic factor in CRC development but also an important biomarker for assessing patient prognosis. The high expression of MORC4 appears to accelerate the malignant progression of CRC, leading to earlier recurrence and metastasis, thus shortening survival time. Similarities with studies in other types of cancer[22,23] suggest that MORC4 may enhance tumor malignancy by affecting tumor cell survival and apoptotic pathways, promoting cancer cell resistance to treatment and anti-apoptotic properties.

CONCLUSION

This study, through the analysis of tissue samples from 143 CRC patients, has for the first time clearly identified the significant overexpression of MORC4 in CRC tissues and elucidated its correlation with clinicopathological characteristics and prognosis. It was found that MORC4 expression in CRC tissues is significantly higher than in adjacent normal tissues, suggesting that it may serve as a potential oncogene in the occurrence and development of CRC. Further analysis showed that high MORC4 expression is significantly associated with tumor differentiation, lymph node metastasis, distant metastasis, TNM stage, and serum CEA levels, indicating that MORC4 may play an important role in regulating tumor invasiveness and metastatic capacity. Survival analysis revealed that the OS of patients with high MORC4 expression was significantly lower than that of patients with low expression, suggesting that high MORC4 expression is closely related to poor prognosis and has significant clinical potential. According to the results of this study, MORC4 may not only play an important role in the diagnosis of CRC but also provide reliable evidence for prognosis evaluation. Moreover, the results of this study provide a theoretical basis for future development of therapies targeting MORC4. MORC4 has the potential to become a new therapeutic target, and inhibiting its expression or blocking its function may offer new hope for CRC treatment. Overall, the findings of this study broaden our understanding of the molecular mechanisms of CRC, especially the function and clinical value of MORC4 in CRC, providing important references for future cancer diagnosis, prognosis evaluation, and targeted therapy. It should be noted that despite the progress made in exploring the relationship between MORC4 and the clinical characteristics and prognosis of CRC, there are still some unavoidable limitations. The following points outline the main shortcomings: (1) Small sample size and regional limitations: This study involved only 143 patients, with a relatively limited sample size, and the cases were sourced from a single region, which may limit the generalizability of the findings to a global population or other cohorts. Future studies should expand the sample size and cover more regions and ethnic backgrounds to improve the general applicability of the results; (2) Insufficient in-depth molecular mechanism research: Although this study revealed the high expression of MORC4 in CRC and its correlation with clinical characteristics, it did not delve into the specific molecular mechanisms by which MORC4 affects CRC. Future studies should combine cell experiments and animal models to analyze in detail the specific roles of MORC4 in signal transduction, gene expression regulation, cell cycle, proliferation, and invasion/metastasis to better understand its function as an oncogene; (3) Lack of prospective research and long-term follow-up data: This is a retrospective study that relies on existing medical records, which may introduce selection bias. Additionally, due to the limited follow-up period, the long-term impact of high MORC4 expression on survival rates was not fully observed. Future studies should be designed prospectively and extend follow-up times to comprehensively assess the impact of MORC4 on the long-term prognosis of CRC patients; (4) Insufficient consideration of other confounding factors: This study did not thoroughly evaluate the potential impact of different treatment methods (e.g., surgery, radiotherapy, chemotherapy) on MORC4 expression, which may limit understanding of MORC4’s role in different treatment contexts. Future studies should include different treatment factors in the analysis to explore the interaction between treatment approaches and MORC4 expression; (5) Limitations of using a single biomarker: Although the high expression of MORC4 and its potential diagnostic and prognostic value in CRC were validated, relying on a single biomarker for cancer diagnosis or prognosis assessment remains limited. Future studies should combine multiple molecular markers (e.g., CEA, carbohydrate antigen 19-9) and use multi-factor models to further enhance diagnostic accuracy and prognostic assessment capabilities in CRC; (6) Lack of comparison with other cancer types: This study focused only on MORC4 expression and function in CRC but did not explore its performance and functional differences in other cancers. MORC4 has been reported to be overexpressed in various malignancies, including breast cancer and gastric cancer. Future studies could conduct comparative analyses of MORC4’s role in different types of tumors to determine whether it has a universal oncogenic function or plays a crucial role only in specific cancers; and (7) Further exploration of therapeutic targeting: Although this study proposed that MORC4 could be a potential therapeutic target for CRC, no experimental validation of MORC4 inhibitors has yet been conducted. Future research should focus on the development of MORC4 inhibitors and their preclinical evaluation. In vitro studies could utilize established CRC cell lines to assess effects on cell proliferation, apoptosis, and invasion, while in vivo studies could employ xenograft models to determine therapeutic efficacy and potential toxicity. These studies would not only validate the feasibility of targeting MORC4 but also bridge the gap between basic research and clinical application, offering promising avenues for translational cancer research. In conclusion, while this study reveals the critical role of MORC4 in CRC, its clinical utility in diagnosis, prognosis, and treatment requires further validation through larger-scale and more comprehensive investigations.