High expression of ECT2 and E2F1 is associated with worse clinical manifestations and prognosis in patients with oral squamous cell carcinoma

Abstract

To detect the expression, prognostic value, and possible effects of epithelial cell transforming sequence 2 (ECT2) and E2F1 in patients with oral squamous cell carcinoma (OSCC). Seventy subjects hospitalized for OSCC surgical treatment in the Fourth Hospital of Hebei Medical University were selected for this study. Expression levels of ECT2 and E2F1 were detected by qRT-PCR, Western blot, and immunohistochemistry. The Kaplan-Meier method and Cox risk proportional regression analysis were used to analyze the relationship between different expression levels of ECT2 or E2F1 and the survival of patients with OSCC in 3 years. Relative expression levels of E2F1 mRNA (1.31 ± 0.15) and ECT2 mRNA (3.95 ± 0.72) in OSCC tissues, compared to adjacent normal tissues (0.87 ± 0.11, 1.03 ± 0.23, all p < 0.05). ECT2 was highly expressed in 42 (60.00%) OSCC samples and E2F1 was highly expressed in 45 (64.29%) samples. The expression of ECT2 and E2F1 was related to clinical stage, lymphatic metastasis, tumor differentiation grade, and tumor diameter in OSCC patients. The higher the expression of ECT2 and E2F1, the lower the 3-year survival rate of patients. ECT2 high expression (HR=2.407, p < 0.001), E2F1 high expression of E2F1 (HR = 2.159, p = 0.013), Clinical stages (III+IV) (HR = 1.362, p = 0.012), medium and low differentiation (HR = 1.522, p = 0.015), lymphatic metastasis (HR = 1.951, p < 0.001), and tumor diameter (≥3 cm) (HR = 1.824, p = 0.002) could be independent factors for 3-year survival of patients with OSCC. The expression of ECT2 and E2F1 in OSCC was significantly up-regulated, which was closely related to clinical stage, lymph node metastasis, tumor size, and 3-year survival of OSCC patients.

Keywords

oral squamous cell carcinoma cell cycle epithelial cell transforming sequence E2F transcription factor prognostic

Introduction

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of head and neck squamous cell carcinoma, accounting for approximately 90% of oral malignancies and 3.5% of systemic tumors.^1,2 Its malignancy is high, locally invasive, prone to early and extensive lymph node metastases, and the 5-year survival rate is still about 50%.^3,4 Therefore, targeting early prevention, exact diagnosis, and effective treatment of OSCC remains an important challenge.

The cell cycle is a fundamental process in the life activities of cells. Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide dissociation exchange factor (Rho GEF). ECT2 can promote GDP dissociation in the bound state, as well as GTP to GDP replacement, thereby activating the Ras analog GTPase (Rho GTPase) of cellular signal transduction pathways. It is this process that influences aspects of malignant transformation, tumor cell growth, invasion, and metastasis.^5–9 Iyoda et al.¹⁰ demonstrated that ECT2 up-regulated in OSCC in vitro and in vivo, which can be an indicator of cellular proliferation.

Cell cycle-related transcription factor E2F transcription factor 1 (E2F1) is the most important transcriptional activator of the Rb/E2F pathway, which regulates the progression from phase G₁ to phase S of the cell cycle.^11–13 E2F1 can participate in apoptosis induction in a p53-dependent or independent manner. Yamazaki et al.¹⁴ found that high levels of E2F1 had shorter recurrence free survival and poor prognosis in patients with OSCC.

Currently, the expression profiles of ECT2 and E2F1 in OSCC patients and the relationship with clinical and pathological characteristics remain incompletely defined. Based on this, the present study was conducted to detect the expression of ECT2 and E2F1 in tissues of OSCC and adjacent tissues of normal tissues, and we also explored the correlation between the expression levels of these two genes. Therefore, the current study may provide valuable clues for further research on the treatment and prognosis of OSCC.

Materials and methods

Study design

This was a cohort study conducted in two hospitals. Seventy OSCC hospitalized subjects for surgical treatment in the Fourth Hospital of Hebei Medical University (Hebei Province, China) between January 2017 and December 2018 were selected for this study. Subjects' inclusion criteria were as follows: (1) surgical treatment was performed for the first time, which required complete clinical data; (2) OSCC confirmed by histopathology; (3) no history of radiation, chemical, and biological therapy for OSCC prior to operation. The subject’s exclusion criteria were as follows: (1) Comorbid other malignancies; (2) Women who were pregnant or lactating; (3) Patients with other malignancies, severe liver and kidney dysfunction, systemic infections, immune system disorders, psychiatric disorders, and severe metabolic disease were included. This study was in accordance with the basic requirements of the Declaration of Helsinki and was approved by the ethics committee of our hospital before the start of the study. All patients signed informed consent before treatment.

Collection and preservation of tissue samples

100 mg of OSCC tissue was collected as a study sample from the surgically resected samples of each patient. And 50–100 mg of adjacent normal tissues that were histomorphometrically confirmed from more than 10 mm outside the tumor border were obtained as the control sample. All samples were snap frozen in liquid nitrogen and stored at −80°C.

Collection of clinical data

Clinical data from OSCC patients were collected including age, gender, tumor diameter, lymphatic metastasis, clinical stage, degree of tumor differentiation, and therapeutic method. Survival within 3 years of the patients was also collected.

Quantitative real-time polymerase chain reaction

The expression levels of ECT2 and E2F1 mRNA expression levels in OSCC and adjacent normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, each sample was collected at 50–100 mg in a homogenizer and ground into a powder with liquid nitrogen. Total RNA was extracted using Trizol reagent (GMS12279, Shanghai Jimei Gene Pharmaceutical Technology Co., Ltd., China). The ECT2 and E2F1 expression levels in OSCC and adjacent normal tissues were determined by qRT-PCR with an ABI Biosystems 7500 (Carlsbad, CA, USA). The PCR reactions were performed in a final volume of 20 μL of a reaction mixture consisting of 2 μL of FirstStart DNA Master SYBR Green I mix, 3 mM MgCl2, and l μM of the primers. The internal reference gene was GAPDH. PCR reaction conditions: polymerase activation: 95°C, 30 s; PCR amplification (40 cycles): 95°C, 5 s; 60°C, 30 s; Dissolution: 68°C, 30 s. Data were analyzed using log2 –Δct. Each sample was tested 3 times and the average value was calculated. The primers used for qRT-PCR were designed and synthesized by BGI Science and Technology Co., Ltd., China, and the primer sequences are shown in Table 1.

Table 1.

Primer sequences for genes used in this study.

Gene	Primer sequences
E2F1
Forward	5′- GCCACCATGGCCTTGGCCGG-3′
Reverse	5′-AAGCCCTGTCAGAAATCCAG-3′
ECT2
Forward	5′-CGGCTCAGTGGAGGGAAG-3′
Reverse	5′-GGAAGGCTGACAAGGGAGG-3′
GAPDH
Forward	5′-GAAGGTGAAGGTCGGAGTC-3′
Reverse	5′-GAAGATGGTGATGGGATTTC-3′

Western blot

The expression of ECT2 and E2F1 protein was detected by Western blot. First, 40 mg of OSSC and adjacent normal tissue were taken respectively, and protein was extracted by electric homogenization after RIPA lysate (Beyotime Biotechnology, Shanghai, China) was added, and 20 μg protein was added to each lane, and then electrophoresis in 8% SDS-PAGE for 1 h. After electrophoresis, the separated protein bands were transferred to the PVDF membrane (Thermo Fisher Scientific, MA, US); Block with the blocking solution containing 5% skimmed milk powder for 1 h, and add anti-ECT2 antibodies (ab236502, Abcam Cambridge, MA, USA), anti-E2F1 antibodies (ab288369, Abcam Cambridge, MA, USA) and β-Actin antibody (ab8226, Abcam Cambridge, MA, USA) respectively. After reacting overnight at 4°C, wash the film with PBST (Beyotime Biotechnology, Shanghai, China) for 3 times, add horseradish peroxidase-labeled Sheep anti-mouse IgG, and react at room temperature for 1 h. After washing the film with PBST 3 times, expose it in the darkroom with ECL luminous solution. Take the target protein band and internal reference β. The ratio of actin band gray value indicates the relative expression level of the target protein. The above experiment was repeated three times. Signal intensities of protein expression were quantitated using the Image J software.

Immunohistochemistry

The expression levels of ECT2 and E2F1 expression levels in OSCC tissues were observed by immunohistochemistry. Tissue sections were placed in xylene for 10 min and immersed again for 10 min after changing xylene. After that, they successively went through absolute ethanol, 95% ethanol, and 75% ethanol for 5 min each. Then it was further incubated with 0.3% hydrogen peroxide for 15 min, microwave-repaired for 15 min, and cooled naturally. After blocking with 10% bovine serum albumin for 30 min, ECT2 antibody (1: 200, ab236502, Abcam, Cambridge, UK) and E2F1 antibody (1:100, ab4070, Abcam, Cambridge, UK) were added. Overnight at 4°C and incubated with horseradish peroxidase-labeled secondary antibodies for 30 min. Finally, hematoxylin counterstaining and neutral gum mounting slides.

Positive immunohistochemical staining of ECT2 showed brown, yellow, or tan in the nucleus with faint expression in some of the cell cytoplasm. On the contrary, the E2F1 protein appears brown yellow or brown yellow in the nucleus.

Ten high-power fields were randomly observed under a light microscope, and the percentage of positive cells and the intensity of combined coloration in 1000 tumor cells were recorded. The corresponding values for the percentage of positive cells were as follows: < 5% was calculated as 0 points, 2–55% as 1 point, 25–50% as 2 points, and >50% as 4 points. The values corresponding to the intensity of binding coloration were as follows: 0 points for none, 1 point for weak (pale yellow), 2 points for moderate (tan), and 3 points for strong staining (brown nuclei). Then, the corresponding value of the intensity of staining multiplied by the corresponding value of the percentage of positive cells was taken as a score. Low expression was defined when the score was <4 points, while high expression was defined when the score was ≥4 points.

Statistical analysis

The enumeration data was expressed as the number and percentage of cases, and the Chi-square test was used for comparison. All measurement data were described by mean ± standard deviation and comparisons between groups were made using the Student’s t test. The correlation between the relative expression levels of E2F1 and ECT2 mRNA in OSCC and adjacent normal tissues was analyzed by Pearson correlation. The Kaplan-Meier method and Cox hazard proportional regression analysis were used to analyze the relationship between different expression levels of ECT2 or E2F1 and the survival of patients with OSCC in 3 years. Statistical analysis was performed using Statistical Product and Service Solutions software (24.0, Chicago, USA). Differences were considered statistically significant when the p value was < 0.05.

Results

Clinical data of OSCC patients

Seventy patients with OSCC who ultimately met the requirements were included, and a total of 70 pairs of tumor tissues and adjacent normal tissues were obtained. The total of subjects included 48 males and 22 females. The age range was 29–79 years with a mean age of 61.37 years. The degree of tumor differentiation was as follows: well-differentiated in 19 cases, moderately differentiated in 48 cases, and poorly differentiated in 3 cases. Thirty-four cases were positive for lymph node metastasis and 36 cases were negative. The clinical stages of the patients were as follows: 11 cases in stage I, 18 cases in stage II, 24 cases in stage III, and 17 cases in stage IV. Site of onset classification: 30 cases of tongue cancer, 6 cases of lip cancer, 14 cases of gingival cancer, 4 cases of palatal cancer, 5 cases of buccal cancer, 3 cases of oral floor cancer, 6 cases of jaw squamous cell carcinoma, 1 case of oropharyngeal cancer and 1 case of maxillary sinus cancer.

Relative expression levels of E2F1mRNA (1.31 ± 0.15) and ECT2 mRNA (3.95 ± 0.72) in OSCC tissues, compared to adjacent normal tissues (0.87 ± 0.11, 1.03 ± 0.23), increased significantly (all p < 0.05), see Figure 1(a) and (b). The results of correlation analysis found that there was a positive correlation between Ect2 and E2F1 expression in OSCC tissues, while no obvious correlation was found in the adjacent normal tissues (Figure 1c and d). The relative expression levels of E2F1 and ECT2 proteins in OSCC tissues were all significantly increased compared with those in adjacent normal tissues (Figure 1e and f).

Figure 1.

Relationship between the level of ECT2 and E2F1 mRNA expression in OSCC and adjacent normal tissues. (a) The level of ECT2 mRNA expression in OSCC tissues was higher than in adjacent normal tissues (p < 0.05). (b) The level of E2F1 mRNA expression in OSCC tissues was higher than in adjacent normal tissues (p < 0.05). (c) Scatter plots and linear regression fit lines showing the correlation between ECT2 and E2F1 mRNA expression in OSCC tissues (r = 0.499, p = 0.001). (d) Scatter plots and linear regression fit lines showing the correlation between ECT2 and E2F1 mRNA expression in adjacent normal tissues (r = 0.374, p = 0.001). The levels of E2F1 (e) and ECT2 (f) expression in OSCC tissues and adjacent normal tissues were detected by Western blot assay and representative bands were shown. The level of E2F1 (e) and ECT2 (f) protein expression in OSCC tissues was higher than in adjacent normal tissues (p < 0.05).

Expression of the ECT2 and E2F1 proteins in OSCC tissues and their correlation with clinical and pathological characteristics

Investigate the relationship between the changes in ECT2 and E2F1 protein levels in OSCC tissues and clinical and pathological characteristics. ECT2 and E2F1 expression were determined by immunohistochemistry in 70 tissues from OSCC patients. The representative images of immunohistochemistry for ECT2 and E2F1 was shown in Figure 2.

Figure 2.

The representative images of immunohistochemistry for 111 and E2F1 genes in OSCC samples and adjacent normal tissues (×400, Scale bars, 10 μm). (a) Negative expression of ECT2 protein in adjacent normal tissues. (b) Positive immunoreaction for ECT2 was detected in the nucleus and cytoplasm in OSCC tissues. (c) Low expression of E2F1 protein expression in adjacent normal tissues. (d) High expression of E2F1 protein expression in OSCC tissues. (e) The mean score of IHC results of ECT2 and ECT2 protein in OSCC tissues was higher than in adjacent normal tissues (p < 0.05).

ECT2 was highly expressed in 42 OSCC samples, while the remaining 28 samples showed low expression. Analysis of the relationship between ECT2 and clinical and pathological characteristics of OSCC patients, the results showed that high expression of ECT2 was closely related to the clinical stage (p = 0.029), lymphatic metastasis (p = 0.025), tumor differentiation grade (p = 0.048) and tumor diameter (p = 0.004), while there was no significant correlation with the gender, age, treatment strategy of the patients (all p > 0.05), see Table 2.

Table 2.

Relationship between the expression of ECT2 and clinical and pathological characteristics of OSCC patients.

Variables	ECT2 high expression group	ECT2 low expression group	p value
n	42	28
Gender, n (%)
Male	26 (61.90)	21 (75.00)	0.253
Female	16 (38.10)	7 (25.00)	0.253
Age, n (%)
<60 year	26 (61.90)	13 (46.43)	0.202
≥60 year	16 (38.10)	15 (53.57)	0.202
Clinical stages, n (%)
I + II	13 (30.95)	16 (57.14)	0.029
III + IV	29 (69.05)	12 (42.86)	0.029
Degree of differentiation, n (%)
High differentiation	10 (23.81)	13 (46.43)	0.048
Medium and low differentiation	32 (76.19)	15 (53.57)	0.048
Lymphatic metastasis, n (%)
Yes	25 (59.52)	9 (32.14)	0.025
No	17 (40.48)	19 (67.86)	0.025
Tumor diameter, n (%)
<3 cm	24 (57.14)	25 (89.29)	0.004
≥3 cm	18 (42.86)	3 (10.71)	0.004
Therapeutic method, n (%)
Surgery	16 (38.1)	10 (35.71)	0.840
Surgery + comprehensive treatment	26 (61.9)	18 (64.29)	0.840

The expression of E2F1 was high in 45 OSCC tissue sections and low in 25 tissues. E2F1 expression was not correlated with patient gender, age, clinical stage, treatment strategy (all p > 0.05) and was associated with lymphatic metastasis (p < 0.001), tumor differentiation grade (p = 0.011) and tumor diameter (p = 0.014) as shown in Table 3.

Table 3.

Relationship between the expression of E2F1 and clinical and pathological characteristics of OSCC patients.

Variables	E2F1 high expression group	E2F1 low expression group	p value
n	45	25
Gender, n (%)
Male	25 (55.56)	22 (88.00)	0.677
Female	20 (44.44)	3 (12.00)	0.677
Age, n (%)
<60 year	31 (68.89)	8 (32.00)	0.834
≥60 year	24 (53.33)	7 (28.00)	0.834
Clinical stages, n (%)
I + II	16 (35.56)	13 (52.00)	0.181
III + IV	29 (64.44)	12 (48.00)	0.181
Degree of differentiation, n (%)
High differentiation	10 (22.22)	13 (52.00)	0.011
Medium and low differentiation	35 (77.78)	12 (48.00)	0.011
Lymphatic metastasis, n (%)
Yes	29 (64.44)	5 (20.00)	<0.001
No	16 (35.56)	20 (80.00)	<0.001
Tumor diameter, n (%)
<3 cm	27 (60.00)	22 (88)	0.014
≥3 cm	18 (40.00)	3 (12)	0.014
Therapeutic method, n (%)
Surgery	15 (33.33)	11 (44)	0.376
Surgery + comprehensive treatment	30 (66.67)	14 (56)	0.376

Relationship between the expression of the ECT2 and E2F1 protein and the prognosis of patients with OSCC

To study whether the expression of ECT2 or E2F1 protein was related to 3-year survival. The expression of ECT2 and E2F1 and clinical data of OSCC patients were analyzed using the Kaplan-Meier method and the Log-rank test. The results showed that the expression of ECT2 and E2F1 was correlated with the prognosis of OSCC patients. The higher the expression of ECT2 and E2F1, the lower the 3-year survival rate of patients (see Figure 3).

Figure 3.

Kaplan-Meier analysis of the expression of ECT2 and E2F1 and 3-year survival of OSCC patients. (a) The 3-year survival of the ECT2 higher expression group was worse than the ECT2 low expression group (p = 0.025). (b) The 3-year survival of the E2F1 higher expression group was worse than the E2F1 low expression group (p= 0.011).

Subsequently, the relationship between ECT2 and E2F1 and the prognosis of patients with OSCC was analyzed using the Cox risk proportional regression model. Univariate analysis showed that the high expression of ECT2, E2F1 and tumor diameter were closely related to 3-year survival and prognosis in patients with OSCC. Multivariate Cox regression analysis showed that high expression of ECT2 high expression (HR = 2.407, p < 0.001), E2F1 high expression of E2F1 (HR = 2.159, p = 0.013), Clinical stages (III+IV) (HR = 1.362, p = 0.012), medium and low differentiation (HR = 1.522, p = 0.015), lymphatic metastasis (HR = 1.951, p < 0.001), and tumor diameter (≥3 cm) (HR = 1.824, p = 0.002) could be independent factors for 3-year survival of OSCC patients (see Table 4).

Table 4.

Cox hazard proportional regression model analysis of factors affecting 3-year survival of OSSC patients.

Parameters	Univariate			Multivariate
Parameters	HR	95% CI	P	HR	95% CI	P
ECT2 high expression (Yes = 1)	2.615	1.410–4.848	0.001	2.407	1.431–4.046	<0.001
E2F1 high expression (Yes = 1)	2.362	1.289–4.328	0.004	2.159	1.178–3.956	0.013
Gender (Male = 1)	1.246	0.661–2.346	0.678	––	––	––
Age (≥60 year = 1)	1.317	0.835–2.075	0.353	––	––	––
Clinical stages (III + IV = 1)	1.729	0.763–3.915	0.284	1.362	1.070–1.733	0.012
Degree of differentiation (medium and low differentiation = 1)	1.573	1.056–2.341	0.030	1.522	1.086–2.132	0.015
Lymphatic metastasis (Yes = 1)	2.461	2.183–2.773	<0.001	1.951	1.551–2.453	<0.001
Tumor diameter (Yes = 1)	1.946	1.433–2.641	<0.001	1.824	1.225–2.715	0.002
Therapeutic method (surgery + comprehensive treatment = 1)	1.016	0.763–1.352	0.927	––	––	––

Discussion

In the present study, the results showed that the expression levels of the ECT2 and E2F1 genes mRNA in OSCC tissues were significantly higher than those of adjacent normal tissues, and the high expression of the ECT2 and E2F1 protein was closely related to the clinical characteristics of the patients and the 3-year survival. The present study will be of some help in studying the role of ECT2 and E2F1 in the initiation and progression of OSCC.

ECT2 is currently believed to function as an oncogene in many tumors in humans. Zhang et al.¹⁵ verified ECT2 as highly expressed in pancreatic cancer cell lines and pancreatic ductal cell carcinoma tissues by qRT-PCR. Jin et al.¹⁶ found that ECT2 was highly expressed in gastric cancer tissues by immunohistochemistry and qPCR, and ECT2 was closely related to tumor grade, degree of invasion, lymph node metastasis, distant metastasis, as well as TNM stage; It was also found that the higher the amount of ECT2 expression, the worse the prognosis of patients with gastric cancer. Chen et al.⁸ found that ECT2 could interact with racgap1, thus stimulating the Rho/ERK signaling pathway, which in turn initiates hepatocarcinogenesis and metastasis. These results suggest that high expression of ECT2 may play an important role in the development and progression of malignant tumors. The relationship between ECT2 and the biological properties of colorectal cancer has not been reported.

The expression level of ECT2 mRNA in OSCC tissues was significantly higher than in adjacent normal tissues. Immunohistochemistry showed that the positive expression rate of ECT2 was 60.0% (42/70), suggesting that there was a high expression of ECT2 in OSCC. This shows that ECT2 is up-regulated not only at the transcriptional level, but also at the post-transcriptional level. ECT2 plays a role in the occurrence and development of OSCC and is also a potential molecular marker for the diagnosis.

E2F1 is an important member of the E2F family. The E2F family is an important transcription factor family, which plays a regulatory role in cell cycle progression, DNA replication, DNA repair, cell differentiation, proliferation, and apoptosis.^11–13 Sun et al.¹⁷ analyzed the expression of E2F1 in patients with lung cancer and found that E2F1 mRNA and protein levels in lung squamous cell carcinoma and lung adenocarcinoma were higher than those in normal tissues. Wang et al.¹⁸ found that E2F1 can promote the epithelial-mesenchymal transformation of small cell lung cancer by regulating and participating in cancer invasion and metastasis. Alonso et al.¹⁹ found that E2F1 plays a dual role in the oncogene and tumor suppressor gene in glioma. Whether E2F1 plays the role of a tumor suppressor gene or oncogene in the process of tumor formation may be related to the tumor inhibition pathway, the specificity of tissues and organs, the activity of cellular proteins, and experimental conditions. In this study, the expression level of E2F1 in OSCC was significantly higher than that of adjacent normal tissues, suggesting that E2F1 may play a role in OSCC. Similarly, a previous study about 43 OSCC patients have shown that E2F1 unregulated the cell cycle and promotes cell cycle progression.¹⁴

This study further showed that the ECT2 and E2F1 proteins were closely related to the clinical stage of OSCC, lymph node metastasis, and tumor size, suggesting that ECT2 and E2F1 may be involved in tumor progression. Furthermore, this study further confirmed that Ect2 and E2F1 can be used as independent prognostic factors for colorectal cancer. On the one hand, univariate and multivariate analysis showed that ECT2 and E2F1 could independently judge the prognosis of patients with colorectal cancer; On the other hand, the survival curve showed that colorectal cancer patients with high expression of ECT2 and E2F1 had a poor prognosis. It also suggests that ECT2 and E2F1 may be involved in the occurrence and development of OSCC, but the specific mechanism is not clear and needs to be further studied.

There are of course some limitations in the present study. (1)The main point is that we did not include healthy control patients. Only two groups of gene differences between OSCC tissues and adjacent normal tissues were compared. The differences between E2F1 and ECT2 and healthy controls were not analyzed. (2) This was a study of matched pairs of OSCC and adjacent carcinoma tissues, but the sample size of patients was not calculated. This is due to the fact that the standard deviation of expectations regarding the differences between E2F1 and ECT2 in the two groups of tissues was affected by several conditions such as reagents used, OSCC stage, etc.^20,21 Because of this, we did not perform the calculation and justification of the sample size.

Conclusion

ECT2 and E2F1 expression in OSCC was significantly up-regulated, which was closely related to the clinical stage, lymph node metastasis, tumor size, and 3-year survival of OSCC patients. This study suggests that ECT2 and E2F1 play an important role in the occurrence and development of OSCC and are expected to become a new target for the treatment of colorectal cancer.

Footnotes

Author contributions

SP and ZC designed the trial. SP, JW, YC, NH, JZ, and XW have conducted the work and are involved in data collection. SP, JW, and YC analyzed the data. SP and ZC interpreted the data. SP wrote the manuscript. All authors revised the manuscript and contributed significantly to this study.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Youth Science and Technology Project of Hebei Provincial Health Commission (No. 20211145).

Ethics approval and consent to participate

All patients signed informed consent before treatment. The study was obtained permission from the Ethics Committee of the Fourth Hospital of Hebei Medical University (2020Ky283).

ORCID iD

Zifeng Cui

Abbreviations

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