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ORIGINAL ARTICLE |
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| Ahead of print
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| The expression of squalene epoxidase in human gastric cancer and its clinical significance |
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Yong-Chao Ma1, Shao-Ju Jin2, Guo-Jian Gu3, Li-Fang Zhao4, Song-Tao Xu5
1 Department of Basic Medicine, Luohe Medical College; Tumor Occurrence and Prevention Research Innovation Team of Henan, Luohe, Henan, PR China
2 Department of Pharmacology, Luohe Medical College; Tumor Occurrence and Prevention Research Innovation Team of Henan, Luohe, Henan, PR Chinax
3 Department of Pathology, Taicang Affiliated Hospital of Soochow University; Department of Pathology, The First People's Hospital of Taicang, Taicang, Jiangsu, PR China
4 Department of Basic Medicine, Luohe Medical College, Luohe, Henan, PR China
5 Tumor Occurrence and Prevention Research Innovation Team of Henan; Clinical, Luohe Medical College; Henan Province Engineering and Technology Research center of Foods for Special Medical Purpose, Luohe, Henan, PR China
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| Date of Submission | 05-Dec-2021 |
| Date of Decision | 19-Jun-2022 |
| Date of Acceptance | 20-Jun-2022 |
| Date of Web Publication | 03-Feb-2023 |
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 Abstract | | |
Context: Squalene epoxidase (SQLE) is overexpressed in a variety of tumors, which may play an important role in their tumorigenesis, development, and prognosis. Aims: The aim of this study is to investigate the expression of SQLE and explore its clinicopathological significance in gastric cancer. Settings and Design: The correlation between its positive expression and the pathological characteristics of patients (such as sex, age, tumor size, survival, tumor differentiation, TNM staging, and lymph node metastasis) was analyzed. Methods and Material: Immunohistochemical method was used to detect its expression in 107 cases of gastric carcinoma and 34 cases of tumor-adjacent tissues. Statistical Analysis Used: Counting data were analyzed by Chi-square test. Its overall survival was analyzed by Kaplan-Meier method and log-rank test. Its hazard factors were analyzed by Cox multivariate analysis. Results: The positive rate of SQLE in gastric cancer is 67.3%, which is higher than that in tumor-adjacent tissues (17.6%), <0.001. Expression of SQLE is closely related to tumor differentiation, TNM staging and lymph node metastasis (P = 0.030, P = 0.009, and P = 0.011, respectively). Furthermore, compared with those low expression of SQLE, the patients of overexpression had worse overall survival by Kaplan–Meier analysis (P = 0.025). Cox multivariate analysis shows that lymph node metastasis, tumor differentiation, SQLE, and TNM staging are independent factors for prognosis of gastric cancer (P = 0.003, 0.020, 0.018, and P = 0.001 respectively). Conclusions: SQLE is overexpressed in gastric cancer. It could be used for the diagnosis and prognosis of the gastric cancer patients.
Keywords: Gastric cancer, immunohistochemistry, protein, SQLE, survival
How to cite this URL:
Ma YC, Jin SJ, Gu GJ, Zhao LF, Xu ST. The expression of squalene epoxidase in human gastric cancer and its clinical significance. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 May 29]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=369082 |
| Introduction | |  |
The gastric cancer is one of the most common gastrointestinal malignancies with poor prognosis, and among all the cancers, its mortality ranks third.[1] 90% of the gastric cancer is adenocarcinoma, which originates from the mucosa, or the glands of the most superficial layer.[2] Nowadays, with the development of science and technology, the level of diagnosis, and treatment of the gastric cancer has made great progress. However, the mortality is still high. It was reported that in 2012, there were 9.51 million new cases in the world, including 723000 deaths.[1] The first choice to treat it is to have surgery, but it is always found in its middle or late stage, because its early symptoms are not obvious, so the prognosis is poor. The 5-year survival rate is about 29%.[3] Therefore, in addition to surgery, other adjuvant treatments are needed, such as chemotherapy, radiotherapy, and targeted therapy. To improve the survival rate of the patients with gastric cancer, at present, targeted therapy is used more frequently in their treatment. So it has a certain clinical practical value to find and study the specific targets for the diagnosis and treatment of the gastric cancer.
Cholesterol is not only an important part of cell membrane, but also widely involved in steroid hormone formation and cell signal transduction.[4] Studies have found that abnormal cholesterol metabolism exists widely in various carcinogenic processes, which speeds up the development of cancer.[5] The human SQLE gene, located on chromosome 8q24.13, is located on the endoplasmic reticulum, or on lipid droplets.[6],[7],[8],[9] Studies have found that it is overexpressed in a variety of tumors, such as lymphomas, prostate carcinoma and lung cancer, and its overexpression leads to a poor prognosis.[8],[10],[11]
However, there are few clinical studies in this field. Therefore, this study comprehensively analyzed its expression in 107 gastric cancer cases, and predicted its clinicopathological significance.
| Subjects and Methods | | |
Patients
The subjects were 107 patients who underwent surgery between January 2, 2016 and January 2, 2021. And 34 cases of tumor-adjacent tissues (>5 cm from tumor margin) were taken from the control group. All the patients were treated by surgical operation for the first time. No radiotherapy or chemotherapy was employed before the operation. The postoperative pathology was gastric adenocarcinoma. This research was approved by the Ethics Committee of the Taicang Affiliated Hospital of Soochow University. All the tissue samples were informed consent of the patients. Among the 107 gastric cancer patients, there were 66 males and 41 females aged from 47 to 80 years. According to histopathological types, there were well (36 cases) and moderately poorly (71 cases). They were divided into I/II (57 cases) and III/IV (50 cases). All the patient data were shown in [Table 1] and [Table 2]. The patients would undergo a CT scan every six months after the surgery, and were followed up for 3 to 60 months by telephone. | Table 1: Expressions of SQLE in human gastric cancer and tumor-adjacent tissue (n=141)
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 | Table 2: Analysis of SQLE expression and related factors in human gastric cancer tissue (n=107)
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Streptavidin peroxidase (SP) immunohistochemical analysis
Immunohistochemical staining was performed according to Xu's literature.[12] In short, paraffin specimens were sliced, dewaxed, and hydrated (3% H2O2 for 10 min). The specimens were immersed in citric acid buffer (Ph 6.0) and heated in microwave oven for 15-20 min for antigen repair. Added the goat serum to seal the antigen for 20 min, then added rabbit polyclonal anti-human-SQLE (1:120; Abcam Trading Co., Ltd, Shanghai, China), and kept it at 4°C overnight. The next day, specimens were washed with phosphate buffer saline (PBS), then added secondary antibody (Beyotime Biotechnology; Shanghai, China) for 60 min. After that they were washed with PBS, followed by further incubation with streptavidin peroxidase (Beyotime Biotechnology; Shanghai, China) for 30 min. They were washed with PBS. 100 μL diaminobenzidine (Beyotime Biotechnology; Shanghai, China) which was used for color rendering. Then they were washed with water to stop the reaction 10 min later. Then we counterstain it with hematoxylin for 2 min, and then washed it with water for 10-15 min. Alcohol was used to dehydrate and xylene was used to transparently process.
Evaluation of SQLE staining
Evaluation of SQL staining was given according to the staining intensity and the percentage of stained tumor cells on the section. Low expression (-, negative) included: (a) the cells were not positive stained. (b) The cells were stained weakly positive (light yellow) or moderately positive (yellow brown) and their percentage <30%. Overexpression (+, positive) included: (a) The cells were stained medium intensity positive (yellow brown) and their percentage ≥30%. (b) The cells were stained strongly positive (brown) and their percentage <30%. In this study, the immunohistochemical results were interpreted by two senior pathologists using a double-blind method.
Statistical analysis
Statistical Product and Service Solutions (SPSS) 23.0 and GraphPad Prism version 6.0 were used for the statistical analyzes of clinical samples. [Table 1] and [Table 2] were analyzed by χ2 test. The data in [Figure 2] were analyzed by Kaplan–Meier method and log-rank tests. The data in [Table 3] were analyzed by Cox proportional hazard model. When P < 0.05, it was considered that the difference was statistically significant. | Table 3: Cox analyzes of different clinicopathologic variables and SQLE expression in gastric cancer tissues (n=107)
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 | Figure 2: Kaplan–Meier survival curves of gastric cancer patients according to SQLE expression
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| Results | | |
The relationship of SQLE expression and clinicopathological parameters
The positive cell of SQLE was the one whose cytoplasm was stained with yellow or brown color [Figure 1]a, and the negative was the one whose cell was not stained [Figure 1]b. Its expression and clinicopathological features of gastric cancer were shown in [Table 1] and [Table 2], the positive rate of SQLE in gastric cancer was 67.3%, which was higher than that in tumor-adjacent tissues (17.6%), <0.001. Expression of SQLE was closely related to tumor differentiation, TNM staging, and lymph node metastasis (P = 0.030, P = 0.009 and P = 0.011, respectively). However, its expression was not associated with sex, age, and tumor size (P = 0.273, P = 0.341, P = 0.671, respectively). | Figure 1: Brown granules in the cytoplasm of gastric cancer cells were the positive expression (200×) (a). SQLE was not expressed in the cytoplasm of gastric cancer tissues (200×) (b)
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Survival analysis
Kaplan–Meier survival curves of gastric cancer patients were based on SQLE expression [Figure 2]. Compared with those low expression of SQLE, the patients with its overexpression had worse overall survival according to Kaplan–Meier analysis (P = 0.025).
Cox multivariate analysis
Cox multivariate analysis showed that lymph node metastasis, tumor differentiation, SQLE, and TNM staging were independent factors for the prognosis of gastric cancer (P = 0.003, 0.020, 0.018, and P = 0.001 respectively) [Table 3].
| Discussion | | |
SQLE is the second rate limiting enzyme in cholesterol synthesis pathway, and it converts squalene to 2,3(S)-oxidosqualene which can synthesize sterol and cholesterol.[7] Its abnormal metabolism will cause abnormal cholesterol metabolism, leading to the accumulation of a large number of metabolites, changing the tumor microenvironment, and resulting in the proliferation and invasion of tumor cells.[13] In lung squamous cell carcinoma, its expression is related to pathological differentiation, TNM staging and lymph node metastasis [Table 4].[11] Its overexpression is closely related to the occurrence, development and prognosis of lung squamous cell carcinoma. It can be regarded as a prognostic marker of lung squamous cell carcinoma.[11],[14] Garcia-Bermudez et al.[8] found SQLE was overexpression in lymphoma cells, and that patients with its overexpression would have poor prognosis. Other scholars have also found that its overexpression in other tumor tissues (lung carcinoma, colorectal cancer, hepatocellular carcinoma), which are negatively correlated to the prognosis of patients.[14],[15],[16] In all, these findings suggest that abnormal expression of SQLE may play an important role in their tumorigenesis, development, and prognosis. | Table 4: Analysis of SQLE expression and related factors in human lung squamous cell carcinoma tissue (n=65)
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Our results showed that the positive expression rate of SQLE in gastric cancer was significantly higher than that in the adjacent gastric cancer, and the positive expression rate was related to tumor differentiation, TNM staging and lymph node metastasis. Our findings were consistent with that of Zhang HY [Table 2] and [Table 4].[11] It might be involved in the occurrence, development, and promoting the metastasis of gastric cancer.
Recent studies had shown that the mechanism of abnormal metabolism of SQLE leading to tumor (including gastric cancer) proliferation and invasion might also be closely related to the process of signal transduction. First, Liang JQ found that it could promote tumorigenesis by affecting the signaling pathways regulated by calcium.[17] Secondly, in terms of the molecular, SQLE regulated protein kinases (ERK) signaling pathway and promoted ERK-phosphorylation, which in turn promoted the promotion and metastasis of carcinoma cells.[18] Thirdly, some scholars have also found that its abnormal expression was related to epithelial-to-mesenchymal transition (EMT), which played a very important role in a variety of tumor metastasis.[19]
Compared with the patients with high expression, the patients with low expression of SQLE in cancer had better prognosis.[15],[16] Our survival curve showed that the survival time of SQLE negative patients was better than that of the positive patients. Through our further study, Cox multivariate analysis also showed that SQLE overexpression was associated with an increased hazard ratio compared with the patients with low expression, and was an independent prognostic marker for gastric cancer. This finding suggested it might be a potential prognostic marker for gastric cancer.
With the development of biotechnology and the further research on the pathogenesis of cancer, at present, the targeted therapy for SQLE in the field of cancer treatment had drawn more and more attention. Some therapeutic intervention is being employed during the treatment. Ge H silenced SQLE by RNA interference and found that the ability of lung carcinoma cells to proliferate and invade was significantly reduced, while overexpression of SQLE showed the opposite.[18] Sui Z found that SQLE was overexpression in the hepatocellular carcinoma. He silenced SQLE by RNA interference and found that the proliferation and migration ability of tumor cells was significantly inhibited.[20] Terbinafinet, SQLE inhibitor was added to breast cancer to reduce the tumorigenicity of breast cancer cell.[21] Combined with the other research studies, SQLE gene may become a new target of gastric cancer gene or drug therapy.
However, the study had its limitations. First, the number of the sample was not large enough, with only 107 patients. Secondly, the detection method was relatively simple. In the future, we intend to expand the number of the patients and perfect the methods of detecting upregulated SQLE, such as mRNA and next generation sequencing (NGS).[11],[22] SQLE is the second rate limiting enzyme in cholesterol synthesis pathway.[7] We assume that molecular basis of SQLE in gastric cancer is to affect cholesterol metabolism, thus affecting the change of tumor microenvironment, or affecting the signal transduction process related to tumor, finally leading to the tumorigenesis.[13],[17],[18],[19] In the future, we can silence SQLE by small interfering RNA (siRNA) or microRNA (miRNA) and detect the changes of related factors in the signal pathway, then find its specific mechanism.
In conclusion, our study found the overexpression of SQLE in gastric cancer by immunohistochemical method, and further confirmed this finding by clinical sample data. It suggested that SQLE might be a potential cancer-promoting gene in gastric cancer, and it could be used as a biomarker for the diagnosis and prognosis in gastric cancer, but its specific mechanism needed further studying.
Acknowledgments
This research was supported by (a) capacity of innovation, entrepreneurship, and improving project in 2021 at Luohe Medical College in Henan Province P. R. China. (2021-LYZTDXM006), (b) capacity of innovation, entrepreneurship, and improving project in 2020 at Luohe Medical College in Henan P. R. China. (2020-LYZKYZD006), and (c) the science and technology project in Henan P. R. China. (No. 182102310348), and (d) the foundation research program of Taicang, Jiangsu, PR China. (no. TC2021JCYL20).
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This research was supported by (a) capacity of innovation, entrepreneurship and improving project in 2021 at Luohe Medical College in Henan Province P. R. China. (2021-LYZTDXM006), (b) capacity of innovation, entrepreneurship and improving project in 2020 at Luohe Medical College in Henan P. R. China. (2020-LYZKYZD006), and (c) the science and technology project in Henan P. R. China. (No. 222102310443), and (d) the foundation research program of Taicang, Jiangsu, PR China. (no. TC2021JCYL20).
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Guo-Jian Gu,
Department of Pathology, Taicang Affiliated Hospital of Soochow University, 58 South Changsheng Road, Taicang 215400, Jiangsu
PR China
Song-Tao Xu,
Department of Clinical, Luohe Medical College, 148 Daxue Road, Luohe 462002, Henan
PR China
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/ijpm.ijpm_1183_21
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4] |
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