Expression and significance of NDRG2, C-MYC, and KI67 in gastric cancer and adjacent tissues

Expression and significance of NDRG2, C-MYC, and KI67 in gastric cancer and adjacent tissues | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression and significance of NDRG2, C-MYC, and KI67 in gastric cancer and adjacent tissues Shiwei zhang, yilin qu, hongliang ji, pan qin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3966089/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: Expressions of NDRG2, C-MYC, and KI67 in gastric cancer (GC) and adjoining tissues were analyzed for their importance in the occurrence and development of the GC. Materials and Methods: Samples from GC and adjacent tissue of patients admitted to the Department of Surgery,XXX Hospital, China, from February 2018 to June 2021, were analyzed for the expression of NDRG2, C-MYC, and KI67 using immunohistochemistry. The association of NDRG2 and degrees of differentiation in gastric cancer were analyzed. Results: There were 41 GC patients in the study. The expression of NDRG2 was significantly lower ( P <0.05), and the C-MYC and KI67 were higher ( P <0.05) in GC versus adjacent tissues. NDRG2 expression varied significantly ( P <0.05) according to the degree differentiation in GC. The expression of KI67 was positively correlated with C-MYC (rs=0.409, P <0.05). Conclusions: There were significant differences in the expression of NDRG2 (low expression), and C-MYC, and KI67 (high expression) in gastric cancer and adjacent tissues. The degree of cancer differentiation was associated with expression of NDRG2 in GC tissue. NDRG2 expression was higher in para-carcinoma tissues and intestinal metaplasia glands. Gastric cancer NDRG2 C-MYC KI67 Figures Figure 1 Main Points NDRG2 protein is an early molecular event in development of gastric cancer. NDRG2 is a tumor suppressor gene for gastric cancer. Intestinal metaplasia has important link in the process of gastric cancer. NDRG2 expression is associated with degree of differentiation of gastric cancer. NDRG2 expression is helpful for the early diagnosis of gastric cancer. Introduction Gastric cancer (GC) is a common malignancy worldwide and responsible for more than 1 million new cases and an estimated death of 769,000 in 2020 [ 1 ]. In China, GC is a public health concern and 2nd common cause of death due to cancer [ 2 ]. The pathogenesis of GC is complex, involving multiple factors, the signaling pathways, inactivation of tumor suppressor genes, activation of proto-oncogenes, and abnormal expression of genes related to apoptosis, etc.[ 3 ]. The MYC (myelocytomatosis) proto-oncogene on chromosome 8 has a significant role in progression of cell cycle and apoptosis. The MYC family of genes consists of C-MYC, L-MYC, and N-MYC. Cellular MYC (C-MYC) drives tumorigenesis by acting as a transcription factor that binds to numerous genomic loci and regulates the expression of a large number of target genes [ 4 ]. The human N-MYC downstream-regulated gene (NDRG) is a new class of MYC suppressor gene. The NDRG2 to regulate cell proliferation, differentiation, and apoptosis, and has important role in inhibition of tumor and its metastasis [ 5 ]. The KI67 acts as a nuclear antigen in cell proliferation and is expressed in all phases (G1, S, G2, and M) of the cell cycle. The KI67 expression signifies the proliferative activity and malignant degree of tumors [ 6 ]. Recent studies show that C-MYC is a proto-oncogene, KI67 is an indicator of proliferation, and NDRG2 is a tumor suppressor gene involved in inhibition and tumor development s [ 7 – 9 ]. This study was designed to analyze NDRG2, C-MYC, and KI67 expression in GC and adjacent tissues, and their role in development of GC. Materials and Methods Gastric cancer patients with a confirmed histopathological diagnosis who were admitted to the Department of Surgery, XXXHospital o, from February 2018 to June 2021 were included in the study. Patients were excluded if they had undergone pre-operative radiotherapy or chemotherapy, or had other malignancy [ 10 ]. The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration. Written informed consent was obtained from individual participants. The study was approved by the Human Ethics Committee of XXX Hospital, China. An Olympus CX31 microscope was used for histopathological examination. Rabbit anti-human NDRG2 monoclonal antibody was obtained from Abcam (UK), rabbit anti-human C-MYC and KI67 monoclonal antibodies, and sheep anti-rabbit secondary antibodies were obtained from China Fuzhou Maixin Biotechnology. The expressions of NDRG2, C-MYC, and KI67 in GC and adjacent normal tissues were assessed by immunohistochemistry. Samples were dewaxed, first with dimethyl benzene, then with 95% ethanol, 85% ethanol, and 75% ethanol, followed by EDTA antigen retrieval solution. Samples were prepared by incubating with an endogenous catalase inhibitor for 10 minutes, and then incubated in primary antibody solution at 37°C for 1 hour. Further incubation was done for 1 hour in a secondary antibody solution at room temperature. Diaminobenzylamine was used for color development. Slides were then stained with hematoxylin, differentiated with 1% hydrochloric acid, and incubated with ammonia. Slides were dehydrated, and then transparently sealed for observation under a microscope. Phosphate buffer was used instead of the primary antibody for control. NDRG2 was positively expressed when there were brown or brownish-yellow particles present in the cytoplasm and cell membrane, while C-MYC and KI67 were positively expressed when there were brown or brownish-yellow particles present in the nucleus. The percentage of cells with staining and the intensity of staining were combined to calculate scoring intensity. Five fields were randomly selected for each section, and stained cells were counted to standardize the expression, i.e., 0 points for no stained cells in the field, 1 for 50% stained. The intensity of staining of cells was standardized, i.e., 0 unstained, 1 light yellow, 2 yellow, and 3 points for of cells stained brown. The percentage of positive cells and staining intensity were used to assign a score, i.e., < 3 cells were considered negative -, 4–6 weak positive +, 7–9 moderate positive ++, and 10–12 strong positive +++. For expression, - or + a low expression, ( + + and +++ a high expression. SPSS 24.0 statistical software was used for data analysis. The Chi-squared test was used for the comparison of data, and Spearman’s Correlation test was used to find out the relationship between NDRG2, C-MYC, and KI67 expression in gastric cancer tissues. A P value of < 0.05 was considered statistically significant. Results There were 41 GC patients (M 30, F 11), age 62.70 ± 8.11 years (range, 39–79 years). There was a poorly differentiated carcinoma in 25 cases and a well differentiated carcinoma in 16. Out of 41 cases, 30 were adenocarcinomas, 8 were mucinous, and others 3. There was one adenosquamous carcinoma and two gastric carcinoma with lymphoid stroma. Adenosquamous carcinoma was classified as well-differentiated carcinoma and gastric carcinoma with lymphoid stroma as poorly-differentiated carcinoma. Lymph node metastasis was present in 28 cases. Depth of invasion up to mucosa or submucosa was seen in 4 cases and up to muscularis or deeper in 37 cases. NDRG2 was expressed in the cell membrane of GC and adjacent tissues, and in some cases in both the cell membrane and cytoplasm. C-MYC and KI67 were expressed in the nucleus of gastric cancer and the adjacent tissues. In GC tissues, NDRG2 protein expression was high in well differentiated and low in poorly differentiated cancer tissues. In adjacent tissues, NDRG2 protein was highly expressed in intestinal metaplasia glands. In gastric cancer tissues, C-MYC and KI67 proteins were highly expressed in poorly differentiated cancer tissues. In adjacent tissues, C-MYC and KI67 were highly expressed in intestinal metaplasia glands but were expressed low in other glands (Fig. 1 ). NDRG2 was positively expressed in 39.02% (16/41) gastric cancer tissues and 92.68% (38/41) in adjacent tissues, difference was statistically significant, χ²= 26.25, P < 0. 05. C-MYC was expressed in nucleus of gastric cancer and adjacent tissues. C-MYC was positively expressed in 73.17% (30/41) of gastric cancer tissues and in 4.88% (2/41) of adjacent tissues, showing a statistically significant difference, χ²=40.18, P < 0. 05. KI67 was positively expressed in 97.56% (40/41) gastric cancer tissues and 24.39% (10/41) in adjacent tissues, difference was statistically significant, χ²= 46.12, P < 0. 05, (Table 1 ). Table 1. Expression of NDRG2, C-MYC, and KI67 in gastric cancer tissues and adjacent tissues Group Cases NDRG2 expression C-MYC expression KI67 expression Low High Positive Low High Positive Low High Positive Gastric cancer tissue 41 25 16 16 (39.02) 11 30 30 (73.17) 1 40 40 (97.56) Adjacent tissues 41 3 38 38 (92.68) 39 2 2 (4.88) 31 10 10 (24.39) χ 2 26.25 40.18 46.12 Ｐ <0.05 <0.05 <0.05 There was significant association of NDRG2 expression with the degree of differentiation of GC ( P 0.05). There was no significant association between C-MYC and KI67 with gender, age, differentiation degree, cancer stage, and lymph node metastasis in GC ( P > 0.05), (Table 2 ). Table 2 Relationship between NDRG2, C-MYC, and KI67 protein expression and clinicopathological features in gastric cancer Clinical features Case (n) Positive cases of NDRG2 protein number of cases and percentage Positive cases of C-MYC protein number of cases and percentage Positive cases of KI67 protein number of cases and percentage Gender Male 30 10/30 (33.33) 22/30 (73.33) 30/30 (100.00) Female 11 6/11 (54.55) 8/11 (72.33) 10/11 (90.91) Age (years) > 60 27 11/27 (40.74) 22/27 (84.28) 27/27 (100.00) ≤ 60 14 5/14 (35.71) 8/14 (57.14) 13/14 (92.86) Differentiation High, moderate 10 5/10 (50.00) 9/10 (90.00) 10/10 (100.00) Poor 31 11/31 (35.48) 21/31 (67.74) 30/31 (96.77) Lymphatic metastasis No 13 6/13 (46.15) 11/13 (84.61) 13/13 (100.00) Yes 28 10/28 (35.71) 19/28 (67.86) 27/28 (100.00) Staging Early 13 7/13 (58.85) 12/14 (85.71) 14/14 (100.00) Advanced 28 9/28 (32.14) 18/27 (66.67) 26/27 (96.30) Correlation analysis of NDRG2, C-MYC, and KI67 in GC showed that NDRG2 expression had no correlation with C-MYC expression (rs = 0.105, P > 0.05). KI67 expression had no significant correlation with C-MYC expression (rs =-0.021, P > 0.05). The expression of KI67 was positively correlated with C-MYC (rs = 0.409, P < 0.05), (Table 3 ). Table 3 Correlation analysis of NDRG2, C-MYC, and KI67 expression in gastric cancer tissues NDRG2 C-MYC KI67 Spearman rho NDRG2 Coefficient of association 1.000 0.105 -0.021 Sig. (one-sided) 0.257 0.449 N 41 41 41 C-MYC Coefficient of association 0.105 1.000 0.409 ** Sig. (one-sided) 0.257 0.004 N 41 41 41 Ki67 Coefficient of association -0.021 0.409 ** 1.000 Sig.(one-sided) 0.449 0.004 N 41 41 41 ** At a confidence level of 0.01(one-sided), the correlation is significant. Discussion In present study, the immunohistochemistry of NDRG2, C-MYC and KI67 performed on gastric cancer tissues and adjacent tissues of gastric cancer patients. We found that In gastric cancer tissues, NDRG2 protein was highly expressed in well differentiated cancer tissues and expressed at lower rates in poorly differentiated cancer tissues. This shows that NDRG2 is a tumor suppressor gene in gastric cancer, which is consistent with the literature[ 11 ] h showing a lack of NDRG2 expression in GC cell line SNU-620 promotes proliferation of GC cells, and thus NDRG2 is seen as a tumor suppressor gene at the cellular level. Our findings suggest that the abnormal expression of the NDRG2 protein is an early molecular event in the development of GC, and the detection of NDRG2 expression is helpful for the early detection of GC. At present, the mechanism of gastric cancer is still unclear. It is generally believed that development of GC is a multi-gene, multi-factor, and multi-stage dynamic process. The N-MYC gene is a member of the same family as the N-MYC downstream regulated gene (NDRG) family, which consists of four members: NDRG1, NDRG2, NDRG3, and NDRG4 [ 12 ]. NDRG2 differentiation related genes. NDRG2 is a novel gene containing acyl-carrying protein-like structures cloned from normal human brain tissue. It is differentially expressed in tumor tissue and normal tissue. NDRG2 is expressed widely and highly in most of the tissues in embryo and adult, especially in highly differentiated tissues, such as brain, heart, and muscles [ 13 – 16 ]. The degree expression of NDRG2 is correlates positively with the degree of maturation of organ and tissue. NDRG2 is abnormally expressed in many human cancer cells, and studies have shown that NDRG2 over expression inhibits proliferation and induces apoptosis in ovarian cancer cells [ 17 ], of thyroid carcinoma [ 18 ] colorectal cancer [ 19 ]., Studies indicate that NDRG2 is a critical regulator of colorectal cancer differentiation and that NDRG2 induction of colorectal cancer cell differentiation is dependent on the repression of E3 ligase Skp2 activity. Studies on esophageal squamous cell carcinoma [ 20 ] found that NDRG2 over expression suppresses tumor by inhibiting cell migration, invasion, and EMT through inhibition of AKT/XIAP signaling pathway. Downregulation of NDRG2 was observed in tumor compared to normal brain after resection of highgrade meningioma [21]. These findings suggest that NDRG2 is a tumor suppressor gene which is consistent with our findings. The development of GC involves many factors. The occurrence of intestinal GC is go through the pattern of “normal gastric mucosa – chronic atrophic gastritis – intestinal metaplasia – dysplasia – gastric cancer (intestinal type)” [ 22 ]. It has also been demonstrated that cancer tissue usually develops from intestinal metaplasia (IM). Intestinal metaplasia is important in the development of GC. The chronic inflammation and injury to the gastric mucosa leads to absorption of normal epithelial cells and replacement of Type I epithelial cells, then goblet cells, and Pan's cells. This a histopathological change in epithelial cells is precancerous in GC lesions [ 23 ]. We found high expression of C-MYC and KI67 in intestinal metaplasia glands in paracancerous tissues, and high expression of NDRG2 in intestinal metaplasia glands and highly differentiated GC, tissues demonstrating role of intestinal metaplasia in the development of GC. This provides clues for further understanding of the pathogenesis of gastric cancer and has significant importance in the diagnosis and treatment of gastric cancer. We found that C-MYC expression correlated positively with KI67 expression. Studies suggest that C-MYC is a proto-oncogene, and KI67 is a measure of proliferation, which increases with the rise in tumor malignancy. At present, it is widely used in clinicopathological diagnosis. We studied NDRG2, C-MYC, and KI67 in the same tissue at the same time, to as comparison to show that NDRG2 is a tumor suppressor gene in GC. Our findings show that NDRG2 is expressed in GC and is related to the degree of differentiation, with high expression in well differentiated gastric cancer tissues, and intestinal metaplasia glands. Declarations Declaration of competing interest The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article. Funding This research was supported by the Hubei Provincial Health and Family Planning Commission of special funding (WJ2021M199). The funders were not involved in the study design, data collection, analysis, and interpretation, or the writing and publication. Author Contribution A：Zhang SW， [email protected] 18963989786B: Panqin， [email protected] 15207193152C: Qu YL， [email protected] 18627105645D: Ji HL， [email protected] 15050564973A and B contributed to manuscript writing and editing, and data collection;C and D to data analysis; C prepared Fig. 1; D prepared Table. 1,2,3;A and B contributed to conceptualization and supervision; all authors have read and approved the final manuscript. Acknowledgments We thank Medjaden Inc. for the scientific editing of this manuscript. References Bray F, Ferlay J, Soerjomataram I, Global Cancer Statistics. 2018: Globocan Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Ndrg2 Down-Regulation and Cd24 up-Regulation Promote Tumor Aggravation and Poor Survival in Patients with Gallbladder Carcinoma. Med Oncol. 2012;29:1879-85. https://doi.10.1007/s12032-011-0110-y. Correa P, Piazuelo MB. The Gastric Precancerous Cascade. J Dig Dis. 2012;13:2–9. https://doi.10.1111/j.1751-2980.2011.00550.x. de Vries AC, Kuipers EJ. Epidemiology of Premalignant Gastric Lesions: Implications for the Development of Screening and Surveillance Strategies. Helicobacter. 2007;12 Suppl 2:22–31. https://doi.10.1111/j.1523-5378.2007.00562.x. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3966089","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273622590,"identity":"459a4149-48e5-4133-a3dc-3f9607bc2f7f","order_by":0,"name":"Shiwei zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shiwei","middleName":"","lastName":"zhang","suffix":""},{"id":273622591,"identity":"a49fc009-16a1-4945-9b54-56a9f781fb3b","order_by":1,"name":"yilin qu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"yilin","middleName":"","lastName":"qu","suffix":""},{"id":273622592,"identity":"03dcd0a1-aee7-47dd-b1b1-8712acbc3092","order_by":2,"name":"hongliang ji","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"hongliang","middleName":"","lastName":"ji","suffix":""},{"id":273622593,"identity":"cadffa6d-0caa-4036-a489-4b09670fa0df","order_by":3,"name":"pan qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIie2PMWrDMBSGbQTK8opWCUrO8IIhjiE0V5EIaDI9g4zBkw/gXqRklNGaNKtLhtp7hmTM0tbt1oKdjBn0IcHj8X/8UhB4PPeKDJbAJlnWqs9+YOYmRU9F6Ry2VD+Kyt7U4yJstBYtdUs0cjwbT3Z1022IMjaNUMIeMLDh6ZwOK0n5vF6oLVWZ2c5ayQ8QE0PEy+uwgjadc1WAysOyb8EDJMZS8jCm7I8/Sn8IzLmUb4BWXlGa3xaMgFLNpbXXlaQ6RgtVyCkH4lCZNYiqzkf/ErN09n4pvmD10WXdxTytGMvr03nsYf3l/3ahGc4PKB6Px+P5yzfuilXTwETiYwAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"pan","middleName":"","lastName":"qin","suffix":""}],"badges":[],"createdAt":"2024-02-18 06:29:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3966089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3966089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51444093,"identity":"6e68ba29-4018-49d9-847e-44681b678e30","added_by":"auto","created_at":"2024-02-21 18:04:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2003787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NDRG2, C-MYC, and KI67 in gastric cancer tissues and adjacent tissues（200 ×, 5μm）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3966089/v1/57f9722922f16fd6af0c4878.jpg"},{"id":51593518,"identity":"1788cd59-9b65-441e-b8cb-d94d3ef8e418","added_by":"auto","created_at":"2024-02-25 02:07:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":573700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3966089/v1/becf9390-51bd-423c-af05-ea93c11a5ea3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression and significance of NDRG2, C-MYC, and KI67 in gastric cancer and adjacent tissues","fulltext":[{"header":"Main Points","content":"\u003cul\u003e\n \u003cli\u003eNDRG2 protein is an early molecular event in development of gastric cancer.\u003c/li\u003e\n \u003cli\u003eNDRG2 is a tumor suppressor gene for gastric cancer.\u003c/li\u003e\n \u003cli\u003eIntestinal metaplasia has important link in the process of gastric cancer.\u003c/li\u003e\n \u003cli\u003eNDRG2 expression is associated with degree of differentiation of gastric cancer.\u003c/li\u003e\n \u003cli\u003eNDRG2 expression is helpful for the early diagnosis of gastric cancer.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is a common malignancy worldwide and responsible for more than 1\u0026nbsp;million new cases and an estimated death of 769,000 in 2020 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In China, GC is a public health concern and 2nd common cause of death due to cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pathogenesis of GC is complex, involving multiple factors, the signaling pathways, inactivation of tumor suppressor genes, activation of proto-oncogenes, and abnormal expression of genes related to apoptosis, etc.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe MYC (myelocytomatosis) proto-oncogene on chromosome 8 has a significant role in progression of cell cycle and apoptosis. The MYC family of genes consists of C-MYC, L-MYC, and N-MYC. Cellular MYC (C-MYC) drives tumorigenesis by acting as a transcription factor that binds to numerous genomic loci and regulates the expression of a large number of target genes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The human N-MYC downstream-regulated gene (NDRG) is a new class of MYC suppressor gene. The NDRG2 to regulate cell proliferation, differentiation, and apoptosis, and has important role in inhibition of tumor and its metastasis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The KI67 acts as a nuclear antigen in cell proliferation and is expressed in all phases (G1, S, G2, and M) of the cell cycle. The KI67 expression signifies the proliferative activity and malignant degree of tumors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies show that C-MYC is a proto-oncogene, KI67 is an indicator of proliferation, and NDRG2 is a tumor suppressor gene involved in inhibition and tumor development s [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study was designed to analyze NDRG2, C-MYC, and KI67 expression in GC and adjacent tissues, and their role in development of GC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eGastric cancer patients with a confirmed histopathological diagnosis who were admitted to the Department of Surgery, XXXHospital o, from February 2018 to June 2021 were included in the study. Patients were excluded if they had undergone pre-operative radiotherapy or chemotherapy, or had other malignancy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration. Written informed consent was obtained from individual participants. The study was approved by the Human Ethics Committee of XXX Hospital, China.\u003c/p\u003e \u003cp\u003eAn Olympus CX31 microscope was used for histopathological examination. Rabbit anti-human NDRG2 monoclonal antibody was obtained from Abcam (UK), rabbit anti-human C-MYC and KI67 monoclonal antibodies, and sheep anti-rabbit secondary antibodies were obtained from China Fuzhou Maixin Biotechnology.\u003c/p\u003e \u003cp\u003eThe expressions of NDRG2, C-MYC, and KI67 in GC and adjacent normal tissues were assessed by immunohistochemistry. Samples were dewaxed, first with dimethyl benzene, then with 95% ethanol, 85% ethanol, and 75% ethanol, followed by EDTA antigen retrieval solution. Samples were prepared by incubating with an endogenous catalase inhibitor for 10 minutes, and then incubated in primary antibody solution at 37\u0026deg;C for 1 hour. Further incubation was done for 1 hour in a secondary antibody solution at room temperature. Diaminobenzylamine was used for color development. Slides were then stained with hematoxylin, differentiated with 1% hydrochloric acid, and incubated with ammonia. Slides were dehydrated, and then transparently sealed for observation under a microscope. Phosphate buffer was used instead of the primary antibody for control.\u003c/p\u003e \u003cp\u003eNDRG2 was positively expressed when there were brown or brownish-yellow particles present in the cytoplasm and cell membrane, while C-MYC and KI67 were positively expressed when there were brown or brownish-yellow particles present in the nucleus. The percentage of cells with staining and the intensity of staining were combined to calculate scoring intensity.\u003c/p\u003e \u003cp\u003eFive fields were randomly selected for each section, and stained cells were counted to standardize the expression, i.e., 0 points for no stained cells in the field, 1 for \u0026lt;\u0026thinsp;10% stained, 2 for 10%-30% stained, 3 for 31%-50% stained, and 4 points for \u0026gt;\u0026thinsp;50% stained. The intensity of staining of cells was standardized, i.e., 0 unstained, 1 light yellow, 2 yellow, and 3 points for of cells stained brown. The percentage of positive cells and staining intensity were used to assign a score, i.e., \u0026lt;\u0026thinsp;3 cells were considered negative -, 4\u0026ndash;6 weak positive +, 7\u0026ndash;9 moderate positive ++, and 10\u0026ndash;12 strong positive +++. For expression, - or +\u0026thinsp;a low expression, (\u0026thinsp;+\u0026thinsp;+\u0026thinsp;and +++ a high expression.\u003c/p\u003e \u003cp\u003eSPSS 24.0 statistical software was used for data analysis. The Chi-squared test was used for the comparison of data, and Spearman\u0026rsquo;s Correlation test was used to find out the relationship between NDRG2, C-MYC, and KI67 expression in gastric cancer tissues. A \u003cem\u003eP\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere were 41 GC patients (M 30, F 11), age 62.70\u0026thinsp;\u0026plusmn;\u0026thinsp;8.11 years (range, 39\u0026ndash;79 years). There was a poorly differentiated carcinoma in 25 cases and a well differentiated carcinoma in 16. Out of 41 cases, 30 were adenocarcinomas, 8 were mucinous, and others 3. There was one adenosquamous carcinoma and two gastric carcinoma with lymphoid stroma. Adenosquamous carcinoma was classified as well-differentiated carcinoma and gastric carcinoma with lymphoid stroma as poorly-differentiated carcinoma. Lymph node metastasis was present in 28 cases. Depth of invasion up to mucosa or submucosa was seen in 4 cases and up to muscularis or deeper in 37 cases.\u003c/p\u003e\n\u003cp\u003eNDRG2 was expressed in the cell membrane of GC and adjacent tissues, and in some cases in both the cell membrane and cytoplasm. C-MYC and KI67 were expressed in the nucleus of gastric cancer and the adjacent tissues.\u003c/p\u003e\n\u003cp\u003eIn GC tissues, NDRG2 protein expression was high in well differentiated and low in poorly differentiated cancer tissues.\u003c/p\u003e\n\u003cp\u003eIn adjacent tissues, NDRG2 protein was highly expressed in intestinal metaplasia glands.\u003c/p\u003e\n\u003cp\u003eIn gastric cancer tissues, C-MYC and KI67 proteins were highly expressed in poorly differentiated cancer tissues.\u003c/p\u003e\n\u003cp\u003eIn adjacent tissues, C-MYC and KI67 were highly expressed in intestinal metaplasia glands but were expressed low in other glands (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eNDRG2 was positively expressed in 39.02% (16/41) gastric cancer tissues and 92.68% (38/41) in adjacent tissues, difference was statistically significant, \u0026chi;\u0026sup2;= 26.25, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0. 05.\u003c/p\u003e\n\u003cp\u003eC-MYC was expressed in nucleus of gastric cancer and adjacent tissues. C-MYC was positively expressed in 73.17% (30/41) of gastric cancer tissues and in 4.88% (2/41) of adjacent tissues, showing a statistically significant difference, \u0026chi;\u0026sup2;=40.18, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0. 05.\u003c/p\u003e\n\u003cp\u003eKI67 was positively expressed in 97.56% (40/41) gastric cancer tissues and 24.39% (10/41) in adjacent tissues, difference was statistically significant, \u0026chi;\u0026sup2;= 46.12, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0. 05, (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTable 1. Expression of NDRG2, C-MYC, and KI67 in gastric cancer tissues and adjacent tissues\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.904936014625228%\" rowspan=\"2\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.678244972577697%\" rowspan=\"2\"\u003e\n \u003cp\u003eCases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.680073126142595%\" colspan=\"3\"\u003e\n \u003cp\u003eNDRG2 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.045703839122485%\" colspan=\"3\"\u003e\n \u003cp\u003eC-MYC expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.691042047531994%\" colspan=\"3\"\u003e\n \u003cp\u003eKI67 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.352517985611511%\"\u003e\n \u003cp\u003eLow\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.872901678657074%\"\u003e\n \u003cp\u003eHigh\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.908872901678658%\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.633093525179856%\"\u003e\n \u003cp\u003eLow\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.071942446043165%\"\u003e\n \u003cp\u003eHigh\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.148681055155876%\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.913669064748201%\"\u003e\n \u003cp\u003eLow\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.551558752997602%\"\u003e\n \u003cp\u003eHigh\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.546762589928058%\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.934065934065934%\"\u003e\n \u003cp\u003eGastric cancer tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.6923076923076925%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.7765567765567765%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.622710622710622%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003cp\u003e(39.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.593406593406593%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.6923076923076925%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.805860805860807%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e(73.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.043956043956044%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.058608058608058%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.637362637362637%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003cp\u003e(97.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.934065934065934%\"\u003e\n \u003cp\u003eAdjacent tissues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.6923076923076925%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.7765567765567765%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.622710622710622%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003cp\u003e(92.68)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.593406593406593%\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.6923076923076925%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.805860805860807%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e(4.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.043956043956044%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.058608058608058%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.637362637362637%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e(24.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.678244972577697%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.680073126142595%\" colspan=\"3\"\u003e\n \u003cp\u003e26.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.045703839122485%\" colspan=\"3\"\u003e\n \u003cp\u003e40.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.691042047531994%\" colspan=\"3\"\u003e\n \u003cp\u003e46.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eＰ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.678244972577697%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.680073126142595%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.045703839122485%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.691042047531994%\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;There was significant association of NDRG2 expression with the degree of differentiation of GC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gender, age, stage of cancer, and lymph node metastasis was not significantly associated with NDRG2 expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was no significant association between C-MYC and KI67 with gender, age, differentiation degree, cancer stage, and lymph node metastasis in GC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRelationship between NDRG2, C-MYC, and KI67 protein expression and clinicopathological features in gastric cancer\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical features\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCase\u003c/p\u003e\n \u003cp\u003e(n)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive cases of NDRG2 protein number of cases and percentage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive cases of C-MYC protein number of cases and percentage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive cases of KI67 protein number of cases and percentage\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/30 (33.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22/30 (73.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30/30 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6/11 (54.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8/11 (72.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/11 (90.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/27 (40.74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22/27 (84.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27/27 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5/14 (35.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8/14 (57.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13/14 (92.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDifferentiation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh, moderate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5/10 (50.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9/10 (90.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/10 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/31 (35.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21/31 (67.74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30/31 (96.77)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphatic metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6/13 (46.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/13 (84.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13/13 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/28 (35.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/28 (67.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27/28 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7/13 (58.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12/14 (85.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14/14 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdvanced\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9/28 (32.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18/27 (66.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26/27 (96.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCorrelation analysis of NDRG2, C-MYC, and KI67 in GC showed that NDRG2 expression had no correlation with C-MYC expression (rs\u0026thinsp;=\u0026thinsp;0.105, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). KI67 expression had no significant correlation with C-MYC expression (rs =-0.021, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The expression of KI67 was positively correlated with C-MYC (rs\u0026thinsp;=\u0026thinsp;0.409, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCorrelation analysis of NDRG2, C-MYC, and KI67 expression in gastric cancer tissues\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNDRG2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC-MYC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKI67\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"9\"\u003e\n \u003cp\u003eSpearman rho\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eNDRG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoefficient of association\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSig. (one-sided)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.449\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eC-MYC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoefficient of association\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.409\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSig. (one-sided)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eKi67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoefficient of association\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.409\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSig.(one-sided)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e** At a confidence level of 0.01(one-sided), the correlation is significant.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn present study, the immunohistochemistry of NDRG2, C-MYC and KI67 performed on gastric cancer tissues and adjacent tissues of gastric cancer patients. We found that In gastric cancer tissues, NDRG2 protein was highly expressed in well differentiated cancer tissues and expressed at lower rates in poorly differentiated cancer tissues. This shows that NDRG2 is a tumor suppressor gene in gastric cancer, which is consistent with the literature[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] h showing a lack of NDRG2 expression in GC cell line SNU-620 promotes proliferation of GC cells, and thus NDRG2 is seen as a tumor suppressor gene at the cellular level. Our findings suggest that the abnormal expression of the NDRG2 protein is an early molecular event in the development of GC, and the detection of NDRG2 expression is helpful for the early detection of GC.\u003c/p\u003e \u003cp\u003eAt present, the mechanism of gastric cancer is still unclear. It is generally believed that development of GC is a multi-gene, multi-factor, and multi-stage dynamic process. The N-MYC gene is a member of the same family as the N-MYC downstream regulated gene (NDRG) family, which consists of four members: NDRG1, NDRG2, NDRG3, and NDRG4 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. NDRG2 differentiation related genes. NDRG2 is a novel gene containing acyl-carrying protein-like structures cloned from normal human brain tissue. It is differentially expressed in tumor tissue and normal tissue. NDRG2 is expressed widely and highly in most of the tissues in embryo and adult, especially in highly differentiated tissues, such as brain, heart, and muscles [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The degree expression of NDRG2 is correlates positively with the degree of maturation of organ and tissue.\u003c/p\u003e \u003cp\u003eNDRG2 is abnormally expressed in many human cancer cells, and studies have shown that NDRG2 over expression inhibits proliferation and induces apoptosis in ovarian cancer cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], of thyroid carcinoma [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] colorectal cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]., Studies indicate that NDRG2 is a critical regulator of colorectal cancer differentiation and that NDRG2 induction of colorectal cancer cell differentiation is dependent on the repression of E3 ligase Skp2 activity. Studies on esophageal squamous cell carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] found that NDRG2 over expression suppresses tumor by inhibiting cell migration, invasion, and EMT through inhibition of AKT/XIAP signaling pathway. Downregulation of NDRG2 was observed in tumor compared to normal brain after resection of highgrade meningioma [21]. These findings suggest that NDRG2 is a tumor suppressor gene which is consistent with our findings.\u003c/p\u003e \u003cp\u003eThe development of GC involves many factors. The occurrence of intestinal GC is go through the pattern of \u0026ldquo;normal gastric mucosa \u0026ndash; chronic atrophic gastritis \u0026ndash; intestinal metaplasia \u0026ndash; dysplasia \u0026ndash; gastric cancer (intestinal type)\u0026rdquo; [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It has also been demonstrated that cancer tissue usually develops from intestinal metaplasia (IM). Intestinal metaplasia is important in the development of GC. The chronic inflammation and injury to the gastric mucosa leads to absorption of normal epithelial cells and replacement of Type I epithelial cells, then goblet cells, and Pan's cells. This a histopathological change in epithelial cells is precancerous in GC lesions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found high expression of C-MYC and KI67 in intestinal metaplasia glands in paracancerous tissues, and high expression of NDRG2 in intestinal metaplasia glands and highly differentiated GC, tissues demonstrating role of intestinal metaplasia in the development of GC. This provides clues for further understanding of the pathogenesis of gastric cancer and has significant importance in the diagnosis and treatment of gastric cancer.\u003c/p\u003e \u003cp\u003eWe found that C-MYC expression correlated positively with KI67 expression. Studies suggest that C-MYC is a proto-oncogene, and KI67 is a measure of proliferation, which increases with the rise in tumor malignancy. At present, it is widely used in clinicopathological diagnosis. We studied NDRG2, C-MYC, and KI67 in the same tissue at the same time, to as comparison to show that NDRG2 is a tumor suppressor gene in GC. Our findings show that NDRG2 is expressed in GC and is related to the degree of differentiation, with high expression in well differentiated gastric cancer tissues, and intestinal metaplasia glands.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This research was supported by the Hubei Provincial Health and Family Planning Commission of special funding (WJ2021M199). The funders were not involved in the study design, data collection, analysis, and interpretation, or the writing and publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA：Zhang SW，[email protected] 18963989786B: Panqin，[email protected] 15207193152C: Qu YL，[email protected] 18627105645D: Ji HL，[email protected] 15050564973A and B contributed to manuscript writing and editing, and data collection;C and D to data analysis; C prepared Fig. 1; D prepared Table. 1,2,3;A and B contributed to conceptualization and supervision; all authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Medjaden Inc. for the scientific editing of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Global Cancer Statistics. 2018: Globocan Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Med Oncol. 2012;29:1879-85. https://doi.10.1007/s12032-011-0110-y.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrea P, Piazuelo MB. The Gastric Precancerous Cascade. J Dig Dis. 2012;13:2\u0026ndash;9. https://doi.10.1111/j.1751-2980.2011.00550.x.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Vries AC, Kuipers EJ. Epidemiology of Premalignant Gastric Lesions: Implications for the Development of Screening and Surveillance Strategies. Helicobacter. 2007;12 Suppl 2:22\u0026ndash;31. https://doi.10.1111/j.1523-5378.2007.00562.x.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, NDRG2, C-MYC, KI67","lastPublishedDoi":"10.21203/rs.3.rs-3966089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3966089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Expressions of NDRG2, C-MYC, and KI67 in gastric cancer (GC) and adjoining tissues were analyzed for their importance in the occurrence and development of the GC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003e Samples from GC and adjacent tissue of patients admitted to the Department of Surgery,XXX Hospital, China, from February 2018 to June 2021, were analyzed for the expression of NDRG2, C-MYC, and KI67 using immunohistochemistry. The association of NDRG2 and degrees of differentiation in gastric cancer were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e There were 41 GC patients in the study. The expression of NDRG2 was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), and the C-MYC and KI67 were higher (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) in GC versus adjacent tissues. NDRG2 expression varied significantly (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) according to the degree differentiation in GC. The expression of KI67 was positively correlated with C-MYC (rs=0.409, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThere were significant differences in the expression of NDRG2 (low expression), and C-MYC, and KI67 (high expression) in gastric cancer and adjacent tissues. The degree of cancer differentiation was associated with expression of NDRG2 in GC tissue. NDRG2 expression was higher in para-carcinoma tissues and intestinal metaplasia glands.\u003c/p\u003e","manuscriptTitle":"Expression and significance of NDRG2, C-MYC, and KI67 in gastric cancer and adjacent tissues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 18:04:40","doi":"10.21203/rs.3.rs-3966089/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8d8cd85-3ae2-4e90-9cdf-e7e7181a17c7","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-08T08:13:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 18:04:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3966089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3966089","identity":"rs-3966089","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}