LRIG1 Expression and Colorectal Cancer Prognosis

LRIG1 Expression and Colorectal Cancer Prognosis | 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 LRIG1 Expression and Colorectal Cancer Prognosis Maryam Bakherad, Mahdieh Salimi, Seyed Abdolhamid Angaji, Frouzandeh Mahjoubi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-118977/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jan, 2021 Read the published version in BMC Medical Genomics → Version 1 posted 2 You are reading this latest preprint version Abstract Background To make the right treatment decisions about colorectal cancer (CRC) patients reliable predictive and prognostic data are needed. However, in many cases this data is not enough. Some studies suggest that LRIG1 gene ( leucine-rich repeats and immunoglobulin-like domains1 ) has prognostic implications in different kinds of cancers. Methods One hundred and two patients with colorectal cancer were retrospectively analyzed for LRIG1 expression at both mRNA and protein levels. SYBR Green Real-Time RT-PCR technique was used for mRNA expression analyses and Glyceraldehyde-3-Phosphate Dehydrogenase gene ( GAPDH ) was considered as a reference gene for data normalization. LRIG1 protein expression was analyzed using Immunohistochemistry (IHC). Additionally, appropriate statistic analyses were used to assess the expression of LRIG1 in test and control groups. The prognostic significance of LRIG1 expression was analyzed using the univariate and multivariate analyses. Results The data revealed that the expression of LRIG1 in both mRNA and protein levels was down regulated in colorectal tumor tissues (P<0.01) but is not clinically relevant prognostic indicator in CRC. Conclusions Therefore, it is suggested that LRIG1 expression analyses may not be considered as an important issue when making informed and individualized clinical decisions regarding the management of colorectal cancer patients. Epigenetics & Genomics Prognostic indicator colorectal cancer CRC LRIG1 gene expression Figures Figure 1 Figure 2 Background CRC (Colorectal Cancer) is considered as one of the most significant cancers worldwide, with about 1 million new cases and more than 550000 deaths per year [ 1 ]. Despite the significant advances in the diagnosis of CRC, the survival rate decreases for patients diagnosed with metastatic and regional disease [ 2 ]. The reported overall median survival time of CRC is only 1.1 years [ 3 ]; therefore, understanding about biological factors with impact on CRC is very important. The lack of predictive and prognostic biomarkers with the ability to predict therapy response and recurrence of the disease is an important issue that needs to be addressed. The LRIG1 ( the leucine-rich repeats and immunoglobulin-like domains 1 ) as an emerging tumor suppressor and its paralogs LRIG2 ( the leucine-rich repeats and immunoglobulin-like domains 2 ) and LRIG3 ( the leucine-rich repeats and immunoglobulin-like domains 3 ), are considered to have prognostic significance in various kinds of cancers, such as head-and-neck [ 4 , 5 ], prostate [ 6 ], breast [ 7 , 8 ], uterine cervical cancer [ 9 – 11 ], and cutaneous squamous cell carcinoma [ 12 ], and glioma [ 13 , 14 ]. The locus of LRIG1 is located on chromosome 3p14.3. The encoding protein is a transmembrane protein consisting of an extracellular region including three immunoglobulin (Ig)-like domains and fifteen leucine-rich repeats. The leucine-rich repeats and immunoglobulin-like domains have interactions with all four extracellular region binding protein B receptor family members leading to the regulation of receptor levels by subsequent lysosomal degradation and increasing ubiquitination, independent of ligands [ 15 – 17 ]. In addition the LRIG1 is considered as a marker of human epithelial stem cells in a quiescent non-proliferative state [ 18 ]. The enhanced proliferation associated with epithelial hyper-proliferation in vivo and stem cell expansion in vitro is the result of the genetic erosion of the leucine-rich repeats and immunoglobulin-like domains [ 18 , 19 ]. It is recommended by lineage tracing that the leucine-rich repeats and immunoglobulin-like domains mark non-cycling, long-lived stem cells of the ‏4 quiescent intestinal stem cell niche in the crypt [ 20 ]; and also progenitor cells in the stomach that are involved in restoring gastric cell mucosa after DMP-777 induced acute damage [ 21 ]. Although the LRIG1 plays vital role in cancers, little is known about its association with clinico-patho-physiology characteristics of CRC patients. Here the LRIG1 expression in the lesions of CRC patients was studied in order to evaluate its relationship with the major clinicohistological predictive factors and its respective impacts on patient prognosis hoping to improve the approaches for colorectal cancer management. Therefore, the main goal of the present study was to compare and analyze the expression levels of the LRIG1 gene in samples of tumor and normal colorectal tissues of CRC patients by quantitative real-time RT-PCR and immunohistochemical (IHC) techniques. Moreover, to estimate the prognostic indicator of the mentioned gene expression levels, we surveyed their correlations with clinicopathological parameters, as well as the overall survival (OS) of patients with CRC. Methods Patient information A total of 102 cases of colorectal cancer from Imam Khomeini Hospital, Tehran, Iran were selected. The average age was 55.0 ± 10.0 years. The inclusion criteria were post-operative diagnosis of primary CRC based on histopathology. The study was approved by the Ethics Committee of the NIGEB based on the Helsinki declaration. All the patients signed informed consent. The ethics code number is IR.NIGEB.1395.11.10.E. The patients' characteristics are presented in Table 1 . Overall survival (OS) was defined as the time from the date of primary treatment to the date of death from any cause or until the date of the last follow-up. Disease-free survival (DFS) for patients with CRC was defined as the time from the date of primary treatment to the date of diagnosis for recurrence or disease or to the date of the last follow-up. Table 1 Baseline characteristics of colorectal cancer patients. Characteristic Number (%) Number of patients 102 (100) Gender Male 49 (48) Female 53 (52) Age (years, mean ± SD) 55.00 ± 10 Pathological Stage Stage1 27 (26) Stage2 29 (28) Stage3 24 (24) Stage4 22 (22) Tumor size < 5 cm 47 (46) 5–8 cm 31 (30) 8–10 cm 14 (14) ≥ 10 cm 10 (10) Lymph nodes metastasis Positive 49 (48) Negative 53 (52) Other metastasis 22 (22) Immunohistochemical analysis Surgical specimens were formaldehyde fixed paraffin embedded and sectioned at a thickness of 4 µm followed by xylene dewaxing, ethanol gradient rehydration and harnessed to high pressure and temperature for antigen retrieval. The slices were incubated in H 2 O 2 harnessed to the primary antibody, rinsed with phosphate buffered saline (PBS), then harnessed to secondary and mouse anti-human LRIG1 monoclonal antibody, respectively. The slices incubated with PBS instead of the primary antibody were used as the negative control. The sections were assessed using an Olympus BX41 light microscope (Olympus, Tokyo, Japan) by a pathologist. The scale based on the reaction intensity were used to assess immunoreactivity in enterocytes or cancer cells of the studied sections (0, no reaction; 10, up to 10%; 30, 11–30%; 60, 31–60%; 80, 61–80%; and 100, > 80%) (Fig. 1 ). RNA extraction, cDNA synthesis and LRIG1 mRNA expression analysis Total RNA was isolated from the colorectal tissue using YTzol kit (Yekta Tajhiz Azma Co, Tehran, Iran) according to the manufacturer’s protocol. cDNA was synthesized following the manufacturer’s instructions (Cinaclon Co, Tehran, Iran) and stored at -20 °C until analyzed. The primer sequences for glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and LRIG1 genes were designed using primer 3 software ( https://primer3.ut.ee/ ) and then blasted using https://www.ncbi.nlm.nih.gov/tools/primer-blast/ . The designed primer sequences are shown in Table 2 . The Real-time RT-PCR amplifications were conducted in a final volume of 15 µl reaction mixture containing 1 µl of cDNA, 7.5 µl RealQ plus 2x master mix green (Ampliqon, Denmark), 0.6 µl (10 µmol/l) of each primer and 5.3 µl sterilized water, using the Rotor-Gene Q System (QIAGEN Hilden, Germany). The cycling conditions were as follows: 15 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s for the LRIG1 and also the GAPDH , which was used as a normalizer. Experiments were performed in triplicates for each data point. The linear standard curve (from 0.1 to 1,000 ng) assessed by ultraviolet spectrophotometer was used for amplification efficiency determination of each primer pair. The standard curves showed good linearity and amplification (100%). The data was presented as the fold change in gene expression normalized to an endogenous reference gene relative to the controls using 2 −△△CT method. Table 2 The primers for real-time quantitative reverse transcription PCR. Gene name Primer sequence Product size (bp) Annealing temperature (°C) LRIG1 F: CTGCATGAGTTGGTCCTGTCC R: TGTGGCTGATGGAATTGTGG 112 60 GAPDH F:GCAGGGGGGAGCCAAAAGGGT R: TGGGTGGCAGTGATGGCATGG 219 60 LRIG1 : leucine-rich repeats and immunoglobulin-like domain-1 ; GAPDH : Glyceraldehyde-3-phosphate dehydrogenase. Statistical analysis Graphpad Prism 8.0.2 (California Corporation, USA) and SAS computer software version 9.1 (SAS Institute Inc., Cary, NC, USA) were used to analyze the data. The Mann-Whitney U test and Kruskal-Wallis test were performed for numerical data and the Chi-square test was used to analyze the relationship between parameter data. Numerical data are presented as the mean ± standard deviation (SD). Differences were considered as statistically significant if 𝑝 <0.05. The Cox proportional-hazards model was used for univariate and multivariate analyses to identify the independent prognostic factors for OS, DFS. Results LRIG1 expression and clinicopathological features LRIG1 mRNA expression The LRIG1 mRNA expression was significantly down-regulated in Colorectal cancerous tissues compared with normal control (P < 0.01). The mean of LRIG1 relative expression in cancerous tissues compared with normal control was 0.57 ± 0.24 with a range of 0.23 to 1.2. About 40% of cancerous samples showed the relative expression < 0.5 that was considered as down-regulation. As shown in Fig. 2 , there were no significant differences between different demographical and clinicopatological characteristics of CRC patients and LRIG1 expression (P > 0.05). LRIG1 protein expression The LRIG1 immunoreactivity was found in the cytoplasm of enterocytes as well as cancer cells of the analyzed tissues (Fig. 1 ). The tumor LRIG1-positive rate in colorectal cancer tumors was 37.4%, which was significantly lower than that in control tissues (57.7%, P < 0.05). There was no significant correlation in the expression of the LRIG1 with tumor size, tumor diameter, tumor differentiation, age, and the number of positive mesenteric lymph nodes or vascular cancer embolus (P > 0.05) (Table 3 ). Table 3 Clinicopathological variables and their correlation with immunohistochemical expression of LRIG1 in primary tumors. Clinicopathological variables CRC patients n (%) Score 57 53 (52) 26(49.1) 27(50.9) Primary tumor location Colon 73 (72) 33(45.2) 40(54.8) 0.321 Rectum 29 (28) 14(48.3) 15(51.7) Differentiation High 12 (12) 5(42.9) 7(57.1) 0.461 Mid-Low 88 (86) 39(44.3) 49(55.7) Unknown 2 (2) 1(50.0) 1(50.0) Positive lymph node 49 (48) 26(53.6) 23(46.4) 0.243 Tumor diameters < 5 cm 47 (46) 22(46.8) 25(53.2) 0.763 5–8 cm 31 (30) 15(48.4) 16(51.6) 8–10 cm 14 (14) 6(42.2) 8(57.8) ≥ 10 cm 10 (10) 4(37.2) 6(62.8) Vascular cancer embolus Yes 65 (64) 34(53.2) 31(46.8) 0.382 No 37 (36) 19(51.3) 18(48.7) Univariate and multivariable analyses of survival impact of LRIG1 expression in patients with CRC The univariate and multivariate analyses were performed to investigate independent prognostic factors for OS (Overall survival) and DFS (Disease-free survival) in patients with colorectal cancer using the Cox proportional-hazards model (Table 4 ). The analysis results demonstrated no significant difference for parameters in terms of OS (P > 0.05) and DFS (P > 0.05). Table 4 Univariate and multivariable analysis of prognostic indicators on overall survival and disease-free survival for the prognostic significance of LRIG1 expression of colorectal cancer patients (N = 102). Parameters Overall survival Disease-free survival Univariate analysis Multivariable analysis Univariate analysis Multivariable analysis HR (95% CI) p-value HR (95% CI) p-value HR (95% CI) p-value HR (95% CI) p-value Age (years) 0.698 (0.376–1.271) 0.243 0.689 (0.303–1.515) 0.354 0.830 (0.453–1.518) 0.555 0.981 (0.439–2.231) 0.991 (≥ 57 vs < 57) Gender 0.944 (0.525–1.658) 0.953 0.859 (0.409–1.755) 0.667 1.029 (0.574–1.806) 0.962 0.923 (0.433–1.921) 0.819 (Male vs Female) Location 0.833 (0.454–1.495) 0.534 0.996 (0.449–2.164) 0.982 0.775 (0.421–1.388) 0.389 1.044 (0.477–2.237) 0.946 Rectum vs Colon Tumor size 0.887 (0.490–1.643) 0.736 1.099 (0.512–2.310) 0.837 0.887 (0.489–1.641) 0.733 1.054 (0.471–2.311) 0.928 (≥ 5 cm vs < 5 cm) LN metastasis 1.073 (0.588–1.923) 0.855 0.998 (0.448–2.181) 0.581 1.091 (0.604–1.965) 0.787 0.824 (0.364–1.817) 0.625 Yes vs No Vascular invasion 1.694 (0.921–2.932) 0.101 1.171 (0.519–2.629) 0.718 1.566 (0.869–2.782) 0.148 1.289 (0.549–3.068) 0.564 Yes vs No LRIG1 expression 1.252 (0.690–2.260) 0.473 1.826 (0.823–4.004) 0.151 1.077 (0.597–1.904) 0.831 1.451 (0.656–.081) 0.271 Positive vs Negative Discussion Cancer is one of the most important and prevalent diseases with poor prognosis and there is no effective method to treat and predict the procedure of tumorigenesis. Finding the appropriate biomarkers of cancer prediction or prognosis will have huge importance in cancer management. Nowadays the new strategies searching for informative biomarkers in cancer management have attracted good attention in the world. The increasing evidences have demonstrated the leucine-rich repeats and immunoglobulin-like domain as an independent prognosis factor and predictive biomarker of clinicopathology in variety of tumors. Due to inconsistency on the effect of the LRIG1 in different types of tumors, the present study was carried out to investigate the prognostic importance of the LRIG1 expression and its relationship with clinicopathological significance in CRC. In the present study, the expression of LRIG1 at both mRNA and protein levels was significantly decreased in CRC tumors compared with normal control but, the high levels of leucine-rich repeats and immunoglobulin-like domains expression were not significantly associated with longer overall survival, which was consistent with the conclusion of subgroup analysis. These results suggested that the LRIG1 was not a prognostic marker in CRC tumors. Meanwhile, the LRIG1 expression was significantly lower in cancer tissues than normal ones and the same result was detected with no heterogeneity in subgroup analysis based on the type of tumor. The higher levels of LRIG1 expression was not also related to positive HPV status and tumor progression assessed by its association with degree of differentiation. Also, there was no association between the LRIG1 expression and lymphatic metastasis. Some genes, such as K-ras and epidermal growth factor receptor ( EGFR ) and human epidermal growth factor receptor 2 ( HER2 ) are reported to be involved in the progress and development of colorectal cancer [ 22 – 25 ], but the LRIG1 roles in colorectal cancer have not been well studied and remained contradictory. Some studies showed that distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features [ 26 , 27 ]. The present data revealed that there was not any correlation between LRIG1 expression and bilateral and peritoneal CRC metastasis (P > 0.05) and also with age, synchronous or metachronous CRC or primary tumor location (P > 0.05). Although, earlier studies proposed that LRIG1 expression was associated with a good prognosis in terms of overall survival (OS) and might act as a predictive factor for characteristics of cancer patients [ 28 ], whether the LRIG1 expression could predict a lower risk of CRC remains doubtful. Conclusions In conclusion, our studies revealed that although LRIG1 was down regulated in CRC and primary tumors of CRC patients but, its expression in both mRNA and protein levels, was not clinically relevant prognostic indicator in CRC. Therefore, it is suggested that LRIG1 expression analyses may not be important when making informed and individualized clinical decisions regarding the management of colorectal cancer patients. Abbreviations CRC: Colorectal cancer GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase LRIG1: Leucine-rich repeats and immunoglobulin-like domains 1 Ig: Immunoglobulin PBS: Phosphate buffered saline OS: Overall survival DFS: Disease-free survival PCR: Polymerase Chain Reaction Declarations Ethics approval and consent to participate This study was approved by the ethics committee of National Institute of Genetic Engineering and Biotechnology (NIGEB), IRAN (#IR.NIGEB.1395.11.10.E.). All individuals included in the study signed a consent form to use their clinical samples and personal data under the supervision of their physician. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors are not aware of any conflict of interest. Funding None. Authors’ contributions MB, acquisition of data and draft the work; MS, conception and designing of the work, analysis and interpretation of data, substantively revise the work; SAA, contributes to the design of the work; FM, contributes to the design of the work and data interpretation; TM, acquisition and analysis of the data. All authors read and approved the final manuscript. Acknowledgments The authors would like to thank all the patients who participated in this study, Dr. B. Mahjoubi and the National Institute of Genetic Engineering and Biotechnology for their support. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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Clinicopathological and Prognostic Significance of Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1 (LRIG1) in Malignant Tumors: A Meta-Analysis. 2018; 9: 2895-2909 Cite Share Download PDF Status: Published Journal Publication published 18 Jan, 2021 Read the published version in BMC Medical Genomics → Version 1 posted Submission checks completed at journal 02 Dec, 2020 Editorial decision: Accept 01 Dec, 2020 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. We do this by developing innovative software and high quality services for the global research community. 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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-118977","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":5744418,"identity":"239a6d9a-8605-4498-8002-12f788309ff3","order_by":0,"name":"Maryam Bakherad","email":"","orcid":"","institution":"Kharazmi University","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Bakherad","suffix":""},{"id":5744419,"identity":"ff5110d1-beb7-4db6-bcfe-5458d4bf73c9","order_by":1,"name":"Mahdieh Salimi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYFAC5oYDDAY2DGwMIMQAI/ECRpCWNBK1AInDRCoGAfn2g42HbhScz+PjX3zsAUONHQOf9AH8WgzOJDYczjG4Xcwm8SzdgOFYMgMbXwIBLQwQLYltEmfMJBjYDjCw8RByWP9DkJZzQC3nv0kw/CNCC8MNsC0HEtv4e9gkGNuI0GJwA2xLMtAWNnODxL5kHiIclnz4c84fu8T5/YefPfjwzU5OvoeQw+BAIoGBAYgI+gQJ8B8gQfEoGAWjYBSMKAAAj5BAEObi6WYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5539-5658","institution":"National Institute of Genetic engineering and Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Mahdieh","middleName":"","lastName":"Salimi","suffix":""},{"id":5744420,"identity":"5dfecbd1-198c-4888-93ac-fa07af6fffea","order_by":2,"name":"Seyed Abdolhamid Angaji","email":"","orcid":"","institution":"Kharazmi University","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Abdolhamid","lastName":"Angaji","suffix":""},{"id":5744421,"identity":"26c1fafb-eca7-4efc-94c3-5f38ac2a3a77","order_by":3,"name":"Frouzandeh Mahjoubi","email":"","orcid":"","institution":"national instiitute of genetic engineering and biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Frouzandeh","middleName":"","lastName":"Mahjoubi","suffix":""},{"id":5744422,"identity":"4723177c-2408-4df0-9103-4379d88e4678","order_by":4,"name":"Tayebeh Majidizadeh","email":"","orcid":"","institution":"National institute of genetic engineering and biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Tayebeh","middleName":"","lastName":"Majidizadeh","suffix":""}],"badges":[],"createdAt":"2020-11-30 20:30:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-118977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-118977/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12920-020-00846-2","type":"published","date":"2021-01-18T15:01:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4073011,"identity":"03527c12-c5da-476b-a588-3bfe942100db","added_by":"auto","created_at":"2020-12-07 17:02:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105680,"visible":true,"origin":"","legend":"Expression of LRIG1 protein in colorectal cancer (CRC) and unchanged colon mucosa as assessed by immunohistochemistry. A; section of unchanged colon mucosa and B; CRC show the immunoreactivity. Magnification, x100.","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-118977/v1/3fa4d27cce9ce380110d9b65.jpg"},{"id":4073012,"identity":"5f3dcdea-065f-4ecf-9e73-9117eda4c442","added_by":"auto","created_at":"2020-12-07 17:02:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113507,"visible":true,"origin":"","legend":"Evaluation of the LRIG1 relative mRNA expression in tumor tissues based on clinopathologic situations.\nLN: Lymph node metastasis, C: normal control","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-118977/v1/64a5faf071609914b4f28461.jpg"},{"id":15671149,"identity":"afcce09f-5f81-46f9-95c0-a15328279c83","added_by":"auto","created_at":"2021-11-18 14:04:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":518821,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-118977/v1/fb24f3da-c3fb-4652-b8ef-d515af9ea779.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eLRIG1\u003c/em\u003e Expression and Colorectal Cancer Prognosis\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eCRC (Colorectal Cancer) is considered as one of the most significant cancers worldwide, with about 1\u0026nbsp;million new cases and more than 550000 deaths per year [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the significant advances in the diagnosis of CRC, the survival rate decreases for patients diagnosed with metastatic and regional disease [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. The reported overall median survival time of CRC is only 1.1\u0026nbsp;years [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]; therefore, understanding about biological factors with impact on CRC is very important.\u003c/p\u003e\n\u003cp\u003eThe lack of predictive and prognostic biomarkers with the ability to predict therapy response and recurrence of the disease is an important issue that needs to be addressed. The \u003cem\u003eLRIG1\u003c/em\u003e (\u003cem\u003ethe leucine-rich repeats and immunoglobulin-like domains 1\u003c/em\u003e) as an emerging tumor suppressor and its paralogs \u003cem\u003eLRIG2\u003c/em\u003e (\u003cem\u003ethe leucine-rich repeats and immunoglobulin-like domains 2\u003c/em\u003e) and \u003cem\u003eLRIG3\u003c/em\u003e (\u003cem\u003ethe leucine-rich repeats and immunoglobulin-like domains 3\u003c/em\u003e), are considered to have prognostic significance in various kinds of cancers, such as head-and-neck [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e], prostate [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], breast [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e], uterine cervical cancer [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], and cutaneous squamous cell carcinoma [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e], and glioma [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe locus of \u003cem\u003eLRIG1\u003c/em\u003e is located on chromosome 3p14.3. The encoding protein is a transmembrane protein consisting of an extracellular region including three immunoglobulin (Ig)-like domains and fifteen leucine-rich repeats. The leucine-rich repeats and immunoglobulin-like domains have interactions with all four extracellular region binding protein B receptor family members leading to the regulation of receptor levels by subsequent lysosomal degradation and increasing ubiquitination, independent of ligands [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition the LRIG1 is considered as a marker of human epithelial stem cells in a quiescent non-proliferative state [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The enhanced proliferation associated with epithelial hyper-proliferation in vivo and stem cell expansion in vitro is the result of the genetic erosion of the leucine-rich repeats and immunoglobulin-like domains [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is recommended by lineage tracing that the leucine-rich repeats and immunoglobulin-like domains mark non-cycling, long-lived stem cells of the \u0026rlm;4 quiescent intestinal stem cell niche in the crypt [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]; and also progenitor cells in the stomach that are involved in restoring gastric cell mucosa after DMP-777 induced acute damage [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAlthough the LRIG1 plays vital role in cancers, little is known about its association with clinico-patho-physiology characteristics of CRC patients. Here the \u003cem\u003eLRIG1\u003c/em\u003e expression in the lesions of CRC patients was studied in order to evaluate its relationship with the major clinicohistological predictive factors and its respective impacts on patient prognosis hoping to improve the approaches for colorectal cancer management. Therefore, the main goal of the present study was to compare and analyze the expression levels of the \u003cem\u003eLRIG1\u003c/em\u003e gene in samples of tumor and normal colorectal tissues of CRC patients by quantitative real-time RT-PCR and immunohistochemical (IHC) techniques. Moreover, to estimate the prognostic indicator of the mentioned gene expression levels, we surveyed their correlations with clinicopathological parameters, as well as the overall survival (OS) of patients with CRC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section3\"\u003e\n\u003ch2\u003ePatient information\u003c/h2\u003e\n\u003cp\u003eA total of 102 cases of colorectal cancer from Imam Khomeini Hospital, Tehran, Iran were selected. The average age was 55.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u0026nbsp;years. The inclusion criteria were post-operative diagnosis of primary CRC based on histopathology. The study was approved by the Ethics Committee of the NIGEB based on the Helsinki declaration. All the patients signed informed consent. The ethics code number is IR.NIGEB.1395.11.10.E. The patients' characteristics are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Overall survival (OS) was defined as the time from the date of primary treatment to the date of death from any cause or until the date of the last follow-up. Disease-free survival (DFS) for patients with CRC was defined as the time from the date of primary treatment to the date of diagnosis for recurrence or disease or to the date of the last follow-up.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eBaseline characteristics of colorectal cancer patients.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNumber (%)\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\u003eNumber of patients\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102 (100)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGender\u003c/p\u003e\n\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\u003e49 (48)\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\u003e53 (52)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge (years, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePathological Stage\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27 (26)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29 (28)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24 (24)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22 (22)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTumor size\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;5\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47 (46)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u0026ndash;8\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31 (30)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u0026ndash;10\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (14)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026ge;\u0026thinsp;10\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (10)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLymph nodes metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49 (48)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53 (52)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOther metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22 (22)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e\n\u003cp\u003eSurgical specimens were formaldehyde fixed paraffin embedded and sectioned at a thickness of 4\u0026nbsp;\u0026micro;m followed by xylene dewaxing, ethanol gradient rehydration and harnessed to high pressure and temperature for antigen retrieval. The slices were incubated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e harnessed to the primary antibody, rinsed with phosphate buffered saline (PBS), then harnessed to secondary and mouse anti-human LRIG1 monoclonal antibody, respectively. The slices incubated with PBS instead of the primary antibody were used as the negative control. The sections were assessed using an Olympus BX41 light microscope (Olympus, Tokyo, Japan) by a pathologist. The scale based on the reaction intensity were used to assess immunoreactivity in enterocytes or cancer cells of the studied sections (0, no reaction; 10, up to 10%; 30, 11\u0026ndash;30%; 60, 31\u0026ndash;60%; 80, 61\u0026ndash;80%; and 100, \u0026gt;\u0026thinsp;80%) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA extraction, cDNA synthesis and LRIG1 mRNA expression analysis\u003c/h2\u003e\n\u003cp\u003eTotal RNA was isolated from the colorectal tissue using YTzol kit (Yekta Tajhiz Azma Co, Tehran, Iran) according to the manufacturer\u0026rsquo;s protocol. cDNA was synthesized following the manufacturer\u0026rsquo;s instructions (Cinaclon Co, Tehran, Iran) and stored at -20\u0026nbsp;\u0026deg;C until analyzed. The primer sequences for \u003cem\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) and \u003cem\u003eLRIG1\u003c/em\u003e genes were designed using primer 3 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primer3.ut.ee/\u003c/span\u003e\u003c/span\u003e) and then blasted using \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/\u003c/span\u003e\u003c/span\u003e. The designed primer sequences are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The Real-time RT-PCR amplifications were conducted in a final volume of 15\u0026nbsp;\u0026micro;l reaction mixture containing 1\u0026nbsp;\u0026micro;l of cDNA, 7.5\u0026nbsp;\u0026micro;l RealQ plus 2x master mix green (Ampliqon, Denmark), 0.6\u0026nbsp;\u0026micro;l (10\u0026nbsp;\u0026micro;mol/l) of each primer and 5.3\u0026nbsp;\u0026micro;l sterilized water, using the Rotor-Gene Q System (QIAGEN Hilden, Germany). The cycling conditions were as follows: 15\u0026nbsp;min at 95\u0026nbsp;\u0026deg;C followed by 40 cycles of denaturation at 95\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s, 60\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s and 72\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s for the \u003cem\u003eLRIG1\u003c/em\u003e and also the \u003cem\u003eGAPDH\u003c/em\u003e, which was used as a normalizer. Experiments were performed in triplicates for each data point. The linear standard curve (from 0.1 to 1,000\u0026nbsp;ng) assessed by ultraviolet spectrophotometer was used for amplification efficiency determination of each primer pair. The standard curves showed good linearity and amplification (100%). The data was presented as the fold change in gene expression normalized to an endogenous reference gene relative to the controls using 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe primers for real-time quantitative reverse transcription PCR.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene name\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimer sequence\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProduct\u003c/p\u003e\n\u003cp\u003esize\u003c/p\u003e\n\u003cp\u003e(bp)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAnnealing\u003c/p\u003e\n\u003cp\u003etemperature\u003c/p\u003e\n\u003cp\u003e(\u0026deg;C)\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\u003e\u003cem\u003eLRIG1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF: CTGCATGAGTTGGTCCTGTCC\u003c/p\u003e\n\u003cp\u003eR: TGTGGCTGATGGAATTGTGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e112\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF:GCAGGGGGGAGCCAAAAGGGT\u003c/p\u003e\n\u003cp\u003eR: TGGGTGGCAGTGATGGCATGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e219\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003cem\u003eLRIG1\u003c/em\u003e: \u003cem\u003eleucine-rich repeats and immunoglobulin-like domain-1\u003c/em\u003e; \u003cem\u003eGAPDH\u003c/em\u003e: \u003cem\u003eGlyceraldehyde-3-phosphate dehydrogenase.\u003c/em\u003e\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eGraphpad Prism 8.0.2 (California Corporation, USA) and SAS computer software version 9.1 (SAS Institute Inc., Cary, NC, USA) were used to analyze the data. The Mann-Whitney U test and Kruskal-Wallis test were performed for numerical data and the Chi-square test was used to analyze the relationship between parameter data. Numerical data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences were considered as statistically significant if 𝑝 \u0026lt;0.05. The Cox proportional-hazards model was used for univariate and multivariate analyses to identify the independent prognostic factors for OS, DFS.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eLRIG1 expression and clinicopathological features\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eLRIG1\u003c/em\u003e mRNA expression\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eLRIG1\u003c/em\u003e mRNA expression was significantly down-regulated in Colorectal cancerous tissues compared with normal control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The mean of \u003cem\u003eLRIG1\u003c/em\u003e relative expression in cancerous tissues compared with normal control was 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 with a range of 0.23 to 1.2. About 40% of cancerous samples showed the relative expression\u0026thinsp;\u0026lt;\u0026thinsp;0.5 that was considered as down-regulation. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, there were no significant differences between different demographical and clinicopatological characteristics of CRC patients and \u003cem\u003eLRIG1\u003c/em\u003e expression (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eLRIG1 protein expression\u003c/h2\u003e\n\u003cp\u003eThe LRIG1 immunoreactivity was found in the cytoplasm of enterocytes as well as cancer cells of the analyzed tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe tumor LRIG1-positive rate in colorectal cancer tumors was 37.4%, which was significantly lower than that in control tissues (57.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant correlation in the expression of the LRIG1 with tumor size, tumor diameter, tumor differentiation, age, and the number of positive mesenteric lymph nodes or vascular cancer embolus (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClinicopathological variables and their correlation with immunohistochemical expression of LRIG1 in primary tumors.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eClinicopathological\u003c/p\u003e\n\u003cp\u003evariables\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCRC patients\u003c/p\u003e\n\u003cp\u003en (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eScore\u0026thinsp;\u0026lt;\u0026thinsp;30\u003c/p\u003e\n\u003cp\u003en (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eScore\u0026thinsp;\u0026ge;\u0026thinsp;30\u003c/p\u003e\n\u003cp\u003en (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep-Value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eAge\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49 (48)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25 (51.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24(49.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.451\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53 (52)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26(49.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27(50.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003ePrimary tumor location\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eColon\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73 (72)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33(45.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40(54.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.321\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRectum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29 (28)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14(48.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15(51.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eDifferentiation\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12 (12)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5(42.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7(57.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e0.461\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMid-Low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e88 (86)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39(44.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49(55.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUnknown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1(50.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1(50.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive lymph node\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49 (48)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26(53.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23(46.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.243\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eTumor diameters\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;5\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47 (46)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22(46.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25(53.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e0.763\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u0026ndash;8\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31 (30)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15(48.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16(51.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u0026ndash;10\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6(42.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8(57.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026ge;\u0026thinsp;10\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (10)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4(37.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6(62.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eVascular cancer embolus\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\u003e65 (64)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34(53.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31(46.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.382\u003c/p\u003e\n\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\u003e37 (36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19(51.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18(48.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eUnivariate and multivariable analyses of survival impact of LRIG1 expression in patients with CRC\u003c/h2\u003e\n\u003cp\u003eThe univariate and multivariate analyses were performed to investigate independent prognostic factors for OS (Overall survival) and DFS (Disease-free survival) in patients with colorectal cancer using the Cox proportional-hazards model (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The analysis results demonstrated no significant difference for parameters in terms of OS (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and DFS (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eUnivariate and multivariable analysis of prognostic indicators on overall survival and disease-free survival for the prognostic significance of LRIG1 expression of colorectal cancer patients (N\u0026thinsp;=\u0026thinsp;102).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eParameters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eOverall survival\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDisease-free survival\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\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eUnivariate analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMultivariable analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eUnivariate analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMultivariable analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR (95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR (95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR (95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR (95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ep-value\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\n\u003cp\u003e0.698 (0.376\u0026ndash;1.271)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.243\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.689 (0.303\u0026ndash;1.515)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.354\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.830 (0.453\u0026ndash;1.518)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.555\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.981 (0.439\u0026ndash;2.231)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.991\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e(\u0026ge;\u0026thinsp;57 vs\u0026thinsp;\u0026lt;\u0026thinsp;57)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGender\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.944 (0.525\u0026ndash;1.658)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.953\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.859 (0.409\u0026ndash;1.755)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.029 (0.574\u0026ndash;1.806)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.962\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.923 (0.433\u0026ndash;1.921)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.819\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e(Male vs Female)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLocation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.833 (0.454\u0026ndash;1.495)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.534\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.996 (0.449\u0026ndash;2.164)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.982\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.775 (0.421\u0026ndash;1.388)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.044 (0.477\u0026ndash;2.237)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.946\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRectum vs Colon\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTumor size\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.887 (0.490\u0026ndash;1.643)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.736\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.099 (0.512\u0026ndash;2.310)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.837\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.887 (0.489\u0026ndash;1.641)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.733\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.054 (0.471\u0026ndash;2.311)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.928\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e(\u0026ge;\u0026thinsp;5\u0026nbsp;cm vs\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026nbsp;cm)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLN metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.073 (0.588\u0026ndash;1.923)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.855\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.998 (0.448\u0026ndash;2.181)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.581\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.091 (0.604\u0026ndash;1.965)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.787\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.824 (0.364\u0026ndash;1.817)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.625\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes vs No\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVascular invasion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.694 (0.921\u0026ndash;2.932)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.101\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.171 (0.519\u0026ndash;2.629)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.718\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.566 (0.869\u0026ndash;2.782)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.148\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.289 (0.549\u0026ndash;3.068)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.564\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes vs No\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"8\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLRIG1 expression\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.252 (0.690\u0026ndash;2.260)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.473\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.826 (0.823\u0026ndash;4.004)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.151\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.077 (0.597\u0026ndash;1.904)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.831\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.451 (0.656\u0026ndash;.081)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.271\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive vs Negative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer is one of the most important and prevalent diseases with poor prognosis and there is no effective method to treat and predict the procedure of tumorigenesis. Finding the appropriate biomarkers of cancer prediction or prognosis will have huge importance in cancer management. Nowadays the new strategies searching for informative biomarkers in cancer management have attracted good attention in the world. The increasing evidences have demonstrated the leucine-rich repeats and immunoglobulin-like domain as an independent prognosis factor and predictive biomarker of clinicopathology in variety of tumors. Due to inconsistency on the effect of the LRIG1 in different types of tumors, the present study was carried out to investigate the prognostic importance of the LRIG1 expression and its relationship with clinicopathological significance in CRC. In the present study, the expression of LRIG1 at both mRNA and protein levels was significantly decreased in CRC tumors compared with normal control but, the high levels of leucine-rich repeats and immunoglobulin-like domains expression were not significantly associated with longer overall survival, which was consistent with the conclusion of subgroup analysis. These results suggested that the LRIG1 was not a prognostic marker in CRC tumors. Meanwhile, the LRIG1 expression was significantly lower in cancer tissues than normal ones and the same result was detected with no heterogeneity in subgroup analysis based on the type of tumor. The higher levels of LRIG1 expression was not also related to positive HPV status and tumor progression assessed by its association with degree of differentiation. Also, there was no association between the LRIG1 expression and lymphatic metastasis. Some genes, such as \u003cem\u003eK-ras\u003c/em\u003e and epidermal growth factor receptor (\u003cem\u003eEGFR\u003c/em\u003e) and human epidermal growth factor receptor 2 (\u003cem\u003eHER2\u003c/em\u003e) are reported to be involved in the progress and development of colorectal cancer [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], but the \u003cem\u003eLRIG1\u003c/em\u003e roles in colorectal cancer have not been well studied and remained contradictory.\u003c/p\u003e\n\u003cp\u003eSome studies showed that distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The present data revealed that there was not any correlation between LRIG1 expression and bilateral and peritoneal CRC metastasis (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and also with age, synchronous or metachronous CRC or primary tumor location (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Although, earlier studies proposed that LRIG1 expression was associated with a good prognosis in terms of overall survival (OS) and might act as a predictive factor for characteristics of cancer patients [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], whether the LRIG1 expression could predict a lower risk of CRC remains doubtful.\u003c/p\u003e"},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, our studies revealed that although \u003cem\u003eLRIG1\u003c/em\u003e was down regulated in CRC and primary tumors of CRC patients but, its expression in both mRNA and protein levels, was not clinically relevant prognostic indicator in CRC. Therefore, it is suggested that \u003cem\u003eLRIG1\u003c/em\u003e expression analyses may not be important when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eCRC: Colorectal cancer\u003c/p\u003e\n\u003cp\u003eGAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase\u003c/p\u003e\n\u003cp\u003eLRIG1: Leucine-rich repeats and immunoglobulin-like domains 1\u003c/p\u003e\n\u003cp\u003eIg: Immunoglobulin\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eOS: Overall survival\u003c/p\u003e\n\u003cp\u003eDFS: Disease-free survival\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eThis study was approved by the ethics committee of National Institute of Genetic Engineering and Biotechnology (NIGEB), IRAN (#IR.NIGEB.1395.11.10.E.). All individuals included in the study signed a consent form to use their clinical samples and personal data under the supervision of their physician.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors are not aware of any conflict of interest.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; contributions\u003c/h3\u003e\n\u003cp\u003eMB, acquisition of data and draft the work; MS, conception and designing of the work, analysis and interpretation of data, substantively revise the work; SAA, contributes to the design of the work; FM, contributes to the design of the work and data interpretation; TM, acquisition and analysis of the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgments\u003c/h3\u003e\n\u003cp\u003eThe authors would like to thank all the patients who participated in this study, Dr. B. Mahjoubi and the National Institute of Genetic Engineering and Biotechnology for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68:394-424.\u003c/li\u003e\n\u003cli\u003eSiegel R, Desantis C, Jemal A. Colorectal cancer statistics. CA Cancer J Clin. 2014; 64:104\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eFeng Q, Pei W, Zheng ZX, Bi JJ, Yuan XH. Clinicopathologic characteristics and prognostic factors of 63 gastric cancer patients with metachronous ovarian metastasis. 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Cytoplasmic LRIG2 expression is associated with poor oligodendroglioma patient survival. Neuropathol\u0026shy;ogy. 2009; 29: 242\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eSegatto O, Anastasi S, Alema S. Regulation of epidermal growth factor receptor signalling by inducible feedback inhibitors. J Cell Sci. 2011; 124: 1785e1793.\u003c/li\u003e\n\u003cli\u003eGur G, Rubin C, Katz M, Amit I, Citri A, Nilsson J, Amariglio N, Henriksson R, Rechavi G, Hedman H, Wides R, Yarden Y. LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. EMBO J. 2004; 23: 3270e3281.\u003c/li\u003e\n\u003cli\u003eLaederich MB, Funes-Duran M, Yen L, Ingalla E, Wu X, Carraway KL. 3rd, Sweeney C: The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J Biol Chem. 2004; 279: Jensen 47050e47056.\u003c/li\u003e\n\u003cli\u003eJensen KB, Watt FM. Single-cell expression profiling of human epidermal stem and transit-amplifying cells: lrig1 is a regulator of stem cell quiescence. Proc Natl Acad Sci U S A. 2006; 103: 11958e11963.\u003c/li\u003e\n\u003cli\u003eJensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S, Watt FM. Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell. 2009; 4: 427e439.\u003c/li\u003e\n\u003cli\u003ePowell AE. Lrig1\u0026rlm; gastric isthmal progenitor cells restore normal gastric lineage cells during damage recovery in adult mouse stomach. Gut. 2018; 67: 1595e1605.\u003c/li\u003e\n\u003cli\u003eChoi E, Lantz TL, Vlacich G, Keeley TM, Samuelson L.C., Coffey, R.J., Goldenring JR, \u003ca href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815959/\"\u003eLrig1+ gastric isthmal progenitor cells restore normal gastric lineage cells during damage recovery in adult mouse stomach\u003c/a\u003e. Gut. 2018; 67: 1595\u0026ndash;1605.\u003c/li\u003e\n\u003cli\u003eLeeWS, Jeong Heum B, Jung Nam L, Woon Kee L. Mutations in K-ras and epidermal growth factor receptor expression in Korean patients with stages III and IV colorectal Cancer. Int J Surg Pathol. 2011; 19: 145\u0026ndash;151.\u003c/li\u003e\n\u003cli\u003eCappuzzo F, Finocchiaro G, Rossi E, Janne PA, Carnaghi C, Calandri C, et al. EGFR FISH assay predicts for response to cetuximab in chemotherapy refractory colorectal cancer patients. Ann Oncol. 2007; 19: 717\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eRego RL, Foster NR, Smyrk TC, Le M, O'Connell MJ, Sargent DJ, et al. Prognostic effect of activated EGFR expression in human colon carcinomas: comparison with EGFR status. Br J Cancer. 2010; 102: 165\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eSawada K, Nakamura Y, Yamanaka T, Kuboki Y, Yamaguchi D, Yuki S, et al. Prognostic and predictive value of HER2 amplification in patients with metastatic colorectal Cancer. 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PLoS One. 2016; 1: e0151865.\u003c/li\u003e\n\u003cli\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Zhang+Q\u0026amp;cauthor_id=30123358\"\u003eZhang\u003c/a\u003e Q ,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Shi+W\u0026amp;cauthor_id=30123358\"\u003e Shi\u003c/a\u003eW,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Wang+Q\u0026amp;cauthor_id=30123358\"\u003eWang\u003c/a\u003e Q,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Zhu+Y\u0026amp;cauthor_id=30123358\"\u003e Zhu\u003c/a\u003e Y,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Zhai+C\u0026amp;cauthor_id=30123358\"\u003e Zhai\u003c/a\u003e C,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Wang+J\u0026amp;cauthor_id=30123358\"\u003e Wang\u003c/a\u003e\u0026nbsp;J,\u0026nbsp;et al. Clinicopathological and Prognostic Significance of Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1 (LRIG1) in Malignant Tumors: A Meta-Analysis. 2018; 9: 2895-2909\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prognostic indicator, colorectal cancer, CRC, LRIG1, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-118977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-118977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003eTo make the right treatment decisions about colorectal cancer (CRC) patients reliable predictive and prognostic data are needed. However, in many cases this data is not enough. Some studies suggest that \u003cem\u003eLRIG1\u003c/em\u003e gene (\u003cem\u003eleucine-rich repeats and immunoglobulin-like domains1\u003c/em\u003e) has prognostic implications in different kinds of cancers. \u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eOne hundred and two patients with colorectal cancer were retrospectively analyzed for LRIG1 expression at both mRNA and protein levels. SYBR Green Real-Time RT-PCR technique was used for mRNA expression analyses and \u003cem\u003eGlyceraldehyde-3-Phosphate Dehydrogenase\u003c/em\u003e gene (\u003cem\u003eGAPDH\u003c/em\u003e) was considered as a reference gene for data normalization. LRIG1 protein expression was analyzed using Immunohistochemistry (IHC). Additionally, appropriate statistic analyses were used to assess the expression of \u003cem\u003eLRIG1\u003c/em\u003e in test and control groups. The prognostic significance of LRIG1\u003cem\u003e \u003c/em\u003eexpression was analyzed using the univariate and multivariate analyses.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eThe data revealed that the expression of\u003cem\u003e LRIG1\u003c/em\u003e in both mRNA and protein levels was down regulated in colorectal tumor tissues (P\u0026lt;0.01) but is not clinically relevant prognostic indicator in CRC. \u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eTherefore, it is suggested that \u003cem\u003eLRIG1\u003c/em\u003e expression analyses may not be considered as an important issue when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.\u003c/p\u003e","manuscriptTitle":"LRIG1 Expression and Colorectal Cancer Prognosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-07 17:00:51","doi":"10.21203/rs.3.rs-118977/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2020-12-02T21:03:10+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2020-12-02T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2c64c17-ab1f-46c9-9e7a-79bf656554de","owner":[],"postedDate":"December 7th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1355609,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-01-26T15:04:08+00:00","versionOfRecord":{"articleIdentity":"rs-118977","link":"https://doi.org/10.1186/s12920-020-00846-2","journal":{"identity":"bmc-medical-genomics","isVorOnly":false,"title":"BMC Medical Genomics"},"publishedOn":"2021-01-18 15:01:55","publishedOnDateReadable":"January 18th, 2021"},"versionCreatedAt":"2020-12-07 17:00:51","video":"","vorDoi":"10.1186/s12920-020-00846-2","vorDoiUrl":"https://doi.org/10.1186/s12920-020-00846-2","workflowStages":[]},"version":"v1","identity":"rs-118977","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-118977","identity":"rs-118977","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}