LRIG1 Modulated Radioresistance of Glioma via Regulating CTLA-4/AKT Signaling Pathway

LRIG1 Modulated Radioresistance of Glioma via Regulating CTLA-4/AKT Signaling Pathway | 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 LRIG1 Modulated Radioresistance of Glioma via Regulating CTLA-4/AKT Signaling Pathway Shiqi Cheng, Xiangqun Huang, Raorao Yuan, Yan Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-131093/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 Background : Radioresistance has a great impact on prognosis of glioma patients. However, the potential mechanism underlying the radioresistance of glioma cells remains largely unknown. Methods : LRIG1 overexpression model was firstly established by using Flag-LRIG1 plasmid. The expression of LRIG1, CTLA-4 proteins were detected by western blot and IHC in cells and human tissue. Real-time PCR was used for deterring mRNA expression. Cell viability and apoptosis were detected using CCK-8 and Annexin-V/propidium iodide (PI), respectively. Co-Immunoprecipitation was used for detecting the combination of LRIG1 and CTLA-4 proteins. Results : LRIG1 was significantly down-regulated in radioresistant glioma cells. Overexpressed LRIG1 could promote the radiosensitivity of glioma cells, meanwhile, inhibit the expression of p-AKT and CTLA-4 protein in radioresistant glioma cells. Furthermore, LRIG1 combined with CTLA-4 and promoted CTLA-4 degradation. In human glioma tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed in glioma tissue. Finally, correlation analysis showed that the expression of LRIG1 was negatively correlated with expression of CTLA-4 and radioresistance of glioma patients. Conclusion : Our findings demonstrated that LRIG1 facilitates radioresistance glioma cells by regulating CTLA4 /AKT signaling pathway. Oncology LRIG1 glioma radioresistance CTLA4 AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Glioma is considered to be one of the most commonly-diagnosed intracranial tumor of the central nervous system, representing 81% of malignant brain tumors in adult [ 1 , 2 ] . Some subtypes of glioma, e.g. glioblastoma, cause significant mortality [ 3 ] . In recent years, the therapeutic interventions for treating glioma are surgery combined with chemotherapy and/or radiotherapy, which provide benefit to patients due to their different merits [ 4 ] . However, even following proper surgical resection, many tumors show a high resistance to irradiation therapy, further cause highly recurrence rate and poor prognosis for glioma patients [ 5 ] . Thus, the potential mechanism underlying resistance to irradiation therapy is urgently needed. Radioresistance of glioma cells has an important effect on prognosis of glioma patients. In numerous clinical studies, improvement of radiosensitivity of tumor cells, e.g., combining PARP inhibitors, using hyperbaric oxygenation, could significantly improve survival outcomes of glioma patients [ 6 , 7 ] . However, due to the heterogeneity of tumor cells, i.e the different self-renewal capacities of each cell population, the radioresistance remains a big challenge in clinical practice [ 8 ] . So far, multiple factors have been identified responsible for radioresistance of tumor cells, e.g. Wnt. [ 9 ] , β-Catenin [ 10 ] , AKT [ 11 ] , STAT3 [ 12 ] , however, a detailed, integrative picture of the molecular network has not yet clarified. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1), a type 1 transmembrane protein whose extracellular domain contains 15 leucine-rich repeats (LRRs) and three immunoglobulin (Ig)-like domains, was firstly cloned by Hedman et al in 2002 [ 13 ] . The gene of the LRIG1 localized in chromosome band 3p14.3, and the protein is well-known as a cell transmembrane protein that widely expressed in human cells [ 14 ] . Over decades, accumulating evidence has suggested that LRIG1 might act as a tumour suppressor, for its expression usually correlates with better patient survival in several human cancers including prostate, bladder, breast, cervical, colon, cervical, non-small-cell lung cancers and gliomas [ 15 – 19 ] . For molecular mechanism, two studies from independent groups [ 20 , 21 ] demonstrated that LRIG1 negatively regulates all four members of the ERBB receptor family (ERBB1/EGFR, ERBB2/HER2/Neu, ERBB3/HER3, and ERBB4/HER4) by heterologous expression. LRIG1 dramatically reduce receptor levels by promoting receptor ubiquitylation and lysosomal degradation [ 22 ] . In addition to ERBBs, LRIG1 also affected other oncogenic signaling molecules, e.g. mutant EGFR (EGFRvIII) [ 23 ] , Stat3 [ 24 ] and TNFα27 [ 25 ] . Despite the large amount of knowledge on LRIG1 in many tumor tissues, little is known about its role in glioma radioresistance. In fact, few papers have been reported that LRIG1 could improve the radiosensitivity of glioma cells by attenuating EGFR/Akt signaling pathway [ 26 ] , or its impression could affect by certain microRNAs, e.g. microRNA-590-3p [ 27 ] , miR-183 [ 28 ] . In order to have a better understanding of LRIG1 and its down-stream factors on glioma radioresistance, in the present study, we tried to further investigate the potential role of LRIG1 in glioma radioresistance and its underlying mechanisms, moreover, the interaction between LRIG1 and cytotoxic T lymphocyte antigen 4 (CTLA-4) / AKT pathway in glioma radioresistance will also be clarified. Our study might set a novel sight into pathologic mechanism of glioma and may provide therapeutic strategies in treating this disease. 2 Materials And Methods 2.1 Cell culture and treatment Human glioma cell line U251 was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). U251 cells were cultured in a standard humidified incubator (5% CO 2 ; 37 °C) with RPMI-1640 (Invitrogen, Carlsbad, CA, USA) culture medium which supplemented with 10% fetal bovine serum (FBS, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). For treatment, as previously described [28] , briefly, U251 cells were irradiated by a linear accelerator 6-MV X-rays with the dose rate of 4 Gy/min. The procedure was performed daily for 5 months, starting with 1 Gy/fraction and ending with 10 Gy/fraction. The X-ray treated U251 cells were labeled as U251R cells. 2.2 Quantitative real time-PCR (qRT-PCR) Total RNA was isolated from glioma cells with an RNA extraction kit (Takara Biotechnology, Japan). Then, Reverse Transcription Kit (Takara Biotechnology, Japan) was used to obtain cDNA and reverse transcribe RNA. The primers used in this study were synthesized by Yingjun Technology (Shanghai, China) which showed in Table 1. qRT-PCR was conducted using the SYBR Green Realtime PCR Master Mix (Toyobo, Japan) and performed on ABI 7900 fast Real‐time PCR Systems (Applied Biosystems, USA). GAPDH were used as internal controls. Table 1 Primers used in the present study Gene Primer sequence LRIG1 forward: 5′-GAAAAGGGACTCTGGTTGGGAT-3′ reverse: 5′-AGGAAGTCATCGCACACGAA-3′ CTLA-4 forward: 5′-AGGTGACTGAAGTCTGTGCG-3′ reverse: 5′-CATGAGCTCCACCTTGCAGA-3′ GAPDH forward: 5'-CCGGGAAACTGTGGCGTGATGG-3' reverse: 5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3' 2.3 Western blot Total proteins from human tissue or glioma cells were extracted by using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein samples were separated using 8% SDS-PAGE. After transferred onto PVDF membrane, the primary antibodies were added: anti-LRIG1 (PA5-52860, 1:500, Invitrogen, Carlsbad, CA, USA), anti-Akt (ab235958, 1:1000, Abcam, UK), anti-Akt (phospho S473) (ab81283, 1:1000, Abcam, UK), anti-CTLA-4(PA5-47547, 1:250, Invitrogen, Carlsbad, CA, USA), anti-Tubulin (ab210797, 1:1000, Abcam, UK). Tubulin served as internal control. After 24 hours of incubation, membrane was incubated along with secondary antibody immunoglobulin G (IgG) at room temperature for 2 hours. 2.4 Cell transfection The LRIG1 was overexpressed or knockdown by using LRIG1 overexpression plasmid (Flag- LRIG1) or Short hairpin RNAs (shRNA) targeting LRIG1 (sh-LRIG1), respectively. The plasmid or empty vectors were purchased from GeneChem Corporation (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection following manufacturer’s instructions. 2.5 CCK-8 assay Transfected U251 cells or U251R cells (2 × 10 5 per well) were seeded into a 96-well plate. CCK-8 Kit (Abcam, UK) and RPMI-1640 were added into each well. After incubation with CCK-8 solution for 2 hours, optical density of cells was measured at 450 nm at different time points by using micro-plate reader (Thermo Fisher, USA). 2.6 Flow cytometry assay Cell apoptosis was detected using Annexin-V-propidium iodide (PI) apoptosis assay. Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA, USA) was used according to the manufacture’s instruction. Briefly, different groups of transfected U251R cells were with propidium iodide (PI) at 37°C for 30min, followed by flow cytometry (Beckman Coulter FC500, CA, USA). 2.7 Co-Immunoprecipitation Co-Immunoprecipitation was performed to detect the combination of LRIG1 and CTLA-4. Briefly, cells were lysed in 500 μl co-IP buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri, USA). Then, 20 μl immobilized protein A/G beads was incubated with cell lysates for 1h at 4 °C. Primary antibody or control Ig G were incubated with lysates for 24 hours. Finally, immobilized protein A/G beads were added, then, the proteins were prepared for western blot. 2.8 Human glioma tissue collection Tissue samples of paired glioma tissues and paracancerous tissues were collected from patients who underwent glioma resection at the second affiliated hospital of Nanchang university. All patients enrolled in this study have given their informed written consents prior to conduct the clinical research related procedure and this study was approved by the second affiliated hospital of Nanchang university Ethical Committee (No. 2019003). All patients have received 5 weeks’ radiation therapy. Tissue samples were collected and immediately snap-frozen in liquid nitrogen after surgery (-80°C) for further use. 2.9 Immunohistochemistry (IHC) The expression of LRIG1 and CTLA-4 proteins was determined in human glioma tissues by IHC. Briefly, paraffin‐embedded sections were dewaxed and dehydrated by xylene and ethanol. Then, after incubation with 50 μl of 10% goat serum for 60min, the sections were probed with primary antibodies: anti-LRIG1 (ab197985, 1:1000, Abcam, UK), anti-CTLA-4 (ab227709, 1:1000, Abcam, UK) for 24 hours. Then, peroxidase‐labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) were added and incubated for 30min. The sections were developed using diaminobenzidine (DAB). 2.10 Statistical analysis All cell experiments were repeated triplicate. The data were exhibited as Mean ± SD. SPSS21.0 (IBM Corp. Armonk, NY) software was used for data analysis. Two-tailed Student's t-test, one-way ANOVA, Pearson correlation coefficient were used in this study. P < 0.05 was considered as statistically significant difference. 3 Results 3.1 LRIG1 was down-regulated in radioresistant glioma cells Firstly, the expression of LRIG1 mRNA was determined in U251 cell or radioresistance U251 cells (U251R) by using qRT-PCR. As shown in Fig. 1A, the expression of LRIG1 mRNA in U251R cells was significantly decreased compared with that in U251 cells ( P <0.05). Subsequently, LRIG1 protein expression in both groups was detected by western blot. Result demonstrated that in U251R cells, the level of LRIG1 protein was much lower compared with that in U251 cells (Fig. 1B). These data suggested that in both mRNA and protein levels, LRIG1 was down-regulated in radioresistant glioma cells. 3.2 Overexpressed LRIG1 increased radiosensitivity of glioma cells To explore the potential role of LRIG1 in radioresistant glioma cells, we firstly used the plasmid to overexpress LRIG1 in U251R cells. Results revealed that LRIG1 protein was highly expressed in Flag-LRIG1 group compared with NC group (Fig. 2A), suggesting that the LRIG1 overexpressed system can be used in further studies. Next, the effect of LRIG1 on cell phenotypes were detected by CCK-8 assay and Flow cytometry assay. We found that the expression of LRIG1 significantly inhibited the cell proliferation (Fig. 2B; P <0.01), meanwhile, in U251R cells, the apoptosis of cells was promoted by overexpression of LRIG1 proteins (Fig. 2C). To sum up, the above results provide the evidence that overexpressed LRIG1 increased radiosensitivity of glioma cells. 3.3 Overexpressed LRIG1 inhibited the expression of p-AKT in U251R cells To further validate the mechanisms of LRIG1 in radioresistant glioma cells, the expression of many proteins was determined in different groups by western blot. First, the expression of LRIG1, p-AKT, AKT proteins was detected in U251 cells or U251R cells. As shown in Fig. 3A, in radioresistant glioma cells, the expression of p-AKT was highly expressed, suggesting that p-AKT was activated. Then, the expression of LRIG1, p-AKT, AKT proteins was also measured in either overexpressed LRIG1 group or NC group. Result demonstrated that the activated p-AKT can be inhibited by overexpression of LRIG1 in U251R cells (Fig 3B). 3.4 Overexpressed LRIG1 inhibited the expression of CTLA-4 protein in U251R cells Subsequently, the effect of overexpression of LRIG1 on CTLA-4 expression was analyzed in U251R cells. We found that overexpression of LRIG1 dramatically suppressed the protein level of CTLA-4 in U251R cells (Fig. 4A). However, in mRNA level, overexpression of LRIG1 was not able to affect the expression of CTLA-4 mRNA (Fig. 4B). These data indicated that LRIG1 only have effect on CTLA-4 post-translational modification, while not change the mRNA level of CTLA-4. 3.5 LRIG1 combined with CTLA-4, and promoted CTLA-4 degradation Furthermore, the relationship between LRIG1 and CTLA-4 was identified by conducting Co-immunoprecipitation and western blot. As shown in Fig 5A, coimmunoprecipitated proteins were analyzed by western blot with indicated antibodies. Results showed that endogenous LRIG1 protein was immunoprecipitated with anti-CTLA-4, meanwhile, CTLA-4 also combined with LRIG1. Moreover, MG132 was used to inhibit the proteasome in each group. In Fig. 5B, results demonstrated that by blocking proteasome, overexpression or knockdown of LRIG1 did not have any effect on expression of CTLA-4 protein in U251R cells. These data indicated that LRIG1 combined with CTLA-4, and the same time, promoted CTLA-4 degradation. 3.6 LRIG1 was down-regulated, while CTLA-4 was highly expressed in glioma tissue Additionally, we had performed experiments on human glioma tissue. The expression of LRIG1 and CTLA-4 protein was detected in human glioma tissue by IHC and western blot. As shown in Fig. 6A, compared with paracancerous tissue, the expression level of LRIG1 was much lower. On the contrary, CTLA-4 was highly expressed in glioma tissue. To better quantify the protein expression, western blot was performed in each group. In tumor tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed compared with paracancerous tissue（Fig. 6B）. 3.7 The expression of LRIG1 was negatively correlated with expression of CTLA-4 and radioresistance of glioma patients Finally, the correlation analysis was performed to better clarify the relationship between LRIG1 and CTLA-4. In Fig 7A, the correlation analysis between expression of LRIG1 and radioresistance of glioma was performed. It was shown that the expression of LRIG1 was negatively correlated with radioresistance of glioma. However, the expression of CTLA-4 was positively correlated with radioresistance of glioma (Fig. 7B). Furthermore, result in Fig 7C showed that the expression of LRIG1 was negatively correlated with expression of CTLA-4 in glioma patients. These data indicated that LRIG1 may have positive effect on glioma in clinic. 4 Discussion Gliomas make up the largest proportion of malignant brain tumors with very limited treatment strategies. Radiotherapy seems to be the most effective nonsurgical treatment for gliomas, however, its efficacy is severely suppressed due to the high intrinsic radioresistance of glioma cells [ 29 ] . Thus, to explore the potential mechanisam underlying gliomas radioresistance is critical to design novel strategies and improve the prognosis of glioma patients. In our study, we firstly found that LRIG1 was down-regulated in radioresistant glioma cells in both mRNA and proteins levels. Due to the fact that expression of LRIG1 can be affected by irradiation, we further established LRIG1 overexpression system in radioresistant glioma cells (U251R) for further studies. The biological effects of irradiation on cells mainly due to DNA lesions, e.g., disruption of the phosphate DNA backbone [ 30 ] . The DNA lesions can be direct caused by interactions between particles or indirect following interactions with reactive oxygen species (ROS) generated by cell water ionization [ 31 ] . These breaks can be either repaired or can lead to cell cycle arrest. As such, we can observe the effect of irradiation which is manifested as viability or apoptosis of tumor cells. Results from our study showed that in radioresistant glioma cells, overexpressed LRIG1 dramatically inhibited the cell proliferation and promote cell apoptosis, indicating that LRIG1 act as a tumor suppressor in radioresistant glioma cells. As an important cell transmembrane protein, LRIG1 was widely studied in gliomas. For instance, a study includes 404 patients with gliomas showed that expression of LRIG1 positively correlated with the prognosis of glioma patients and negatively correlated with WHO histological grade [ 32 ] . Moreover, in Ye et al ’s study, after clinical surgical resections of astrocytoma tumor tissue, the results showed that LRIG1 expression was significantly decreased in tumor tissue compared to the corresponding surrounding non-tumor tissues [ 18 ] . In molecular level, the underlying mechanism of LRIG1 on glioma radioresistance was further clarified in our studies. We found that in radioresistant glioma cells, overexpression of LRIG1 could inhibit the expression of phosphorylated AKT (p-AKT), but not AKT, suggesting that LRIG1 might play a role in AKT activation. AKT is a serine/threonine kinase which can be activated by translocation to the plasma membrane or phosphorylation [ 33 ] . Activation of AKT, i.e, high level of p-AKT has been reported to correlate with a poor prognosis for patients with glioma [ 34 ] . AKT-related pathways have been also widely studied in molecular level in the oncogenic and development of cancers cells [ 35 – 37 ] . Previously in our group, we found that the glioblastoma radioresistance can be promoted by overexpressed miR-183, further, expression of LRIG1 and activation of EFGR/Akt pathway can be regulated by miR-183 [ 28 ] . In the present study, we further clarified that overexpression of LRIG1 inhibited the expression of CTLA-4 protein, but not CTLA-4 mRNA. In addition, results also confirmed that LRIG1 could combined with CTLA-4, and promoted CTLA-4 degradation. CTLA-4, known as a member of a family of Immunoglobulin-related receptors, has been confirmed play a key role in tumorigenesis [ 38 ] . For its machoism, CTLA-4 can bind to phosphatidylinositol 3-kinase (PI3K) as well as phosphatases PP2A and SHP-2, which considered to be the key factors in AKT-related pathway [ 39 , 40 ] . However, whether CTLA-4 involved in the resistant to irradiation was still un-documented. To the best of our knowledge, our study was the first one to clarify the relationship between LRIG1 and CTLA4/PI3K/AKT pathway in glioma radioresistance. We found that in human glioma tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed. By conducting correlation analysis, results shown that the expression of LRIG1 was negatively correlated with radioresistance of glioma and expression of CTLA-4. However, the expression of CTLA-4 was positively correlated with radioresistance of glioma. To sum up, our data provided information that LRIG1 could increase the radiosensitivity of glioma cells, overexpressed LRIG1 had a significantly impact on activation of AKT and CTLA-4 expression. These findings provided a novel insight into the mechanism of radiotherapy in glioma and could be the potential targets for treating gliomas in the future. Declarations Competing interests The authors declare that they have no financial or other conflicts of interest. Acknowledgments This study has received funding from the Educational department foundation of Jiangxi Province, China (Grant No. GJJ190145) Funding No applicable. Availability of data and materials The data used and analyzed in this paper are available from the corresponding author under reasonable request. Authors' contributions SQ and XQ wrote the manuscript and performed most of the experiments. RR participated in the study data acquisition and analysis. YZ edited and revised final manuscript. Ethics approval and consent to participate Ethical approval was obtained from the Biomedical Ethics Committee of The Second Affiliated Hospital of Nanchang University. Consent for publication Patients provided their consent for publication. References Ostrom, Q.T., et al., The epidemiology of glioma in adults: a "state of the science" review. Neuro Oncol, 2014. 16(7): p. 896-913. Ostrom, Q.T., et al., Epidemiology of gliomas. Cancer Treat Res, 2015. 163: p. 1-14. Omuro, A. and L.M. DeAngelis, Glioblastoma and other malignant gliomas: a clinical review. Jama, 2013. 310(17): p. 1842-50. 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Science, 1998. 282(5397): p. 2263-6. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-131093","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":6695491,"identity":"4a3b9954-2e29-4615-b303-cb0dc4f3f429","order_by":0,"name":"Shiqi Cheng","email":"","orcid":"https://orcid.org/0000-0003-1984-4169","institution":"The second affiliated hospital of Nanchang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shiqi","middleName":"","lastName":"Cheng","suffix":""},{"id":6695492,"identity":"6227e988-01b8-40ba-87a8-c354d091fada","order_by":1,"name":"Xiangqun Huang","email":"","orcid":"","institution":"Nanchang University Second Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangqun","middleName":"","lastName":"Huang","suffix":""},{"id":6695493,"identity":"68563aa8-0da0-4c98-bf4d-9c3107eb3277","order_by":2,"name":"Raorao Yuan","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Raorao","middleName":"","lastName":"Yuan","suffix":""},{"id":6695494,"identity":"e014c818-d257-42a2-8a1d-c43691d495f0","order_by":3,"name":"Yan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDCCAxAqAcq1AWLmBrgwDi2MDUha0hgY2BhJ03KYsBa+483PH3zcU5fHP7v9msTPHeflDO43Nj74cIZBnl8Muz7JM8cMG2c8YyuWuHOmTLL3zG1jg2OMzYYzbjAYzpydgFWLwY0cxmaeAzyJDTdy0iR4224nbjjG2CbN84EhweA2Xi0SifOBWiT/tp0jWotB4oYb6cekedsOQLXcwK0F5JeZMw4kJG68kcNsLduWbCx5LBHolzMSOP0CDLEHHz4cqEucdyP94c23bXZyfIcPH3zw4ZiNPL80di1IgMdEAokngVMdEmB//IEYZaNgFIyCUTDyAACYQm4AMJ0UswAAAABJRU5ErkJggg==","orcid":"","institution":"The second affiliated hospital of Nanchang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2020-12-17 23:02:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-131093/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-131093/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4420595,"identity":"1cc17368-d53c-4c87-8e44-e214e3ed0ef8","added_by":"auto","created_at":"2020-12-21 18:38:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25951,"visible":true,"origin":"","legend":"The expression of LRIG1 in radioresistant glioma cells.\n(A) The relative expression level of LRIG1 mRNA in U251 and U251R cells were\ndetermined by qRT-PCR. (B) The expression of LRIG1 protein in U251 and U251R cells\nwere checked by western blot. Tublin served as internal control. Data were expressed as mean\n± SD. (*P \u003c 0.05).","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/86e4eea830a2c490ab76463a.jpg"},{"id":4420597,"identity":"64928cc2-c4a3-4ef8-8328-c733e115077c","added_by":"auto","created_at":"2020-12-21 18:38:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69800,"visible":true,"origin":"","legend":"Effects of LRIG1 on radioresistance of glioma cells.\n(A) The expression of LRIG1 protein in LRIG1 overexpressed or control group were\ndetermined by western blot. (B) The effect of overexpressed LRIG1 on cell viability were\ndetected by CCK-8 assay. (C) The cell apoptosis of glioma cells was determined in LRIG1\noverexpressed or control group by flow cytometry assay. Data were expressed as mean ± SD.\n(**P ＜ 0.01, NC vs p-LRIG1; NC + X ray vs p-LRIG1 + X ray)","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/39c7eb3feb6bff70630fc158.jpg"},{"id":4420706,"identity":"e36fd169-1e58-4ff8-9a3a-55fa68444f0b","added_by":"auto","created_at":"2020-12-21 18:41:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31665,"visible":true,"origin":"","legend":"Effects of LRIG1 on expression of p-AKT and AKT in glioma cells.\n(A) The expression of protein levels of LRIG1, p-AKT, AKT in U251 and U251R cells were\ndetermined by western blot. (B) The expression of protein levels of LRIG1, p-AKT, AKT in\nLRIG1 overexpressed or control group were determined by western blot.","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/14d99760bcc2154d39317dbb.jpg"},{"id":4421208,"identity":"fe644956-cddb-41f1-9d78-bd394bd02cbc","added_by":"auto","created_at":"2020-12-21 18:47:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33370,"visible":true,"origin":"","legend":"Effects of LRIG1 on expression of CTLA-4 in U251R cells.\nThe effect of overexpressed LRIG1 on expression of CTLA-4 protein (A) and CTLA-4\nmRNA (B) were determined by western blot and qRT-PCR, respectively. Data were expressed\nas mean ± SD. (** P \u003c 0.01 vs NC; NS, P \u003e 0.05 vs NC)","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/4ef86b34d01d75e9d9a8f971.jpg"},{"id":4420596,"identity":"05e5e3a0-cae5-4fa2-b07a-56f1cf7d929e","added_by":"auto","created_at":"2020-12-21 18:38:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37411,"visible":true,"origin":"","legend":"LRIG1 affects the degradation of CTLA-4 protein.\n(A) The binding of LRIG1 to CTLA-4 protein was determined by immunoprecipitation assay.\n(B) The effect of LRIG1 on the expression of CTLA-4 protein was determined by western\nblot after blocking the ubiquitin-proteasome.","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/e59763cba864747142c54ebd.jpg"},{"id":4420967,"identity":"2ab4a357-bfb1-4411-bfe6-485fa9c84193","added_by":"auto","created_at":"2020-12-21 18:44:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70003,"visible":true,"origin":"","legend":"The expression of LRIG1 and CTLA-4 in glioma tissue.\nThe expression of LRIG1 and CTLA-4 were detected in tumor tissue or paracancerous tissue\nby immunochemistry (A) and western blot (B), respectively.","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/6b8714decd20f741a98b2683.jpg"},{"id":4420601,"identity":"c7d80242-6ea5-406d-81d8-fccbc87be228","added_by":"auto","created_at":"2020-12-21 18:38:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":56493,"visible":true,"origin":"","legend":"Correlation analysis of LRIG1, CTLA-4 and radioresistance of glioma.\nCorrelation analysis between LRIG1 and radioresistance of glioma (A), CTLA-4 and\nradioresistance of glioma (B), LRIG1 and CTLA-4 were conducted by Pearson correlation\nanalysis.","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/ba51959df7b399da04d996a6.jpg"},{"id":13639288,"identity":"3aca8b39-9022-471d-bb1a-8f9a8298c6e7","added_by":"auto","created_at":"2021-09-17 08:55:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":668889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-131093/v1/4b603088-ab99-4e7f-b3de-621d2f6fe63c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eLRIG1 Modulated Radioresistance of Glioma via Regulating CTLA-4/AKT Signaling Pathway\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGlioma is considered to be one of the most commonly-diagnosed intracranial tumor of the central nervous system, representing 81% of malignant brain tumors in adult \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Some subtypes of glioma, e.g. glioblastoma, cause significant mortality \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In recent years, the therapeutic interventions for treating glioma are surgery combined with chemotherapy and/or radiotherapy, which provide benefit to patients due to their different merits \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, even following proper surgical resection, many tumors show a high resistance to irradiation therapy, further cause highly recurrence rate and poor prognosis for glioma patients \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Thus, the potential mechanism underlying resistance to irradiation therapy is urgently needed.\u003c/p\u003e\n\u003cp\u003eRadioresistance of glioma cells has an important effect on prognosis of glioma patients. In numerous clinical studies, improvement of radiosensitivity of tumor cells, e.g., combining PARP inhibitors, using hyperbaric oxygenation, could significantly improve survival outcomes of glioma patients \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. However, due to the heterogeneity of tumor cells, i.e the different self-renewal capacities of each cell population, the radioresistance remains a big challenge in clinical practice \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. So far, multiple factors have been identified responsible for radioresistance of tumor cells, e.g. Wnt.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, \u0026beta;-Catenin\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, AKT \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, STAT3 \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, however, a detailed, integrative picture of the molecular network has not yet clarified.\u003c/p\u003e\n\u003cp\u003eLeucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1), a type 1 transmembrane protein whose extracellular domain contains 15 leucine-rich repeats (LRRs) and three immunoglobulin (Ig)-like domains, was firstly cloned by Hedman \u003cem\u003eet al\u003c/em\u003e in 2002 \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The gene of the LRIG1 localized in chromosome band 3p14.3, and the protein is well-known as a cell transmembrane protein that widely expressed in human cells \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Over decades, accumulating evidence has suggested that LRIG1 might act as a tumour suppressor, for its expression usually correlates with better patient survival in several human cancers including prostate, bladder, breast, cervical, colon, cervical, non-small-cell lung cancers and gliomas \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. For molecular mechanism, two studies from independent groups \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e demonstrated that LRIG1 negatively regulates all four members of the ERBB receptor family (ERBB1/EGFR, ERBB2/HER2/Neu, ERBB3/HER3, and ERBB4/HER4) by heterologous expression. LRIG1 dramatically reduce receptor levels by promoting receptor ubiquitylation and lysosomal degradation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In addition to ERBBs, LRIG1 also affected other oncogenic signaling molecules, e.g. mutant EGFR (EGFRvIII)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, Stat3\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e and TNF\u0026alpha;27\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDespite the large amount of knowledge on LRIG1 in many tumor tissues, little is known about its role in glioma radioresistance. In fact, few papers have been reported that LRIG1 could improve the radiosensitivity of glioma cells by attenuating EGFR/Akt signaling pathway\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, or its impression could affect by certain microRNAs, e.g. microRNA-590-3p \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, miR-183\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In order to have a better understanding of LRIG1 and its down-stream factors on glioma radioresistance, in the present study, we tried to further investigate the potential role of LRIG1 in glioma radioresistance and its underlying mechanisms, moreover, the interaction between LRIG1 and cytotoxic T lymphocyte antigen 4 (CTLA-4) / AKT pathway in glioma radioresistance will also be clarified. Our study might set a novel sight into pathologic mechanism of glioma and may provide therapeutic strategies in treating this disease.\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 \u003c/strong\u003e\u003cstrong\u003eCell culture and treatment \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman glioma cell line U251 was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). U251 cells were cultured in a standard humidified incubator (5% CO\u003csub\u003e2\u003c/sub\u003e; 37\u0026thinsp;\u0026deg;C) with RPMI-1640 (Invitrogen, Carlsbad, CA, USA) culture medium which supplemented with 10% fetal bovine serum (FBS, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n\u003cp\u003eFor treatment, as previously described \u003csup\u003e[28]\u003c/sup\u003e, briefly, U251 cells were irradiated by a linear accelerator 6-MV X-rays with the dose rate of 4\u0026thinsp;Gy/min. The procedure was performed daily for 5 months, starting with 1\u0026thinsp;Gy/fraction and ending with 10\u0026thinsp;Gy/fraction. The X-ray treated U251 cells were labeled as U251R cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 \u003c/strong\u003e\u003cstrong\u003eQuantitative real time-PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from glioma cells with an RNA extraction kit (Takara Biotechnology, Japan). Then, Reverse Transcription Kit (Takara Biotechnology, Japan) was used to obtain cDNA and reverse transcribe RNA. The primers used in this study were synthesized by Yingjun Technology (Shanghai, China) which showed in Table 1. qRT-PCR was conducted using the SYBR Green Realtime PCR Master Mix (Toyobo, Japan) and performed on ABI 7900 fast Real‐time PCR Systems (Applied Biosystems, USA). GAPDH were used as internal controls.\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\u003ePrimers used in the present study\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\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimer sequence\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLRIG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eforward: 5\u0026prime;-GAAAAGGGACTCTGGTTGGGAT-3\u0026prime;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ereverse: 5\u0026prime;-AGGAAGTCATCGCACACGAA-3\u0026prime;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCTLA-4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eforward: 5\u0026prime;-AGGTGACTGAAGTCTGTGCG-3\u0026prime;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ereverse: 5\u0026prime;-CATGAGCTCCACCTTGCAGA-3\u0026prime;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGAPDH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eforward: 5'-CCGGGAAACTGTGGCGTGATGG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ereverse: 5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3'\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\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 \u003c/strong\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins from human tissue or glioma cells were extracted by using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein samples were separated using 8% SDS-PAGE. After transferred onto PVDF membrane, the primary antibodies were added: anti-LRIG1 (PA5-52860, 1:500, Invitrogen, Carlsbad, CA, USA), anti-Akt (ab235958, 1:1000, Abcam, UK), anti-Akt (phospho S473) (ab81283, 1:1000, Abcam, UK), anti-CTLA-4(PA5-47547, 1:250, Invitrogen, Carlsbad, CA, USA), anti-Tubulin (ab210797, 1:1000, Abcam, UK). Tubulin served as internal control. After 24 hours of incubation, membrane was incubated along with secondary antibody immunoglobulin G (IgG) at room temperature for 2 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Cell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LRIG1 was overexpressed or knockdown by using LRIG1 overexpression plasmid (Flag- LRIG1) or Short hairpin RNAs (shRNA) targeting LRIG1 (sh-LRIG1), respectively. The plasmid or empty vectors were purchased from GeneChem Corporation (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection following manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 CCK-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected U251 cells or U251R cells (2\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e per well) were seeded into a 96-well plate. CCK-8 Kit (Abcam, UK) and RPMI-1640 were added into each well. After incubation with CCK-8 solution for 2 hours, optical density of cells was measured at 450 nm at different time points by using micro-plate reader (Thermo Fisher, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 \u003c/strong\u003e\u003cstrong\u003eFlow cytometry assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell apoptosis was detected using Annexin-V-propidium iodide (PI) apoptosis assay. Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA, USA) was used according to the manufacture\u0026rsquo;s instruction. Briefly, different groups of transfected U251R cells were with propidium iodide (PI) at 37\u0026deg;C for 30min, followed by flow cytometry (Beckman Coulter FC500, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Co-Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-Immunoprecipitation was performed to detect the combination of LRIG1 and CTLA-4. Briefly, cells were lysed in 500 \u0026mu;l co-IP buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri, USA). Then, 20 \u0026mu;l immobilized protein A/G beads was incubated with cell lysates for 1h at 4 \u0026deg;C. Primary antibody or control Ig G were incubated with lysates for 24 hours. Finally, immobilized protein A/G beads were added, then, the proteins were prepared for western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Human glioma tissue collection \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples of paired glioma tissues and paracancerous tissues were collected from patients who underwent glioma resection at the second affiliated hospital of Nanchang university. All patients enrolled in this study have given their informed written consents prior to conduct the clinical research related procedure and this study was approved by the second affiliated hospital of Nanchang university Ethical Committee (No. 2019003). All patients have received 5 weeks\u0026rsquo; radiation therapy. Tissue samples were collected and immediately snap-frozen in liquid nitrogen after surgery (-80\u0026deg;C) for further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Immunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of LRIG1 and CTLA-4 proteins was determined in human glioma tissues by IHC. Briefly, paraffin‐embedded sections were dewaxed and dehydrated by xylene and ethanol. Then, after incubation with 50\u0026thinsp;\u0026mu;l of 10% goat serum for 60min, the sections were probed with primary antibodies: anti-LRIG1 (ab197985, 1:1000, Abcam, UK), anti-CTLA-4 (ab227709, 1:1000, Abcam, UK) for 24 hours. Then, peroxidase‐labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) were added and incubated for 30min. The sections were developed using diaminobenzidine (DAB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cell experiments were repeated triplicate. The data were exhibited as Mean \u0026plusmn; SD. SPSS21.0 (IBM Corp. Armonk, NY) software was used for data analysis. Two-tailed Student's t-test, one-way ANOVA, Pearson correlation coefficient were used in this study. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant difference.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 LRIG1 was down-regulated in radioresistant glioma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the expression of LRIG1 mRNA was determined in U251 cell or radioresistance U251 cells (U251R) by using qRT-PCR. As shown in Fig. 1A, the expression of LRIG1 mRNA in U251R cells was significantly decreased compared with that in U251 cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Subsequently, LRIG1 protein expression in both groups was detected by western blot. Result demonstrated that in U251R cells, the level of LRIG1 protein was much lower compared with that in U251 cells (Fig. 1B). These data suggested that in both mRNA and protein levels, LRIG1 was down-regulated in radioresistant glioma cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Overexpressed LRIG1 increased radiosensitivity of glioma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the potential role of LRIG1 in radioresistant glioma cells, we firstly used the plasmid to overexpress LRIG1 in U251R cells. Results revealed that LRIG1 protein was highly expressed in Flag-LRIG1 group compared with NC group (Fig. 2A), suggesting that the LRIG1 overexpressed system can be used in further studies. Next, the effect of LRIG1 on cell phenotypes were detected by CCK-8 assay and Flow cytometry assay. We found that the expression of LRIG1 significantly inhibited the cell proliferation (Fig. 2B; \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), meanwhile, in U251R cells, the apoptosis of cells was promoted by overexpression of LRIG1 proteins (Fig. 2C). To sum up, the above results provide the evidence that overexpressed LRIG1 increased radiosensitivity of glioma cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Overexpressed LRIG1 inhibited the expression of p-AKT in U251R cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the mechanisms of LRIG1 in radioresistant glioma cells, the expression of many proteins was determined in different groups by western blot. First, the expression of LRIG1, p-AKT, AKT proteins was detected in U251 cells or U251R cells. As shown in Fig. 3A, in radioresistant glioma cells, the expression of p-AKT was highly expressed, suggesting that p-AKT was activated. Then, the expression of LRIG1, p-AKT, AKT proteins was also measured in either overexpressed LRIG1 group or NC group. Result demonstrated that the activated p-AKT can be inhibited by overexpression of LRIG1 in U251R cells (Fig 3B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Overexpressed LRIG1 inhibited the expression of CTLA-4 protein in U251R cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, the effect of overexpression of LRIG1 on CTLA-4 expression was analyzed in U251R cells. We found that overexpression of LRIG1 dramatically suppressed the protein level of CTLA-4 in U251R cells (Fig. 4A). However, in mRNA level, overexpression of LRIG1 was not able to affect the expression of CTLA-4 mRNA (Fig. 4B). These data indicated that LRIG1 only have effect on CTLA-4 post-translational modification, while not change the mRNA level of CTLA-4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 LRIG1 combined with CTLA-4, and promoted CTLA-4 degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, the relationship between LRIG1 and CTLA-4 was identified by conducting Co-immunoprecipitation and western blot. As shown in Fig 5A, coimmunoprecipitated proteins were analyzed by western blot with indicated antibodies. Results showed that endogenous LRIG1 protein was immunoprecipitated with anti-CTLA-4, meanwhile, CTLA-4 also combined with LRIG1. Moreover, MG132 was used to inhibit the proteasome in each group. In Fig. 5B, results demonstrated that by blocking proteasome, overexpression or knockdown of LRIG1 did not have any effect on expression of CTLA-4 protein in U251R cells. These data indicated that LRIG1 combined with CTLA-4, and the same time, promoted CTLA-4 degradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 LRIG1 was down-regulated, while CTLA-4 was highly expressed in glioma tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, we had performed experiments on human glioma tissue. The expression of LRIG1 and CTLA-4 protein was detected in human glioma tissue by IHC and western blot. As shown in Fig. 6A, compared with paracancerous tissue, the expression level of LRIG1 was much lower. On the contrary, CTLA-4 was highly expressed in glioma tissue. To better quantify the protein expression, western blot was performed in each group. In tumor tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed compared with paracancerous tissue（Fig. 6B）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 The expression of LRIG1 was negatively correlated with expression of CTLA-4 and radioresistance of glioma patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, the correlation analysis was performed to better clarify the relationship between LRIG1 and CTLA-4. In Fig 7A, the correlation analysis between expression of LRIG1 and radioresistance of glioma was performed. It was shown that the expression of LRIG1 was negatively correlated with radioresistance of glioma. However, the expression of CTLA-4 was positively correlated with radioresistance of glioma (Fig. 7B). Furthermore, result in Fig 7C showed that the expression of LRIG1 was negatively correlated with expression of CTLA-4 in glioma patients. These data indicated that LRIG1 may have positive effect on glioma in clinic.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eGliomas make up the largest proportion of malignant brain tumors with very limited treatment strategies. Radiotherapy seems to be the most effective nonsurgical treatment for gliomas, however, its efficacy is severely suppressed due to the high intrinsic radioresistance of glioma cells \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Thus, to explore the potential mechanisam underlying gliomas radioresistance is critical to design novel strategies and improve the prognosis of glioma patients. In our study, we firstly found that LRIG1 was down-regulated in radioresistant glioma cells in both mRNA and proteins levels. Due to the fact that expression of LRIG1 can be affected by irradiation, we further established LRIG1 overexpression system in radioresistant glioma cells (U251R) for further studies.\u003c/p\u003e\n\u003cp\u003eThe biological effects of irradiation on cells mainly due to DNA lesions, e.g., disruption of the phosphate DNA backbone \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The DNA lesions can be direct caused by interactions between particles or indirect following interactions with reactive oxygen species (ROS) generated by cell water ionization \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. These breaks can be either repaired or can lead to cell cycle arrest. As such, we can observe the effect of irradiation which is manifested as viability or apoptosis of tumor cells. Results from our study showed that in radioresistant glioma cells, overexpressed LRIG1 dramatically inhibited the cell proliferation and promote cell apoptosis, indicating that LRIG1 act as a tumor suppressor in radioresistant glioma cells. As an important cell transmembrane protein, LRIG1 was widely studied in gliomas. For instance, a study includes 404 patients with gliomas showed that expression of LRIG1 positively correlated with the prognosis of glioma patients and negatively correlated with WHO histological grade \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Moreover, in Ye \u003cem\u003eet al\u003c/em\u003e\u0026rsquo;s study, after clinical surgical resections of astrocytoma tumor tissue, the results showed that LRIG1 expression was significantly decreased in tumor tissue compared to the corresponding surrounding non-tumor tissues \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn molecular level, the underlying mechanism of LRIG1 on glioma radioresistance was further clarified in our studies. We found that in radioresistant glioma cells, overexpression of LRIG1 could inhibit the expression of phosphorylated AKT (p-AKT), but not AKT, suggesting that LRIG1 might play a role in AKT activation. AKT is a serine/threonine kinase which can be activated by translocation to the plasma membrane or phosphorylation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Activation of AKT, i.e, high level of p-AKT has been reported to correlate with a poor prognosis for patients with glioma \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. AKT-related pathways have been also widely studied in molecular level in the oncogenic and development of cancers cells \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Previously in our group, we found that the glioblastoma radioresistance can be promoted by overexpressed miR-183, further, expression of LRIG1 and activation of EFGR/Akt pathway can be regulated by miR-183 \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In the present study, we further clarified that overexpression of LRIG1 inhibited the expression of CTLA-4 protein, but not CTLA-4 mRNA. In addition, results also confirmed that LRIG1 could combined with CTLA-4, and promoted CTLA-4 degradation. CTLA-4, known as a member of a family of Immunoglobulin-related receptors, has been confirmed play a key role in tumorigenesis \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. For its machoism, CTLA-4 can bind to phosphatidylinositol 3-kinase (PI3K) as well as phosphatases PP2A and SHP-2, which considered to be the key factors in AKT-related pathway \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. However, whether CTLA-4 involved in the resistant to irradiation was still un-documented.\u003c/p\u003e\n\u003cp\u003eTo the best of our knowledge, our study was the first one to clarify the relationship between LRIG1 and CTLA4/PI3K/AKT pathway in glioma radioresistance. We found that in human glioma tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed. By conducting correlation analysis, results shown that the expression of LRIG1 was negatively correlated with radioresistance of glioma and expression of CTLA-4. However, the expression of CTLA-4 was positively correlated with radioresistance of glioma.\u003c/p\u003e\n\u003cp\u003eTo sum up, our data provided information that LRIG1 could increase the radiosensitivity of glioma cells, overexpressed LRIG1 had a significantly impact on activation of AKT and CTLA-4 expression. These findings provided a novel insight into the mechanism of radiotherapy in glioma and could be the potential targets for treating gliomas in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or other conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has received funding from the Educational department foundation of Jiangxi Province, China (Grant No. GJJ190145)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and analyzed in this paper are available from the corresponding author under reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSQ and XQ wrote the manuscript and performed most of the experiments. RR participated in the study data acquisition and analysis. YZ edited and revised final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the Biomedical Ethics Committee of The Second Affiliated Hospital of Nanchang University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients provided their consent for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOstrom, Q.T., et al., \u003cem\u003eThe epidemiology of glioma in adults: a \"state of the science\" review.\u003c/em\u003e Neuro Oncol, 2014. 16(7): p. 896-913.\u003c/li\u003e\n\u003cli\u003eOstrom, Q.T., et al., \u003cem\u003eEpidemiology of gliomas.\u003c/em\u003e Cancer Treat Res, 2015. 163: p. 1-14.\u003c/li\u003e\n\u003cli\u003eOmuro, A. and L.M. 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Mo, \u003cem\u003eAcidosis promotes tumorigenesis by activating AKT/NF-\u0026kappa;B signaling.\u003c/em\u003e Cancer Metastasis Rev, 2019. 38(1-2): p. 179-188.\u003c/li\u003e\n\u003cli\u003eGuo, J., et al., \u003cem\u003eAKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions.\u003c/em\u003e Nat Cell Biol, 2019. 21(2): p. 226-237.\u003c/li\u003e\n\u003cli\u003eMao, H., et al., \u003cem\u003eNew insights of CTLA-4 into its biological function in breast cancer.\u003c/em\u003e Curr Cancer Drug Targets, 2010. 10(7): p. 728-36.\u003c/li\u003e\n\u003cli\u003eSchneider, H., et al., \u003cem\u003eCTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.\u003c/em\u003e J Exp Med, 1995. 181(1): p. 351-5.\u003c/li\u003e\n\u003cli\u003eLee, K.M., et al., \u003cem\u003eMolecular basis of T cell inactivation by CTLA-4.\u003c/em\u003e Science, 1998. 282(5397): p. 2263-6.\u003c/li\u003e\n\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":"LRIG1, glioma, radioresistance, CTLA4, AKT","lastPublishedDoi":"10.21203/rs.3.rs-131093/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-131093/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Radioresistance has a great impact on prognosis of glioma patients. However, the potential mechanism underlying the radioresistance of glioma cells remains largely unknown. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: LRIG1 overexpression model was firstly established by using Flag-LRIG1 plasmid. The expression of LRIG1, CTLA-4 proteins were detected by western blot and IHC in cells and human tissue. Real-time PCR was used for deterring mRNA expression. Cell viability and apoptosis were detected using CCK-8 and Annexin-V/propidium iodide (PI), respectively. Co-Immunoprecipitation was used for detecting the combination of LRIG1 and CTLA-4 proteins. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: LRIG1 was significantly down-regulated in radioresistant glioma cells. Overexpressed LRIG1 could promote the radiosensitivity of glioma cells, meanwhile, inhibit the expression of p-AKT and CTLA-4 protein in radioresistant glioma cells. Furthermore, LRIG1 combined with CTLA-4 and promoted CTLA-4 degradation. In human glioma tissue, LRIG1 was down-regulated, while CTLA-4 was highly expressed in glioma tissue. Finally, correlation analysis showed that the expression of LRIG1 was negatively correlated with expression of CTLA-4 and radioresistance of glioma patients. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Our findings demonstrated that LRIG1 facilitates radioresistance glioma cells by regulating CTLA4 /AKT signaling pathway.\u003c/p\u003e","manuscriptTitle":"LRIG1 Modulated Radioresistance of Glioma via Regulating CTLA-4/AKT Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-21 18:38:54","doi":"10.21203/rs.3.rs-131093/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":"89acd2e7-9ea5-400b-a1bc-a742ab1ed866","owner":[],"postedDate":"December 21st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1573560,"name":"Oncology"}],"tags":[],"updatedAt":"2020-12-21T18:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-21 18:38:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-131093","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-131093","identity":"rs-131093","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}