Upregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer

Upregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Upregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer Qiaohong Nong, Shubin Wang, Zhu Li, Gangling Tong, Shaokang Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-95730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract OBJECTIVE: Recent studies suggest that abnormal snoRNA expression may play crucial role in tumor development and progression. In the study, we aim to explore the clinical significance and functional role of Small nucleolar RNA 78 (SNORD78) in breast cancer. PATIENTS AND METHODS: The expression of SNORD78 in 92 breast cancer and adjacent non tumor tissues was analyzed by quantitative real time polymerase chain reaction (qRT-PCR). The association between SNORD78 expression and clinicopathological factors was evaluated. The biological functions in vitro were examined by MTT cell proliferation, colony formation and transwell invasion assays. QRT-PCR and Western blot assays were used to analyze the mRNA and protein expression of WNT1, GSK3β and β-catenin. RESULTS: SNORD78 expression showed a significant increase in breast cancer tissues and cell lines. Higher SNORD78 expression positively was related to lymph node metastasis and TNM stage in patients. Furthermore, knockdown of SNORD78 inhibited the abilities of cell proliferation, colony formation, and cell invasion in MDA-MB-231 and SKBR-3 cells. Moreover, we demonstrated that knockdown of SNORD78 inhibited WNT/β-catenin signaling pathway in MDA-MB-231 and SKBR-3 cells via downregulating the relative protein expression of WNT1 and β-catenin and upregulating the GSK3β expression. CONCLUSION : Our results indicated that SNORD78 may be potential therapeutic target for breast cancer. Oncology Pathology Breast cancer Small nucleolar RNA SNORD78 prognosis cell proliferation Introduction Breast cancer is the most common malignancy in women worldwide. In 2015, 231,840 women were diagnosed with breast cancer, and approximately 40,290 died of the disease [ 1 , 2 ]. Tumor recurrence and metastasis cause larger cancer related morbidity and mortality [ 3 ]. In recent decades, large advances in the diagnosis and therapy for breast cancer have been improved, however, patients who are diagnosed at advanced stage present poor outcome [ 4 ]. Therefore, to investigate tumor makers of early detection or therapeutic target of breast cancer is important. Small nucleolar RNAs (snoRNAs) are a class of RNA without protein coding capacity and about 60–300 nucleotides in length [ 5 ]. Recent studies have indicated that snoRNAs were involved in some disease process including tumor development [ 6 ]. For instance, small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer [ 7 ]. SNORD76, not its host gene, is selectively downregulated in glioblastoma (WHO grade IV) and acts as a tumor suppressor in glioblastoma [ 8 ]. SNORD113-1 suppresses tumorigenesis in hepatocellular carcinoma by inactivating the phosphorylation of ERK1/2 and SMAD2/3 in MAPK/ERK and TGF-β pathways [ 9 ]. However, the clinical role and potential functions of small nucleolar RNA 78 (SNORD78) in breast cancer remain unknown. In the study, we demonstrated that SNORD78 expression was significantly upregulated in breast cancer. SNORD78 silencing obviously inhibited breast cancer cells proliferation, cell invasion and Wnt/β-catenin signaling pathway. Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer. Patients And Methods Human patient tissue specimens Freshly breast tissue specimens obtained from breast cancer patients who underwent curative surgical treatment between April 2010 and March 2012 were immediately frozen in liquid nitrogen for further RNA extraction. All of patients were selected at Department of Oncology, Peking University Shenzhen Hospital. The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients. Cell lines culture Three human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All of breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) added with 10% fetal bovine serum at 37 °C with 5% CO 2 . Quantitative real-time polymerase chain reaction (QRT-PCR) The TRIzol® (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction from tissues and cells according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using a Prime Script RT Regent kit (Takara, Dalian, China). The SYBR Green PCR Master Mix (Takara, Dalian, China) was used for qRT-PCR assay on ABI PRISM 7900 Real-time PCR system (Applied Biosystems, Foster City, CA). GAPDH were used for normalization. The primer sequences were as follows: SNORD78(Forward:5’-GTGTAATGATGTTGATCAAATGTCTGAC-3’;Reverse: 5’-CACATTACTACAACTAGTTTACAGACTGG-3’),GAPDH(Forward:5’-CTCAAGGGCATCCTGGGCTAC-3’;Reverse:5’-CAGCCCCAGCGTCAAAGGT-3’).QRT-PCR results were analyzed using 2 △△CT Methods. RNA interference and cell transfection Two siRNAs against SNORD78 was designed to knockdown SNORD78 expression and one negative control (si-NC) was purchased from Ribobio (Guangzhou, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The siRNAs targeted SNORD78 sequences were as follow: si-SNORD78-1(5’-GTTGATCAAATGTCTGACCTG-3’), si-SNORD78-2 (5’-GACCTGAAATGAGCATGTAGA-3’). MTT assay Cell growth rate was investigated using a MTT assay. 3 × 10 3 transfected cells/well were seeded into 96-well plates. At indicated time 0, 24, 48, and 72 h, cells were incubated with 20 µl MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and cultured for 4 h at 37 °C with 5% CO 2 . The cell proliferation was analyzed using an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the absorbance at 490 nm. Cell clonogenic assay 300 transfected cells were placed in 12-well plates cultured in medium added with 10% fetal bovine serum at 37 °C with 5% CO 2 for 14 days. Colonies were fixed with 100% methanol, stained with 0.1% crystal violet, and then were counted under a microscope. Cell invasion assays Transwell chambers with an 8 µm pore polycarbonate membrane (Costar; Corning Incorporated, Corning, NY, USA) were used to assess cell invasion ability. 1 × 10 5 cells were added on the upper chamber supplemented with 300 µl culture medium without FBS and 500 µl culture medium supplemented with 20% FBS was added on the lower chamber. After cell cultured for 24 h, cells were fixed with 100% methanol, and then stained with 0.1% crystal violet. Cells on the lower chamber were counted in five random fields under a light microscope. Western blot analysis Cells were lysed using radioimmunoassay precipitation assay (RAPA) lysis buffer with a proteinase inhibitor. The quantity of approximately 40 µg proteins in the lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with anti-WNT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GSK3β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 12 h at 4 °C and then washed three times for 5 min. Following horseradish peroxidase-conjugated secondary antibodies were incubated. The blot was visualized by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Statistical analysis All of results are presented as means ± SD from at least three or more independent experiments. The association between SNORD78 expression and clinical characteristics was evaluated using the chi-square test. Differences between groups were compared using Student’s t -test or an analysis of variance (ANOVA). A p < 0.05 were considered statistically significant. Results SNORD78 expression is upregulated in breast cancer tissues and associates with clinicopathologic features In the study, SNORD78 expression was determined by qRT-PCR in 92 breast cancer tissues compared with adjacent normal tissues. As shown in Fig. 1 A, results analysis demonstrated that SNORD78 expression in breast cancer tissues was significantly higher compared with adjacent normal tissues ( p < 0.05). According to the median expression rate of SNORD78 expression in breast cancer tissues, we divided patients into higher expression group and lower expression group. The association between the SNORD78 expression and the clinical characteristics of breast cancer patients were analyzed. We demonstrated that higher SNORD78 expression was significantly associated with the lymph node status ( p = 0.001, Table I) and advanced TNM stage ( p = 0.023, Table I), but was not associated with the other characteristics in breast cancer patients ( p > 0.05, Table I). Table I Association of SNORD78 expression with clinicopathological feathers Clinicopathological feathers Total (n = 92) Lower SNORD78 (n = 44) Higher SNORD78 (n = 48) p -value Age (year) 0.548 ≤ 50 43 22 21 > 50 49 22 27 Tumor size (cm) 0.714 2 cm 52 24 28 Tumor grade 0.676 G1-G2 65 32 33 G3 27 12 15 HER-2 status 0.064 Negative 49 19 30 Positive 43 25 18 Lymph node status 0 .001* Negative 43 29 14 Positive 49 15 34 Distant metastasis 0.944 No 75 36 39 Yes 17 8 9 PR 0.485 Negative 53 27 26 Positive 39 17 22 TNM stage 0.023* I-II 58 33 25 III 34 11 23 * p < 0.05. Downregulation of SNORD78 expression suppresses cell growth and invasion in MDA-MB-231 and SKBR-3 cells in vitro Additionally, we evaluated the expression of SNORD78 in three human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A). As shown in Fig. 1 B, the expression of SNORD78 was higher in three human breast cancer cell lines than MCF-10A cells ( p < 0.05). To further analyze the biological effects of on cell growth and invasion, two siRNAs targeting SNORD78 oligos were transfected into MDA-MB-231 and SKBR-3 cells (Fig. 2 A- 2 B). The si-SNORD78-1 was chosen to knock down SNORD78 expression in following experiments due to its higher silencing efficiency. MTT analysis results showed that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably suppressed cell proliferation at 48 h and 72 h, compared with control group (Fig. 2 C- 2 D, p < 0.05). Cell colony formation results demonstrated that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably reduced cell colony formation ability at 14 days, compared with control group (Fig. 3 A- 3 B, p < 0.05). Moreover, cell invasion assay results also showed that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably reduced cell invasion ability after cell transfection at 48 h, compared with control group (Fig. 3 C- 3 D, p < 0.05). These results indicated that downregulation of SNORD78 expression suppressed cell growth and invasion in MDA-MB-231 and SKBR-3 cells. Downregulation of SNORD78 expression inhibits Wnt/β-catenin signaling pathway in MDA-MB-231 and SKBR-3 cells It has been reported that abnormal Wnt/β-catenin signaling pathway functions as a crucial signaling cascade in breast cancer [ 10 ]. After knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells at 48 h, we performed qRT-PCR and western blot analysis to examine the mRNA and protein expression of WNT1, GSK3β and β-catenin. The results demonstrated mRNA and protein expression levels of WNT1 and β-catenin was significantly downregulated, while GSK3β expression was upregulated after knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells at 48 h (Fig. 4 A- 4 D, p < 0.05). Thus, these results indicated that downregulation of SNORD78 expression inhibited Wnt/β-catenin signaling pathway in breast cancer. Discussion Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes and increasing evidence indicates that dysregulation of small nucleolar RNAs (snoRNAs) were associated with tumorigenesis [ 11 ]. Such as, integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression [ 12 ]. SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types [ 13 ]. Small nucleolar RNA 47 promotes tumorigenesis by regulating EMT markers in hepatocellular carcinoma [ 14 ]. Small nucleolar RNA ACA11 promotes cell proliferation, migration and invasion in hepatocellular carcinoma by targeting the PI3K/AKT signaling pathway [ 15 ]. In the study, we found that SNORD78 expression in breast cancer tissues was significantly higher compared with adjacent normal tissues. Higher SNORD78 expression was significantly associated with the lymph node status and advanced TNM stage. Moreover, knockdown of SNORD78 inhibited cell proliferation, colony formation and cell invasion capacities in breast cancer. Consistent with our findings, SNORD78 was observed to be upregulated in NSCLC tissues compared to their adjacent normal tissues. SNORD78 promoted cell proliferation and invasion of NSCLC cells, which indicated SNORD78 function as an oncogene [ 16 ]. Furthermore, we demonstrated that WNT/β-catenin signaling pathway related protein expression levels of WNT1 and β-catenin was significantly downregulated, but GSK3β expression were upregulated when SNORD78 was knocked down in MDA-MB-231 and SKBR-3 cells. The WNT pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis [ 10 ]. Aberrantly activated Wnt/β-catenin signaling could affect diverse biological processes including cell motility, migration, differentiation, proliferation, and invasion [ 17 ]. Overexpression of Wnt/β-catenin protein Lrp6 or stabilized β-catenin, or loss of APC, resulted in mouse mammary hyperplasia or tumors [ 18 ]. To elucidate the underlying mechanisms of SNORD78 affecting cell proliferation and invasion, we demonstrated that knockdown of SNORD78 inhibited cell proliferation and invasion by regulating WNT/β-catenin signaling pathway. Conclusions In conclusion, our results found that SNORD78 expression was significantly upregulated in breast cancer. Knockdown of SNORD78 inhibited cell proliferation, invasion and WNT/β-catenin signaling pathway. Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer. Declarations Ethics approval and consent to participate The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients. Consent for publication All the authors agreed that the article should be published. Availability of data and material All data generated or analyzed during this study are included in this published article. Competing interests The authors declare no conflict of interest. Funding N/A Authors' contributions Qiaohong Nong and Shubin Wang carried out the study concepts, study design, manuscript editing and manuscript review; Zhu Li and Gangling Tong was dedicated to the literature research, data acquisition and data analysis; Shaokang Yu and Hui Hu was involved in the manuscript preparation and manuscript editing. All authors read and approved the final manuscript. Acknowledgements N/A References Leong SP. Paradigm shift of staging and treatment for early breast cancer in the sentinel lymph node era. Breast J. 2006;12:S128-133. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5-29. Weigelt B, Peterse JL, van 't Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer. 2005;5:591-602. Marquette C, Nabell L. Chemotherapy-resistant metastatic breast cancer. Curr Treat Options Oncol. 2012;13:263-275. Thorenoor N, Slaby O. Small nucleolar RNAs functioning and potential roles in cancer. Tumour Biol. 2015;36:41-53. Mannoor K, Shen J, Liao J, Liu Z, Jiang F. Small nucleolar RNA signatures of lung tumor-initiating cells. Mol Cancer. 2014;13:104. Popov A, Szabo A, Mandys V. Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer. BMC Cancer. 2015;15:774. Chen L, Han L, Wei J, Zhang K, Shi Z, Duan R, et al. SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma. Sci Rep. 2015;5:8588. Xu G, Yang F, Ding CL, Zhao LJ, Ren H, Zhao P, et al. Small nucleolar RNA 113-1 suppresses tumorigenesis in hepatocellular carcinoma. Mol Cancer. 2014;13:216. Yu QC, Verheyen EM, Zeng YA. Mammary Development and Breast Cancer: A Wnt Perspective. Cancers (Basel). 2016;8. Stepanov GA, Filippova JA, Komissarov AB, Kuligina EV, Richter VA, Semenov DV. Regulatory role of small nucleolar RNAs in human diseases. Biomed Res Int. 2015;2015:206849. Crea F, Quagliata L, Michael A, Liu HH, Frumento P, Azad AA, et al. Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression. Mol Oncol. 2016;10:693-703. Siprashvili Z, Webster DE1, Johnston D1, Shenoy RM1, Ungewickell AJ, Bhaduri A, et al. The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nat Genet. 2016;48:53-58. Li G, He Y, Liu X, Zheng Z, Zhang M, Qin F, et al. Small nucleolar RNA 47 promotes tumorigenesis by regulating EMT markers in hepatocellular carcinoma. Minerva Med. 2017;108:396-404. Wu L, Zheng J, Chen P, Liu Q, Yuan Y. Small nucleolar RNA ACA11 promotes proliferation, migration and invasion in hepatocellular carcinoma by targeting the PI3K/AKT signaling pathway. Biomed Pharmacother. 2017;90:705-712. Zheng D, Zhang J, Ni J, Luo J, Wang J, Tang L, et al. Small nucleolar RNA 78 promotes the tumorigenesis in non-small cell lung cancer. J Exp Clin Cancer Res. 2015;34:49. Song JL, Nigam P, Tektas SS, Selva E. microRNA regulation of Wnt signaling pathways in development and disease. Cell Signal. 2015;27:1380-1391. Incassati A, Chandramouli A, Eelkema R, Cowin P. Key signaling nodes in mammary gland development and cancer: beta-catenin. Breast Cancer Res. 2010;12:213. Figures Figure 1 to 4 are missing in this version of the manuscript. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-95730","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":3748043,"identity":"64b80902-8ff4-4344-b613-3d79b2687632","order_by":0,"name":"Qiaohong Nong","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaohong","middleName":"","lastName":"Nong","suffix":""},{"id":3748044,"identity":"222893c5-985b-409d-a06e-cfe15cc47548","order_by":1,"name":"Shubin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDACCQY2hgQwi7HxQUJFDUlamJsNHpw5RqQWCGBvk3zYwkxYh/zsHrMHD3ccljfnX9hWkdjAxsDf3p2AV4vBnTPmBolnDhvunPGw7UbiDhkGiTNnN+DXIpFjJpHYdptxw42DQC1n2IAiufi1yM+AaLEHaSlIbGMmrIXhBkRL4obzjW0MRGkxuJFWbpDY9j95ww3GZomEM8d4CPpFfkbytoc/29JsN5w//vDjj4oaOf72XgIOgwOJBDDFQ6RyEOA/QILiUTAKRsEoGFEAAPBaUTvwtpzjAAAAAElFTkSuQmCC","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shubin","middleName":"","lastName":"Wang","suffix":""},{"id":3748045,"identity":"ec6b4d97-0e74-4e1e-9c71-40aeb97d890d","order_by":2,"name":"Zhu Li","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Li","suffix":""},{"id":3748046,"identity":"af8e473a-c32d-46c9-aa31-92d5bb5dbfdd","order_by":3,"name":"Gangling Tong","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gangling","middleName":"","lastName":"Tong","suffix":""},{"id":3748047,"identity":"93f64d02-2913-4d7a-a054-cfc90caaa734","order_by":4,"name":"Shaokang Yu","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaokang","middleName":"","lastName":"Yu","suffix":""},{"id":3748048,"identity":"a405ecfe-6719-4d80-9074-557e5b546203","order_by":5,"name":"Hui Hu","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2020-10-20 22:22:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-95730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-95730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":13605673,"identity":"be084a72-7163-4332-a4ef-0c15e1e71d9d","added_by":"auto","created_at":"2021-09-17 06:04:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":372322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-95730/v1/19dfe3a9-3ea3-4161-9e8b-ecf6181bec65.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eUpregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eBreast cancer is the most common malignancy in women worldwide. In 2015, 231,840 women were diagnosed with breast cancer, and approximately 40,290 died of the disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Tumor recurrence and metastasis cause larger cancer related morbidity and mortality [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In recent decades, large advances in the diagnosis and therapy for breast cancer have been improved, however, patients who are diagnosed at advanced stage present poor outcome [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, to investigate tumor makers of early detection or therapeutic target of breast cancer is important.\u003c/p\u003e \u003cp\u003eSmall nucleolar RNAs (snoRNAs) are a class of RNA without protein coding capacity and about 60\u0026ndash;300 nucleotides in length [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent studies have indicated that snoRNAs were involved in some disease process including tumor development [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SNORD76, not its host gene, is selectively downregulated in glioblastoma (WHO grade IV) and acts as a tumor suppressor in glioblastoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. SNORD113-1 suppresses tumorigenesis in hepatocellular carcinoma by inactivating the phosphorylation of ERK1/2 and SMAD2/3 in MAPK/ERK and TGF-β pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the clinical role and potential functions of small nucleolar RNA 78 (SNORD78) in breast cancer remain unknown.\u003c/p\u003e \u003cp\u003eIn the study, we demonstrated that SNORD78 expression was significantly upregulated in breast cancer. SNORD78 silencing obviously inhibited breast cancer cells proliferation, cell invasion and Wnt/β-catenin signaling pathway. Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer.\u003c/p\u003e "},{"header":"Patients And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman patient tissue specimens\u003c/h2\u003e \u003cp\u003eFreshly breast tissue specimens obtained from breast cancer patients who underwent curative surgical treatment between April 2010 and March 2012 were immediately frozen in liquid nitrogen for further RNA extraction. All of patients were selected at Department of Oncology, Peking University Shenzhen Hospital. The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines culture\u003c/h2\u003e \u003cp\u003eThree human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All of breast cancer cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Gibco, Grand Island, NY, USA) added with 10% fetal bovine serum at 37\u0026nbsp;\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (QRT-PCR)\u003c/h2\u003e \u003cp\u003eThe TRIzol\u0026reg; (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction from tissues and cells according to the manufacturer\u0026rsquo;s instructions. RNA was subjected to cDNA synthesis using a Prime Script RT Regent kit (Takara, Dalian, China). The SYBR Green PCR Master Mix (Takara, Dalian, China) was used for qRT-PCR assay on ABI PRISM 7900 Real-time PCR system (Applied Biosystems, Foster City, CA). GAPDH were used for normalization. The primer sequences were as follows: SNORD78(Forward:5\u0026rsquo;-GTGTAATGATGTTGATCAAATGTCTGAC-3\u0026rsquo;;Reverse: 5\u0026rsquo;-CACATTACTACAACTAGTTTACAGACTGG-3\u0026rsquo;),GAPDH(Forward:5\u0026rsquo;-CTCAAGGGCATCCTGGGCTAC-3\u0026rsquo;;Reverse:5\u0026rsquo;-CAGCCCCAGCGTCAAAGGT-3\u0026rsquo;).QRT-PCR results were analyzed using 2\u003csup\u003e△△CT\u003c/sup\u003e Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference and cell transfection\u003c/h2\u003e \u003cp\u003eTwo siRNAs against SNORD78 was designed to knockdown SNORD78 expression and one negative control (si-NC) was purchased from Ribobio (Guangzhou, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol. The siRNAs targeted SNORD78 sequences were as follow: si-SNORD78-1(5\u0026rsquo;-GTTGATCAAATGTCTGACCTG-3\u0026rsquo;), si-SNORD78-2 (5\u0026rsquo;-GACCTGAAATGAGCATGTAGA-3\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eCell growth rate was investigated using a MTT assay. 3\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e transfected cells/well were seeded into 96-well plates. At indicated time 0, 24, 48, and 72\u0026nbsp;h, cells were incubated with 20\u0026nbsp;\u0026micro;l MTT (5\u0026nbsp;mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and cultured for 4\u0026nbsp;h at 37\u0026nbsp;\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cell proliferation was analyzed using an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the absorbance at 490\u0026nbsp;nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell clonogenic assay\u003c/h2\u003e \u003cp\u003e300 transfected cells were placed in 12-well plates cultured in medium added with 10% fetal bovine serum at 37\u0026nbsp;\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 14 days. Colonies were fixed with 100% methanol, stained with 0.1% crystal violet, and then were counted under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion assays\u003c/h2\u003e \u003cp\u003eTranswell chambers with an 8\u0026nbsp;\u0026micro;m pore polycarbonate membrane (Costar; Corning Incorporated, Corning, NY, USA) were used to assess cell invasion ability. 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells were added on the upper chamber supplemented with 300\u0026nbsp;\u0026micro;l culture medium without FBS and 500\u0026nbsp;\u0026micro;l culture medium supplemented with 20% FBS was added on the lower chamber. After cell cultured for 24\u0026nbsp;h, cells were fixed with 100% methanol, and then stained with 0.1% crystal violet. Cells on the lower chamber were counted in five random fields under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells were lysed using radioimmunoassay precipitation assay (RAPA) lysis buffer with a proteinase inhibitor. The quantity of approximately 40\u0026nbsp;\u0026micro;g proteins in the lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with anti-WNT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GSK3β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 12\u0026nbsp;h at 4\u0026nbsp;\u0026deg;C and then washed three times for 5\u0026nbsp;min. Following horseradish peroxidase-conjugated secondary antibodies were incubated. The blot was visualized by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll of results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three or more independent experiments. The association between SNORD78 expression and clinical characteristics was evaluated using the chi-square test. Differences between groups were compared using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or an analysis of variance (ANOVA). A \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSNORD78 expression is upregulated in breast cancer tissues and associates with clinicopathologic features\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the study, SNORD78 expression was determined by qRT-PCR in 92 breast cancer tissues compared with adjacent normal tissues. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, results analysis demonstrated that SNORD78 expression in breast cancer tissues was significantly higher compared with adjacent normal tissues (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). According to the median expression rate of SNORD78 expression in breast cancer tissues, we divided patients into higher expression group and lower expression group. The association between the SNORD78 expression and the clinical characteristics of breast cancer patients were analyzed. We demonstrated that higher SNORD78 expression was significantly associated with the lymph node status (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Table I) and advanced TNM stage (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023, Table I), but was not associated with the other characteristics in breast cancer patients (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05, Table I).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cp\u003eTable I\u0026nbsp;\u003cbr /\u003eAssociation of SNORD78 expression with clinicopathological feathers\u003c/p\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eClinicopathological\u003c/p\u003e\n\u003cp\u003efeathers\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;92)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLower SNORD78\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;44)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHigher SNORD78\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;48)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\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\u003eAge (year)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.548\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTumor size (cm)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.714\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;2\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2\u0026nbsp;cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTumor grade\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.676\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG1-G2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHER-2 status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.064\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=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLymph node status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0 .001*\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=\"char\" char=\".\"\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDistant metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.944\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=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.485\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=\"char\" char=\".\"\u003e\n\u003cp\u003e53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTNM stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.023*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eI-II\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIII\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003e*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDownregulation of SNORD78 expression suppresses cell growth and invasion in\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e MDA-MB-231 and SKBR-3 cells in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, we evaluated the expression of SNORD78 in three human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the expression of SNORD78 was higher in three human breast cancer cell lines than MCF-10A cells (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). To further analyze the biological effects of on cell growth and invasion, two siRNAs targeting SNORD78 oligos were transfected into MDA-MB-231 and SKBR-3 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The si-SNORD78-1 was chosen to knock down SNORD78 expression in following experiments due to its higher silencing efficiency. MTT analysis results showed that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably suppressed cell proliferation at 48\u0026nbsp;h and 72\u0026nbsp;h, compared with control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Cell colony formation results demonstrated that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably reduced cell colony formation ability at 14 days, compared with control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, cell invasion assay results also showed that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably reduced cell invasion ability after cell transfection at 48\u0026nbsp;h, compared with control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicated that downregulation of SNORD78 expression suppressed cell growth and invasion in MDA-MB-231 and SKBR-3 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDownregulation of SNORD78 expression inhibits\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e Wnt/\u0026beta;-catenin signaling pathway in MDA-MB-231 and SKBR-3 cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003cp\u003eIt has been reported that abnormal Wnt/\u0026beta;-catenin signaling pathway functions as a crucial signaling cascade in breast cancer [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. After knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells at 48\u0026nbsp;h, we performed qRT-PCR and western blot analysis to examine the mRNA and protein expression of WNT1, GSK3\u0026beta; and \u0026beta;-catenin. The results demonstrated mRNA and protein expression levels of WNT1 and \u0026beta;-catenin was significantly downregulated, while GSK3\u0026beta; expression was upregulated after knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells at 48\u0026nbsp;h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, these results indicated that downregulation of SNORD78 expression inhibited Wnt/\u0026beta;-catenin signaling pathway in breast cancer.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eSmall nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes and increasing evidence indicates that dysregulation of small nucleolar RNAs (snoRNAs) were associated with tumorigenesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Such as, integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Small nucleolar RNA 47 promotes tumorigenesis by regulating EMT markers in hepatocellular carcinoma [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Small nucleolar RNA ACA11 promotes cell proliferation, migration and invasion in hepatocellular carcinoma by targeting the PI3K/AKT signaling pathway [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the study, we found that SNORD78 expression in breast cancer tissues was significantly higher compared with adjacent normal tissues. Higher SNORD78 expression was significantly associated with the lymph node status and advanced TNM stage. Moreover, knockdown of SNORD78 inhibited cell proliferation, colony formation and cell invasion capacities in breast cancer. Consistent with our findings, SNORD78 was observed to be upregulated in NSCLC tissues compared to their adjacent normal tissues. SNORD78 promoted cell proliferation and invasion of NSCLC cells, which indicated SNORD78 function as an oncogene [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, we demonstrated that WNT/β-catenin signaling pathway related protein expression levels of WNT1 and β-catenin was significantly downregulated, but GSK3β expression were upregulated when SNORD78 was knocked down in MDA-MB-231 and SKBR-3 cells. The WNT pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Aberrantly activated Wnt/β-catenin signaling could affect diverse biological processes including cell motility, migration, differentiation, proliferation, and invasion [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Overexpression of Wnt/β-catenin protein Lrp6 or stabilized β-catenin, or loss of APC, resulted in mouse mammary hyperplasia or tumors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To elucidate the underlying mechanisms of SNORD78 affecting cell proliferation and invasion, we demonstrated that knockdown of SNORD78 inhibited cell proliferation and invasion by regulating WNT/β-catenin signaling pathway.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, our results found that SNORD78 expression was significantly upregulated in breast cancer. Knockdown of SNORD78 inhibited cell proliferation, invasion and WNT/β-catenin signaling pathway. Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors agreed that the article should be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiaohong Nong and Shubin Wang carried out the study concepts, study design, manuscript editing and manuscript review; Zhu Li and Gangling Tong was dedicated to the literature research, data acquisition and data analysis; Shaokang Yu and Hui Hu was involved in the manuscript preparation and manuscript editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeong SP. Paradigm shift of staging and treatment for early breast cancer in the sentinel lymph node era. Breast J. 2006;12:S128-133.\u003c/li\u003e\n\u003cli\u003eSiegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5-29.\u003c/li\u003e\n\u003cli\u003eWeigelt B, Peterse JL, van 't Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer. 2005;5:591-602.\u003c/li\u003e\n\u003cli\u003eMarquette C, Nabell L. Chemotherapy-resistant metastatic breast cancer. Curr Treat Options Oncol. 2012;13:263-275.\u003c/li\u003e\n\u003cli\u003eThorenoor N, Slaby O. Small nucleolar RNAs functioning and potential roles in cancer. Tumour Biol. 2015;36:41-53.\u003c/li\u003e\n\u003cli\u003eMannoor K, Shen J, Liao J, Liu Z, Jiang F. Small nucleolar RNA signatures of lung tumor-initiating cells. Mol Cancer. 2014;13:104.\u003c/li\u003e\n\u003cli\u003ePopov A, Szabo A, Mandys V. Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer. BMC Cancer. 2015;15:774.\u003c/li\u003e\n\u003cli\u003eChen L, Han L, Wei J, Zhang K, Shi Z, Duan R, et al. SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma. Sci Rep. 2015;5:8588.\u003c/li\u003e\n\u003cli\u003eXu G, Yang F, Ding CL, Zhao LJ, Ren H, Zhao P, et al. Small nucleolar RNA 113-1 suppresses tumorigenesis in hepatocellular carcinoma. Mol Cancer. 2014;13:216.\u003c/li\u003e\n\u003cli\u003eYu QC, Verheyen EM, Zeng YA. Mammary Development and Breast Cancer: A Wnt Perspective. Cancers (Basel). 2016;8.\u003c/li\u003e\n\u003cli\u003eStepanov GA, Filippova JA, Komissarov AB, Kuligina EV, Richter VA, Semenov DV. Regulatory role of small nucleolar RNAs in human diseases. Biomed Res Int. 2015;2015:206849.\u003c/li\u003e\n\u003cli\u003eCrea F, Quagliata L, Michael A, Liu HH, Frumento P, Azad AA, et al. Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression. Mol Oncol. 2016;10:693-703.\u003c/li\u003e\n\u003cli\u003eSiprashvili Z, Webster DE1, Johnston D1, Shenoy RM1, Ungewickell AJ, Bhaduri A, et al. The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nat Genet. 2016;48:53-58.\u003c/li\u003e\n\u003cli\u003eLi G, He Y, Liu X, Zheng Z, Zhang M, Qin F, et al. Small nucleolar RNA 47 promotes tumorigenesis by regulating EMT markers in hepatocellular carcinoma. Minerva Med. 2017;108:396-404.\u003c/li\u003e\n\u003cli\u003eWu L, Zheng J, Chen P, Liu Q, Yuan Y. Small nucleolar RNA ACA11 promotes proliferation, migration and invasion in hepatocellular carcinoma by targeting the PI3K/AKT signaling pathway. Biomed Pharmacother. 2017;90:705-712.\u003c/li\u003e\n\u003cli\u003eZheng D, Zhang J, Ni J, Luo J, Wang J, Tang L, et al. Small nucleolar RNA 78 promotes the tumorigenesis in non-small cell lung cancer. J Exp Clin Cancer Res. 2015;34:49.\u003c/li\u003e\n\u003cli\u003eSong JL, Nigam P, Tektas SS, Selva E. microRNA regulation of Wnt signaling pathways in development and disease. Cell Signal. 2015;27:1380-1391.\u003c/li\u003e\n\u003cli\u003eIncassati A, Chandramouli A, Eelkema R, Cowin P. Key signaling nodes in mammary gland development and cancer: beta-catenin. Breast Cancer Res. 2010;12:213.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Figures","content":"\u003cp\u003eFigure 1 to 4 are missing in this version of the manuscript.\u003c/p\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":"Breast cancer, Small nucleolar RNA, SNORD78, prognosis, cell proliferation","lastPublishedDoi":"10.21203/rs.3.rs-95730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-95730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eOBJECTIVE: \u003c/strong\u003eRecent studies suggest that abnormal snoRNA expression may play crucial role in tumor development and progression. In the study, we aim to explore the clinical significance and functional role of Small nucleolar RNA 78 (SNORD78) in breast cancer.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePATIENTS AND METHODS:\u003c/strong\u003e The expression of SNORD78 in 92 breast cancer and adjacent non tumor tissues was analyzed by quantitative real time polymerase chain reaction (qRT-PCR). The association between SNORD78 expression and clinicopathological factors was evaluated. The biological functions in vitro were examined by MTT cell proliferation, colony formation and transwell invasion assays. QRT-PCR and Western blot assays were used to analyze the mRNA and protein expression of WNT1, GSK3β and β-catenin.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRESULTS: \u003c/strong\u003eSNORD78 expression showed a significant increase in breast cancer tissues and cell lines. Higher SNORD78 expression positively was related to lymph node metastasis and TNM stage in patients. Furthermore, knockdown of SNORD78 inhibited the abilities of cell proliferation, colony formation, and cell invasion in MDA-MB-231 and SKBR-3 cells. Moreover, we demonstrated that knockdown of SNORD78 inhibited WNT/β-catenin signaling pathway in MDA-MB-231 and SKBR-3 cells via downregulating the relative protein expression of WNT1 and β-catenin and upregulating the GSK3β expression.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCONCLUSION\u003c/strong\u003e: Our results indicated that SNORD78 may be potential therapeutic target for breast cancer.\u003c/p\u003e","manuscriptTitle":"Upregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-23 20:09:17","doi":"10.21203/rs.3.rs-95730/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":"2ee18b2c-4d7c-4d3c-a11a-cdf4707e30a7","owner":[],"postedDate":"October 23rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":867441,"name":"Oncology"},{"id":867442,"name":"Pathology"}],"tags":[],"updatedAt":"2020-11-13T18:04:21+00:00","versionOfRecord":[],"versionCreatedAt":"2020-10-23 20:09:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-95730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-95730","identity":"rs-95730","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}