MUS81 Inhibits Cell Proliferation, Migration and Invasion in Breast Cancer

MUS81 Inhibits Cell Proliferation, Migration and Invasion in 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MUS81 Inhibits Cell Proliferation, Migration and Invasion in Breast Cancer Rong Xie, Xinyu Su, Yonggang Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3638911/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 Researchers have demonstrated that MUS81 is a powerful tumor suppressor, and plays in important role in DNA damage repair and maintenance of chromosomal stability in mice. To explore its role and expression pattern in human breast cancer (BC), which currently are unknown, we carried out this study to examine the correlation between MUS81 expression and the proliferation and invasiveness of BC cells. The expression of MUS81 in BC was predicted by bioinformatics. QRT-PCR and Western Blot were used to determine MUS81 expression levels in BC. Stable transfection of MUS81 overexpression lentivirus (MUS81) or MUS81 interference lentivirus (MUS81 RNAi) to establish its overexpression or knockdown in BC cells. CCK8 and colony formation experiments were used to determine the proliferation ability of the cells; scratch and transwell experiments were performed to detect the invasiveness of the cells. MUS81 overexpression cells were inoculated subcutaneously at the dorsal flanks of nude mice to form subcutaneous xenografts and evaluate the functional impact of MUS81 on the growth of BC. The expression of MUS81 and Ki-67 in xenografts were also detected by immunohistochemistry. Both MUS81 mRNA and protein expression levels were reduced significantly in BC tissues and cells compared with the corresponding normal tissues and MCF-10A. In vitro experiments indicated overexpression or silencing of MUS81 respectively decreased and facilitated proliferation and invasion of BC cells than control groups. In vivo experiment revealed a significant reduction in the growth of BC cells and tumor weight of their formed subcutaneous xenografts in nude mice was noted upon overexpression of MUS81. The expression level of Ki 67 was also dramatically reduced following the overexpression of MUS81. MUS81 was significantly low-expressed in breast cancer, and the low expression was correlated with the enhanced proliferation and aggressiveness of breast cancer cells, suggesting that MUS81 as a candidate tumor suppressor gene. MUS81 breast cancer proliferation invasiveness Figures Figure 1 Figure 2 Figure 3 Introduction Breast cancer (BC) is the most frequently cancer in women worldwide and remains the most common cause of death from cancer among women 1 . In 2020, the incidence rate of breast cancer has surpassed lung cancer, becoming the most prevalent form of cancer globally. Breast cancer is curable in about 70–80% of patients with early-stage, non-metastatic disease, while advanced breast cancer with distant metastases is considered incurable with currently available therapies 2,3 . On the molecular level, breast cancer is a heterogeneous disease. According to molecular features, breast cancer is distinguished into four subtypes: luminal A and luminal B (expressing the oestrogen receptor (ER) and/or progesterone receptor (PR)), human epidermal growth factor receptor 2 (HER2) - enriched and triple negative breast cancer (without expressing ER, PR and HER2) 4 . Previous studies have reported that the most frequently mutated and/or amplified genes in the tumor cells such as TP53 (41% of tumors), PIK3CA (30%), MYC (20%), PTEN (16%), CCND1 (16%), ERBB2 (13%), FGFR1 (11%) and GATA3 (10%) were closely related to breast cancer 5 . However, in order to improve the therapeutic efficacy of breast cancer, further investigations are meaningful to identify gene alterations as new markers for estimating cancer progression. The MUS81 gene encoding product is a structure-specific DNA endonuclease that resolve Holliday junctions (HJs) by constituting a heterodimer with EME1/MMs4, playing a crucial role in the repair of DNA double strand breaks (DSBs) and maintenance of chromosomal integrity 6-8 . In 2004, McPherson et al. found that 73% of MUS81 -/- mice and 50% of MUS81 +/- mice died of various spontaneous tumors such as lymphoma, breast cancer and prostate cancer through gene knockout technology, which indicates that MUS81 is a candidate tumor suppressor gene in mice 9 . Recently, an in vitro study revealed that the haploinsufficiency of MUS81 in human colon cancer cell line HCT116 could activate the intra-S-phase and G2 ⁄ M checkpoints and promote cellular replication, suggesting the involvement of MUS81 in the carcinogenesis of colorectal cancer 10,11 . Moreover, Wu et al. reported MUS81 mRNA and protein expression levels were decreased significantly in hepatocellular carcinoma tissues, and low-expression of MUS8l was closely related to poor differentiation, metastasis and poor prognosis of liver cancer patients 12 . Although these studies have indicated the potential role of MUS81 in human malignancies, the role of MUS81 and the evidence for its expression pattern in human breast cancer remain largely unknown. Thus, we carried out this study to determine the expression pattern of MUS81 in breast cancer and to explore the functions of MUS81 on proliferation, invasion and migration of breast cancer. Materials and methods Patients and tissues Specimens of breast cancer tissues and adjacent normal specimens were obtained from 16 patients who had undergone operation at the Breast and Thyroid Surgery, Central Hospital of Wuhan between 2022 and 2023. All the patients did not receive neoadjuvant therapy (including chemotherapy, targeted therapy and endocrine therapy) before surgery. All tissues were collected and frozen in liquid nitrogen immediately after surgery and then stored at ﹣80℃ until analysis. This study was approved by the Medical Research Ethics Committee of Central Hospital of Wuhan. Prior written informed consent was obtained from all patients. Cell lines Human breast cancer MDA-MB-231, MDA-MB-468, BT549, MCF-7, BT474, T47D cells and normal breast epithelial cell MCF-10A were purchased from the American Model Culture Repository (ATCC). MDA-MB-231 was cultured in L15 medium containing 10% Fetal bovine serum (FBS); MDA-MB-468, BT549 and T47D were cultured in RPMI-1640 medium, supplemented with 10% FBS; MCF-7 and BT474 were cultured in DMEM medium containing 10% FBS; MCF-10A was cultured in DME/F12 medium, supplemented with 10% FBS, 10ug/ml insulin, 20ng/ml epidermal growth factor, 100ng/ml cholera toxin and 0.5ug/ml hydrocortisone. MDA-MB-231 was incubated at 37 ℃ constant temperature incubator without CO 2 ; All other cells at 37 ℃ in a humidified incubator with 5% CO 2 . In vivo experiment The animal experiment was approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. 10 nude mice (BALB/c-nu, 3-4 weeks old) were purchased from Molebao (Wuhan) Biotechnology Co., Ltd, and randomly divided into two groups. For the transplantation tumor experiment, cell suspension(100uL) of stable transduced MDA-MB-231 (1×10 7 ) was injected subcutaneously into the left axilla of each group. The longest and shortest diameter of the tumor were measured using a caliper, tumor volumes were calculated and recorded (Volume=length × width 2 /2) every 5 days. After 4-5 weeks of feeding, the implanted tumors were stripped, fixed with 4% Formaldehyde#Forms, and then prepared for immunohistochemical staining. RNA extraction and Quantitative real-time polymerase chain reaction (QRT-PCR) Total RNA was extracted from cells and tissues by the Trizol reagent (Invitrogen, USA), and then was reverse transcribed into cDNA utilizing the PrimeScript® RT reagent Kit (Takara, Japan). QRT-PCR was performed using a TB Green Premix Ex Taq II kit (Takara Bio Inc., Japan). MUS81 primers for PCR were as follows: forward 5’-TGTGTGGACATTGGCGAGAC-3’ and reverse 5’-CTGCAAAGGTCATCCAGTC G-3’. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was measured and used as an internal control. The transcript levels of MUS81 were normalized to GAPDH and analyzed by 2 －△△ Ct method. Gene over-expression and knockdown Lentivirus over-expressing MUS81 gene and lentivirus expressing RNAi specific for MUS81 gene were purchased from Jikai Gene Chemical Technology Co., Ltd, Shanghai, China. The above lentivirus were transfected into breast cancer cells in cell culture medium with HitransG P (25×). After screening with puromycin (Sigma-Aldrich, MO, USA), stable transduced cells were established. The overexpression and knockdown efficiency of the lentivirus was determined by western blot and PCR. Western blot Protein of cells or tissues was extracted using 1× cell lysis buffer (Biosharp). Total protein was separated by SDS-PAGE and then transferred onto PVDF membrane (Merck Millipore). The membranes were probed with primary antibodies specific for Mus81(ab14387, Abcam, 1:1000 dilution) and GAPDH (ab8245, Abcam, 1:3000 dilution) and then the corresponding secondary antibody in order. GAPDH protein was determined as a loading control. Cell proliferative experiments The cells were seeded in a 96-well plate, five wells per group and each well contained 3×10 3 cells. After culturing for 1, 2, 3, 4 and 5 days, 10 µL of CCK-8 (cell counting kit-8, Dojindo, Japan) reagent was added in each well. Absorbance value was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). For the colony formation assay, the cells (1×10 3 per well, six wells per group) were cultured in 6-well plates. Incubated for 14 days until visible clones appeared, the clones were stained with crystal violet dye (Aspen) for 30 min after fixed with 4% paraformaldehyde for 20 min. The number of colonies were observed under a light microscope. All experiments were repeated for three times. Cell invasion assays 200uL tumor cell suspension containing 0.1% serum (2×10 4 per well) were added to upper chamber of Transwell insert with 8.0-μm pores (Corning, New York, USA), 600uL culture medium containing 20% serum was added to the upper chamber. Allowed to invade for 24~48h, invaded cells were stained with 0.1% crystal violet for 10 min, and counted under the microscope. For the scratch experiment, the cells were cultured in 6-well plates, and the appropriate number of cells should be overspread to the bottom of the plate after adhering to the wall. A straight line was drew on each group of cells using the pipette suction head of 10uL, detached cells were washed off with PBS. 2mLof serum-free culture medium to each group of cells, and recorded the scratch width of each group of cells under an inverted microscope for 0 and 48 hours. Immunohistochemistry staining Immunohistochemistry (IHC) was used to detect the expression of Mus81 and Ki67 in xenograft tumors. Briefly, the paraffin sections of xenograft tumors were dewaxed, rehydrated, and subjected to microwave heat-induced antigen recovery in citrate buffer (pH 6.0). Subsequently, the sections were incubated with 3% H 2 O 2 to block endogenous peroxidase. After rinsing with TBS, the sections were blocked with 10% goat serum and incubated overnight at 4℃ with primary antibody. On the second day, the sections were incubated with the corresponding HRP-conjugated second antibody for 1 hour at room temperature. Then, all sections were visualized by reacted with diaminobenzidine and re stained with hematoxylin. Next, the sections were dehydrated and dried with gradient alcohol. Finally, the sections were transparent with xylene and sealed with neutral gum, followed by subjected to microscopic examination and photography. Statistical analysis We used the Mann–Whitney U -test to analyze the MUS81 expression levels in the breast cancer tissues and the corresponding normal tissues. Student’s t test was applied for comparing the differences between the groups. All statistical analyses were performed using SPSS Version 22.0. and a P-value of <0.05 was considered statistically significant. Results Down-regulation of MUS81 in breast cancer. To investigate the expression pattern of MUS81 in breast cancer, we first analyzed the mRNA sequencing data of 1085 breast cancer tissues in TCGA database and 291 normal breast tissues in GTEx database through the GEPIA2 website (http://gepia2.cancer-pku.cn/). We identified the expression of MUS81 mRNA was lower in breast cancer tissues (Fig 1a). Western blot and real-time quantitative RT-PCR (qRT-PCR) were utilized to verify this result at the cellular and tissue levels. The relative expression of MUS81 in breast cancer cells was significantly lower than that in MCF-10A (Fig 1b, d). In addition, western blot and qRT-PCR revealed lower MUS81 levels in breast cancer tissues, than those of corresponding para-cancerous tissues (Fig 1c, e). These data suggested down-regulation of MUS81 in breast cancer. Fig.1 a GEPIA2 website (http://gepia2.cancer-pku.cn/) revealed the low expression of MUS81 in breast cancer to normal controls. b, c QRT-PCR indicated that the expression level of MUS81 mRNA in BC cells (MDA-MB-231、MDA-MB-468、BT549、MCF-7、BT474、T47D) and BC tissues (n=16) was significantly reduced compared to MCF-10A and corresponding para-cancerous tissues. d, e Western blot showed that the expression level of MUS81 protein in BC cells (MDA-MB-231MDA-MB-468、BT549、BT474、T47D) and BC tissues (n=10) was significantly lower than that in MCF-10A and corresponding para-cancerous tissues Knockdown of MUS81 facilitates the proliferation and invasion of breast cancer cells. Since the low expression of MUS81 in breast cancer, we explored the functional impact of MUS81 overexpression or knockdown on breast cancer cell lines representing low or high expression levels. Depletion of MUS81 using interference lentivirus (MUS81 RNAi) in MCF-10A and T47D cells resulted in a dramatic decrease in MUS81 expression levels (Fig 2a). CCK-8 and the colony formation assays indicated MCF-10A and T47D cells with stably suppressed MUS81 exhibited enhanced proliferation compared to those transfected with negative control (RNAi NC; Fig 2b, c). Scratch and Transwell experiments revealed that the invasiveness of breast cells was enhanced following stable transfection of MUS81 RNAi (Fig 2d, e). Fig.2 a Western blot and qRT-PCR assays indicated the expression of MUS81 in MCF-10A and T47D stably transfected with RNAi NC or MUS81 RNAi. b, c CCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the downregulation of MUS81 expression. d, e Scratch and Transwell experiments showed the invasive capability of BC cells stably transfected with RNAi NC or MUS81 RNAi Overexpression of MUS81 inhibits the proliferation and invasion of breast cancer cells. Stable transfection of Lentivirus over-expressing MUS81 (MUS81) led to its overexpression in MDA-MB-231 and BT549 cells. The transfection efficiency was confirmed by western blot and qRT-PCR (Figure 3A). CCK-8 and the colony formation assays showed overexpression of MUS81 decreased the growth of breast cancer cells, than those transfected by empty vector (Mock; Fig 3b, c). Migratory potential of breast cancer cells transfected with MUS81 was significantly reduced compared to the control groups (Fig 3d, e). Moreover, in vivo experiment indicated that a significant reduction in the growth of MDA-MB-231 cells and tumor weight of their formed subcutaneous xenografts in nude mice was noted upon stable overexpression MUS81 (Fig 3f). Immunohistochemistry was performed to detect the expression of MUS81 and proliferation index Ki 67 in subcutaneous implanted tumors. The results showed that the expression level of Ki 67 was dramatically reduced following the overexpression of MUS81 (Fig 3g). Together, these results suggested that MUS81 inhibited the growth and aggressiveness of breast cancer cell lines. Fig.3 a Western blot and qRT-PCR assays indicated the expression of MUS81 in MDA-MB-231 and BT549 stably transfected with Mock or MUS81. b, c CCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the overexpression of MUS81. d, e Scratch and Transwell experiments showed the invasive capability of BC cells stably transfected with Mock or MUS81. f In vivo growth curves (left panel) and representative images (right panel) of xenografts formed by subcutaneous injection of MDA-MB-231 stably transfected with Mock or MUS81. g Immunohistochemistry staining of subcutaneous xenografts showed the expression of MUS81 and Ki 67 Discussion MUS81 is a structure specific DNA endonuclease that was first discovered by Interthal et al. in 2000 13 . MUS81 is able to restart stagnant replication forks and resolve HJs by forming heterodimers with EME1 or EME2, and plays a crucial role in repairing DSBs and maintaining chromosome integrity 6 , 14 . A recent study has reported that the low expression of MUS81 in human hepatocellular carcinoma (HCC) and demonstrated its relevance to poor prognosis, which provided the first evidence for the expression pattern and role of MUS81 in human solid tumor 12 . Another research suggests the downregulation of MUS81 in colorectal cancer, which is not only consistent with the expression pattern of MUS81 in HCC, but also with the view that MUS81 might be a candidate as a potent tumor suppressor 11 . At present, few scholars have explored the impact of MUS81 on the occurrence and development of human malignant tumors. The expression pattern and role of MUS81 in human breast cancer (BC) have not been reported. In this study, we first predicted the expression pattern of MUS81 in BC through bioinformatics, and found that MUS81 was low expressed in BC. Subsequently, the decreased of MUS81 in BC tissues and BC cell lines was verified by qRT-PCR and western blot experiments. Therefore, we infered that MUS81 plays an anti-tumor role in BC. So as to confirm this inference, we proved that MUS81 could inhibit the proliferation, invasion and migration of BC through a series of cell experiments and animal experiments. In conclusion, the results of our study indicate that MUS81 is down-regulated in human BC, and its low-expression is correlated with the growth and aggressiveness of BC. However, it is unclear whether MUS81 is related to the prognosis in patients with BC. The molecular mechanism of this gene's anti-tumor effect in BC is still unknown. Moreover, further studies are required to determine how and why MUS81 expression is down-regulated in BC. Declarations Author Contribution Rong Xie performed partial experiments, wrote the main manuscript text and prepared figures 1-3. Xinyu Su collected specimens and performed partial experiments. Yonggang Yang revised the manuscript. All authors reviewed the manuscript. References Harbeck N, Penault-Llorca F, Cortes J et al (2019) Breast cancer. Nat Rev Dis Primers 23;5(1):66. 10.1038/s41572-019-0111-2 DeSantis C, Ma J, Bryan L, Jemal A et al (2014) Breast cancer statistics, 2013. CA Cancer J Clin 64(1):52–62. 10.3322/caac.21203 Harbeck N, Gnant M (2017) Breast cancer. Lancet 18;389(10074):1134–1150. 10.1016/S0140-6736(16)31891-8 Perou CM, Sørlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 17(6797):747–752. 10.1038/35021093 Nik-Zainal S, Davies H, Staaf J et al (2019) Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 566(7742):E1. 10.1038/s41586-019-0883-2 Boddy MN, Gaillard PHL, McDonald WH et al (2001) Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 16(4):537–548. 10.1016/s0092-8674(01)00536-0 Chen XB, Melchionna R, Denis CM et al (2001) Human Mus81-associated endonuclease cleaves Holliday junctions in vitro. Mol Cell 8(5):1117–1127. 10.1016/s1097-2765(01)00375-6 Bastin-Shanower SA, Fricke WM, Mullen JR, Brill SJ (2003) The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10. Mol Cell Biol 23(10):3487–3496. 10.1128/MCB.23.10.3487-3496 McPherson JP, Lemmers B, Chahwan R et al (2004) Involvement of mammalian Mus81 in genome integrity and tumor suppression. Science 18(5678):1822–1826. 10.1126/science.1094557 Hiyama T, Katsura M, Yoshihara T et al (2006) Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells. Nucleic Acids Res 2;34(3):880 – 92. 10.1093/nar/gkj495 Wu F, Shirahata A, Sakuraba K et al (2011) Downregulation of Mus81 as a novel prognostic biomarker for patients with colorectal carcinoma. Cancer Sci 102(2):472–477. 10.1111/j.1349-7006.2010.01790.x Wu F, Liu SY, Tao YM et al (2008) Decreased expression of methyl methansulfonate and ultraviolet-sensitive gene clone 81 (Mus81) is correlated with a poor prognosis in patients with hepatocellular carcinoma. Cancer 1;112(9):2002-10. 10.1002/cncr.23396 Interthal H, Heyer WD (2000) MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae. Mol Gen Genet 263(5):812–827. 10.1007/s004380000241 Ciccia A, McDonald N, West SC (2008) Structural and functional relationships of the XPF/MUS81 family of proteins. Annu Rev Biochem 77:259–287. 10.1146/annurev.biochem.77.070306.102408 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3638911","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":251723695,"identity":"ba3f876f-59fa-4606-b083-0f6cef0722f8","order_by":0,"name":"Rong Xie","email":"","orcid":"","institution":"The Central Hospital of Wuhan","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Xie","suffix":""},{"id":251723696,"identity":"0bd1f60a-bb32-4ba8-8e10-1d04e326550f","order_by":1,"name":"Xinyu Su","email":"","orcid":"","institution":"The Central Hospital of 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10:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3638911/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3638911/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47063049,"identity":"6bc4b06d-112a-4f02-9fb2-726f0f57f120","added_by":"auto","created_at":"2023-11-25 12:56:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eGEPIA2 website (\u003ca href=\"http://gepia2.cancer-pku.cn/\"\u003ehttp://gepia2.cancer-pku.cn/\u003c/a\u003e) revealed the low expression of MUS81 in breast cancer to normal controls. \u003cstrong\u003eb, c \u003c/strong\u003eQRT-PCR indicated that the expression level of MUS81 mRNA in BC cells (MDA-MB-231、MDA-MB-468、BT549、MCF-7、BT474、T47D) and BC tissues (n=16) was significantly reduced compared to MCF-10A and corresponding para-cancerous tissues. \u003cstrong\u003ed, e \u003c/strong\u003eWestern blot showed that the expression level of MUS81 protein in BC cells (MDA-MB-231MDA-MB-468、BT549、BT474、T47D) and BC tissues (n=10) was significantly lower than that in MCF-10A and corresponding para-cancerous tissues\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3638911/v1/334831fc707f72fbfd9d4e2d.jpg"},{"id":47063047,"identity":"a84a33e2-a4dc-4396-95fa-4b3db4dfe6c7","added_by":"auto","created_at":"2023-11-25 12:56:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":280416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eWestern blot and qRT-PCR assays indicated the expression of MUS81 in MCF-10A and T47D stably transfected with RNAi NC or MUS81 RNAi. \u003cstrong\u003eb, c \u003c/strong\u003eCCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the downregulation of MUS81 expression. \u003cstrong\u003ed, e \u003c/strong\u003eScratch and Transwell experiments showed the invasive capability of BC cells stably transfected with RNAi NC or MUS81 RNAi\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3638911/v1/b5ee0af25c7e080949da969d.jpg"},{"id":47063048,"identity":"4dcd12ad-60a2-40ff-a888-cb2a967e1059","added_by":"auto","created_at":"2023-11-25 12:56:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":372779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eWestern blot and qRT-PCR assays indicated the expression of MUS81 in MDA-MB-231 and BT549 stably transfected with Mock or MUS81. \u003cstrong\u003eb, c \u003c/strong\u003eCCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the overexpression of MUS81. \u003cstrong\u003ed, e \u003c/strong\u003eScratch and Transwell experiments showed the invasive capability of BC cells stably transfected with Mock or MUS81. \u003cstrong\u003ef\u003c/strong\u003eIn vivo growth curves (left panel) and representative images (right panel) of xenografts formed by subcutaneous injection of MDA-MB-231 stably transfected with Mock or MUS81. \u003cstrong\u003eg \u003c/strong\u003eImmunohistochemistry staining of subcutaneous xenografts showed the expression of MUS81 and Ki 67\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3638911/v1/04ba3cc80c75d3fa8a40d2b0.jpg"},{"id":49068332,"identity":"8fd4efde-7bae-4fd5-acde-11abedfbe815","added_by":"auto","created_at":"2024-01-02 16:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":673513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3638911/v1/525f023b-98e1-4248-8ed7-ec0029ada659.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMUS81 Inhibits Cell Proliferation, Migration and Invasion in Breast Cancer\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most frequently cancer in women worldwide and remains the most common cause of death from cancer among women\u003csup\u003e1\u003c/sup\u003e. In 2020, the incidence rate of breast cancer has surpassed lung cancer, becoming the most prevalent form of cancer globally. Breast cancer is curable in about 70\u0026ndash;80% of patients with early-stage, non-metastatic disease, while advanced breast cancer with distant metastases is considered incurable with currently available therapies\u003csup\u003e2,3\u003c/sup\u003e. On the molecular level, breast cancer is a heterogeneous disease. According to molecular\u0026nbsp;features,\u0026nbsp;breast cancer is distinguished into four subtypes:\u0026nbsp;luminal A and luminal B (expressing the oestrogen receptor (ER) and/or progesterone receptor (PR)),\u0026nbsp;human epidermal growth factor receptor 2 (HER2) - enriched and triple negative breast cancer (without expressing ER, PR and HER2)\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;Previous studies have reported that the most frequently mutated and/or amplified genes in the tumor cells such as \u003cem\u003eTP53\u003c/em\u003e (41% of tumors), \u003cem\u003ePIK3CA\u0026nbsp;\u003c/em\u003e(30%), \u003cem\u003eMYC\u003c/em\u003e (20%), \u003cem\u003ePTEN\u003c/em\u003e (16%), \u003cem\u003eCCND1\u003c/em\u003e (16%), \u003cem\u003eERBB2\u0026nbsp;\u003c/em\u003e(13%), \u003cem\u003eFGFR1\u003c/em\u003e (11%) and \u003cem\u003eGATA3\u003c/em\u003e (10%) were closely related to breast cancer\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;However, in order to improve the therapeutic efficacy of breast cancer, further investigations are meaningful to identify gene alterations as new markers for estimating cancer progression.\u003c/p\u003e\n\u003cp\u003eThe MUS81 gene encoding product is a structure-specific DNA endonuclease that resolve Holliday junctions (HJs) by constituting a heterodimer with EME1/MMs4, playing a crucial role in the repair of DNA double strand breaks (DSBs) and maintenance of chromosomal integrity\u003csup\u003e6-8\u003c/sup\u003e.\u0026nbsp;In 2004, McPherson et al. found that 73% of MUS81\u003csup\u003e-/-\u003c/sup\u003e mice and 50% of MUS81\u003csup\u003e+/-\u003c/sup\u003e mice died of various spontaneous tumors such as lymphoma, breast cancer and prostate cancer through gene knockout technology, which indicates that MUS81 is a candidate tumor suppressor gene in mice\u003csup\u003e9\u003c/sup\u003e.\u0026nbsp;Recently, an in vitro study revealed that the haploinsufficiency of MUS81 in human colon cancer cell line HCT116 could activate the intra-S-phase and G2 \u0026frasl; M checkpoints and promote cellular replication, suggesting the involvement of MUS81 in the carcinogenesis of colorectal cancer\u003csup\u003e10,11\u003c/sup\u003e. Moreover, Wu et al. reported MUS81 mRNA and protein expression levels were decreased significantly in hepatocellular carcinoma tissues,\u0026nbsp;and low-expression of MUS8l was closely related to poor differentiation, metastasis and poor prognosis of liver cancer patients\u003csup\u003e12\u003c/sup\u003e. Although these studies have indicated the potential role of MUS81 in human malignancies, the role of MUS81 and the evidence for its expression pattern in human breast cancer remain largely unknown. Thus, we carried out this study to determine the expression pattern of MUS81 in breast cancer and to explore the functions of MUS81 on proliferation, invasion and migration of breast cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePatients and tissues\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens of breast cancer tissues and adjacent normal specimens were obtained from 16 patients who had undergone operation at the Breast and Thyroid Surgery, Central Hospital of Wuhan between 2022 and 2023. All the patients did not receive neoadjuvant therapy (including chemotherapy, targeted therapy and endocrine therapy) before surgery. All tissues were collected and frozen in liquid nitrogen immediately after surgery and then stored at\u0026nbsp;﹣80℃\u0026nbsp;until analysis. This study was approved by the Medical Research Ethics Committee of Central Hospital of Wuhan. Prior written informed consent was obtained from all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell lines\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman breast cancer MDA-MB-231, MDA-MB-468, BT549, MCF-7, BT474, T47D cells and normal breast epithelial cell MCF-10A were purchased from the American Model Culture Repository (ATCC). MDA-MB-231 was cultured in L15 medium containing 10% Fetal bovine serum (FBS);\u0026nbsp;MDA-MB-468, BT549 and T47D were cultured in RPMI-1640 medium,\u0026nbsp;supplemented with 10% FBS;\u0026nbsp;MCF-7 and BT474 were cultured in DMEM medium containing 10% FBS; MCF-10A was cultured in\u0026nbsp;DME/F12 medium, supplemented with 10% FBS, 10ug/ml insulin, 20ng/ml epidermal growth factor, 100ng/ml cholera toxin and 0.5ug/ml hydrocortisone. MDA-MB-231 was incubated at 37 ℃ constant temperature incubator without CO\u003csub\u003e2\u003c/sub\u003e; All other cells at 37 ℃ in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo experiment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiment was approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. 10\u0026nbsp;nude mice (BALB/c-nu, 3-4 weeks old) were purchased from Molebao (Wuhan) Biotechnology Co., Ltd,\u0026nbsp;and randomly divided into two groups.\u0026nbsp;For the transplantation tumor experiment, cell suspension(100uL) of stable transduced\u0026nbsp;MDA-MB-231 (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) was injected subcutaneously into the left axilla of each group. The longest and shortest diameter of the tumor were measured using a caliper,\u0026nbsp;tumor volumes were calculated and recorded (Volume=length \u0026times; width\u003csup\u003e2\u003c/sup\u003e /2) every 5 days.\u0026nbsp;After 4-5 weeks of feeding, the implanted tumors were stripped, fixed with 4% Formaldehyde#Forms, and then prepared for immunohistochemical staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA extraction and Quantitative real-time polymerase chain reaction (QRT-PCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells and tissues by the Trizol reagent (Invitrogen, USA), and then was reverse transcribed into cDNA utilizing the PrimeScript\u0026reg; RT reagent Kit (Takara, Japan). QRT-PCR was performed using\u0026nbsp;a TB Green Premix Ex Taq II kit (Takara Bio Inc., Japan). MUS81 primers for PCR were as follows: forward 5\u0026rsquo;-TGTGTGGACATTGGCGAGAC-3\u0026rsquo; and reverse 5\u0026rsquo;-CTGCAAAGGTCATCCAGTC\u003c/p\u003e\n\u003cp\u003eG-3\u0026rsquo;.\u0026nbsp;The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was measured and used as an internal control. The transcript levels of MUS81 were normalized to GAPDH and analyzed by 2\u003csup\u003e－△△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGene over-expression and knockdown\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentivirus over-expressing MUS81 gene and lentivirus expressing RNAi specific for MUS81 gene were purchased from Jikai Gene Chemical Technology Co., Ltd, Shanghai, China. The above lentivirus were transfected into breast cancer cells in cell culture medium with\u0026nbsp;HitransG P (25\u0026times;).\u0026nbsp;After screening with puromycin (Sigma-Aldrich, MO, USA),\u0026nbsp;stable transduced cells were established.\u0026nbsp;The overexpression and knockdown efficiency of the\u0026nbsp;lentivirus\u0026nbsp;was determined by western blot and PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein of cells or tissues was extracted using 1\u0026times; cell lysis buffer (Biosharp).\u0026nbsp;Total protein was separated by SDS-PAGE and then transferred onto PVDF membrane (Merck Millipore).\u0026nbsp;The membranes were probed with primary antibodies specific for Mus81(ab14387, Abcam, 1:1000 dilution) and GAPDH (ab8245, Abcam, 1:3000 dilution)\u0026nbsp;and then the corresponding secondary antibody in order.\u0026nbsp;GAPDH protein was determined as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell proliferative experiments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were seeded in a 96-well plate, five wells per group and each well contained 3\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells. After culturing for 1, 2, 3, 4 and 5 days, 10 \u0026micro;L of CCK-8 (cell counting kit-8, Dojindo, Japan)\u0026nbsp;reagent was added in each well. Absorbance value was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). For the colony formation assay, the cells (1\u0026times;10\u003csup\u003e3\u003c/sup\u003e per well, six wells per group) were cultured in 6-well plates.\u0026nbsp;Incubated for 14 days until visible clones appeared, the clones were stained with crystal violet dye (Aspen) for 30 min after fixed with 4% paraformaldehyde for 20 min. The number of colonies were observed under a light microscope. All experiments were repeated for three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell invasion assays\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e200uL tumor cell suspension containing 0.1% serum (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e per well) were added to upper chamber of Transwell insert with 8.0-\u0026mu;m pores (Corning, New York, USA), 600uL culture medium containing 20% serum was added to the upper chamber.\u0026nbsp;Allowed to invade for 24~48h, invaded cells were stained with 0.1% crystal violet for 10 min, and counted under the microscope. For the scratch experiment, the cells were cultured in 6-well plates, and the appropriate number of cells should be overspread to the bottom of the plate after adhering to the wall. A straight line was drew on each group of cells using the pipette suction head of 10uL,\u0026nbsp;detached cells were washed off with PBS. 2mLof serum-free culture medium to each group of cells, and recorded the scratch width of each group of cells under an inverted microscope for 0 and 48 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry staining\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry (IHC) was used to detect the expression of Mus81 and Ki67 in xenograft tumors. Briefly, the paraffin sections of xenograft tumors were dewaxed, rehydrated, and subjected to microwave heat-induced antigen recovery in citrate buffer (pH 6.0). Subsequently, the sections were incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to block endogenous peroxidase. After rinsing with TBS, the sections were blocked with 10% goat serum and incubated overnight at 4℃ with primary antibody. On the second day, the sections were incubated with the corresponding HRP-conjugated second antibody for 1 hour at room temperature. Then, all sections were visualized by reacted with diaminobenzidine and re stained with hematoxylin. Next, the sections were dehydrated and dried with gradient alcohol. Finally, the sections were transparent with xylene and sealed with neutral gum, followed by subjected to microscopic examination and photography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test to analyze the MUS81 expression levels in the breast cancer tissues and the corresponding normal tissues. Student\u0026rsquo;s t test was applied for comparing the differences between the groups.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eAll statistical analyses were performed using SPSS Version 22.0. and a P-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDown-regulation of MUS81 in breast cancer.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the expression pattern of MUS81 in breast cancer, we first analyzed the mRNA sequencing data of 1085 breast cancer tissues in TCGA database and 291 normal breast tissues in GTEx database through the GEPIA2 website (http://gepia2.cancer-pku.cn/). We identified the expression of MUS81 mRNA was lower in breast cancer tissues (Fig 1a). Western blot and real-time quantitative RT-PCR (qRT-PCR) were utilized to verify this result at the cellular and tissue levels. The relative expression of MUS81 in breast cancer cells was significantly lower than that in MCF-10A (Fig 1b, d). In addition, western blot and qRT-PCR revealed lower MUS81 levels in breast cancer tissues, than those of corresponding para-cancerous tissues (Fig 1c, e). These data suggested down-regulation of MUS81 in breast cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.1 a\u0026nbsp;\u003c/strong\u003eGEPIA2 website (http://gepia2.cancer-pku.cn/) revealed the low expression of MUS81 in breast cancer to normal controls. \u003cstrong\u003eb, c\u0026nbsp;\u003c/strong\u003eQRT-PCR indicated that the expression level of MUS81 mRNA in BC cells (MDA-MB-231、MDA-MB-468、BT549、MCF-7、BT474、T47D) and BC tissues (n=16) was significantly reduced compared to MCF-10A and corresponding para-cancerous tissues. \u003cstrong\u003ed, e\u0026nbsp;\u003c/strong\u003eWestern blot showed that the expression level of MUS81 protein in BC cells (MDA-MB-231MDA-MB-468、BT549、BT474、T47D) and BC tissues (n=10) was significantly lower than that in MCF-10A and corresponding para-cancerous tissues\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eKnockdown of MUS81 facilitates the proliferation and invasion of breast cancer cells.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the low expression of MUS81 in breast cancer, we explored the functional impact of MUS81 overexpression or knockdown on breast cancer cell lines representing low or high expression levels.\u003c/p\u003e\n\u003cp\u003eDepletion of MUS81 using interference lentivirus (MUS81 RNAi) in MCF-10A and T47D cells resulted in a dramatic decrease in MUS81 expression levels (Fig 2a). CCK-8 and the colony formation assays indicated MCF-10A and T47D cells with stably suppressed MUS81 exhibited enhanced proliferation compared to those transfected with negative control (RNAi NC; Fig 2b, c). Scratch and Transwell experiments revealed that the invasiveness of breast cells was enhanced following stable transfection of MUS81 RNAi (Fig 2d, e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.2 a\u0026nbsp;\u003c/strong\u003eWestern blot and qRT-PCR assays indicated the expression of MUS81 in MCF-10A and T47D stably transfected with RNAi NC or MUS81 RNAi. \u003cstrong\u003eb, c\u0026nbsp;\u003c/strong\u003eCCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the downregulation of MUS81 expression. \u003cstrong\u003ed, e\u0026nbsp;\u003c/strong\u003eScratch and Transwell experiments showed the invasive capability of BC cells stably transfected with RNAi NC or MUS81 RNAi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOverexpression of MUS81 inhibits the proliferation and invasion of breast cancer cells.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable transfection of Lentivirus over-expressing MUS81 (MUS81) led to its overexpression in MDA-MB-231 and BT549 cells. The transfection efficiency was confirmed by western blot and qRT-PCR (Figure 3A). CCK-8 and the colony formation assays showed overexpression of MUS81 decreased the growth of breast cancer cells, than those transfected by empty vector (Mock; Fig 3b, c). Migratory potential of breast cancer cells transfected with MUS81 was significantly reduced compared to the control groups (Fig 3d, e).\u003c/p\u003e\n\u003cp\u003eMoreover, in vivo experiment indicated that a significant reduction in the growth of MDA-MB-231 cells and tumor weight of their formed subcutaneous xenografts in nude mice was noted upon stable overexpression MUS81 (Fig 3f). \u0026nbsp;Immunohistochemistry was performed to detect the expression of MUS81 and proliferation index Ki 67 in subcutaneous implanted tumors. The results showed that the expression level of Ki 67 was dramatically reduced following the overexpression of MUS81 (Fig 3g). Together, these results suggested that MUS81 inhibited the growth and aggressiveness of breast cancer cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.3 a\u0026nbsp;\u003c/strong\u003eWestern blot and qRT-PCR assays indicated the expression of MUS81 in MDA-MB-231 and BT549 stably transfected with Mock or MUS81. \u003cstrong\u003eb, c\u0026nbsp;\u003c/strong\u003eCCK-8 and the colony formation assays depicted the change in proliferation of BC cells following the overexpression of MUS81. \u003cstrong\u003ed, e\u0026nbsp;\u003c/strong\u003eScratch and Transwell experiments showed the invasive capability of BC cells stably transfected with Mock or MUS81. \u003cstrong\u003ef\u003c/strong\u003e In vivo growth curves (left panel) and representative images (right panel) of xenografts formed by subcutaneous injection of MDA-MB-231 stably transfected with Mock or MUS81. \u003cstrong\u003eg\u0026nbsp;\u003c/strong\u003eImmunohistochemistry staining of subcutaneous xenografts showed the expression of MUS81 and Ki 67\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMUS81 is a structure specific DNA endonuclease that was first discovered by Interthal et al. in 2000\u003csup\u003e13\u003c/sup\u003e. MUS81 is able to restart stagnant replication forks and resolve HJs by forming heterodimers with EME1 or EME2, and plays a crucial role in repairing DSBs and maintaining chromosome integrity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A recent study has reported that the low expression of MUS81 in human hepatocellular carcinoma (HCC) and demonstrated its relevance to poor prognosis, which provided the first evidence for the expression pattern and role of MUS81 in human solid tumor\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Another research suggests the downregulation of MUS81 in colorectal cancer, which is not only consistent with the expression pattern of MUS81 in HCC, but also with the view that MUS81 might be a candidate as a potent tumor suppressor\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. At present, few scholars have explored the impact of MUS81 on the occurrence and development of human malignant tumors. The expression pattern and role of MUS81 in human breast cancer (BC) have not been reported.\u003c/p\u003e \u003cp\u003eIn this study, we first predicted the expression pattern of MUS81 in BC through bioinformatics, and found that MUS81 was low expressed in BC. Subsequently, the decreased of MUS81 in BC tissues and BC cell lines was verified by qRT-PCR and western blot experiments. Therefore, we infered that MUS81 plays an anti-tumor role in BC. So as to confirm this inference, we proved that MUS81 could inhibit the proliferation, invasion and migration of BC through a series of cell experiments and animal experiments.\u003c/p\u003e \u003cp\u003eIn conclusion, the results of our study indicate that MUS81 is down-regulated in human BC, and its low-expression is correlated with the growth and aggressiveness of BC. However, it is unclear whether MUS81 is related to the prognosis in patients with BC. The molecular mechanism of this gene's anti-tumor effect in BC is still unknown. Moreover, further studies are required to determine how and why MUS81 expression is down-regulated in BC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRong Xie performed partial experiments, wrote the main manuscript text and prepared figures 1-3. Xinyu Su collected specimens and performed partial experiments. Yonggang Yang revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarbeck N, Penault-Llorca F, Cortes J et al (2019) Breast cancer. Nat Rev Dis Primers 23;5(1):66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41572-019-0111-2\u003c/span\u003e\u003cspan address=\"10.1038/s41572-019-0111-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeSantis C, Ma J, Bryan L, Jemal A et al (2014) Breast cancer statistics, 2013. CA Cancer J Clin 64(1):52\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21203\u003c/span\u003e\u003cspan address=\"10.3322/caac.21203\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbeck N, Gnant M (2017) Breast cancer. 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Annu Rev Biochem 77:259\u0026ndash;287. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.biochem.77.070306.102408\u003c/span\u003e\u003cspan address=\"10.1146/annurev.biochem.77.070306.102408\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MUS81, breast cancer, proliferation, invasiveness","lastPublishedDoi":"10.21203/rs.3.rs-3638911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3638911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResearchers have demonstrated that MUS81 is a powerful tumor suppressor, and plays in important role in DNA damage repair and maintenance of chromosomal stability in mice. To explore its role and expression pattern in human breast cancer (BC), which currently are unknown, we carried out this study to examine the correlation between MUS81 expression and the proliferation and invasiveness of BC cells. The expression of MUS81 in BC was predicted by bioinformatics. QRT-PCR and Western Blot were used to determine MUS81 expression levels in BC. Stable transfection of MUS81 overexpression lentivirus (MUS81) or MUS81 interference lentivirus (MUS81 RNAi) to establish its overexpression or knockdown in BC cells. CCK8 and colony formation experiments were used to determine the proliferation ability of the cells; scratch and transwell experiments were performed to detect the invasiveness of the cells. MUS81 overexpression cells were inoculated subcutaneously at the dorsal flanks of nude mice to form subcutaneous xenografts and evaluate the functional impact of MUS81 on the growth of BC. The expression of MUS81 and Ki-67 in xenografts were also detected by immunohistochemistry. Both MUS81 mRNA and protein expression levels were reduced significantly in BC tissues and cells compared with the corresponding normal tissues and MCF-10A. In vitro experiments indicated overexpression or silencing of MUS81 respectively decreased and facilitated proliferation and invasion of BC cells than control groups. In vivo experiment revealed a significant reduction in the growth of BC cells and tumor weight of their formed subcutaneous xenografts in nude mice was noted upon overexpression of MUS81. The expression level of Ki 67 was also dramatically reduced following the overexpression of MUS81. MUS81 was significantly low-expressed in breast cancer, and the low expression was correlated with the enhanced proliferation and aggressiveness of breast cancer cells, suggesting that MUS81 as a candidate tumor suppressor gene.\u003c/p\u003e","manuscriptTitle":"MUS81 Inhibits Cell Proliferation, Migration and Invasion in Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-25 12:56:12","doi":"10.21203/rs.3.rs-3638911/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":"14385c7c-d995-49cf-bc96-f94d9c940141","owner":[],"postedDate":"November 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-02T15:59:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-25 12:56:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3638911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3638911","identity":"rs-3638911","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}