Shugoshin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas

Glioblastomas are the most common type of adult primary brain neoplasms. Clinically, it is helpful to identify biomarkers to predict the survival of patients with gliomas due to its poor outcome. Shugoshin 2 (SGO2) is critical in cell division and cell cycle progression in eukaryotes. However, the association of SGO2 with pathological grading and survival in patients with gliomas remains unclear. We analyzed the association between SGO2 expression and clinical outcomes from Gene Expression Omnibus (GEO) dataset profiles, The Cancer Genome Atlas (TCGA), and Chinese Glioma Genome Atlas (CGGA). SGO2 mRNA and protein expression in normal brain tissue and glioma cell lines were investigated via quantitative RT-PCR, Western blot, and IHC staining. The roles of SGO2 in proliferation, migration, and apoptosis of GBM cells were studied with wound-healing assay, BrdU assay, cell cycle analysis, and JC-1 assay. The protein–protein interaction (PPI) was analyzed via Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). SGO2 mRNA expression predicted higher grade gliomas than non-tumor brain tissues. Kaplan–Meier survival analysis showed that patients with high-grade gliomas with a higher SGO2 expression had worse survival outcomes. SGO2 mRNA and protein expression were upper regulated in gliomas than in normal brain tissue. Inhibition of SGO2 suppressed cell proliferation and migration. Also, PPI result showed SGO2 to be a potential hub protein, which was related to the expression of AURKB and FOXM1. SGO2 expression positively correlates with WHO pathological grading and patient survival, suggesting that SGO2 is a biomarker that is predictive of disease progression in patients with gliomas.

www.nature.com/scientificreports/ the development, differentiation, and recurrence of GBM to provide new directions in diagnosis and treatment will be a major focus of future studies on GBM. Shugoshin 2 (SGO2), which is a conserved centromeric protein belonging to the Shugoshin family, plays an important role during cell division in eukaryotes 10 . SGO2 functions as a guardian that protects centromeric cohesion from precocious dissociation, resulting in an early separation of sister chromatids, via the Shugoshin-serine/ threonine protein phosphatase 2A (PP2A) interaction 11 . During meiosis, SGO2 maintains normal gametogenesis by preventing the premature release of REC8-cohesin complex from the centromere 12 . Huang et al. have found that SGO2 is critical for the correct attachment of kinetochore to the centromere and that SGO2-deficient cells are defective in kinetochore attachment, which results in lagging chromosomal formation during anaphase 13 .
In this study, we hypothesized that SGO2 is overexpressed in patients with high-grade gliomas. First, we investigated the relationship between SGO2 expression and survival in patients with gliomas and attempted to investigate the association of SGO2 expression with WHO pathological grading of human gliomas. Then, the Gene Expression Omnibus (GEO) dataset profiles, The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), RT-PCR, and Western blotting analysis suggested that SGO2 might be a new prognostic biomarker for human gliomas. Further, we explored the biological role of SGO2 in glioma cell migration, proliferation, apoptosis, and protein-protein interaction. These resulted indicated that SGO2 has potential to be the target for new treatment design.

Materials and methods
SGO2 gene expression, survival outcome, and pathological grading in human gliomas. The methodology for the analyses of functional genomic databases was as previously described [14][15][16] . In brief, 100 sheets of de-linked data (GDS1816/230165_at/SGO2) on SGO2 mRNA expression, sex, age, pathologic grading, and survival rates of patients with primary high-grade gliomas were obtained from NCBI (available online: https:// www. ncbi. nlm. nih. gov/ geo/ tools/ profi leGra ph. cgi? ID= GDS18 16: 230165_ at). Twenty-three sheets of data without detailed information on age and survival times were excluded; thus, a total of 77 sheets were included in the statistical analyses. An additional database (GDS1962/230165_at/SGO2) that contained 180 sheets from 81 patients with grade IV gliomas, 19 with grade III gliomas, seven with grade II gliomas, 23 without tumors (non-tumor control) (Available online: https:// www. ncbi. nlm. nih. gov/ geo/ tools/ profi leGra ph. cgi? ID= GDS19 62: 230165_ at) and included. 38 with grade II oligodendroglioma and 12 with grade III oligodendroglioma were excluded. Also, we analyzed the The Cancer Genome Atlas (TCGA), and Chinese Glioma Genome Atlas (CGGA, http:// www. cgga. org. cn) 17 database to obtain the glioma overall survival and gene expression. The TCGA dataset was acquired through the cBio Cancer Genomics Portal (http:// cbiop ortal. org) 18 , which containing 343 patients with gliomas included 61 panels of grade II gliomas, 130 panels of grade III gliomas, and 152 panels of grade IV gliomas. The CGGA dataset comprised 211 patients with 75 panels of grade II gliomas, 28 panels of grade III gliomas, and 108 panels of grade IV gliomas.
The Kaplan-Meier method was used to analyze the overall survival rates and cohorts of low-vs. high-SGO2 expressions in high-grade gliomas from the GEO profile (GDS1816/230165_at/SGO2), TCGA, and CGGA. SGO2 expression cutoff point was decided using statistical analysis. The GraphPad Prism 5 software was used to generate the figures, and P < 0.05 was defined as statistical significance.
RNA isolation and quantitative RT-PCR. Total RNA was extracted using EasyPure Total RNA reagent (Bioman, Taipei, Taiwan) according to the manufacturer's protocol. For cDNA synthesis, 1.0 μg RNA was reverse transcribed into cDNA using Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA). Normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA).
Cell lysate preparation and Western blot. Cells were lysed using RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% Triton-X-100) at 4 °C for 10 min, and cell lysates were harvested by centrifugation at 15,000 rpm for 10 min to obtain the supernatants. Normal brain lysates were purchased from Origene Technologies. Thirty-microgram cell lysates from each group were applied to 10% sodium dodecyl sulfate www.nature.com/scientificreports/ polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA) and blocked with 5% skim milk in TBST for 1 h at room temperature. Anti-SGO2 antibody (Atlas Antibodies AB, Stockholm. Sweden) was diluted at a ratio of 1:1000 with SignalBoost Immunoreaction Enhancer Kit following the protocol of manufacturer. Band were detected using enhanced chemiluminescence and X-ray film (GE Healthcare, Piscataway, NJ, USA).
Cell proliferation, cell cycle analysis, cell apoptosis, and flow cytometry analysis. For cell counting assay, we seeded LN229 and GBM8401 cells (2.5 × 10 4 per well) in a 12-well plate. Cells were transfected with 25 nM siRNA on the next day. The cells were counted at 24, 48, and 72 h after transfection. Before counting, cells were mixed with trypan blue for 3-5 min according to the previous description 26 . The differences in growth rate between the experimental groups and the control groups was detected in five independent experiments. For cell proliferation analysis, LN229 and GBM8401 cells were transfected with siRNA then processed with the FITC-BrdU Flow Kits according to the manufacturer's instructions (BD Biosciences). To analyze the distribution of cell cycle stage after RNA interference, we detected the DNA content by fluorescence activated cell sorting (FACS) as previous description 27 . The cells in experimental and control group were fixed in 70% ethanol at 4 °C and kept at − 20 °C overnight. Then the cells were washed twice with cold phosphate-buffered saline (PBS), and stained with propidium iodide (PI) solution (50 μg/ml PI in PBS, 1% Tween 20 and 10 μg/ml RNase A) for 30 min in the dark. Then the DNA content was analyzed by fluorescence activated cell sorting (BD Biosciences, San Jose, CA, USA) in two independent experiments. For apoptosis, we used JC-1 assay accordance with previous description [28][29][30] . Briefly, cells were seeded in 6-well plate then transfected with siSGO2 or siControl on next day. Cells were collected to proceed the protocol as the manufacturer's guide (BM MitoScreen). All these samples were analyzed by FACSCalibur flow cytometer (BD Biosciences) and Cell Quest Pro software (BD Biosciences).
Cell migration assay. We used wound-healing assay for cell migration analysis according to previous studies 31, 32 . In brief, LN229 and GMB8401 (2 × 10 5 ) were seeded into 12-well plates and grown at 37 °C in a 5% CO 2 incubator. RNA interference was performed on the next day. On the day 3, we removed the medium when cell confluence reached 90% and made a wound in the monolayer with a pipette tip. Then, we washed the plate for three times to remove the non-adherent cells. The wound area was photographed immediately after wounding (0 h) and at 16 h post wounding. The migration rates were computed according to the change of wound area measured by ImageJ software (NIH, Bethesda, MD).
Protein-protein network and signaling pathways analysis. Known

SGO2 mRNA and protein expression is increased in human glioma cells.
We further investigated the expression of SGO2 mRNA amount normal brain, WHO grade IV glioma cell lines including LN229, U87MG, GBM8401, and U118MG. The results as showed in Fig. 3a, revealed that the expression of SGO2 was significantly increased in glioma cells comparing with normal brain tissue. Using western blot, we found the expression of SGO2 protein revealed higher in LN229 and GBM8401 (Fig. 3b) then in normal brain.

SGO2 protein expression is increased in human high-grade gliomas. To investigate the SGO2
protein expression in non-tumor brain tissues and human gliomas tissues, IHC staining of two human tissue microarrays were conducted (Fig. 4a-f). We found that the immunohistochemical staining score of SGO2 was higher in high-grade (WHO IV) gliomas than in low-grade (WHO grade I, II, and III) gliomas (nucleus: p = 0.0815; cytoplasm: p = 0.3904; cytoplasm and nucleus: p = 0.4558). Also, the SGO2 immunohistochemical staining score was higher in high-grade gliomas then in normal brain (nucleus: p = 0.1761; cytoplasm: p = 0.0498; cytoplasm and nucleus: p = 0.0283, respectively). Moreover, SGO2 immunostain score was higher in low-grade   www.nature.com/scientificreports/ Protein lysates of glioma cell lines, including U87MG, LN229, GBM8401, and U118MG were applied to SDS-PAGE and Western blot analysis to quantitate SGO2 protein expression (full length blot is presented in Supplementary Fig. 1). GAPDH served as a loading control. www.nature.com/scientificreports/ gliomas than in normal brain (nucleus: p = 0.2390; cytoplasm: p = 0.0991; cytoplasm and nucleus: 0.0456, p adjusted by Bonferroni method, Fig. 4g-i). The result suggested that SGO2 protein overexpression in high-grade gliomas compared with non-tumor brain tissues.

SGO2 down regulation inhibits cell proliferation in glioma cells.
To explore the effect of SGO2 in glioma tumorigenesis, we used siRNA to knock down SGO2 expression in LN229 and GBM8401 cells (Fig. 5a). SGO2 has been reported to protect centromeric cohesion during cell division 34 . Thus, we investigated the effect of SGO2 in glioma cell proliferation. Cell counting of LN229 and GBM8401 decreased after SGO2 down regulation (Fig. 5b). Using BrdU assay, we found that SGO2 knockdown can resulted in decreased the proportion of active cell proliferation compared with siControl glioma cells (Fig. 5c). Furthermore, cell cycle analysis showed that SGO2 down regulation leaded to G1 phase arrest in LN229 and GBM8401 cells (Fig. 5d). Then we further investigate the relationship between SGO2 and cell apoptosis. In JC-1 assay, we found that the proportion of cell apoptosis revealed no different between siSGO2 and siControl glioma cells (Fig. 5e). Based on these results and the biological function of SGO2, we believe that SGO2 may have crucial role in glioma cells proliferation. www.nature.com/scientificreports/

SGO2 plays an important role in glioma cell migration.
To investigate effect of SGO2 in glioma cell migration, we performed wound healing and migration assays. The results showed that the ability of LN229 and GBM8401 cell migration revealed significant decreased in siSGO2 compared with siControl. (Fig. 6a,b).

SGO2 hubs the protein-protein interactions.
To further understand the protein-protein interaction (PPI) network of SGO2-regulated oncogenesis, we use Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The network showed that SGO2 had interactions with Aurora B kinase (AURKB) and BUB1 (Fig. 7a,b). Also, SGO2 may have relationship in FOXM1 regulation (Fig. 7b). Further we investigated the expression of AURKB and FOXM1 by Western blot (Fig. 7c,d). The data showed that AURKB and FOXM1 revealed decreased protein expression after SGO2 knockdown, which indicated that SGO2 hubs may the protein-protein interaction.

Discussion
Till date, no studies have investigated the role of SGO2 in GBM. This is the first study to investigate SGO2 expression according to the WHO pathological grading of human gliomas and the association between SGO2 expression and clinical outcomes. In this study, we found a significantly higher SGO2 expression in patients with high-grade gliomas than in non-tumor brain tissue controls. We also found that high SGO2 expression predicts poor survival outcomes in patients with gliomas. Furthermore, SGO2 overexpression in high grade gliomas was confirmed using qRT-PCR, Western blot, and IHC staining. Together with these results, we thought that SGO2 has the potential to be a new biomarker for clinical specialists to predict survival outcomes in patients with GBM.
According to the analysis of TCGA database from the Human Protein Atlas (HPA), SGO2 is not considered prognostic in GBM (https:// www. prote inatl as. org/ ENSG0 00001 63535-SGO2/ patho logy). However, the analysis of TCGA in HPA only enrolled the cases of GBM (n = 153) but not that of low-grade gliomas (LGG). When analyzed the TCGA database, we enrolled both cases of GBM and LGG so that the n = 343 (grade 2: n = 61, grade 3: n = 130, grade 4: n = 152). Although the case number of GBM is different in our dataset (n = 152) and in HPA dataset (n = 153), but we thought that this is the inter-databased difference. Our result showed that SGO2 has prognostic value in gliomas.
An accurate chromosomal segregation results in proper cell division. The key processes involved in chromosomal segregation are the formation of sister chromatid cohesion, correct assemblage of spindle, and well linkage between sister kinetochores and microtubules [35][36][37] . SGO2 has been found to be associated with centromeric cohesion protection and chromosome alignment. SGO2 can help chromosomal passenger complex loading on to centromere during the M phase 38 . Huang et al. have found that SGO2 can recruit mitotic centromere-associated kinesin, which is a microtubule depolymerase, at centromeres to modify the microtubule dynamics, and SGO2deficient HeLa cells revealed chromosome steady during anaphase 13 . These results suggest an important role Figure 6. The effect of SGO2 knockdown on cell migration detected by wound-healing assays. Images and Quantitative analysis of LN229 (a) and GBM8401 (b) cells in the wound-healing assay. Data are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01.  39 . The expression of Aurora B kinase is related to poor clinical survival outcomes in patients with GBM 40 . The inhibition of Aurora A and B kinase can enhance the sensitivity to temozolomide and radiotherapy in glioblastoma cell lines 41 . Furthermore, malignant human glioma cells with the inhibition of Aurora A and B have revealed G2/M lagging and caspase-related cell death 42 . On the other hand, the transcription factor, FOXM1, which is known as human proto-oncogene, can maintain the activity of glioma stem cells (GSCs) and can promote the activity of β-catenin to regulate Wnt target gene expression in GSCs 43 . FOXM1 can also interact with MELK to regulate GSC mitosis 44 . FOXM1 can activate the STAT3 signaling pathway to enhance the selfrenewing and tumorigenesis of GSCs 45 .

Scientific Reports
In the protein-protein interaction network, we found possible associations among SGO2, Aurora B kinase, and FOXM1, but the exact roles of these proteins in the functioning of GSCs remain unknown. To know their relationship will be helpful to understand the mechanism of GSC tumorigenicity and identifying new treatment targets in patients with glioblastomas. In budding yeast Saccharomyces pombe, SGO2 has been found to be localized at subtelomeres to form a chromatin domain during the G2 phase. Furthermore, SGO2 can regulate the expression of genes and cell replication timing localized at subtelomeres 34 . In our current study, the downregulation of Aurora B kinase and FOXM1 might be mediated through the gene regulation by SGO2. Further studies assessing whether SGO2 has another chromosomal localization during the cell cycle in glioblastoma cells and the relationship between SGO2 and gene expression in glioblastoma are areas of future research.
This study had several limitations. First, it was difficult to collect a large sample of non-tumor brain tissue and low-grade human gliomas to validate SGO2 expression. We performed a large-scale analysis of GEO profiles, TCGA, and CGGA data sets, to reveal that SGO2 is a biomarker related to WHO pathological grading and survival outcome. We further confirmed the data analyzed from the three independent cohort studies though wet lab approaches, such as qRT-PCR and Western blot. Second, the true value of SGO2 in the prediction of survival outcomes in patients with gliomas should be further investigated. Third, more research efforts should be invested in investigating detailed mechanisms to explain the influence of SGO2 on the survival rates of patients with gliomas.
In conclusion, this is the first study investigating the relationship between SGO2, which is a conserved centromeric protein, and gliomas. SGO2 expression revealed a positive correlation with WHO pathological grades of gliomas. Higher SGO2 expression was associated with worse survival outcomes in patients with high-grade gliomas. Regulation of SGO2 signal interfere the expression of mitosis related protein AURKB and FOXM1. Thus, The STRING dataset also predicted the association between SGO2, ARUKB, and FOXM1. (c) Protein lysates of LN229 and GBM8401 were applied to SDS-PAGE and Western blot to investigate the protein expression of AURKB and FOXM1(full length blot is presented in Supplementary Fig. 2). α-actinin served as a loading control. www.nature.com/scientificreports/ we suggest that SGO2 is not only a potential biomarker for disease prediction in patients with gliomas, but also has potential to be the new target of glioma treatment.