Expression and prognosis analyses of BUB1, BUB1B and BUB3 in human sarcoma

Results

Transcriptional levels of BUBs in patients with sarcoma

Three BUB family members have been identified in patients with different types of sarcoma: BUB1, BUB1B and BUB3. They are overexpressed in all samples of sarcoma compared to normal tissue. The transcriptional levels of BUBs were compared between sarcoma and normal samples using Oncomine databases (Figure 1). The mRNA levels of these BUBs were significantly upregulated in patients with sarcoma in two datasets.

Figure 1. The transcription levels of BUB factors in different types of cancers (Oncomine). The BUB family of genes are overexpressed in many types of cancers including Sarcoma. The red cells represent evidence of gene overexpression. The blue cells represent evidence of decreased gene expression. The number in each cell represents the amount of evidence. The deeper the color, the higher the significance.

In the Detwiller Sarcoma dataset, BUB1 was upregulated in malignant fibrous histiocytoma, fibrosarcoma, leiomyosarcoma, round cell liposarcoma, and pleomorphic liposarcoma with 12.013 (P = 1.59E-09), 14.408 (P = 2.70E-08), 11.066 (P = 3.42E-07), 9.248 (P = 2.85E-06), and 10.159 (P = 6.81E-05) fold changes, respectively. BUB1B was upregulated in fibrosarcoma, pleomorphic liposarcoma, malignant fibrous histiocytoma, round cell liposarcoma, synovial sarcoma, and leiomyosarcoma. The fold changes for upregulation of each sarcoma are as follows: 8.908 (P = 2.52E-07), 4.687 (P = 6.25E-06), 5.849 (P = 3.01E-07), 4.734 (P = 1.99E-06), 3.933 (P = 1.13E-05), and 5.471 (P = 2.31E-05), respectively. BUB3 was upregulated in synovial sarcoma, fibrosarcoma, and malignant fibrous histiocytoma. The fold changes are 2.999 (P = 1.14E-05), 2.164 (P = 2.64E-05), and 2.350 (P = 7.05E-05), respectively. In the Barretina Sarcoma dataset, BUB1 was upregulated in myxofibrosarcoma, pleomorphic liposarcoma, and leiomyosarcoma with 3.085 (P = 6.53E-14), 2.926 (P = 1.22E-09), and 2.878 (P = 5.95E-09) fold changes, respectively. BUB1B was upregulated in dedifferentiated liposarcoma, myxofibrosarcoma, pleomorphic liposarcoma, leiomyosarcoma, and myxoid/round cell liposarcoma with fold changes of 3.628 (P = 2.20E-17), 7.544 (P = 1.11E-17), 5.121 (P = 8.66E-13), 4.838 (P = 1.46E-10), and 2.842 (P = 1.88E-10), respectively. BUB3 was upregulated in myxofibrosarcoma with a 2.514 (P = 1.57E-12) fold change (Table 1).

Table 1. The significant changes of BUBs expression in transcription level between different types of sarcomas (ONCOMINE database).

Gene ID	Types of Sarcoma versus Normal	Fold Change	p-Value	t-Test	References
BUB1	Malignant Fibrous Histiocytoma vs. Normal	12.013	1.59E–09	9.949	Detwiller Sarcoma
	Fibrosarcoma vs. Normal	14.408	2.70E–08	10.002	Detwiller Sarcoma
	Leiomyosarcoma vs. Normal	11.066	3.42E–07	9.000	Detwiller Sarcoma
	Round Cell Liposarcoma vs. Normal	9.248	2.85E–06	8.909	Detwiller Sarcoma
	Pleomorphic Liposarcoma vs. Normal	10.159	6.81E–05	8.788	Detwiller Sarcoma
	Myxofibrosarcoma vs. Normal	3.085	6.53E–14	11.255	Barretina Sarcoma
	Pleomorphic Liposarcoma vs. Normal	2.926	1.22E–09	8.750	Barretina Sarcoma
	Leiomyosarcoma vs. Normal	2.878	5.95E–09	7.802	Barretina Sarcoma
BUB1B	Dedifferentiated Liposarcoma vs. Normal	3.628	2.20E–17	12.579	Barretina Sarcoma
	Myxofibrosarcoma vs. Normal	7.544	1.11E–17	16.533	Barretina Sarcoma
	Pleomorphic Liposarcoma vs. Normal	5.121	8.66E–13	13.250	Barretina Sarcoma
	Leiomyosarcoma vs. Normal	4.838	1.46E–10	9.806	Barretina Sarcoma
	Myxoid/Round Cell Liposarcoma vs. Normal	2.842	1.88E–10	10.778	Barretina Sarcoma
	Fibrosarcoma vs. Normal	8.908	2.52E–07	7.540	Detwiller Sarcoma
	Pleomorphic Liposarcoma vs. Normal	4.687	6.25E–06	6.348	Detwiller Sarcoma
	Malignant Fibrous Histiocytoma vs. Normal	5.849	3.01E–07	7.079	Detwiller Sarcoma
	Round Cell Liposarcoma vs. Normal	4.734	1.99E–06	6.976	Detwiller Sarcoma
	Synovial Sarcoma vs. Normal	3.933	1.13E–05	5.772	Detwiller Sarcoma
	Leiomyosarcoma vs. Normal	5.471	2.31E–05	5.623	Detwiller Sarcoma
BUB3	Myxofibrosarcoma vs. Normal	2.514	1.57E–12	10.808	Barretina Sarcoma
	Synovial Sarcoma vs. Normal	2.999	1.14E–05	5.854	Detwiller Sarcoma
	Fibrosarcoma vs. Normal	2.164	2.64E–05	5.391	Detwiller Sarcoma
	Malignant Fibrous Histiocytoma vs. Normal	2.350	7.05E–05	4.687	Detwiller Sarcoma

The expression patterns of BUBs in sarcoma samples were further compared to normal tissue using the GEPIA database. Notably, the expressions of BUB1 and BUB1B are significantly higher in sarcomas compared to normal tissues (Figure 2A–2E). The overexpression of BUB3 does not reach significance, possibly due to the low number of patients in the control group.

Figure 2. The expression of BUBs in different types of cancers (GEPIA). The expression of (A) BUB1, (B) BUB1B, and (C) BUB3 in different types of cancers. (D, E) The expression of BUBs in sarcomas. Each dot represents a sample. Cancer names were abbreviated. Details were listed in Supplementary Materials. ^*P < 0.05.

Transcriptional level of BUBs in sarcoma cell lines

To further explore the expression level of BUBs in sarcoma cell lines, we queried the CCLE database. The expressions of BUB1, BUB1B and BUB3 were highly upregulated (Figure 3) compared to normal control cell lines.

Figure 3. The expression of BUBs in cancer cell lines (CCLE). The mRNA expression of (A) BUB1, (B) BUB1B, and (C) BUB3 in sarcoma cell lines, analyzed by CCLE. Lineages are composed of a number of cell lines from the same area or system of the body. The number next to the lineage name indicates how many cell lines are in the lineage. The highest average distribution is on the left and is colored red. The dashed line within a box is the mean.

The prognostic values of BUBs in sarcoma

To investigate the clinical prognostic value of BUBs in sarcoma, we analyzed survival data of sarcoma patients using Kaplan-Meier Plotter and the GEPIA database (Figure 4). Both overall survival and disease-free survival of sarcoma patients are negatively associated with BUBs in the datasets. In the GEPIA dataset, the hazard ratios of BUB1 are 1.4 (P = 0.096) for overall survival and 1.6 (P = 0.015) for disease-free survival. As for BUB1B, they are 1.8 (P = 0.0038) and 1.6 (P = 0.0064) for overall and disease-free survivals, respectively. For BUB3, they are 1.5 (P = 0.046) and 1.6 (P = 0.015) for overall and disease-free survivals, respectively. In the Kaplan-Meier Plotter dataset, the hazard ratios of BUB1 are 2.18 (P = 0.0023) for overall survival and 2.26 (P = 0.0012) for disease-free survival. As for BUB1B, they are 2 (P = 7e-04) and 2.54 (P = 0.00083) for overall and disease-free survivals, respectively. For BUB3, they are 2.16 (P = 0.0048) and 1.96 (P = 0.027) for overall and disease-free survivals, respectively.

Figure 4. The prognostic value of mRNA level of BUB factors in sarcoma patients (GEPIA and kaplan meier plotter). The prognostic value of mRNA level of BUB factors in sarcoma patients was analyzed by (A) GEPIA and (B) Kaplan Meier Plotter. Higher expression of BUBs is associated with worse survival. HR, hazard ratio; TPM, Transaction per million.

Co-expression profile of BUBs in sarcoma

Genes co-expressed with BUBs in sarcoma were analyzed using the Oncomine database. In the Chibon Sarcoma datasets [22], we found BUB1 expression level was positively correlated with levels of CCNB1, AURKA, UBE2C, DLGAP5, NCAPG, CENPA, BIRC5, KIF4A, CCNB2, TPX2, KIF18B, KIF2C, CDC20, CDCA5, CENPE, CKS2, and MELK. For BUB1B, its expression level was positively correlated with levels of CACS5, NUSAP1, KIF20A, TOP2A, PRC1, KIF14, PTTG1, RACGAP1, CKS2, MELK, CENPE, CDCA5, BUB1, CCNB1, and AURKA. In the Schaefer Sarcoma dataset, we found that BUB3 expression level was positively correlated with levels of DLAT, PRPS1, MRPS7, TIMM23, PPIF, MAPK1, TFDP1, NUP98, ATP6V1A, MED6, CAPRIN1, CYP51P2, TMX1, EXOC5, MAT2A, and ACLY (Figure 5A). The association between BUB1, BUB1B, and BUB3 was analyzed using the GEPIA (Figure 5B) and Linked Omics databases (Figure 5C). The correlation coefficients of BUB1 and BUB1B were 0.8 (GEPIA) and 0.8988 (Linked Omics). For BUB1 and BUB3, they were 0.62 (GEPIA) and 0.5179 (Linked Omics). For BUB1B and BUB3, they were 0.6 (GEPIA) and 0.5293 (Linked Omics). All correlations were found to be statistically significant.

Figure 5. Co-expressed genes of BUBs, correlations between BUBs (ONCOMINE, GEPIA, and LinkedOmics). Co-expressed genes of BUBs in sarcomas, analyzed by (A) ONCOMINE. For each BUB factor, the top twenty co-expressed genes were displayed. The correlations between BUBs in sarcomas were analyzed by (B) GEPIA and (C) LinkedOmics. BUB1, BUB1B and BUB3 are positively correlated with each other. TPM, Transaction per million.

Predicted functions and pathways of BUBs in sarcomas

The functional roles of BUBs were evaluated from three perspectives: biological processes, molecular functions, and cellular components. We found that BUBs are involved in the biological processes of meiotic sister chromatid cohesion, meiotic cell cycle, cell division, cellular protein metabolic process, anaphase-promoting complex-dependent catabolic process, cell population proliferation, mitotic spindle assembly checkpoint, apoptotic process, protein phosphorylation, sister chromatid segregation, and nuclear division. Regarding molecular function, protein serine/threonine kinase activity was found to be significantly controlled by BUBs. Cellular components including protein-containing complex, nuclear lumen, cytosol, condensed, chromosome outer kinetochore, condensed nuclear chromosome kinetochore were all found to be associated with the function of BUBs. KEGG pathways analysis showed that BUBs are involved in Human T-lymphotropic virus 1 infection and cell cycle pathways (Table 2 and Figure 6).

Table 2. The functions of BUBs and genes significantly associated with BUBs alterations.

Category	Term	Name	Count	FDR
BP	GO:0051754	meiotic sister chromatid cohesion, centromeric	2	1.42e–06
BP	GO:0051321	meiotic cell cycle	3	4.97e–06
BP	GO:0051301	cell division	3	4.22e–05
BP	GO:0044267	cellular protein metabolic process	3	0.0107
BP	GO:0031145	anaphase-promoting complex-dependent catabolic process	2	3.01e–05
BP	GO:0008283	cell population proliferation	2	0.0062
BP	GO:0007094	mitotic spindle assembly checkpoint	3	2.16e–07
BP	GO:0006915	apoptotic process	2	0.0107
BP	GO:0006468	protein phosphorylation	2	0.0108
BP	GO:0000819	sister chromatid segregation	3	1.51e–06
BP	GO:0000280	nuclear division	3	8.96e–06
MF	GO:0004674	protein serine/threonine kinase activity	2	0.0429
CC	GO:0032991	protein-containing complex	3	0.0386
CC	GO:0031981	nuclear lumen	3	0.0277
CC	GO:0005829	cytosol	3	0.0402
CC	GO:0000940	condensed chromosome outer kinetochore	2	1.03e–05
CC	GO:0000778	condensed nuclear chromosome kinetochore	2	1.03e–05
CC	GO:0000777	condensed chromosome kinetochore	3	6.68e–06
KEGG Pathways	hsa05166	HTLV-I infection	2	0.00098
KEGG Pathways	hsa04110	Cell cycle	3	1.04e–06
The functions of BUBs and the genes significantly associated with BUBs alterations were predicted by analyzing Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) using Database for Annotation, Visualization and Integrated Discovery (DAVID). Biological Processes (BP), Molecular Functions (MF), Cellular Components (CC).

Figure 6. Cell cycle regulated by the BUBs alteration in sarcomas. The cell cycle regulated by the BUBs alteration in sarcomas are shown. Figure was downloaded from the KEGG database.

Materials and Methods

Ethics statement

The current study was approved by the Academic Committee of Central South University and was conducted in accordance with the principles of the Declaration of Helsinki. All data sets are retrieved from published literature, all written informed consents have been obtained.

Data collection and processing

We first researched the expression level of the BUB gene family in tumors, especially sarcoma and normal tissues using the Oncomine database (https://www.oncomine.org/), Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/) and Cancer Cell Line Encyclopedia (CCLE, https://www.broadinstitute.org/ccle). Then, the prognostic value of BUBs was analyzed using GEPIA and Kaplan-Meier Plotter (https://kmplot.com/analysis/). Next, we analyzed the co-expression of BUBs in Oncomine and the correlations among BUBs in GEPIA and LinkedOmics (http://www.linkedomics.org/login.php). Lastly, the predicted functions and pathways of BUBs were analyzed in the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/), Gene Ontology (GO, http://geneontology.org/), and Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.ncifcrf.gov) (Figure 7).

Figure 7. The flowchart of this study.

ONCOMINE analysis

The transcription levels of the BUB family in different sarcomas were analyzed using Oncomine gene expression array datasets (https://www.oncomine.org/), an online cancer microarray database. Student’s t test was conducted to compare the mRNA expressions of the BUB family in clinical cancer specimens and normal controls [42]. The cutoff of fold change was defined as > 2. Statistical significance was set at P value < 0.01. Co-expressed genes of BUBs in sarcoma were analyzed using ONCOMINE.

GEPIA dataset

Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/) is an interactive web platform that uses standard processing procedures to analyze the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the Cancer Genome Atlas and Genotype Tissue Expression Project [43]. The expression levels of BUB family members were compared between tumor tissues and normal tissues. P < 0.05 was considered statistically significant.

CCLE dataset

The Cancer Cell Line Encyclopedia (CCLE, https://www.broadinstitute.org/ccle) project is a collaboration between the Broad Institute and the Novartis Institute of Biomedicine and the Institute of Genomics of the Novartis Research Foundation to carry out detailed genetics and pharmacologic characterization on a large group of human cancer models, to develop integrated computational analysis linking different pharmacological weaknesses with genomic patterns, and to transform cell line genomics into cancer patient stratification. CCLE provides public access to genomic data, analysis and visualization for approximately 1,000 cell lines [44]. The BUB family’s expressions in cancer cell lines were confirmed by the CCLE database.

Kaplan-Meier plotter

Kaplan Meier Plotter (https://kmplot.com/analysis/) contained the expression data of 54000 genes and the survival information of 21 cancer types including sarcoma. In order to analyze the overall survival and relapse-free survival of patients with sarcoma, the samples were divided into two groups according to median expression (BUBs high and low expression) and assessed by the Kaplan-Meier survival chart, based on hazard ratios (HR) and log-rank P-value [45].

KEGG and GO enrichment analyses of DEGs

The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.ncifcrf.gov) (version 6.7) is an online biological information database that provides a comprehensive set of functional annotation information of genes and proteins [46]. The Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) is a database resource which contains large-scale molecular datasets of high-level functions and biological systems generated by high-throughput experimental technologies [47]. Gene ontology (GO) (http://geneontology.org/) is a bioinformatics tool which can annotate genes and analyze their biological processes [48]. To further analyze the function of BUBs, pathway analyses were also performed using KEGG online database.

Abbreviations

BUB: Budding Uninhibited By Benzimidazoles; APC/C: Anaphase-Promoting Complex/Cyclosome; AURKB: Aurora Kinase B; GEPIA: Gene Expression Profiling Interactive Analysis; CCLE: Cancer Cell Line Encyclopedia; DAVID: Database for Annotation, Visualization and Integrated Discovery; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene ontology; DEGs: Different expressed genes.

Author Contributions

Conceptualization, W.G., L.-Z.L., T.-Q.Y.; Investigation, W.G., W. T., T.-Q.Y.; Writing, L.-Z.L., C.-L.A., T.-Q.Y., W.-W.C.; Review & Editing, L.-Z.L., W.G., C.-L.A., W. T., W.-W.C.; Visualization, L.-Z.L., W.-W.C., W. T.; Supervision, W.-W.C.; Funding Acquisition, W.-W.C.

Acknowledgments

I would like to express my gratitude to all the authors that contributed to the present manuscript. This study would not be finished without the efforts of all my co-authors.

Conflicts of Interest

The authors declare no competing interests.

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