Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

BUB1 and BUBIB are differentially expressed in pan-cancerCompared with BUB1 gene expression in normal tissues, BUB1 gene was expressed at higher levels in BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), SKCM (skin cutaneous melanoma), STAD (stomach adenocarcinoma), TGCT (testicular germ cell tumor), UCEC (uterine corpus endometrial carcinoma) (P < 0.001), CESE (cervical squamous cell carcinoma and endocervical adenocarcinoma), and PCPG (pheochromocytoma and paraganglioma) (P < 0.01). Only PAAD (pancreatic adenocarcinoma) showed no difference in BUB1 gene expression (Fig. 1A). BUB1B gene was highly expressed in BLCA, BRCA, CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA, UCEC (P < 0.001), KICH, CESC, PCPG, and SKCM (P < 0.01); only PAAD showed no difference in BUB1 gene expression (Fig. 1B).Figure 1Analysis of BUB1 and BUB1B gene differential expression in tumors. (A) Analysis of BUB1 gene expression in tumors in TIMER 2.0 database; (B) Analysis of BUB1 gene expression analysis in tumors in GEPIA2 database; (C) Analysis of BUB1B gene expression analysis in tumors in TIMER 2.0 database; (D) Analysis of BUB1B gene expression analysis in tumors in GEPIA2 database. *P < 0.05.BUB1 was highly expressed in DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), OV (ovarian cancer), SARC (sarcoma), THYM (thymoma) and USC (uterine carcinosarcoma) compared with normal tissues; no difference was observed in ACC (adrenocortical carcinoma) and LGG (brain lower grade glioma) (Fig. 1C). BUB1B was highly expressed in DLBC, OV, SARC, THYM and USC (P < 0.05); no difference was observed in ACC and LGG (Fig. 1D).BUB1 and BUBIB are highly expressed in ECBUB1 and BUB1B mRNAs were upregulated in EC tissues, as determined by XiaoTao tool analysis (Fig. 2A,C). IHC showed that BUB1 and BUBIB were positively expressed in EC tissues (P < 0.05) and barely expressed in paracancerous tissues, consistent with the bioinformatics analysis (Fig. 2B,D). BUB1 and BUB1B proteins were mainly located in the cytoplasm.Figure 2BUB1 and BUB1B expression in EC. (A) BUB1 expression in normal endometrium and EC (XianTao database); (B) BUB1 expression in normal endometrium and EC (IHC); (C) BUB1B expression in normal endometrium and EC (XiaoTao database); (D) BUB1B expression in normal endometrium and EC (IHC); (E) BUB1 ROC curve in EC; (F) BUB1B ROC curve in EC.ROC curve showed that the high expressions of BUB1 (AUC = 0.972, CI 0.947–0.997) and BUB1B (AUC = 0.953, CI 0.912–0.994) had high accuracy in the prediction of EC (Fig. 2E,F).High expression of BUB1 and BUBIB leads to poor prognosis in pan-cancerGEPIA2 analysis indicated that high BUB1 expression was associated with OS in ACC, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, and PAAD (P < 0.05) (Fig. S1A) and associated with DFS in ACC, KIRC, KIRP, LGG, LIHC, MESO, PAAD PRAD, SARC and THCA (P < 0.05) (Fig. S1B). High BUB1B expression was associated with OS in ACC, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, and SARC (P < 0.05) (Fig. S1C) and associated with DFS in ACC, CHOL, KIRC, KIRP, LGG, LIHC, LUAD, PAAD, PRAD, SARC and THCA (P < 0.05) (Fig. S1D).High expression of BUB1 and BUBIB leads to poor prognosis in ECHigh BUB1 mRNA expression was associated with poor OS (P = 0.00036) and RFS (P = 0.0011) in EC. High BUB1B mRNA was associated with poor OS (P = 0.0024) but had no effect on RFS (P = 0.064) in EC (Fig. S2A,B).BUB1 and BUBIB variation tumors frequently show other genetic variations in pan-cancerThe frequency of BUB1 gene alterations in melanoma, bladder cancer, and EC was high (frequency > 5%) and the gene alterations mainly included mutations (Fig. S3A), including missense mutations, amplifications and profound deletions. The most common CNVs were diploidy, gain and shallow deletion (Fig. S3B). ARAP-AS1, TTN, MUC16, LRP1B, FLG, CSMD3, TP53, SLTL2-IT1, MT1F, and PTPRQ genetic alterations were more common in the BUB1 variant group (Fig. S3C).BUB1B gene alterations were more frequent in EC and melanoma (frequency > 5%) and the alterations were predominantly mutations (Fig. S3D). All PM (pleural mesothelioma) (frequency > 4%) had BUB1B gene deep deletion. Missense mutations, deep deletions and amplification were the main types of variations. The most common CNVs were diploidy, gain and shallow deletion (Fig. S3E). KIZ-ASL, LNCNEF, linc01721, linc00261 LINC00656, LINC01727, LINC01427, LINC01432, LINC01431, and CST13P were common in the BUB1B gene alteration group (Fig. S3F).BUB1 and BUBIB genetic variation had no effect on survival of EC patientsBUB1 gene alteration occurred in 6% (33/509) of EC patients and the main type was missense mutation (Fig. 3A). The effect on OS (P = 0.534) (Fig. 3B) and PFS was not significant (P = 0.0789) (Fig. 3C). BUB1B gene alteration occurred in 6% (33/509) of EC patients, and the main type was missense mutation (Fig. 3D); it did not have a significant effect on OS (P = 0.219) (Fig. 3E) and PFS (P = 0.0790) (Fig. 3F).Figure 3BUB1 and BUB1B gene variations in EC. (A) BUB1 gene alteration in EC; (B) BUB1 gene alteration in relation to OS in EC; (C) BUB1 gene alteration in relation to PFS in EC. (D) BUB1B gene alteration in EC; € BUB1B gene alteration in relation to OS in EC; F. BUB1B gene alteration in relation to PFS in EC.BUB1 and BUBIB were associated with multiple immune infiltrations in pan-cancerWe used TIMER, CBERSORT, TIDE, XCELL, MCPCOUNTER, and QUANTISEQEPIC databases to explore the correlation between BUB1and BUB1B and cancer-associated fibroblast (CAF), endothelial cell and neutrophil infiltration levels in different tumors in TCGA database. BUB1 expression level was negatively correlated with CAF infiltration of BRCA and TGCT and endothelial cell infiltration of BRCA, KIRC, LUAD, STAD, THCA, and THYM and positively correlated with neutrophil infiltration of COAD and endothelial cell infiltration of KIRP and LGG (Fig. S4A–C). BUB1B expression level was negatively correlated with the CAF infiltration of BRCA, HNSC-HPV + (HPV-associated head and neck squamous cell carcinoma), TGCT, and THYM and endothelium infiltration of BRCA, KIRC, LUAD, STAD, and THYM (Fig. S4D–F).BUB1 and BUBIB was associated with immune infiltration in ECBUB1 was associated with abundance of a variety of tumor-infiltrating lymphocytes (TILs) and mostly showed a negative correlation (Fig. 4A). BUB1 level was associated with act-CD8 + T cells (rho = − 0.157), act-CD4 + T cells (rho = 0.5676, act-B cells (rho = − 0.240), NK cells (rho = − 0.155), macrophages (rho = − 0.312), eosinophils (rho = − 0.38), monocytes (rho = − 0.318), and neutrophils (rho = − 0.344) (P < 0.001). BUB1 was significantly associated with immunomodulators and immunosuppressive agents in EC in TISIDB database online analysis (Fig. 4B). For example, BUB1 was associated with ADORA2A (rho = − 0.232), SLAMF4 (rho = − 0.251), CSF1R (rho = − 0.263), LGALS9 (rho = − 0.298), TGFB1 (rho = − 0.251), and CD160 (rho = − 0.209) (P < 0.001). BUB1 was also significantly associated with multiple immunostimulatory factors, including CD27 (rho = − 0.26), CD40LG (rho = − 0.301), HHLA2 (rho = − 0.286), NT5E (rho = − 0.324), TNFRSF14 (rho = − 0.389), and TNFRSF4 (rho = − 0.311) (P < 0.001) (Fig. 4C).Figure 4BUB1 or BUB1B and EC immune filtration. (A) BUB1 expression was associated with TIL abundance in EC; (B) BUB1 expression was associated with immunosuppressants in EC; (C) BUB1 expression and immunostimulants in EC. (D) BUB1B expression was associated with TIL abundance in EC; E. BUB1B expression was associated with immunosuppressants in EC; (F) BUB1B expression and immunostimulants in EC.BUB1B also correlated strongly with the abundance of multiple TILs (Fig. 4D). BUB1B expression levels correlated with act-CD8 + T cells (rho = − 0.146), act-CD4 + T cells (rho = 0.556), act-B cells (rho = − 0.237), NK cells (rho = − 2.76), macrophages (rho = − 0.347), eosinophils (rho = − 0.381), monocytes (rho = − 0.356), neutrophils (rho = − 0.388), and act-dendritic cells (rho = -0.159) (P < 0.001). BUB1B was significantly associated with multiple immunosuppressive factors (Fig. 4E), including ADORA2A (rho = − 0.283), CD244 (rho = − 0.232), CSF1R (rho = − 0.357), PVRL2 (rho = − 0.248), TGFB1 (rho = − 0.272), and LGALS9 (rho = − 0.319) (P < 0.001). BUB1B was also significantly associated with multiple immunostimulatory factors, including C10orf54 (rho = − 0.317), RAET1E (rho = − 0.29), TNFRSF14 (rho = − 0.379), TNFSF14 (rho = − 0.377), TNFRSF4 (rho = − 0.318), and TMEM173 (rho = − 0.286) (P < 0.001) (Fig. 4F).BUB1 and BUBIB affects multiple pathways in tumorsWe used the STRING database and obtained 50 BUB1-binding and BUB1B-binding proteins (Fig. S5A,D). The top five genes most strongly associated with BUB1 were NCAPH (r = 0.93), SGOL1 (r = 0.93), DLGAP5 (r = 0.94), CKAP2L (r = 0.94), and KIF11 genes (r = 0.93) (Fig. S5B). The top five genes most strongly associated with BUB1B were NUSAP1(r = 0.93), BUB1 (r = 0.93), OIP5 (r = 0.92), ARHGAP11A (r = 0.92), and KIF11 genes (r = 0.93) (Fig. S5E).The GO/KEGG enrichment analysis revealed that the main pathways affected by BUB1 and related proteins in tumors were protein homogenization, nuclear chromosome segregation, response to topologically incorrect proteins, response to unfolded proteins, and ER-associated misfolded proteolysis protein catabolic process. The pathways affected by BUB1 and related proteins included protein metabolic processes in tumors (Fig. S5C, Table S2). The pathways mainly affected by BUB1B and related proteins in tumors were cell cycle, DNA replication, microtubule motor activity, microtubule binding, ATPase activity, nuclear division, chromosome segregation, and mitotic nuclear division (Fig. S5F, Table S3). BUB1B and related proteins mainly affect cell cycle proteins in tumors.BUB1 and BUBIB expressions are related to clinicopathologic factorsBUB1 and BUB1B expressions were significantly higher in EC in all cancer stages (P < 0.01), with higher expression in middle to late stage cancer than early-stage cancer. BUB1 and BUB1B were highly expressed in EC in pre-menopausal, peri-menopausal and post-menopausal stages (P < 0.01). BUB1 and BUB1B expressions were significantly higher in serous cases compared with endometrioid tissue cases (P < 0.01). BUB1 and BUB1B expressions were higher in TP53-mutated EC compared with TP53 wild-type EC (Table 1).
Table 1 The relation between BUB1 and BUB1B expression with clinicopathological characteristics in EC.Knockdown of BUB1 and BUBIB influences proliferation, migration, and invasion of EC cellsWe used siRNA to downregulate BUB1 and BUB1B in EC cells. The relative expression of BUB1 mRNA in the BUB1-siRNA group was significantly lower than the blank group and negative control group (P < 0.001), and the relative expression of BUB1B mRNA in the BUB1B-siRNA group was significantly lower than the blank group and NC group (P < 0.05), indicating the efficacy of gene knockdown (Fig. 5A).Figure 5Influence of BUB1 and BUBIB knockdown on EC cell phenotype. (A) The growth of Ishikawa cells in each group; (B) The migration rates of Ishikawa cells in each group: blank group (50.99 ± 1.02)%, NC-siRNA group (66.50 ± 7.74)%, BUB1-siRNA group (34.87 ± 4.26)%, and BUB1B-siRNA group (31.96 ± 4.72)%; (C) The number of cells that crossed the membrane in each group: blank group (127.3 ± 4.8), NC-siRNA group (125.3 ± 3.9), BUB1-siRNA group (48.6 ± 3.2), and BUB1B-siRNA group (35.0 ± 2.1). *P < 0.05, **P < 0.01, ***P < 0.001.Cell growth in the siRNA-BUB1 and siRNA-BUB1B groups was significantly inhibited compared with the blank group and NC group (P < 0.05) (Fig. 5B). In scratch assays, the wound healing rates of the BUB1-siRNA group (34.87 ± 4.26)% and BUB1B-siRNA group (31.96 ± 4.72)% after 24 h were significantly decreased compared with the blank group (50.99 ± 1.02)% and NC-siRNA group (66.50 ± 7.74)% (P < 0.05) (Fig. 5D). In invasion assays, the numbers of invaded cells in the si-BUB1 group and si-BUB1B group were 48.6 ± 3.2 and 35.0 ± 2.1, respectively, which were less than those in the blank group and negative group (127.3 ± 4.8 and 125.3 ± 3.9, respectively), indicating that invasion ability was significantly decreased upon BUB1 and BUB1B knockdown (P < 0.05) (Fig. 5C1–4).