The highlight of our study was that it comprehensively demonstrated the up-regulation of BUB1B mRNA in NPC from multiple databases based on 308 NPC samples and 51 controls. Meanwhile, we applied IHC to verify that BUB1B protein expression was also highly expressed in another independent cohort with 110 cases of NPC and 66 cases of non-NPC controls. Furthermore, our study shed light on the underlying upstream transcription factors of BUB1B and its potential molecular mechanisms in NPC.
Before our study, only two studies focused on the role of BUB1B expression levels in NPC, both based on limited sample sizes(Huang, Tang, Zhang et al. 2012; Yue, Zhu, Luo et al. 2022). The shortcomings of the previous research by Huang et al. (Huang, Tang, Zhang et al. 2012) and Yue et al. (Yue, Zhu, Luo et al. 2022) could be solved in our current study. Combining public high throughput data and in-house IHC, we showed the consistent up-regulation of BUB1B in NPC. The case number was 10 folds more than that of Huang et al. with 418 cases of NPC and 113 cases of controls being involved. Various detecting methods being used also enhanced the convincingness of the results, including mRNA and protein levels of BUB1B. Moreover, the samples were from different areas of the world, including China, USA, UK and Singapore. Our study conducted experiments with different detection methods on cases from multiple regions, whose results verified that the expression of BUB1B was up-regulated, suggesting that BUB1B has a certain oncogenic effect in the occurrence of NPC. This effect is suitable for all NPC patients, without regional difference.
To help understand the underlying mechanisms of BUB1B in NPC, the intersecting genes of BUB1B co-expressed genes and DEGs were used to demonstrate the potential signaling pathways related to BUB1B in NPC and to obtain other NPC biomarkers. In the mechanism analysis of this study, BUB1B was positively correlated with the following 10 hub genes involving in the cell cycle pathway: CDC6, MCM2, CDC45, MCM3, CHEK2, CCNB1, MCM7, CDK1, MCM4 and CHEK1. Among them, CHEK1, CDK1, CCNB1, and CDC6 (Wang, Chang, Lai et al. 2018; Yu, Liu, Yin et al. 2019; Luo, He, Liu et al. 2020; Qian, Zheng, Chen et al. 2020) in NPC have been studied to some extent. Considering the important role of BUB1B in mitotic checkpoint signaling and chromosome assembly, BUB1B imbalance often leads to aneuploidy and chromosomal instability (Simmons, Park, Sterling et al. 2019), which may lead to an increase in the incidence of cancer. Consistent with this, the knockdown of BUB1B inhibited brain tumor-initiating cells -driven tumor formation (Ding, Hubert, Herman et al. 2013). In addition, the over-expressed BUB1B found in prostate cancer, lung cancer, and breast cancer was associated with the proliferation and metastasis of cancer cells (Chen, Lee, Kljavin et al. 2015; Fu, Chen, Cai et al. 2016; Cai, Mei, Xiao et al. 2019). We speculate that the up-regulation of BUB1B expression will promote the expression of the 10 hub genes, thereby accelerating the progress of the cell cycle, and finally enabling the proliferation and invasion of NPC cells.
Interestingly, when predicting the upstream TF of BUB1B, Histone Deacetylase 2 (HDAC2) was achieved by in-silico method. HDAC2 is a class I isoform of histone deacetylases, which could remove acetyl groups from histones, resulting in a tighter chromatin structure that regulates large amounts of gene transcription(Lin, Yao, Wu et al. 2020). Previous studies shown that HDAC2 was upregulated in breast, colorectal and prostate cancers(Jang, Hwang and Choi 2018; Zhang, Qiu, Yin et al. 2020; Qi, Yalikong, Zhang et al. 2021), and HDAC2 was associated with cancer-promoting molecular events. In addition, Lee et al. pointed out that HDAC2/3 bound to acetylated BUB1B(Park, Kwon, Paik et al. 2017), leading to ubiquitin and degradation of BUB1B to participate in the cell mitosis process, which is consistent with our pathway analysis. However, no researches focused on the regulatory axis of HDAC2-BUB1B in cancer. Our study revealed that HDAC2 was upregulated in NPC, meanwhile, public ChIP-seq data strongly supported the regulatory role of HDAC2 in BUB1B. In conclusion, we hypothesize that the upregulated HDAC2 in NPC induced cell cycle disorders by regulating BUB1B, and ultimately promotes the development of cancer.
Some limitations of this study should not be ignored. First, due to lack of data, the influence of BUB1B on the prognosis of NPC patients was not evaluated. Second, the included data sets were highly heterogeneous, and the influence analysis showed no distinct differences. This may be due to the different study design in the datasets and samples from different countries: China, the United States of America, Singapore and United Kingdom. Third, the precise molecular mechanism of BUB1B in NPC remains to be further studied. Finally, the role of BUB1B in NPC treatment strategies needs to be verified by in vitro and in vivo experiments.