CENPA is highly expressed in human HCC tissues and correlated with poor prognosis in HCC.
To investigate the potential role of the CENPs family in HCC, we first analyzed the mRNA expression patterns of the CENPs family using RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) database. In the CENPs family genes, CENPA, CENPE, CENPF, CENPI, CENPK, CENPL, CENPM, CENPU, and CENPW significantly up-regulated in the HCC tissues compared with the adjacent non-tumor tissues (Figure. 1A). The cox regression analysis showed that the expression level of these nine CENPs family members were risk factors for HCC prognosis (Figure. 1B; Supplementary Figure. S1A). Among these genes, CENPA showed higher expression and the highest hazard ratio (HR) for OS in HCC (Figure. 1A and 1B; Supplementary Figure. S1A), so we concentrate on the function of CENPA in the development process of HCC.
We further validated the expression pattern of CENPA in GSE22058 and GSE14520 datasets and found that the expression level of CENPA in HCC was significantly higher than in adjacent non-tumor tissues (Figure. 1C). Then, after performing real-time quantitative PCR (qRT-PCR) as well as immunoblotting assays of 100 paired HCC samples from Tongji hospital, we found that the mRNA and protein level of CENPA were elevated in tumor tissues when compared to nearby non-tumor tissues (Figure. 1D and 1E). In addition, immunohistochemical (IHC) staining in the TME cohort showed the same results (Figure. 1F). Moreover, we found that CENPA mRNA was typically highly expressed in HCC cell lines profiled in the Cancer Cell Line Encyclopedia project (Supplementary Figure. S1B).
We also analyzed the correlation between CENPA expression and the HCC tumor stage, and discovered that CENPA expression was positively linked with the stage (Figure. 1G). Further analysis of the relationship between CENPA expression and prognosis showed that the patients with high CENPA mRNA expression had shorter OS than those with low expression (Figure. 1H). The receiver operating characteristic (ROC) curve indicated CENPA expression level showed a high correlation with HCC diagnosis (Figure.1I). These findings revealed that CENPA is highly expressed in HCC and highly expressed CENPA predicted poor prognosis for HCC patients.
CENPA promotes HCC cell proliferation and tumor growth.
To explicit the biological function of CENPA in HCC, both gain- and loss- of function studies were performed. We first detected the CENPA expression level of HCC cell lines and constructed stable CENPA knockdown cells in Huh7 and overexpression cells in MHCC-97H and HLF cells separately (Supplementary Figure. S2A and S2B). After the knockdown of CENPA, the growth and colony formation capabilities of the Huh7 cells were remarkably inhibited (Figure. 2A and 2B), whereas overexpression of CENPA showed the opposite results (Figure. 2A and 2B; Supplementary Figure. S2C and S2D). The EdU assay further demonstrated that CENPA was essential for cell proliferation, as CENPA knockdown showed a pronounced reduction in DNA synthesis and vice versa (Figure. 2C). Since DNA synthesis is closely correlated with the cell cycle, we examined the change of cell cycle procession by flow cytometry. Analysis suggested that CENPA raised the proportion of HCC cells in the G2/M phase while decreasing the proportion of cells in the G0/G1 phase, and CENPA knockdown resulted in the opposite phenotypic changes (Figure. 2D; Supplementary Figure. S2E).
We then used heterotopic xenograft and orthotopic xenograft animal models to further detect the function of CENPA in HCC cell growth. Results showed that in comparison to the control group, HCC tumor size and weight of the CENPA knockdown group were significantly reduced after subcutaneous injection (Figure. 2E; Supplementary Figure. S2F). The contrary results were found in the MHCC-97H cells (Figure. 2F; Supplementary Figure. S2G). The orthotopic xenograft tumor model showed that compared with the control group, CENPA knockdown significantly reduced the volume of orthotopic liver tumor, while the overexpression of CENPA had the opposite effect (Figure. 2G and 2H; Supplementary Figure. S2H and 2I). IHC staining results showed that, compared with controls, the positive proportion of Ki67 was lower in tumors with CENPA knockdown but higher in tumors with CENPA overexpression (Supplementary Figure. S2F-S2I). All these results suggested that CENPA promotes HCC cell proliferation and tumor growth.
CENPA interacts with YY1 and collaborates as co-transcriptional factors.
To study the mechanism by which CENPA promotes HCC proliferation, we first conducted co-immunoprecipitation (co-IP) assay to enrich potential proteins interacting with CENPA. By mass spectrometry analysis, we found that YY1, as one of the top candidates, interacted with CENPA (Figure. 3A and 3B; Supplementary Table 2). Co-IP experiments in HEK293T cells showed that exogenous CENPA coprecipitated with YY1 (Figure. 3C). We further performed co-IP experiments with whole-cell lysate from Huh7 cells to confirm the protein-protein interaction between the endogenous CENPA and YY1 (Figure. 3D). In addition, confocal microscopy experiments revealed that CENPA colocalized with YY1 in HCC cells (Figure. 3E). YY1 consists of transcriptional activation domain (residues 1-154), transcriptional repression domain (residues 155–226), spacer domain (residues 227–295) and DNA-binding domain (residues 296–414) [24]. To identify the functional domain of YY1 that interacts with CENPA, YY1 deletion mutants (YY1 Δ1, YY1 Δ2, YY1 Δ3, YY1 Δ4) were constructed. We found that YY1 Δ4 (in which the zinc finger region was deleted) lost the ability to interact with CENPA (Figure. 3F), indicating that the binding of YY1 to CENPA depended on the zinc finger region.
Since YY1 is a member of the GL-Kruppel family of zinc finger DNA binding proteins that can regulate gene expression relying on chromatin structure, promoter context, and partner proteins [25]. Recent studies also reported that CENPA might function as a transcription regulator [18]. Considering the previous studies, we hypothesize whether CENPA interacts with YY1 forming the co-transcription factor complex. To show the co-binding landscape of CENPA and YY1, we conducted Flag-CENPA ChIP-sequencing (ChIP-seq) in HEK293T and obtained YY1 ChIP-seq data in HEK293T from the Cistrome database. Venn diagram showed that CENPA and YY1 have 5287 co-binding sites/regions (Figure. 3G). The majority of the sites/regions (~ 68%) were located in the transcriptional start elements, including the promoter, 5’-UTR, and 1st exon, followed by the distal intergenic region (8.44%) (Figure. 3H). In those sites/regions, we observed a closer spatial distance between the binding tracks (Figure. 3I) and similar distribution patterns in CENPA and YY1 (Supplementary Figure. S3A). Moreover, KEGG/GO analysis showed that these co-binding genes are related to multiple cancer-related signaling pathways (Supplementary Figure. S3B). Thus, we demonstrated that CENPA interacts with YY1, forming the transcriptional complex, to co-regulate a group of genes involved in tumor progression.
YY1 is essential for CENPA-mediated HCC progression.
As a transcription factor, YY1 plays the dual role of transcriptional activator and repressor in different tumors [26]. To detect the specific role of YY1 in HCC, we silenced or overexpressed the expression of YY1 in Huh7 and MHCC-97H (Supplementary Figure. S4A and S4B). In vitro experiments with HCC cell lines showed that knockdown of YY1 reduced cell viability and clonogenic capacity (Fig. 4A-C) and vice versa (Supplementary Figure. S4C). Consistent results were obtained in vivo with subcutaneous tumors (Figure. 4D; Supplementary Figure. S4D).
To explore whether the oncogenic role of CENPA was through YY1, we constructed YY1 overexpression cell line in CENPA knockdown Huh7 cell (Supplementary Figure. S4E). YY1 overexpression significantly reversed the inhibition of CENPA knockdown on HCC cell proliferation in vitro (Figure. 4E and 4F). Also, YY1 knockdown through siRNA significantly weakened the effect of CENPA overexpression on promoting HCC cell proliferation in vitro (Figure. 4E and 4F). Similar results were observed in vivo with subcutaneous tumors (Figure. 4G; Supplementary Figure. S4F). Therefore, these results suggest that YY1 acts as an essential mediator of CENPA to regulate the progression of HCC.
CCND1 and NRP2 may serve as the key candidate genes regulated by CENPA and YY1
To further explore the downstream genes involved in the mechanism by which CENPA and YY1 work together to enhance HCC growth, we performed RNA-seq in CENPA or YY1 knockdown Huh7 cells. Integrative analysis of transcriptome and ChIP-seq data revealed that 12 genes were potentially highly related direct target genes that down-regulated in HCC cells after knockdown of CENPA and YY1 (Figure. 5A). We further confirmed that CCND1 and NRP2 were the main target genes of CENPA/YY1 complex in HCC cells (Supplementary Figure. S5A and S5B). With further research, we discovered the co-binding sites of CENPA and YY1 on the promoters of CCND1 and NRP2 (Figure. 5B). The qRT-PCR and western blotting assays further confirmed that CCND1 and NRP2 were up-regulated by both CENPA and YY1 (Figure. 5C and 5D; Supplementary Figure. S5C). Luciferase and ChIP-qPCR analyses demonstrated that CENPA and YY1 promoted the transcription efficiency of CCND1 and NRP2 via binding at their promoter regions (Figure. 5E and 5F). These results validated that CCND1 and NRP2 are the target genes of the CENPA/YY1 complex.
We further explored the biological effect of CCND1 and NRP2 in HCC cell lines. Functionally, the knockdown of CCND1 and NRP2 suppressed the HCC cell growth of Huh7 and MHCC-97H (Figure. 5G-5I). Clinically, correlation analysis of indicated mRNA data from Tongji cohort showed significant positive correlations between the expression of CENPA, YY1, CCND1, and NRP2 (Figure. 5J). Based on the TCGA database, we found that the mRNA expression level of CCND1 and NRP2 was substantially increased in the HCC (Figure. 5K). Collectively, these findings suggest that CENPA and YY1 collaborate to promote HCC cell proliferation via transcriptional activation of CCND1 and NRP2.
Besides CCND1 and NRP2, YY1 was also a potential target gene that down-regulated in HCC cells after the knockdown of CENPA and YY1 (Figure. 5A). To detect whether CENPA also regulated YY1, we employed qRT-PCR and western blotting for experimental validation, and found that knockdown of CENPA reduced the expression of YY1 at both mRNA and protein level and vice versa (Figure. 5C and 5D; Supplementary Figure. S5C). Furthermore, the luciferase reporter assay results showed that CENPA knockdown reduced YY1 promoter activity, indicating that CENPA regulates YY1 expression at the transcriptional level (Supplementary Figure. S5D). Sequence analysis revealed the presence of three potential CENPA-binding sites in the YY1 promoter. Site-directed mutagenesis and serial deletion showed that the second CENPA-binding site is vital for CENPA-induced YY1 transactivation (Supplementary Figure. S5D). ChIP-qPCR results further validated the direct binding of CENPA to the YY1 promoter (Supplementary Figure. S5E). These results further confirmed that YY1 was the target gene of CENPA and played a crucial role in regulating HCC proliferation by CENPA.
The K124 lactylation enhances CENPA transcriptional activity.
PTMs of histones play essential roles in maintaining homeostasis through regulating DNA-dependent processes, including DNA repair, replication, transcription, and so on [27–29]. Several types of PTMs on CENPA have proven to be critical to its function. For example, ubiquitylation has been reported to be essential for CENPA deposition at the centromere [19, 30]. We are keen on investigating the new PTM modification that modulates the function of CENPA, such as whether there is direct lactylation of CENPA. To verify whether CENPA can be lactylated in cells, an immunoprecipitation (IP) test was conducted, which showed that pan-Kla was indeed present in CENPA, and lactic acid (LA) increased the lactylation level of CENPA (Figure. 6A). Moreover, CENPA lactylation also can be detected in HCC cell lines, which could be enhanced by 25-mM LA (Figure. 6B). Collectively, these data suggested that lactylation modification did exist on CENPA.
Combined with the previous studies that lysine lactylation (Kla) was identified as the novel PTM of histone and research over the PTM sites of CENPA [22], we speculated that lysine 124 amino acid may be lactylated in CENPA. To further confirm the modified site, we constructed IP assays which transfected Flag-CENPA plasmid or Flag-CENPA K124 mutation plasmid respectively. Results showed that K124R of CENPA extensively diminished the overall lactylation level of CENPA (Figure. 6C).
Since previous research showed that histone lactylation may influence the transcription efficiency of downstream genes [23, 31], and CENPA is centromeric histone H3 variant centromere protein A, we speculated whether K124 lactylation of CENPA enhances its transcriptional activity. To test this hypothesis, we transfected Flag-NC, Flag-WT, and Flag-K124R with 25 mM LA into HCC cell lines. Notably, we identified the target genes of CENPA whose expression was significantly decreased by transfecting Flag-K124R compared to Flag-CENPA (Figure. 6D). Likewise, 2-deoxy-D-glucose (2-DG), a non-metabolizable glucose analog, significantly reduced the effect of CENPA overexpression on promoting the mRNA and protein expression level of its target genes in HCC cells (Figure. 6E). Luciferase assay showed the same results that 2-DG reduced the effect of CENPA overexpression on promoting the transcription activity of its target genes (Figure. 6F). Additionally, CENPA-K124R mutant markedly reduced the binding of CENPA to the promoters of YY1, CCND1, and NRP2 in Huh7 cell by ChIP-qPCR (Figure. 6G). These data indicated that the lactylation of CENPA K124 enhanced its transcriptional activation ability in HCC cells.
CENPA expression is positively correlated with YY1 expression in human HCC tissues.
To determine the relevance in HCC, we performed IHC and western blotting staining to examine the abundance of YY1 and CENPA. In 50 pairs of HCC samples, YY1 was highly expressed in HCC as determined by qRT-PCR and western blotting (Figure. 7A and 7B). The correlation analysis showed that CENPA was strongly positively correlated with YY1 in HCC tissues (Figure. 7C). Consistent result was obtained by IHC staining (Figure. 7D). Moreover, HCC patients with high CENPA expression were more prone to have high expression of YY1 than those with low CENPA expression (chi-square, P = 0.0105, Figure. 7E). Additionally, we analyzed the prognostic significance of CENPA and YY1 expression in HCC patients. Patients with high expression of both CENPA and YY1 had the worst OS and RFS rates among all patients analyzed (Figure. 7F). These results indicate that both CENPA and YY1 are highly expressed in HCC and have similar expression characteristics. High expression of both CENPA and YY1 suggests a poor prognosis.