KAT2A/E2F1 Promotes Cell Proliferation and Migration via Upregulating the Expression of UBE2C in Pan-Cancer

Various studies have shown that lysine acetyltransferase 2A (KAT2A), E2F transcription factor 1 (E2F1), and ubiquitin conjugating enzyme E2 C (UBE2C) genes regulated the proliferation and migration of tumor cells through regulating the cell cycle. However, there is a lack of in-depth and systematic research on their mechanisms of action. This study analyzed The Cancer Genome Atlas (TCGA) to screen potential candidate genes and the regulation network of KAT2A and E2F1 complex in pan-cancer. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB), cell phenotype detection, immunofluorescence co-localization, chromatin immunoprecipitation assay (ChIP), and RNA-Seq techniques were used to explore the functional of a candidate gene, UBE2C. We found that the expression of these three genes was significantly higher in more than 10 tumor types compared to normal tissue. Moreover, UBE2C was mainly expressed in tumor cells, which highlighted the impacts of UBE2C as a specific therapeutic strategy. Moreover, KAT2A and E2F1 could promote cell proliferation and the migration of cancer cells by enhancing the expression of UBE2C. Mechanically, KAT2A was found to cooperate with E2F1 and be recruited by E2F1 to the UBE2C promoter for elevating the expression of UBE2C by increasing the acetylation level of H3K9.


Introduction
Many cancer treatment strategies have been developed based on targeting specific molecules related to specific gene mutation or specific gene expression, and breaking progress is being made with these cancer treatment strategies, and the potential is infinite [1][2][3]. Therefore, there is an urgent need to identify efficient therapeutical targets in tumors. Currently, many common molecular mechanisms across pan-cancer have been discovered by various studies [4][5][6]. High-throughput transcriptome was an important and effective data to identify the candidate targets or pathways [7][8][9]. Although tumors have great heterogeneity, there are commonalities among different types of tumors, so using the transcriptome data of large clinical pan-cancer samples to authenticate potential candidate targets upstream may be a good option. However, whether it will shed light on future cancer treatment still needs further comprehensive and in-depth research on pan-cancer.
Previous studies have shown that there were abnormalities of histone acetylation modification in various cancers, including liver, lung, and breast [10,11]. Histone acetylation mediates the expression and activation of genes related to cell proliferation, differentiation,

Identification of Differentially Expressed Genes of Pan-Cancer from TCGA
The samples of different types of cancers in the TCGA databases were separated into 30% each of KAT2A, E2F1, and UBE2C high and low groups to obtain DEGs using the "DESeq2" package (version 1.28.1) in R language (version 4.0.2). |Fold Change| > 1.5 and FDR < 0.05 were set as the statistical threshold value of DEGs. Using the transcriptome data of 11 tumor types (with normal tissues more than 30) in TCGA, which contains the significantly highly expressed level of KAT2A, overlapping DEGs were screened according to the following conditions: each tumor KAT2A and E2F1 were grouped according to the 30% high and low groups to obtain: the DEGs (FC > 1.2, FDR < 0.05, KAT2A 30%-Up, and E2F1 30%-Up), respectively; the correlation coefficient with KAT2A and E2F1 > 0. 3, respectively; and DEGs that were highly expressed in tumor tissues compared with normal tissues.

Spearman Correlation among KAT2A, E2F1, and UBE2C
Spearman's correlation coefficient analysis was performed to explore the correlation among KAT2A, E2F1, and UBE2C in 11 out of 33 cancers with more than 200 tumor tissues from the TCGA database, and the corresponding cell lines in the CCLE database.

Functional Enrichment Analysis
Functional enrichment analysis, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted by the R package (clusterProfiler, version 3.16.1) to explore the different molecular mechanisms and involved pathways between high and low UBE2C expression. The protein-protein interaction (PPI) network of DEGs was obtained from the STRING (version 11.0) database [38]. The results from the TCGA RNA transcriptome data and the existing GEO dataset were combined to obtain the overlapping DEGs, and the key genes or target proteins and signal pathways regulated by UBE2C were searched. The common mechanism of KAT2A/E2F1/UBE2C affecting tumor cell proliferation, cell cycle, and apoptosis in different tumors were explored through GO and KEGG function enrichment analyses.

Single-Cell RNA-Seq Data Processing
The single-cell RNA-Seq data were analyzed as described previously [39] Expression data were extracted from a previous study [39] and violin plots were drawn using R.

Real-Time Quantitative Polymerase Chain Reaction (qPCR)
Real-time quantitative PCR (qPCR) analysis was performed according to the user's manual using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green PCR Master Mix (Applied Biosystems) kits. All samples were analyzed in triplicate, and the expression of KAT2A, E2F1, and UBE2C was normalized relative to that of GAPDH, which was used as an internal loading control. The primers for qPCR are listed in Table S2.

Cellular Immunofluorescence
The detailed immunohistochemistry procedures were performed as described before [40]. Cells were seeded into the 35 mm laser confocal petri dishes under normal culture conditions to reach 60% density without any treatment to prepare for performing cell immunofluorescence. After incubating with KAT2A and E2F1 primary antibodies (Invitrogen, Thermo Fisher Scientific, 1:1000, MA5-14884, MA1-23202), the cells were then incubated with the corresponding diluted IgG fluorescent secondary antibodies (Invitrogen, Thermo Fisher Scientific, 1:5000, A11029, A11012) for 1 h at room temperature in the dark. Then, the cells were stained with nucleus DAPI dropwise and incubated for 5 min in the dark. The mounting solution containing anti-fluorescence quencher (Invitrogen, Thermo Fisher Scientific, P36971) was dropped, followed by observing and collecting the image under a fluorescence microscope (Wetzlar, Germany, Leica TCS SP8 X).

Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (Co-IP) Assay
ChIP was performed according to the instructions of the Pierce Agarose ChIP Kit (Thermo Fisher Scientific, 26156). The E2F1 binding sites on the UBE2C promoter were analyzed using the JASPAR online tool (http://jaspar.genereg.net/, accessed on 8 June 2020). ChIP-qPCR data were shown as the percentage of input following normalization with no antibody (mock). The primers for ChIP-qPCR are listed in Table S2. Co-IP was performed using indicated antibodies and IgG (Invitrogen) according to the manufacturer's instructions. In brief, cell lysates were incubated with antibody-conjugated beads at 4 • C for 2 h. Then, the beads were washed extensively and boiled in SDS loading buffer. A total of 4% of total protein was used per IP, about 50 µg/100 µg.

Luciferase Assay
The luciferase assay was performed as described previously [41].

Cell Proliferation Assay
Cell proliferation assays were performed using the Cell Counting Kit-8 (CCK-8; Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Briefly, cells were seeded onto 96-well plates (3 × 10 3 cells per well) and transfected when they reached 70-80% of confluence according to the protocol of Lipofectamine™ 3000, and were then added with 10 µL of CCK-8 solution and cultured for 1 h at 37 • C in air with 5% CO 2 on designated days. The absorbance was measured at 450 nm using TECAN infinite M200 (Softmax Pro., Molecular Devices, Sunnyvale, CA, USA). For EdU assay, the cells were treated for 48 h, followed by using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime Biotechnology, C0078S) according to the manufacturer's protocol.

Clone Formation Experiment
The stable KAT2A overexpression NCI-H460 cells were seeded in a 6-well plate with 2000 cells per well. After culturing for 14 days, the culture medium was discarded, was washed carefully with 1 × phosphate-buffered saline (PBS) (Gibco, Grand Island, NY, USA) once, and 1 mL of methanol solution was added to each well to fix the cells for 30 min. The methanol solution was aspirated, 1 mL of crystal violet stain solution was added to each well, and it was left at room temperature for 30 min. The crystal violet was recycled and each well was washed with distilled water, and the culture plate was placed upside down on absorbent paper to absorb the water. It was dried naturally, and pictures were taken using a digital camera. The number of clones with more than 10 cells under the microscope (4 × magnification) was counted. Finally, we calculated the clone formation rate = (number of clones/number of inoculated cells) × 100%.

Cell Migration and Invasion Assay
A wound healing assay was performed to detect the migration of three kinds of cancer cell lines after treatment. Cells growing the in log phase were trypsinized and seeded in 24-well plates until confluent. A total of 1 × 10 5 cells per well were seeded in 24-well plates. After 24 h, the cells were transfected with shKAT2A-1, siE2F1-2, and shUBE2C-3, and the corresponding control shNC and siNC using Lipofectamine TM 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. After transfection, cells were incubated at 37 • C and 95% confluent cells were used for a wound healing assay. Wounds were made using a 10 µL sterile tip. After incubation for 0, 24, 48, 72, and 96 h, the cells were photographed under an inverted microscope, respectively. The distance between the two edges of the scratch (wound width) was measured at 8 sites using ImageJ in each image (40× magnification). MCF-7, 786-O, and NCI-H460 cells were transiently transfected with shUBE2C-3 for 48 h, followed by using Falcon ® Permeable Support for a 24-well Plate with 8.0 µm Transparent PET Membrane (Corning, NY, USA) for measuring cell migration and invasion. After taking pictures of the cells, we added 1 mL absolute ethanol. With sufficient shaking, the light absorbance was measured at 570 nm. The optical absorbance (OD) value was used to plot the dilution of the samples, and the curves of the standard product and the samples were compared.

Cell Apoptosis Assay
The cells were treated with shUBE2C-3 and shNC for 48 h, respectively. Afterwards, the cells were digested with trypsin without EDTA. After termination, the cells were centrifuged at 1000 rpm for 5 min at 4 • C to remove the supernatant containing trypsin, and were then washed 2 times with precooled PBS. The cells were regenerated with serum-free medium, and cell apoptosis was analyzed using an Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1062M) according to the instructions.

Cell Cycle Assay
After 48 h treatment of shNC and shUBE2C, the cells were washed twice with precooled PBS, centrifuged at 1200 rpm 4 • C for 5 min, fixed with 70% ethanol, and then stained with 10 µg/mL propidium iodide in a solution containing 100 µg/mL RNase in PBS; the cell pellet was slowly and fully resuspended, and was incubated at 37 • C for 30 min in the dark. Then, the cells were analyzed on a BD FACSCalibur (Becton, Dickinson and Company, San Jose, CA, USA).

RNA-Seq
Total RNA was extracted from the cells transfected for 48 h using Trizol reagent (Takara, Japan) and stored at −80 • C. The complementary DNA (cDNA) libraries of each pooled RNA sample for single-end sequencing were generated using the NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, E7530L) according to the manufacturer's instructions. The cDNA libraries were subjected to the NovaSeq 6000 system (Illumina), according to commercially available protocols. The changed RNAs were validated by quantitative PCR using the primers listed in Table S2.

RNA-Seq Analysis
The quality control of raw sequencing data was conducted using fastp [42] The clean reads from RNA-seq were aligned to the human reference genome sequence, GRCH38.p13, using the HISAT2 program (v2.2.3) [43] The gene expression level was determined by the featurecounts function of subread software with the genome annotation file from GENCODE (v36) [44][45][46].

Statistical Analysis
Data were showed as the mean values with the standard error of the mean. Statistical differences were determined using Student's t-test or one-way ANOVA. p < 0.05 was considered to indicate statistical significance.

The Highly Expressed KAT2A and E2F1 Could Promote Cancer Progress in Pan-Cancer
From the GEPIA online analysis, we found that compared with the expression level distributed in a normal person, KAT2A was slightly higher in cancer patients, and E2F1 was obviously higher expressed in cancer patients ( Figure 1A,C) [47,48]. As a result of the TCGA pan-cancer transcriptome data, the expression levels of KAT2A and E2F1 were significantly up-regulated in 16 and 20 kinds of tumor tissues compared with normal tissues, respectively. Both the expression levels of KAT2A and E2F1 were significantly upregulated in the same 16 cancers, and E2F1 was also higher expressed in CESC, GBM, KICH, and UCEC cancers ( Figure 1B,D). In addition, the expression levels of KAT2A and E2F1 in pathological stages were remarkably higher expressed in stage III and IV in 10 cancers ( Figure 1E,F). These findings suggested that KAT2A and E2F1 may play an important role in tumor development.
From the results of CCK-8 and wound-healing assays, the proliferation of MCF-7, 786-O, and NCI-H460 cells (Figure 2A-F), and the cell migration ability of NCI-H460 cells ( Figure 2G,H) were suppressed with the knockdown of either KAT2A or E2F1, compared with the control group. Moreover, the knockdown of KAT2A significantly inhibited the colony-forming ability of NCI-H460 cells ( Figure 2I).

UBE2C May Be a Downstream Gene That Is Co-Regulated by KAT2A/E2F1 in Pan-Cancer
In order to explore the potential target genes co-regulated by KAT2A and E2F1, the differentially expressed genes (DEGs) of KAT2A and E2F1 were screened according to the conditions. A total of 9 to 1202 overlapping DEGs were obtained from different cancer types ( Figure S1A-K). Among 11 cancers, there were 222 genes that appeared in more than five cancer types at the same time ( Figure S1L), including UBE2C, which appeared in six types of cancer (Table S3). The GO analysis of the overlapping 222 DEGs revealed that the biological pathways (BP) were related to DNA replication, nuclear division, the regulation of cell cycle phase transition and cell cycle checkpoint, and so on ( Figure  S2A); the enriched cell components (CC) included chromosomal region, chromosome, centromeric region, spindle, and kinetochore ( Figure S2B); and the molecular function (MF) was involved in the action of catalytic activity, acting on DNA, deoxyribonuclease activity, and so on ( Figure S2C). The KEGG pathway analysis indicated that these genes were significantly enriched in the cell cycle, DNA replication, homologous recombination, base excision repair and nucleotide excision repair, and so on ( Figure S2D). From the result of correlation analysis in tumor tissue samples across 11 cancer types and cell line samples in CCLE, we found that the correlation coefficient between the two in KAT2A, E2F1, and UBE2C was 0.037-0.82 ( Figure 3A). Considering the analysis results of the transcriptional data of various cancers in the TCGA database ( Figure S1), it is suggested that UBE2C may be a downstream gene that is co-regulated by KAT2A and E2F1. Correspondingly, we found that both the mRNA and the protein levels of UBE2C were significantly suppressed after the knockdown of KAT2A or E2F1 in MCF-7, 786-O, and NCI-H460, respectively ( Figure 3B-H). Moreover, the mRNA/protein changes of E2F1 were significantly inhibited after knocking down KAT2A ( Figure 3B-D). Furthermore, the knockdown of UBE2C caused an extreme decrease of the KAT2A protein level ( Figure 3H).
Additionally, the results of cellular immunofluorescence showed the nuclear localization of KAT2A and E2F1 in MCF-7, 786-O, and NCI-H460 cells ( Figure 4A). Moreover, ChIP-qPCR showed that KAT2A and E2F1 can bind to the −322 to +39 region of the UBE2C promoter (with more than one-fold enrichment), not −1081 to −819, or +11 to +215 regions (with less than one-fold enrichment) ( Figure 4B-G). Additionally, the dual luciferase reporter gene experiment also confirmed that E2F1 could bind to the −273 to −266 region of the UBE2C promoter ( Figure S3A-F). Moreover, the ChIP-qPCR assay demonstrated that the KAT2A bound to the promoter region of UBE2C, and increased the H3K9 acetylation level in this promoter region, which suggested that KAT2A and E2F1 may cooperate to regulate UBE2C gene transactivation via histone modification. Moreover, the Co-IP assays showed that KAT2A, E2F1, and H3K9ac could bind to each other ( Figure S3G). These results demonstrated that KAT2A may promote the expression of UBE2C through combining with E2F1 to the E2F1 binding site on UBE2C promoter −322/+39 region to increase the acetylation level of H3K9, and consequently stimulated cancer cell proliferation and migration.

UBE2C Highly Expresses in Pan-Cancer
The results of the GEPIA online analysis and TCGA database showed that the expression of UBE2C was higher in many types of tumors than normal tissues, especially in brain, lung, and breast ( Figure 5A,B). Additionally, the result of the expression levels of UBE2C in pathological stages showed that it was significantly highly expressed in stage III and IV in 10 types of cancer ( Figure 5C). In addition, based on our previous study [39], the analysis of tumor tissue single-cell transcriptome data showed that the expression of UBE2C has a significant up-regulation trend in cancer cell clusters (CS), such as CS4 of CRC, CS2 of LC, CS4 of OV, CS3 of PDAC, and CS5 of SCC ( Figure S4). Moreover, the survival analyses revealed that the high expression of UBE2C was significantly associated with a poor prognosis in patients of nine cancer types ( Figure 5D). The above results suggested that UBE2C was up-regulated in a variety of cancer tissues/cells, and it may play a pivotal role in the development of pan-cancer. Additionally, the results of cellular immunofluorescence showed the nuclear localization of KAT2A and E2F1 in MCF-7, 786-O, and NCI-H460 cells ( Figure 4A). Moreover, results demonstrated that KAT2A may promote the expression of UBE2C through combining with E2F1 to the E2F1 binding site on UBE2C promoter −322/+39 region to increase the acetylation level of H3K9, and consequently stimulated cancer cell proliferation and migration.

UBE2C Highly Expresses in Pan-Cancer
The results of the GEPIA online analysis and TCGA database showed that the expression of UBE2C was higher in many types of tumors than normal tissues, especially in brain, lung, and breast ( Figure 5A,B). Additionally, the result of the expression levels of UBE2C in pathological stages showed that it was significantly highly expressed in stage III and IV in 10 types of cancer ( Figure 5C). In addition, based on our previous study [39], the analysis of tumor tissue single-cell transcriptome data showed that the expression of UBE2C has a significant up-regulation trend in cancer cell clusters (CS), such as CS4 of

Knockdown of UBE2C Significantly Inhibits Cancer Cell Proliferation and Migration, and Promotes Cell Apoptosis
To investigate the function of UBE2C in MCF-7, 786-O, and NCI-H460 cells, we transfected the cell lines with UBE2C shRNA (shUBE2C), and the qRT-PCR results showed that the expression of UBE2C in shUBE2C-treated group was significantly lower than that in the negative control group (shNC) ( Figure 6A). Additionally, the results from the CCK-8 assay showed that the proliferation of different cancer cell lines was markedly suppressed with knocking down UBE2C in MCF-7, 786-O, and NCI-H460 cells compared with the control group ( Figure 6B-D). As a key downstream gene of KAT2A and E2F1, we also found that UBE2C significantly inhibited the proliferation of the liver cancer cell line, HepG2, and pancreatic cancer cell line, BxPC3, through a CCK-8 assay ( Figure S5), which suggested that UBE2C plays a critical role in pan-cancer. The EdU experiment suggested that the number of proliferating cells was significantly reduced after the knockdown of UBE2C at 48 h ( Figure 6E). In addition, we found that the cell proliferation ability was significantly inhibited after co-interfering with KAT2A and UBE2C, E2F1, and UBE2C, compared to that of only knocking down KAT2A or E2F1 in NCI-H460 cells ( Figure 6F,G). Moreover, compared with the control group (pLVX-puro), the overexpression of UBE2C (pLVX-UBE2C) significantly promoted cell proliferation, whereas knocking down KAT2A and E2F1 caused an inhibition on cell proliferation, which can be restored by the overexpression of UBE2C ( Figure 6H,I). Moreover, the overexpression of KAT2A (pLVX-KAT2A) also significantly promoted cell proliferation, whereas knocking down UBE2C caused an inhibition on cell proliferation, which can be restored by the overexpression of KAT2A ( Figure 6J). These results not only suggested that the downregulation of UBE2C could significantly inhibit the proliferation of tumor cells, but also confirmed that KAT2A and E2F1 could indeed affect the proliferation of tumor cells through regulating the expression of UBE2C.  Figure S4). Moreover, survival analyses revealed that the high expression of UBE2C was significantly associat with a poor prognosis in patients of nine cancer types ( Figure 5D). The above results su gested that UBE2C was up-regulated in a variety of cancer tissues/cells, and it may pla pivotal role in the development of pan-cancer.

Knockdown of UBE2C Significantly Inhibits Cancer Cell Proliferation and Migration, and Promotes Cell Apoptosis
To investigate the function of UBE2C in MCF-7, 786-O, and NCI-H460 cells, we tra

UBE2C Promotes the Development of Pan-Cancer through Influencing the Cell Cycle
To better understand the molecular signatures after the knockdown of UBE2C in NCI-H460 and MCF-7 cells, we performed RNA-seq analysis and analyzed the DEGs. As a result, a total of 5539 and 1756 DEGs in NCI-H460 and MCF-7 cancer cells was screened with |FC| > 1.2 and FDR < 0.05, respectively ( Figure 7A,B). As expected, the significantly enriched pathways of GO_BP of NCI-H460 were translational initiation, regulation of cell growth, cell cycle arrest, cell cycle checkpoint, and so on (p.adjust < 0.05) ( Figure 7C). The KEGG pathways of NCI-H460 were the included ribosome, cell cycle, adherens junction, protein processing in endoplasmic reticulum, and apoptosis ( Figure 7D). Additionally, the GO analysis showed that the DEGs of MCF-7 were highly associated with cell cycle G1/S phase transition, the intrinsic apoptotic signaling pathway, histone modification, and DNA replication initiation ( Figure 7E). The KEGG analysis showed that the DEGs of MCF-7 were significantly related to protein processing in the endoplasmic reticulum, DNA replication, and the cell cycle ( Figure 7F). These results suggested that UBE2C may promote the development of pan-cancer through influencing the cell cycle.
Notably  [49][50][51][52][53][54][55][56]. Intriguingly, the GO analysis showed that the overlap genes were highly associated with the G1/S transition of the mitotic cell cycle, intrinsic apoptotic signaling pathway, and negative regulation of growth and DNA replication initiation ( Figure 7G), and the KEGG analysis showed that they were significantly related to cell cycle and protein processing in the endoplasmic reticulum ( Figure 7H). Moreover, the heat map showed that the DEGs in two cancer cell lines with interfering UBE2C was involved in the cell cycle ( Figure 7I), suggesting that UBE2C participating in the cell cycle may be a common regulatory mechanism of pan-cancer development. Therefore, we performed cell cycle experiments and found that the cell cycle was significantly blocked in the G1 phase after the knockdown of UBE2C, suggesting that UBE2C may affect the cell cycle by regulating the G1/S phase transition ( Figure S9).
To further validate how UBE2C affects the progress of cancer development through the cell cycle, the DEGs were obtained based on the 30% of high and low expression levels of UBE2C in 11 types of cancer tissues. A total of 1514 DEGs, which appeared in five types of cancer, were screened. A total of 596 DEGs was obtained from 1514 DEGs overlapped with the DEGs from the GSE173127 dataset. The GO analysis of these 596 DEGs revealed that they were mainly involved in organelle fission, DNA replication, and the cell cycle checkpoint ( Figure S10A-C), and the KEGG analysis showed that they were also involved in the cell cycle, DNA replication, and p53 signaling pathway ( Figure S10D). The PPI analysis results of the above 596 DEGs showed that UBE2C can form an interaction network with Tumor-Transforming Protein 1 (PTTG1), Polo Like Kinase 1 (PLK1), Cyclin Dependent Kinase 1 (CDK1), Cell Division Cycle 20 (CDC20), cyclin B2 (CCNB2), cyclin B1 (CCNB1), cyclin A2 (CCNA2), and Breast and Ovarian Cancer Susceptibility Protein 1 (BRCA1) ( Figure S10E). Genes regulated by UBE2C can also form an interaction network with TP53 ( Figure S10F). Furthermore, hub genes contained kinesin family members 11 and 20A (KIF11, KIF20A), BUB1 mitotic checkpoint serine/threonine kinase (BUB1), Aurora Kinase B (AURKB), CCNA2, CCNB2, CDC20, CDK1, topoisomerase (DNA) II α (TOP2A), and DLG associated protein 5 (DLGAP5) ( Figure S10G).

Discussion
Multiple studies have revealed that KAT2A was highly expressed in a variety of cancers compared with adjacent tissues, such as liver cancer [57], colon adenocarcinoma tissues [58], and non-small cell lung cancer tissues [16]. The downregulation of KAT2A can significantly reduce the proliferation and migration of cancer cells and the growth of xenograft tumors [57,59]. In this study, we found that KAT2A was generally highly expressed in seven cancer tissues compared with normal tissues, including BLCA, CHOL, ESCA, and HNSC, KIRP, STAD, and THCA. As a transcription factor, high E2F1 levels were commonly associated with aggressive cancer and poor patient prognosis for multiple cancer types. E2F1 transcription factor was a key regulator of genes required for cell cycle progression, cell proliferation, and differentiation [21,60], and played a key regulatory role in the invasion-metastasis cascade of certain cancer types [61,62]. Previous studies have revealed that E2F1 can induce cell metastasis by inducing chemoresistance, angiogenesis, secondary site extravasation, and EMT [63][64][65][66][67][68][69] This study found that E2F1 was significantly highly expressed in 20 cancers, and it was significantly related to the poor prognosis in patients of 9 different cancers.
By screening the transcriptome data of 11 cancer tissues of TCGA, it was exposed that the gene set potentially co-regulated by KAT2A and E2F1 was significantly enriched in the cell cycle, and participated in DNA replication, base excision repair, and nucleotide excision repair. It was suggested that the regulatory network of KAT2A and E2F1 involved in the cancer process was very complex, and it could regulate the expression level of genes involved in the cancer development process.
Previous studies have shown that KAT2A could increase the chromatin accessibility of E2F1, DNA Damage Inducible Transcript 3 (DDIT3), and other transcription factors, and form protein complexes with them, and then be recruited to the promoter regions of related genes, consequently enhancing their expression through increasing the acetylation level of H3K9 on these gene-promoting regions and regulating the development of cancer. In this study, the ChIP-qPCR and Co-IP results have demonstrated that KAT2A may interact with E2F1 and form a complex to bind the UBE2C promoter in MCF-7, 786-O, and NCI-H460 cells. The complex could increase the level of H3K9ac, thereby promoting the expression of UBE2C. Moreover, we uncovered that the E2F1 binding site region (−322/+39) of the UBE2C promoter was consistent with the results of a previous study [70].
This study explored the regulatory relationship between KAT2A/E2F1 and UBE2C, and found that KAT2A can regulate the expression of UBE2C through interacting with E2F1. KAT2A can also affect the expression of E2F1, which is in agreement with previous studies [16,71]. E2F1 can also regulate the expression of KAT2A, but whether it affects the expression of KAT2A through the E2F1 binding site on the KAT2A promoter or other mechanisms still needs further in-depth research. Interestingly, the downregulation of UBE2C can inhibit KAT2A protein expression levels, indicating that UBE2C can regulate the expression of KAT2A and form feedback regulation. Previous studies have reported that the SCF-Cyclin F ubiquitin ligase complex participates in the ubiquitination and degradation of E2F1 [72], but whether its degradation required the participation of UBE2C has not been reported. If it was required for the involvement of UBE2C, it can be explained that the E2F1 protein was significantly upregulated in 786-O and NCI-H460 after the knockdown of UBE2C for the reason that the degradation of the E2F1 protein was reduced after the knockdown of UBE2C. However, what role these up-regulated E2F1 proteins may play need further research.
Given the clinical and functional significance of KAT2A/E2F1/UBE2C in pan-cancer, we concluded that KAT2A/E2F1/UBE2C and its associated pathway were crucial for cancer carcinogenesis, and targeting this pathway may be pivotal in the prevention or treatment of pan-cancer.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes13101817/s1, Figure S1. Potential target genes co-regulated by KAT2A and E2F1; Figure S2. GO and KEGG enrichments of KAT2A and E2F1 target genes; Figure S3. KAT2A and E2F1 regulate the transcription of UBE2C through E2F1 binding site; Figure S4. The expression level of UBE2C in different cell types in cancer tissues; Figure S5. Interference with UBE2C significantly inhibits cancer cell proliferation; Figure S6. Interference with UBE2C significantly inhibited cancer cell migration; Figure S7. Interfering UBE2C could remarkably repress cancer cell invasion; Figure S8. Interference with UBE2C significantly promoted cancer cell apoptosis; Figure S9. Interference with UBE2C significantly induced cell cycle arrest; Figure S10. Top 15 of GO terms, top 12 of KEGG signaling pathways and PPI network of 596 DEGs that are regulated by UBE2C; Table S1. Summary of cancer and normal samples analyzed in this study; Table S2. Primers and interfering sequence for target genes used in this study; Table S3. A total of 222 DEGs regulated by KAT2A and E2F1 appeared in more than five cancer types among 11 different types of cancer at the same time.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data analyzed during the present study are available from the corresponding author upon reasonable request.