WHSC1 is involved in DNA damage, cellular senescence and immune response in hepatocellular carcinoma progression

Abstract Wolf–Hirschhorn syndrome candidate 1 (WHSC1) is a transcriptional regulatory protein that encodes a histone methyltransferase to control H3K36me2 modification. WHSC1 was upregulated and associated with poor prognosis in HCC. The elevated WHSC1 likely due to the alterations of DNA methylation or RNA modification. WHSC1 perhaps form a chromatin cross talk with H3K27me3 and DNA methylation to regulate transcription factors expression in HCC. Functional analysis indicated that WHSC1 was involved in DNA damage repair, cell cycle, cellular senescence and immune regulations. Furthermore, WHSC1 was associated with the infiltrating levels of B cell, CD4+, Tregs and macrophage cells. Therefore, our findings suggested that WHSC1 might function as a promotor regulator to affect the development and progression of HCC. Thus, WHSC1 could be a potential biomarker in predicting the prognosis and therapeutic target for patients with HCC.

WHSC1 perhaps form a chromatin cross talk with H3K27me3 and DNA methylation to regulate transcription factors expression in HCC. Functional analysis indicated that WHSC1 was involved in DNA damage repair, cell cycle, cellular senescence and immune regulations. Furthermore, WHSC1 was associated with the infiltrating levels of B cell, CD4+, Tregs and macrophage cells. Therefore, our findings suggested that WHSC1 might function as a promotor regulator to affect the development and progression of HCC. Thus, WHSC1 could be a potential biomarker in predicting the prognosis and therapeutic target for patients with HCC. domain-containing histone methyltransferase, which could catalyse the di-methylation of H3K36 to involve in gene transcriptional regulation. 4 However, further study is necessary for the function and carcinogenic mechanism of WHSC1 in HCC. To fully assess the role of WHSC1 in HCC, we comprehensively investigated the gene expression, prognosis, epigenetic regulation and functions of WHSC1 in HCC.

| Gene expression and survival analysis
The mRNA and protein level of WHSC1 was confirmed based on UALCAN-LIHC and HPA data. 5 Kaplan-Meier survival analysis was used to assess the prognosis of WHSC1 in HCC.

| The functional enrichment analysis of WHSC1
The GSEA was used to evaluate the KEGG and GO enrichment of WHSC1. The proteins interaction networks of WHSC1-related genes were constructed using the Cytoscape base on the STRING data. 6

| Cell function assays
The assay was performed in accordance with the manufacturer's protocol. The proliferation of HCC cell was evaluated using the CCK-8 assay (Mei5bio, China). The cell cycle was detected using flow cytometer.

| Immune infiltration analysis
The TIMER, CAMOIP and GSCA were used to calculate the abundance of tumour infiltrating immune cells in tumour tissues of LIHC. 7

| Statistical analysis
All the experiment was independently repeated three times. Data were summarized as the mean ± SD. The results were considered to be statistically significant when the value of p was <0.05.

| Relationships between the clinicopathological and prognostic features of WHSC1 in HCC
In order to evaluate the role of WHSC1 in cancers, we assessed the mRNA level of WHSC1 and found it showed lowest level in liver tissues ( Figure S1). However, it was abnormally upregulated in tumour tissues with the highest level at the stage III of HCC ( Figure 1A,B).
The immunohistochemistry results indicated that protein level of WHSC1 was also increased ( Figure 1C), and the elevated WHSC1 was significantly associated with poor prognosis in HCC ( Figure 1D,E).

| WHSC1 was involved in DNA damage, cell cycle, cellular senescence in HCC
The functional enrichment analysis was performed based on GSEA assay. We found that WHSC1 was significantly enriched in mismatch repair, cell cycle and DNA replication pathways ( Figure 1F). Furthermore, we detected the expression of WHSC1 in cell cycle. The protein interaction network of WHSC1 associated genes in DNA damage, mismatch repair, cell cycle and cellular senescence pathways. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The p < 0.05 is considered statistically significant.
Its expression was increased in the S and G2 stage ( Figure 1G,H).
To confirm function of WHSC1, the shRNA was transfected to downregulate WHSC1 level in HCC. The CCK-8 assay showed that cell proliferation and cycle was abnormal arrested ( Figure 1I-L).
Moreover, the yH2AX, a biomarker of DSB, was increased in downregulated WHSC1 HepG2 cell ( Figure 2N). Therefore, WHSC1 was involved in cell cycle and DNA damage in HCC.
To further confirm role of WHSC1, we obtained all the WHSC1associated genes in LIHC based on RNA-seq results. KEGG analysis indicated that these genes were involved in cell cycle, DNA replication, mismatch repair and cellular senescence ( Figure 1N).

| Relationship between WHSC1 and tumour immune microenvironment
We further analysed the relationships of WHSC1 and MSI or stemness. The strong correlations were found between WHSC1 expression and MSI or stemness ( Figure S4A,B). Moreover, stemnessrelated genes were associated with PD-L1 pathways. Therefore, we evaluated the function of WHSC1 in immune response. GO analysis showed that WHSC1 was mainly involved in several immune pathways (Table S1). WHSC1 expression was significantly positively associated with macrophages, monocyte, CD4 + T, Treg, neutrophil and dendritic cells infiltration (Figure 2A). It was increased in wound healing and inflammatory immune subtypes ( Figure 2B). Moreover, the prognosis of higher level WHSC1 with high immune cells infiltration, including MDSC, monocyte, macrophage was significantly worse than that in low-infiltrating groups ( Figure 2C

| DISCUSS ION
It is well known that DNA damage repair pathways is correlated with variability in HCC clinical outcomes. WHSC1 specifically catalyses H3K36me2, which will result in conformation of chromatin, transcriptional regulation to control cell growth, DNA damage and apoptosis in several cancers. 4 It has been reported that WHSC1-mediated di-methylation of PTEN at K349 is recognized by the tudor domain of 53BP1 to recruit PTEN to DNA damage sites and result in dephosphorylation of γH2AX for governing efficient repair of DSBs. 8 Recently, studies have indicated that WHSC1 is involved in antitumor immunity. 9,10 The down-regulated WHSC1 expression will diminish MHC-I levels, impair antitumor immunity and blunt the effect of immune checkpoint blockade. 9 WHSC1 directly interact with NLRC5 to promote MHC-I gene expression and results in impairing the IFNγ-stimulated antitumor immunity. 10 It also acts as a critical epigenetic regulator for follicular helper T cell differentiation and Treg recruitment. 11,12 Compelling evidence has shown that DNA damage response deficiency is associated with activation of anticancer immunity.
The alterations of DDR genes can serve as biomarkers for the selection of suitable patients to receive specific therapeutics. 13 It has been reported that cGAS-cGAMP-STING pathway connects DNA damage to inflammation and cellular senescence. 14 Our results indicated that WHSC1 perhaps serve as a link from DNA damage to cellular senescence and immune response. In mechanism, WHSC1 likely results in H3K36me2 modification to combined with PRC2related H3K27me3, or DNMTs-related DNA methylation to affect the transcriptional regulation of DNA damage, cellular senescence or immunity-related genes expression in HCC. (equal); methodology (equal); resources (equal); software (equal); supervision (equal). ke xin Li: Data curation (equal); formal analysis (equal); resources (equal); software (equal). zhi min Zhao: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); visualization (equal). yu min Gao: Data curation (lead); project administration (equal); supervision (equal); validation (equal). xiu ling Deng: Formal analysis (supporting); methodology (supporting); software (equal); supervision (supporting). chang shan wang:

ACK N O WLE D G E M ENTS
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declared that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used in this article are publicly available as described in Materials and Methods. All data generated during this study are included in this published article and its supplementary information files. The data available in TCGA (https://portal.gdc.cancer.gov/) databases. The datasets analysed during the current study are available from the corresponding author on reasonable request.