Introduction
Oral squamous cell carcinoma (OSCC) is a highly prevalent head and neck malignancy worldwide [
1,
2]. Tobacco smoking, alcohol consumption, HPV infection and genetic factors, etc. are risk factors for OSCC [
1,
3]. The five-year survival for patients with Tumor-Node-Metastasis (TNM) stage I/II OSCC is ~ 90%, whereas the five-year survival for patients with TNM stage III/IV OSCC is only ~ 30% [
4,
5. Most OSCC patients (> 66%) are diagnosed at TNM stage III/IV [
6]. Cervical lymph nodes and distant metastases are the major causes of poor prognosis in advanced OSCC [
3,
7]. Patients with OSCC are often treated with a multidisciplinary approach, including surgery, chemotherapy, biotherapy and radiotherapy [
6]. Despite recent advances in the diagnosis and treatment of OSCC, the overall survival of OSCC still has not improved significantly. Therefore, the development of effective treatment options for OSCC is urgently needed.
DNA damage leads to apoptosis, autophagy, senescence and other responses [
8,
9]. DNA repair pathways include five main types, including base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining [
10]. Abnormal DNA damage repair capacity of tumor cells contributes the maintenance of malignant phenotype, and the acquisition of resistance to radiotherapy and chemotherapy [
11]. Therefore, blocking DNA repair pathway is an important approach for tumor treatment. For instance, cisplatin, the first-line chemotherapeutic drug for OSCC, induces DNA damage through the formation of cisplatin-DNA adducts, leading to cell cycle arrest and apoptosis [
12]. Nucleotide excision repair is a key pathway to resist damage caused by cisplatin [
13]. ERCC excision repair 1, endonuclease non-catalytic subunit (ERCC1) is an important factor of nucleotide excision repair pathway [
13]. It was found that high ERCC1 expression is associated with cisplatin resistance and poor prognosis in head and neck squamous carcinoma [
14]. DNA double-strand breaks are a serious form of DNA damage [
15]. γ-H2AX marking is observed at DNA double-strand break sites, and the level of γ-H2AX is positively correlated with the degree of DNA damage [
16]. γ-H2AX recruits DNA repair-related proteins, particularly BRCA1 DNA repair associated (BRCA1) and RAD51 recombinase (RAD51), to activate the homologous recombination repair pathway [
16]. Wang et al. found that palbociclib inhibits DNA damage repair in OSCC cells by suppressing RAD51 expression [
17]. Despite the progress made in the study of DNA repair in OSCC, the mechanisms of DNA repair in OSCC cells and their relationship with the malignant phenotype remain to be clarified. Exploring the mechanisms of DNA repair in OSCC cells may provide guidance to improve the effectiveness of OSCC treatment.
Enhancers are typically 200–1500 bp in size which bind to transcription factors and cofactors to
cis-regulate the expression of target genes [
18]. Enhancers can be located at the 3’-end, 5’-end and intron of target genes. In addition, enhancers can remotely regulate the expression of target genes. Enhancer activation is characterized by high levels of acetylation of histone 3 at lysine 27 (H3K27ac) and histone 3 lysine 4 monomethylation (H3K4me1), and is cell-specific [
18,
19]. In contrast, poised state enhancers repress target gene transcription and generally characterized by both H3K4me1 and histone 3 lysine 27 trimethylation (H3K27me3) histone tags [
20‐
23]. Therefore, H3K27ac is defined as a specific enhancer activation signal. Enhancers are closely associated with the development of many cancers, including OSCC [
24]. To our best knowledge, the mechanisms by which enhancers regulate genes related to DNA repair in OSCC are still incompletely understood.
The overall aim of the current study was to uncover the key enhancer drivers affecting DNA repair in OSCC cells. We analyzed the relationship between DNA repair-related genes and clinical features. Subsequently, DNA repair-related genes regulated by metastasis-specific enhancers as well as their key transcription factor with prognostic value were screened. Finally, the function of the key enhancer driver was explored in vitro. This study is expected to identify novel DNA repair blocking targets to support the advancement of OSCC clinical treatment efficiency.
Methods
Data collection
Gene expression and the corresponding clinical data of 28 metastatic OSCC (metastatic recurrence occurred within 5 years following surgery) and 47 non-metastatic OSCC (without metastasis within 5 years following surgery) patients were downloaded from The Cancer Genome Atlas database (TCGA,
https://portal.gdc.cancer.gov/). The “edgeR” package in R software was employed to normalize the gene expression data. H3K27ac ChIP-seq data of GSE120634 cohort were downloaded from the Gene Expression Omnibus database (GEO,
http://www.ncbi.nlm.nih.gov/geo/). Immunohistochemical data of OSCC and normal control tissues were downloaded from The Human Protein Atlas database (
https://www.proteinatlas.org/).
Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA)
GSEA (
http://www.broadinstitute.org/gsea/index.jsp) was performed to unearth the underlying relationship among DNA repair and metastatic phenotype of OSCC using data downloaded from TCGA. Six DNA repair-related gene sets (“DNA repair”, “base excision repair”, “nucleotide excision repair”, “mismatch repair”, “homologous recombination” and “non-homologous end-joining”) were obtained from the Molecular Signatures Database (MSigDB,
https://www.gsea-msigdb.org/gsea/msigdb/index.jsp). Normalized (NOM) P < 0.05, normalized enrichment score (NES) ≥ 1 and false discovery rate (FDR) q-value ≤ 0.25 was considered as screening criteria for significant enrichment.
Hierarchical clustering was applied to cluster OSCC samples from TCGA based on the expression of DNA repair-related genes. Hierarchical clustering was shown with a dendrograms in heatmaps. DNA repair pathway enrichment scores for each cluster were calculated using “GSVA” package in R software. Comparisons of enrichment scores among clusters were performed using one-way analysis of variance (ANOVA) followed by Turky’s test.
Analysis of differentially expressed genes
DNA repair-related genes in cluster 2 and cluster 3 were analyzed for differential expressed based on TCGA-OSCC data. Differential gene expression analysis was performed using the “limma” package in R software. Genes with |log2 fold change| ≥1.0 and Benjamini-Hochberg adjusted P < 0.05 were retained as the differentially expressed genes.
Prognostic analysis of different clusters and transcription factors
Prognostic analysis was performed by Kaplan-Meier plots and log-rank tests using data downloaded from TCGA. Disease-free survival (DFS) analysis was performed in each cluster using the “survival” R package. Transcription factors were predicted using The Toolkit for Cistrome Data Browser (
http://dbtoolkit.cistrome.org/). Transcripts per million (TPM) < 1 was used to reject transcription factors which were low expressed in OSCC. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using the “survival” R package. OSCC patients were divided into high and low expression groups based on the quartiles. Overall survival was evaluated using the “survival” R package.
Enhancer identification
H3K27ac ChIP-seq data of HN120Pri and HN120Met cells from GSE120634 data set were analyzed for enhancer identification using “findPeaks” tool in HOMER software. Integrative Genomics Viewer (
https://igv.org) was applied to visualize H3K27ac peaks. In this study, the gene closest to an enhancer locus on the genome was identified as an enhancer-controlled gene. The “annotatePeaks” tool in HOMER software was conducted to screen the enhancer-controlled genes.
Cell lines and cell culture
BHY and HSC3 were two human metastatic OSCC cell lines, which were purchased from the Japanese Collection of Research Bioresources (JCRB). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) at 37 °C and 5% CO2.
Cell transfection
SiRNA specifically targeting high mobility group 20 A (HMG20A) (si-HMG20A) and the control si-RNA (si-NC) were synthesized by Gene Pharma (Shanghai, China). BHY and HSC3 cells were seeded into 6-well plates at the density of 1 × 10
5 cells per well, and transfected with si-HMG20A or si-NC using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s instructions. The transfection concentration of si-RNAs was 50 nM/1 × 10
5 cells. The sequence of si-RNAs were as follows: si-HMG20A, 5′-AGGCAAAUCUCAUAGGCAA-3′ [
25; si-NC, 5′-GCACAAGCUGGAGUACAACUACATT-3′. The transfected cells were processed for subsequent studies 48 h after transfection.
Patient collection
A total of 72 patients with pathologically confirmed OSCC at the Fourth Affiliated Hospital of Hebei Medical University were collected in this study (January 2015 to December 2016). These OSCC patients were further divided into non-metastatic OSCC patients (n = 40) and metastatic OSCC patients (n = 32) according to whether metastasis occurred within 5 years after surgery. All included patients had not undergone any cancer-related treatment prior to surgery. OSCC tissue and adjacent control tissue samples from all participants were preserved in the form of frozen specimens.
This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Hebei Medical University (Shijiazhuang, China). All participants were provided with written informed consent prior to the start of the study.
qRT-PCR
Total RNA was extracted from cells and tissues using TRIzol reagent (Invitrogen, USA). 1 µg of total RNA was used to synthesize cDNA by High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA). qRT-PCR was conducted using the Power SYBRs Green PCR Master Mix (Thermo Fisher, USA), and executed by ABI 7900 real-time PCR system (Applied Biosystems, USA). The 2−ΔΔCt method was applied to determine the relative expression levels with GAPDH as the endogenous control. Primers used in qRT-PCR as following: ADRM1 26S proteasome ubiquitin receptor (ADRM1)-F: 5’-GGCGGGAAAGATGTCCCTG-3’; ADRM1-R: 5’-GTCGTCCGTCTGCTGAATGT-3’. Solute carrier family 12 member 7 (SLC12A7)-F: 5’-CTGGCGGGTCCTACTACATGA-3’; SLC12A7-R: 5’-AAAATCTCGATGGTCCCCAAAAT-3’. HMG20A-F: 5’-ATGACTAGCTCCACCCTACCG-3’; HMG20A-R: 5’- CTCGTTTACTTCGTTGCTCATCT-3’. GAPDH-F: 5’-GGAGCGAGATCCCTCCAAAAT-3’; GAPDH-R: 5’-GGCTGTTGTCATACTTCTCATGG-3’.
Chromatin immunoprecipitation (ChIP)-qPCR
HSC3 and BHY cells transfected with si-NC or si-HMG20A were cross-linked by 1% formaldehyde for 10 min at room temperature. The cross-linked cells were lysed with cell lysis solution for 20 min, and then sonicated using Covaris E220 (Woburn, USA) followed by immunoprecipitation with anti-H3K27ac (ab4729, Abcam, USA) at 4°C overnight. Gel Extraction Kit (Omega Bio-Tek, USA) was used to purify DNA. Finally, the purified products were subjected to qRT-PCR. The primers of enhancer regions of ADRM1 and SLC12A7 as following: ADRM1-E1-F: 5’- CCTCACAGCACAAGCTCAGA-3’; ADRM1-E1-R: 5’- ACCTTAATGGCTGCAGGACC-3’. SLC12A7-E1-F: 5’- CGGATGGAAGGGCCTAAGAG-3’; SLC12A7-E1-R: 5’- CCATCCTGGCTCCAAATCCC-3’.
Western blotting
Total protein of BHY and HSC3 cells was extracted using RIPA buffer (Sigma-Aldrich, USA). Protein concentration was measured using the Pierce BCA protein assay (Thermo Fisher, USA). Proteins were subjected to SDS-PAGE for separation, followed by transfer to PVDF membranes (Millipore, USA). After immersion in 5% nonfat milk for 1 h, the membranes were cut according to the molecular weight of the protein and incubated with primary antibodies, anti-HMG20A (1:2000, 12085-2-AP, Proteintech, USA), anti-β-actin (1:2000, ab8226, Abcam, USA), anti-ERCC1 (1:2000, ab129267, Abcam, USA) and anti-γ-H2AX (1:2000, ab81299, Abcam, USA), overnight at 4 °C, followed by incubating with goat anti-rabbit IgG H&L (1:5000, ab96899, Abcam, USA) at room temperature for 1 h. Protein bands were detected using an enhanced chemiluminescence system (Thermo Fisher, USA). The grey scale of protein bands was analyzed using Image J software (National Institutes of Health, Bethesda, USA).
Cell counting Kit-8 (CCK8) assay
For drug cytotoxicity assay, BHY and HSC3 cells transfected with si-NC or si-HMG20A were seeded into 96-well plates with 1, 2, 4, 8, 16 and 32 µM cisplatin treatment for 48 h. Then, 10 µl of the CCK8 solution (Solarbio, China) was added into each well and maintained at 37 °C for 2 h. Absorbance was measured at 450 nm by a microplate reader (Thermo Fisher, USA), and then the half-maximal inhibitory concentration (IC50) to cisplatin was calculated. For cell proliferation assay, 10 µl of the CCK8 solution was added into each well at 0, 24, 48 and 72 h. Absorbance at each time point were measured at 450 nm by a microplate reader (Thermo Fisher, USA).
Transwell assay
Matrigel chambers (BD Biosciences, USA) were constructed according to manufacturer’s instructions. BHY and HSC3 cells transfected with si-NC or si-HMG20A were cultured in serum-free DMEM medium with 5 µM cisplatin, and then shifted to the upper Matrigel chambers (50 µL). Lower chambers were supplemented with DMEM containing 1% fetal bovine serum and 5 µM cisplatin (600 µL). After 48 h incubation at 37 °C, the cells on the upper surface of the membrane were removed, while the invaded cells on the lower surface were stained with 0.1% crystal violet for 20 min.
Statistical analysis
All experiments were performed at least three replications. Data was presented as mean ± standard deviation. Statistical data were analyzed using GraphPad Prism 9.1.0. Student’s t-test was employed to compare two different groups. ANOVA followed by Turky’s test was employed to evaluate difference among multiple groups. The cutoff of statistical significance was P < 0.05.
Discussion
Factors contributing to DNA damage can be divided into two categories, endogenous factors and exogenous factors [
25]. Endogenous factors refer to DNA damage caused by by-products of cellular metabolism (e.g. reactive oxygen species) and factors such as base mismatches, insertions or deletion during DNA replication [
25]. Exogenous factors mainly include the ultraviolet light, ionizing radiation, chemotherapeutic drugs, etc. [
25]. DNA repair is essential to maintain cellular homeostasis. Many anti-cancer drugs achieve the purpose of treatment by inducing DNA damage of tumor cells. However, tumor cells can activate DNA repair mechanism to repair the damage, resulting in drug resistant [
11,
26]. Blocking DNA repair pathways is crucial to improve the therapeutic efficiency of OSCC. Elucidating the molecular regulatory mechanisms of DNA repair has implications for improving the efficacy of tumor chemotherapy. In this study, we analyzed the relationship between the expression of DNA repair-related genes and metastasis, prognosis, staging, differentiation, and risk factors (alcohol consumption and smoking) of OSCC. Metastatic-specific enhancer-regulated DNA repair-related genes were screened, and transcription factors of these genes were predicted. Furthermore, the effects of the key transcription factor on metastatic-specific enhancers-controlled target genes were verified, and the impacts of the key enhancer driver on DNA repair, proliferation and invasion of OSCC cells under cisplatin treatment were explored in vitro.
The main DNA repair pathways triggered by DNA damage include base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining [
10]. It is well known that metastasis is one of the major reasons for the poor prognosis of OSCC [
3,
7]. Therefore, we analyzed the enrichment of metastatic OSCC and non-metastatic OSCC in the above five DNA repair pathways using GSEA, and found that all DNA repair pathways were positively correlated with the metastatic phenotype of OSCC, although the correlation between non-homologous end-joining and metastatic phenotype was not significant. OSCC patients were clustered according to the expression of DNA repair-related genes resulted in 4 clusters (C1, C2, C3 and C4). Since the sample size of C1 and C4 was small, we focused on the two clusters with a large sample size, C2 (cluster with high expression of DNA repair-related genes) and C3 (cluster with low expression of DNA repair-related genes). Base excision repair score, nucleotide excision repair score, mismatch repair score, homologous recombination score and non-homologous end-joining score of C2 were higher than those of C3. DFS of patients in C2 was significantly worse than that in C3. Tumor stage and differentiation are important prognostic factors [
27,
28]. We analyzed the distribution of tumor stage and differentiation of the four clusters, and found that the proportions of patients with advanced stage and low-differentiation were higher in C2 than in C3. In addition, we also considered two important risk factors, alcohol consumption and smoking [
1,
3]. The results showed that the proportion of patients with alcohol consumption and smoking was higher in C2 than in C3. These results suggested that alcohol consumption and smoking may affect DNA repair of OSCC cells, and abnormal DNA repair of OSCC cells was closely related to metastasis, tumor stage, differentiation, and adversely affects the prognosis of OSCC patients.
To further investigate the regulatory mechanisms of DNA repair, we identified differentially expressed DNA repair-related genes between C2 and C3. A total of 390 differentially expressed genes were screened, of which 283 were upregulated in C2. Aberrant regulation of gene expression by enhancers is a key regulatory mechanism in cancer progression. Enhancers are DNA sequences that regulate gene expression, and contain sequence-specific transcription factor recognition and binding sites, which bind to transcription factors and initiate transcription of target genes [
18]. In recent years, the mechanism of action of enhancers has been continuously explored. Active enhancers are located in open chromatin regions, with the high degree of H3K27ac modification being one of their distinguishing features [
29,
30]. In the present study, we identified enhancer-controlled upregulated DNA repair-related genes in primary OSCC cells and metastatic OSCC cells. A total of 17 metastatic-specific enhancer-controlled upregulated DNA repair-related genes were screened out. Function of enhancers in promoting the expression of target genes is dependent on the binding of transcription factors [
18]. To explore the regulatory mechanisms of the 17 metastatic-specific enhancer-controlled upregulated genes, we predicted their transcription factors, and analyzed the impacts of transcription factors on the overall survival of OSCC patients. We found that only the expression of HMG20A had a significant impact on the overall survival of OSCC patients, exhibiting that high expression of HMG20A corresponded with a poor overall survival. The expression of HMG20A was significantly higher in OSCC tissues than in normal control tissues. In addition, HMG20A expression was upregulated in metastatic OSCC tissues compared to non-metastatic OSCC tissues.
High mobility group 20 A (HMG20A), also known as HMGX1 or HMGXB1, maps to chromosome 15q24 and is homologous to HMG20B [
31,
32]. HMG20B is the core subunit of the Lys-specific demethylase 1/REST co-repressor 1 (LSD1-CoREST) histone demethylase complex, and HMG20A can function in place of HMG20B [
32]. It has been reported that HMG20A has important biological functions such as promoting functional maturation of pancreatic β-cells, promoting neuronal differentiation, regulating inflammatory responses and epithelial mesenchymal transition [
32‐
34]. A prognostic model of SUMOylation-regulated genes involving HMG20A could predict the prognosis of OSCC [
35]. However, the role and mechanism by which HMG20A regulates OSCC remains largely unknown. In this study, we selected two genes, ADRM1 and SLC12A7, which were the top two upregulated genes in C2, for validation. ADRM1 is involved in proteasome composition and acts as a ubiquitin receptor to recruit deubiquitinating enzymes [
36,
37]. Aberrant expression of ADRM1 is associated with a variety of cancers [
38‐
41]. SLC12A7, also known as KCC4, is involved in cell volume homeostasis, inorganic ion homeostasis and transmembrane transport [
42‐
44]. In the present study, we found the presence of metastatic-specific enhancer regions of ADRM1 and SLC12A7 locus in metastatic OSCC cells, which were absent in primary OSCC cells. Furthermore, we verified the binding of HMG20A to metastatic-specific enhancers of ADRM1 and SLC12A7 by ChIP-qPCR. Knockdown of HMG20A inhibited the expression of ADRM1 and SLC12A7 in metastatic OSCC cells.
Cisplatin-based chemotherapy is the first-line chemotherapy agent for OSCC [
12]. Cisplatin covalently binds to the N7 position of the purine base of DNA, forming adducts such as intra-strand cross links, which trigger cytotoxicity [
45]. In the present study, we found that cisplatin sensitivity of metastatic OSCC cells was enhanced after knockdown of HMG20A. Excision of cisplatin-DNA adducts by the nucleotide excision repair pathway is the primary method of cellular repair of DNA damage caused by cisplatin [
13]. ERCC1 is an essential factor in nucleotide excision repair pathway [
13]. Expression of ERCC1 is associated with chemoresistance in many cancers [
46,
47]. In the present study, we demonstrated that HMG20A knockdown inhibited ERCC1 protein expression in BHY and HSC3 cells under cisplatin treatment. In addition, DNA damage caused by cisplatin can be repaired by homologous recombination and non-homologous end-joining, among which homologous recombination repair has a more stringent repair mechanism, thus ensuring a high degree of accuracy [
48‐
50]. When a DNA double strand break occurs, γ-H2AX is enriched at the break sites [
16]. Subsequently, RAD51 was recruited to the γ-H2AX-labeled fracture site mediated by BRCA1 [
51]. RAD51 searches for homologous DNA sequences along the sister chromatids, mediates the linking of sister chromatids by single-stranded nucleotides at the 3’ end, and then DNA polymerase resynthesizes the excised nucleotide sequence using the sister chromatids as a template [
51]. In the present study, we found that knockdown of HMG20A resulted in upregulation of γ-H2AX. These results suggested that HMG20A expression facilitates OSCC cells to resist DNA damage caused by cisplatin. Furthermore, we demonstrated that knockdown of HMG20A inhibited the proliferation and invasion of OSCC cells under cisplatin treatment. The detailed mechanism by which HMG20A regulates DNA repair in OSCC cells will be explored in further study.
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