HMGN1 is a potential chemotherapeutic target for lung adenocarcinoma.

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Xianli Wu, Geqi Cai, Jing Feng, Wenchu Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3669863/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 May, 2024 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract The high mobility group nucleosome binding (HMGN) family, constitutes a large family of non-histone protein family known to bind the acidic patch of the nucleosomes with various key cellular functions. Several studies have highlighted the pivotal roles of HMGNs in the pathogenic process of various cancer types. However, the roles of HMGN family in lung adenocarcinoma (LUAD) have not been fully elucidated. Herein, integrative analyses of multiple-omics data revealed that HMGNs frequently exhibit dysregulation in LUAD. Subsequent analysis of the clinical relevance of HMGN1 demonstrated its association with poor prognosis in LUAD and its potential as a diagnostic marker to differentiate LUAD from heathy controls. Additionally, functional enrichment analysis suggested that HMGN1 was mainly involved in DNA repair. To corroborate these findings, cellular experiments were conducted, confirming HMGN1’s crucial involvement in homologous recombination repair and its potential to enhance the sensitivity of LUAD cells to standard chemotherapeutic drugs. This study proposes HMGN1 as a novel prognostic biomarker and a promising target for chemotherapy in lung adenocarcinoma. Biological sciences/Cancer/Lung cancer Biological sciences/Cancer/Tumour biomarkers Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Molecular biology Health sciences/Biomarkers Health sciences/Molecular medicine Health sciences/Oncology LUAD HMGN1 biomarker DNA repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lung cancer, recognized as the leading cause of cancer-related deaths globally [ 1 ], is primarily categorized into non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC), with NSCLC further subdivided into lung adenocarcinoma (LUAD), squamous cell carcinoma, and large cell carcinoma based on pathological classification [ 2 ]. Notably, LUAD is the most prevalent subtype, accounting for 40% of all new lung cancer cases diagnosed annually worldwide. Despite considerable advancements in therapeutic strategies for LUAD in recent decades, the long-term prognosis for patients remains a significant clinical challenge [ 3 ]. Consequently, the development of early diagnostic methods and the implementation of effective targeted therapies are imperative for this devastating disease. DNA, the carrier of storing and transmitting genetic information, is organized into chromatin in eukaryotic cells through stably interactions with histone and non-histone proteins. The chromatin structure is a dynamic entity, continuously modified by nuclear factors, which compact the genome for efficient accommodation within the nucleus [ 4 ]. Critical DNA-dependent events, including transcription, replication, and DNA repair, are intricately linked to the chromatin, necessitating precise spatial and temporal regulation of its structure [ 5 ]. High mobility group N (HMGN), a group of chromatin-binding architectural proteins, binds specifically to nucleosomes without preference for any DNA sequence [ 6 ]. The association of HMGN with nucleosomes impedes histone H1 binding, thereby influencing chromatin accessibility and remodeling [ 7 ]. To date, five HMGN family members have been discovered in human genome. All of them possess a positively charged nucleosome binding domain, a bipartite nuclear localization signal (NLS), and an acidic C-terminal chromatin regulatory domain [ 8 ]. HMGN1 and HMGN2 are ubiquitously expressed in mammalian cells, while HMGN3, 4, and 5 have more restricted expression patterns [ 7 ]. The interaction of HMGN proteins with nucleosomes destabilizes the high-order chromatin structure, modulates the pattern of histone post-translational modification, and enhances the accessibility of non-histone proteins such as transcription factors, thereby significantly influencing cellular phenotypes [ 9 ]. Increasing evidence has demonstrated that HMGN genes are implicated in development, immunological processes, and the etiology of disease, including cancer [ 10 ]. For example, appropriate expression levels of HMGN family proteins are crucial in both maintenance of the pluripotent identity of stem cells and facilitating cellular differentiation during embryogenesis [ 11 , 12 ]. HMGN1, acting as an alarmin, fosters antitumor immunity by inducing maturation in human dendritic cells via Toll-like receptor 4 (TLR-4) [ 13 ]. Additionally, high HMGN1 levels correlate with increased peri-tumor infiltration of lymphocytes in Her2-positive breast cancer [ 14 ]. Moreover, recent studies in head and neck carcinoma have linked elevated cytoplasmic HMGN1 levels with increasing tumor-infiltrating lymphocytes [ 15 ], indicating potential immunotherapeutic applications. HMGN2 promotes breast cancer progression by facilitating STAT5 access to the promoter region of its targets [ 16 ]. Elevated HMGN4 expression is associated with high grade tumors and poor outcomes in hepatocellular carcinoma [ 17 ]. Despite growing evidence of aberrant HMGN family gene expression across multiple tumor lineages, their specific roles in lung adenocarcinoma, the predominant type of lung cancer, remain largely unexplored. Given the crucial role of HMGN proteins in chromatin de-condensation and posttranslational modifications in histone tails, which are integral to DNA damage repair, it is plausible that HMGN family protein may be involved in DNA damage response. Indeed, studies show that HMGN1 −/− mice and cells are hypersensitive to UV radiation due to impaired accessibility to UV-damaged sites in chromatin [ 18 ]. Loss of HMGN1 also impairs ionizing radiation (IR)-induced ATM auto-phosphorylation and the activation of several ATM targets in mouse embryonic fibroblasts [ 19 ]. However, the mechanisms by which HMGN proteins modulate DNA repair processes are yet to be fully understood, warranting systematic analysis and exploration. Prior research has indicated aberrant expressions of HMGNs and their prognostic value in some members of HMGN family [ 20 ]. Nevertheless, the role of distinct HMGN family members in the development and progression of lung adenocarcinoma have not been clearly defined. Through comprehensive analyses of multi-level omics data, this study investigated the expression and prognostic significance of HMGNs in lung adenocarcinoma. We discovered that HMGN1 was upregulated in LUAD, and its expression was associated with clinical outcomes. Function enrichment analysis further revealed a strong association between HMGN1 expression six DNA damage repair pathways. Finally, the roles of HMGN1 in the ATR-ChK1 and homologous recombination repair pathways was manifested in vitro in lung adenocarcinoma cells. Materials and methods 1. Data collection 1.1 LUAD cohor Clinical information and RNA-seq data of LUAD patients were sourced from the TCGA database [ 21 ] ( https://portal.gdc.cancer.gov ). Additionally, gene chips numbered GSE11969, GSE10072, GSE13213, GSE19804, and GSE116959 were selected from the GEO database ( http://www.ncbi.nlm.nih.gov/geo/ ) [ 22 ]. Transcriptome data of the 75 LUAD cell lines were obtained from the CCLE database [ 23 ]. 1.2. Human protein atlas Human Protein Atlas database ( http://www.proteinatlas.org/ ) were utilized to analyze HMGNs expression in LUAD tissues compared to adjacent normal tissues at the protein level [ 24 ]. 1.3. Ualcan dataset Ualcan dataset ( http://ualcan.path.uab.edu ) was employed to examine the protein expression of HMGNs in LUAD tissues versus normal tissues from Clinical Proteomic Tumor Analysis Consortium (CPTAC). Additionally, this dataset provided DNA promoter methylation profiles of HMGN family in LUAD from TCGA [ 25 ]. 1.4. cBioportal Mutation profiles of HMGN family genes were analyzed by a standard processing pipeline in the cBioPortal ( https://www.cbioportal.org ) [ 26 ]. 2. Weighted gene co-expression network analysis (WGCNA) WGCNA was used to analysis gene association patterns in LUAD patients from TCGA. Initially, a correlation coefficient was calculated between two genes based on their expression patterns, followed by the construction of a gene network based on these coefficients. Optimal soft thresholding was determined using the 'pick soft threshold' function to categorize genes with high correlation into modules. The module containing HMGN1 was identified as 'brown', and genes in this module were subjected to GO analysis using the DAVID dataset ( https://david.ncifcrf.gov/summary.jsp ). The top 8 channels with P value less than 0.05 were selected [ 27 ]. 3. Gene Set Enrichment Analysis(GSEA) GSEA was conducted using the clusterProfiler (version 4.4.4) and enrichplot (version 1.16.1) R packages. RNA-Seq data from 516 lung adenocarcinoma specimens and pre-defined gene sets based on the Human MSigDB v2022.1 were used to identify signatures associated with HMGN1 expression in LUAD [ 28 ]. 4. Cell culture LUAD cell lines A549 and PC9 were maintained in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO 2 .Regular testing for bacterial and mycoplasma contamination was conducted. 5. RNA interference and stable cell line construction HMGN1 siRNAs sequences, referenced from relevant literature [ 29 , 30 ] and synthesized by General Biosystems (Hefei, China). Cells at 50% density confluency in 6-well plates were transfected with HMGN1 siRNAs or NC-siRNA using the Effectine transfection agent, following the manufacturer’s instruction. After 48 hours of culture, cells were harvested for further analysis. For stable cell line construction, the short hairpin RNA (shRNA) oligonucleotides of HMGN1 were synthesized based on siRNA sequence and cloned into the pLKO.1 vector (Sigma). Resulting constructs were packaged into lentivirus for transduction into LUAD cancer cells. Stable cell lines were established by puromycin selection over one week. 6. RNA preparation and quantitative real-time PCR Total RNA was extracted from cultured cells using the Trizol-up plus RNA kit (Thermo Scientific), following the manufacturer’s protocol. cDNA synthesis was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time quantitative PCR (RT-qPCR) was conducted using ChamQ SYBR qPCR Master Mix (Vazyme) in a Roche LC96 Real-Time PCR System. mRNA expression levels were calculated using the 2 −ΔΔCt method. The primers used were as follows: HMGN1-F: 5’-CGGGAAAGGATAAAGCATCA-3’ HMGN1-R: 5’-TGGACTCTGGTTTTCCGTCT-3’ β-actin-F: 5’-CATGTACGTTGCTATCCAGGC-3’ β-actin-R: 5’-CTCCTTAATGTCACGCACGA-3’ RAD51-F: 5’-CAACCCATTTCACGGTTAGAGC-3’ RAD51-R: 5’-TTCTTTGGCGCATAGGCAACA-3’ 7. Western blot Standard Western blotting protocols were followed as previously described [ 31 ]. Primary antibodies used included RAD51 (1:1000, Abcam ab133534), γH2AX (1:1000, CST 2577), p-CHK1 (1:1000, Ser317, CST 12302), CHK1 (1:1000, CST 2G1D5), β-actin (1:1000, TransGen HC201-02), p-RPA2 (1:1000,Ser4, Ser8, NBP1-23017), RPA2 (1:1000, Abcam ab2175), HMGN1 (1:1000,proteintech 11695-1-AP). Secondary antibodies were Rabbit IgG (1:3000, CST 7074) and mouse IgG (1:3000, CST 7076). 8. Colony formation assay Stable knockdown cells were cultured in six-well plates until colonies reached appropriate size (approximate 7–10 days). Cells were fixed with methanol for 5–10 minutes, then stained with 0.1% crystal violet solution for 15 min. After rinsing and drying, colonies were photographed. 9. Statistical analysis All in vitro analyses were replicated at least three times. P-value < 0.05 was considered statistically significant. Data were analyzed using two-tailed unpaired Student’s t tests with GraphPad Prism software. Data availability: The datasets generated during and/or analyzed during the current study are available in the TCGA repository, https://tcgadata.nci.nih.gov/tcga; UALCAN repository, http://ualcan.path.uab.edu; Human Protein Atlas repository, https://www.proteinatlas.org; cBioPortal repository, https://www.cbioportal.org; KMplot repository, https://.kmplot.com . Results Expression levels of HMGNs in LUAD. To investigate the deregulation of HMGNs in lung adenocarcinoma (LUAD), the RNA-sequencing data of five HMGN members in LUAD and normal lung tissues were extracted from the TCGA database and two GSE data sets and analyzed. Volcano plots analysis revealed a significant upregulation of HMGN1 (shown in Fig. 1 A and supplement Fig. 1 A). The up-regulation of HMGN1 was confirmed in the GSE19804 data set (shown in Fig. 1 B) and the GSE10072 (shown in Fig. 1 C) data sets, while HMGN3 and HMGN5 did not exhibit a similar trend. Analysis of gene expression of LUAD and matched adjacent non-tumor tissues from three cohorts showed consistent upregulation of HMGN1 in LUAD across all cohorts, with HMGN5 downregulated in two of them (shown in Fig. 1 D-F). Finally, pan-cancer analysis across various cancers, including breast, colon, liver, and stomach cancers, also indicated high expression of HMGN1 (shown in supplement Fig. 1 C). Protein expression of HMGNs in LUAD was then assessed using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) mass-spectrometry-based proteomics dataset. The results were in line with mRNA expression data, showing increased protein levels of HMGN1 and decreased levels of HMGN4/5 in LUAD (shown in Fig. 1 G). Additionally, the Human Protein Atlas was used to corroborate these findings at the protein level (shown in supplement Fig. 1 B). In short, these findings indicate that abnormal expression of HMGN family members is associated with patients with LUAD. 2. Genetic and epigenetic alterations of HMGNs in LUAD The aberrant expression of HMGNs might be due to genetic alterations that occur in coding regions and dysregulation of epigenetics at the promoter region. Therefore, we characterized the mutation patterns of HMGNs in LUAD using the cBioPortal online tool. In general, the mutation rates of HMGNs are relatively low. HMGN genes were altered in 25 of 507 specimens (5%). Two alterations were simultaneously detected in almost 1/20 of the samples (shown in Fig. 2 A). Since genomic structural variations are the most genetic alterations in LUAD, the MEXPRESS online tools were applied to assess the transcriptional consequences of HMGN copy number variation (CNV). As shown in Figure. 2B, a significantly greater percentage of copy number gain and losses of HMGN1/2/3/4/5 in LUAD was observed. Increasing evidence has shown that DNA methylation level at promoter region is a critical factor determining transcription. Thereby the promoter methylation of HMGNs in LUAD was analyzed using the UALCAN portal. As shown in Figure.2C, reduced methylation levels of HMGN1/2/4 were found in primary lung tumors. And promoter methylation of HMGN3/5 were upregulated in tumor. These results implied that HMGNs gene were mutated infrequently, and the deregulation of HMGNs might be at least partly due to copy number variation and abnormal promoter methylation. 3. Potential of HMGN1 as a biomarker and prognosis in LUAD We selected HMGN1 for subsequent assessment of prognostic values and function enrichment analysis in LUAD since HMGN1 displays most prominent changes in expression, copy number variation promoter methylation. First, the relationship between HMGN1 mRNA levels and clinicopathological subgroups of LUAD patients was evaluated in the TCGA lung cancer and GSE11969 cohorts. As shown in Figure.3A, the expression of HMGN1 in all tumor stages (I-IV) was significantly higher than that in normal group. In addition, the expression of HMGN1 in different subtypes of LUAD was significantly higher than that in normal group (shown in Fig. 3 B − 3C). Furthermore, HMGN1 expression in four major subtypes of lung cancer was significantly higher than normal control (shown in Fig. 3 D). However, no difference was seen between different tumor stages/subtypes (shown in Fig. 3 B-D). To further seek the possibility of HMGN1 as a biomarker, we analyzed the diagnostic efficiency of HMGN1 expression in discriminating LUAD patients from healthy individuals using receiver operating characteristic (ROC) curves from TCGA and GEO datasets. The data showed that HMGN1 had a very high area under the curve (AUC = 0.91) value. Similar high AUC values were observed in other three lung cancer cohorts (AUC = 0.89 in GSE10072, AUC = 0.98 in GSE11969, and AUC = 0.87 in GSE19804) (shown in Fig. 3 E-H). And we analyzed the diagnostic efficiency of HMGN1 expression in discriminating LUAD patients from other lung cancer using ROC curves from GEO dataset. The data showed that HMGN1 had no high area under the curve value (shown in supplement Fig. 2 A). To date, the potential prognostic value of HMGN family members remains unclear. Therefore, the prognostic significance of HMGN1 mRNA expression were evaluated by using three public available datasets. Kaplan–Meier (KM) curve analysis showed that HMGN1 expression correlated significantly with overall survival (OS, shown in Fig. 3 I) and disease-specific survival (DSS, shown in Fig. 3 J) in LUAD patients in the TCGA cohort. Moreover, the association of HMGN1 mRNA expression with worse survival in LUAD was also seen in two other cohorts (shown in Fig. 3 K − 3L). However, the association of HMGN1 mRNA expression with worse survival in LUAD was also seen no significantly in TCGA cohort (shown in supplement Fig. 2 B). And we analyzed data from the KM ploter website and found similar results in three survival analyses for OS, FPS, and PPS (shown in supplement Fig. 2 C). Furthermore, the univariate and multivariate Cox analyses were performed to investigate the relationship between HMGN1 expression and OS or DSS. As indicated in Tables 1 and 2, the analysis shown that T stage, N stage, HMGN1 expression were associated with OS and DSS in LUAD patients. These results indicated that the expression of HMGN1 might be helpful in the prognosis of LUAD patients. 4. Functional enrichment analysis of the HMGN1 in LUAD To investigate the function of HMGN1 in lung adenocarcinoma, we first explored the potential co-expression genes of HMGN1 using the weighted correlation network analysis (WGCNA) followed by GO analysis. The network constructed on 1157 genes led to the identification of 100 modules comprising genes with similar expression patterns (shown in Fig. 4 A). GO analysis further pointed out that cell division, protein ubiquitination, intracellular protein, cell cycle, and DNA repair are the top five modules which were related to the expression of HMGN1 in LUAD (shown in Fig. 4 B). To further verify the physiological functions of HMGN1, Gene Set Enrichment Analysis (GSEA) analysis was then undertaken to investigate hallmarks associated with HMGN1 expression. The results confirmed the association of HMGN1 with DNA repair pathways (shown in Fig. 4 C). Additionally, DNA repair were the top two gene sets with significant association with HMGN1 in multiple LUAD cohorts (shown in Fig. 4 D-F andsupplement Fig. 3 A). Notably, GSEA analyses of HMGN1 in several other cancer types have also emphasized the essential roles of HMGN1 in DNA repair (shown in supplement Fig. 3 B-E). The enrichment degree of specific pathways related to DNA repair is shown in supplement Fig. 3 F. And as displayed in supplement Fig. 3 G, the changes of marker genes in DNA repair pathway were analyzed according to different expressions of HMGN1.DNA repair pathways primarily consist of six modalities: base excision repair (BER), nucleotide excision repair (NER), crypto-chrome/photolyase family (CPF), Fanconi Anemia DNA Repair Pathway (FA), non-homologous end joining (NHEJ), and homologous recombination repair (HRR). Correlation analysis indicated that HMGN1 expression was linked to all of these six DNA damage repair pathways, especially HRR (shown in Fig. 4 G-L). HMGN1 loss reduces the DNA repair response . The mRNA expression of RAD51, a key recombinase in homologous recombination repair, showed positive correlation with HMGN1 in the TCGA LUAD cohorts (shown in Fig. 5 A). Since HMGN1 function in HRR remains largely unknown, this data and above analysis prompted us to test whether HMGN1 is involved in homologous recombination repair in lung adenocarcinoma cells. Efficient depletion HMGN1 expression using siRNA was confirmed at the RNA and protein level in A549 and PC9 cells (shown in Fig. 5 B). We then assessed the effect of HMGN1 knockdown on Rad51 expression. The results showed that RAD51 expression was remarkably decreased at the mRNA and protein level (shown in Fig. 5 C-E). Importantly, p-CHK1, an essential component of the ATR-ChK1 pathway, was markedly reduced. At the same time, p-RPA2, a marker monitoring the activity of DNA end resection that is prerequisite for homologous recombination, was remarkably accumulated after knocking down HMGN1. In agreement with the western results for Rad51, p-CHK1, and p-RPA2, HMGN1 depletion led to robust accumulation of γH2AX, a classic DSB marker (shown in Fig. 5 F). Similar phenomenon was also seen in HMGN1 stably knockdown A549 and PC9 cells (shown in Fig. 5 G). In sum, these data suggest that HMGN1 inhibition attenuates the ATR-ChK1 pathway and subsequent Rad51-mediated recombination, thereby impairing homologous recombination capacity. 6. HMGN1 depleted cells are more sensitive to HU treatment The indispensable role of HMGN1 in HRR prompted us to test the potential of HMGN1 inhibition to sensitize lung adenocarcinoma cells to DNA damage agents, which is frequently applied in clinical setting. First, we investigated the effect of HMGN1 knockdown on the sensitivity of hydroxyurea (HU). As shown in Figure.6A, HMGN1-deficent A549 and PC9 cells were more much more sensitive to HU treatment, which was consistent with the role of HMGN1 in the ATR-ChK1 pathway. Moreover, long-term colony formation assays demonstrated that HMGN1 could protect the cells from HU-induced toxicity (shown in Fig. 6 B). To explore the chemosensitizing potential of targeting HMGN1, we treated A549 and PC9 cells with a range of concentrations of cisplatin to assess cell survival in the absence of HMGN1. As displayed in Figure.6C, HMGN1 silencing could enhance the cytotoxic activity of cisplatin, a commonly used chemotherapeutic drug in the clinic. Conclusion Lung cancer, as the most common cause of cancer-related deaths globally, with lung adenocarcinoma (LUAD) being its most common histological subtype, continue to present significant challenges. Despite advances in diagnosis and treatment in the past decades, there remains a critical need for novel prognostic markers and therapeutic strategies for LUAD, which is characterized by an extremely poor 5-year survival rate. Aberrant epigenetic regulation, alongside genetic alterations, has been implicated in every step of lung cancer. Among epigenetic factors, the High Mobility Group Nucleosome Binding (HMGN) family, known for its specific interaction with nucleosomes in chromatin, particularly in enhancers and promoters, is crucial in establishing cell-type-specific gene expression programs. Although HMGNs' involvement in various cancers has been noted, their roles in LUAD remain unclear. This study utilized publicly available datasets and bioinformatics approaches to comprehensively analyze the expression and prognostic value of HMGNs in LUAD. We focused on HMGN1 due to its significant differential gene expression. Our results suggest HMGN1's potential as a prognostic marker for distinguishing LUAD patients from healthy individuals. Functional enrichment analysis and cellular experiments emphasized HMGN1's critical role in the DNA damage response, particularly in Homologous Recombination (HR). We observed that HMGN1 were highly expressed in LUAD tissues compared with normal tissues at the RNA and protein level, while HMGN3/5 were expressed at a lower level in the tumor tissues compared to normal controls. Mutation profile and promoter methylation analysis indicate that both copy number variation and promoter methylation might contribute the dysregulation of HMGNs, though the underlying mechanisms lead to these alterations remain under investigation. Interestingly, HMGNs show a relatively low mutation rate in lung adenocarcinoma, this phenomenon has been observed for a number of epigenetic factors. We speculated that HMGN1 might be too critical to be mutated due to its importance in modulation of chromatin structure and histone modifications. To identify potential signaling pathways regulated by HMGN1, we constructed a module-centric co-expression network based on weighted correlation network analysis (WGCNA) and analyzed the possible functions of the correlated gene set by GO analysis. The results highlighted that cell division, protein ubiquitination, intracellular protein, cell cycle, and DNA repair were significantly associated with HMGN1 expression in LUAD. Moreover, functional enrichment analysis by GSEA confirmed that HMGN1 was highly involved in the regulation of the DNA repair signaling, which is in line with the previous findings in UV and irradiation-treated cells. Since the roles of HMGN1 in specific DNA repair pathways are not well characterized. We set to correlate the specific DNA repair pathways with HMGN1 expression and found that HRR and NER were the top two pathways among the six classic DNA repair pathways. Subsequent in vitro experiments using PC9 and A549 cells confirmed that silencing HMGN1 attenuated HR repair capacity. Indeed, several studies suggest that HMGN1 manifests anti-tumor effects by promoting DNA repair and genome stability [ 32 ]. Interestingly, high HMGN1 expression correlates with poor prognosis in LUAD, despite its role in promoting DNA repair. Two recent studies have demonstrated thatHMGN1 acts chromatin architectural protein to compete with histone H1 for nucleosome binding sites [ 7 , 33 ]. HMGN1 supports repair of DNA lesions, it would be expected that histone H1 will have an opposite effect on DNA damage repair. Indeed, a number of studies suggest that histone H1 has an inhibitory effect on repair of DNA lesions [ 33 , 34 ]. Another investigation indicates that histone H1 suppresses repair of DSB in vitro when present in high concentrations [ 35 ]. The above observations suggest that the balance between the relative amounts of HMGN1 and histone H1, as well as their chromatin binding abilities, may be essential for the regulation of the cellular response to DNA damage [ 36 ]. Therefore, how HMGN1 ultimately participates in the progression of lung adenocarcinoma remains to be explored. However, there are some limitations to this study. First, although our experimental data showed the functional connection between HMGN1 and the DNA damage response pathway, the underlying mechanisms leading to the down-regulation of RAD51 and CHK1 phosphorylation remain to be determined. Perturbation of HMGN expression might interfere with the expression of some DNA damage response genes including RAD51 on the basis of the preferential association of HMGN proteins with chromatin regulatory sites including enhancers and promoters. Second, only one pathway of DNA repair has been verified, and other enrichment pathways need to be further addressed. In summary, our systematic analysis positions HMGN1 as a diagnostic and prognostic marker for lung adenocarcinoma. Moreover, HMGN1 holds great potential as an epigenetic therapeutic target given that HMGN1 plays critical roles in the DNA damage response, especially in HRR. Given the pivotal role of HMGN1 in peri-tumor infiltration of lymphocytes, combining HMGN1 targeting with immunotherapy might achieve better therapeutic outcomes for lung adenocarcinoma. Abbreviations LUAD：Lung adenocarcinoma HMGN: The high mobility group nucleosome-binding protein CNV: DNA copy number variation ROC: The receiver operating characteristic curve AUC: Area under the curve HR: Hazard ratio OS: Overall survival PFS: Progression survival DFS: Disease-free survival DSS: Disease-specific survival HU: Hydroxyurea Declarations Conflict of Interest Statement: The authors declare that they have no conflicts of interests and do not exist any commercial or financial relationships, and acknowledgment that all authors have contributed significantly, and that all authors are in agree with the content of the manuscript. And The manuscript has not been published nor submitted for publication elsewhere except as a brief abstract in the proceedings of a scientific meeting or symposium. Funding Source: This study was supported by the National Natural Science Foundation of China (81972191 and 81672647), Science and Technology Major Project of Anhui Province (18030801140), and the 100-Talent Program of Chinese Academy of Sciences. A portion of this work was supported by the High Magnetic Field Laboratory of Anhui Province. Author contributions: Conceptualization and design: WX, CG, FJ, LW; Development of methodology and investigation, WX, CG; Writing, review, and/or revision of the manuscript: WX, CG, FJ, LW. All authors read and approved the final manuscript. Data availability statement: The datasets generated during and/or analysed during the current study are available in the TCGA repository, https://tcgadata.nci.nih.gov/tcga; MEXPRESS repository, https://mexpress.be; UALCAN repository, http://ualcan.path.uab.edu; Human Protein Atlas repository, https://www.proteinatlas.org; cBioPortal repository, https://www.cbioportal.org; KMplot repository, https://.kmplot.com References Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73(1):17–48. https://doi.org/10.3322/caac.21763 . Relli V, Trerotola M, Guerra E, Alberti S. Abandoning the Notion of Non-Small Cell Lung Cancer. Trends Mol Med. 2019;25(7):585–594. https://doi.org/10.1016/j.molmed.2019.04.012 . Lahiri A, Maji A, Potdar PD, Singh N, Parikh P, Bisht B, Mukherjee A, Paul MK. Lung cancer immunotherapy: progress, pitfalls, and promises. 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Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. https://doi.org/10.1073/pnas.0506580102 Apelt K, Zoutendijk I, Gout DY, Wondergem AP, van den Heuvel D, Luijsterburg MS. Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair. Sci Rep. 2020;10(1):4332. https://doi.org/10.1038/s41598-020-61343-4 . Zhu N, Hansen U. HMGN1 modulates estrogen-mediated transcriptional activation through interactions with specific DNA-binding transcription factors. Mol Cell Biol. 2007;27(24):8859–73. https://doi.org/10.1128/MCB.01724-07 . Ma L, Bian X, Lin W. The dual HDAC-PI3K inhibitor CUDC-907 displays single-agent activity and synergizes with PARP inhibitor olaparib in small cell lung cancer. J Exp Clin Cancer Res. 2020;39(1):219. https://doi.org/10.1186/s13046-020-01728-2 . Nie Y, Yang D, Oppenheim JJ. Alarmins and Antitumor Immunity. Clin Ther. 2016;38(5):1042–53. https://doi.org/10.1016/j.clinthera.2016.03.021 . Rosidi B, Wang M, Wu W, Sharma A, Wang H, Iliakis G. Histone H1 functions as a stimulatory factor in backup pathways of NHEJ. Nucleic Acids Res. 2008;36(5):1610–23. https://doi.org/10.1093/nar/gkn013 . Wang S, Meyer DH, Schumacher B. Inheritance of paternal DNA damage by histone-mediated repair restriction. Nature. 2023;613(7943):365–374. https://doi.org/10.1038/s41586-022-05544-w . Kysela B, Chovanec M, Jeggo PA. Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1. Proc Natl Acad Sci U S A. 2005;102(6):1877–82. https://doi.org/10.1073/pnas.0401179102 . Gerlitz G. HMGNs, DNA repair and cancer. Biochim Biophys Acta. 2010 Jan-Feb;1799(1–2):80 – 5. https://doi.org/10.1016/j.bbagrm.2009.10.007 . Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table.pdf S1.pdf S2.pdf S3.pdf Cite Share Download PDF Status: Published Journal Publication published 06 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Jan, 2024 Reviews received at journal 29 Dec, 2023 Reviewers agreed at journal 20 Dec, 2023 Reviewers agreed at journal 19 Dec, 2023 Reviewers invited by journal 17 Dec, 2023 Editor assigned by journal 11 Dec, 2023 Editor invited by journal 28 Nov, 2023 Submission checks completed at journal 28 Nov, 2023 First submitted to journal 26 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3669863","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":253869115,"identity":"dc109004-e737-4460-a306-4a9c1cdf1c75","order_by":0,"name":"Xianli Wu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianli","middleName":"","lastName":"Wu","suffix":""},{"id":253869116,"identity":"586050b0-3265-44eb-bc99-c815677106da","order_by":1,"name":"Geqi Cai","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Geqi","middleName":"","lastName":"Cai","suffix":""},{"id":253869117,"identity":"de1fd601-add1-4a1e-b89c-0e5518a93961","order_by":2,"name":"Jing Feng","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Feng","suffix":""},{"id":253869121,"identity":"7bb5cb13-a53e-4fe4-ae02-0b8643336168","order_by":3,"name":"Wenchu Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACPmYIncDA3gAVOkBACxtcC88BqGqCWhhgWiQSiNXCzntM4uMOuzyDm2+MP3+oYZDju5HA+LkAr8P40iRnnkkuNridYyZx4BiDseSNBGbpGXi18Jjd5m07kLgBqIXhYAND4oYbCUBBQlr+grTcPGP8AailnjgtjCAtN3gMJIBaEgyI0GL+s7ctOXHmmbQyiTPHJAxnnnnYLI1PCz//GWODn212iX3HD2/+UFFjI893PPngZ3xa0IEEEDM2kKBhFIyCUTAKRgE2AAD4QUtxu4UrkwAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wenchu","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2023-11-27 02:29:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3669863/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3669863/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-60352-8","type":"published","date":"2024-05-06T21:18:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":47452640,"identity":"39d6072b-5e08-4ca1-b137-27f32baed138","added_by":"auto","created_at":"2023-12-01 17:22:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":476990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrant expression of HMGN family in lung adenocarcinoma.\u0026nbsp; \u003c/strong\u003e(A-C)\u003cstrong\u003e \u003c/strong\u003eVolcano plots illustrating differential HMGN expression in lung adenocarcinoma versus normal tissues from the TCGA database (A), GSE19804 (B), and GSE10072 (C). Expression values were presented as log\u003csub\u003e2\u003c/sub\u003e (FPKM+1). Fold changes were presented as log2 (fold change). (D-F) Transcriptional expression of HMGNs in 57 paired lung adenocarcinoma and adjacent normal tissues from TCGA (D); 60 pairs from GSE19804 (E) and 33 pairs from GSE10072 (F). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. ns: not statistically significant. (G) Protein expression levels of HMGNs in lung cancer versus normal lung tissues\u003cstrong\u003e \u003c/strong\u003efrom the CPATC dataset.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/6adfb9712f5e535fcbbe35cb.jpg"},{"id":47455071,"identity":"0ccd5a93-ece2-4afc-bb3e-8735a4177357","added_by":"auto","created_at":"2023-12-01 17:38:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":566648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic and epigenetic alterations of HMGN family in lung adenocarcinoma.\u003c/strong\u003e(A) OncoPrint from the cBioPortal database showing the distribution and proportion of samples with genetic alterations in HMGN genes in LUAD. (B) Correlation between copy number variation and gene expression variation for HMGNs in LUAD. (C) The contribution of promoter methylation level of HMGNs to the expression of corresponding genes in LUAD.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/f6112b613af850ba46f920a6.jpg"},{"id":47452642,"identity":"74c05f2c-fdb1-4384-9fcd-915b2b307747","added_by":"auto","created_at":"2023-12-01 17:22:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":698743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical significance of HMGN1 expression in lung adenocarcinoma.\u003c/strong\u003e (A) Boxplot depictng HMGN1 expression association with tumor stages in the TCGA dataset. （B）Boxplot illustrating the association between HMGN1 expression and tumor clinicopathological classification in the TCGA dataset. (C) Boxplot showing HMGN1 expression in different invasive tumor subtypes from the TCGA database, with types 1-7 representing various subtypes: 1, lung bronchioloalveolar carcinoma mucinous; 2, lung bronchioloalveolar carcinoma nonmucinous; 3, lung acinr adenocarcinoma; 4, lung micropapillary adenocarcinoma; 5, lung papillary adenocarcinoma; 6, lung solid pattern predominant adenocarcinoma; \u0026nbsp;7, mucinous (colloid) carcinoma. (D) Boxplot demonstrating HMGN1 expression in different lung cancer subtypes from the GSE11969 dataset. from GSE11969 dataset. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. (E-H) Receiver operating characteristic curve (ROC) analysis of HMGN1 expression in LUAD from TCGA (E) and three GEO datasets (F-H). (I-J) Kaplan–Meier curves for overall survival and disease-specific survival in LUAD patients, associated with HMGN1 expression. (K-L) Overall survival analysis of HMGN1 in LUAD from two GEO datasets.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/4ab967841afc0507ebf9044a.jpg"},{"id":47452645,"identity":"6c247b0d-3c9e-408e-adbd-219f1c002b36","added_by":"auto","created_at":"2023-12-01 17:22:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":821011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of HMGN1 in LUAD. \u003c/strong\u003e(A) WGCNA cluster dendrogram showing co-expression gene modules in LUAD; the ‘brown’ module contains genes co-expressed with HMGN1. (B) Bubble diagram of GO enrichment analysis for HMGN1 co-expressed genes. (C-F) Bubble map of GSEA for HMGN1 from TCGA (C) and three GEO datasets (D-F). (G-L) Lollipop charts correlating HMGN1 with genes in different DNA repair pathways; CPF: check point factors; FA: Fanconi anemia; NER: nucleotide excision repair; BER: base excision repair; NHEJ: Non-homologous end joining; HRR: homologous recombination repair.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/afd23515489a265ef2303044.jpg"},{"id":47452646,"identity":"8030f39c-aa67-4324-907a-7da6473a677e","added_by":"auto","created_at":"2023-12-01 17:22:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":500732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of HMGN1 knockdown on DNA damage response in lung adenocarcinoma.\u003c/strong\u003e (A) RT–qPCR analysis pf HMGN1 silencing in A549 and PC9 cells and its effect on RAD51 expression. (B) Western blot analysis of Rad51, p-Rpa2, γH2AX and p-Chk1 proteins following HMGN1 depletion\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/411a9c1314466e16dd17696a.jpg"},{"id":47452644,"identity":"dd12b052-a895-4b52-a6e6-8444b54d61d4","added_by":"auto","created_at":"2023-12-01 17:22:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":655788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of HMGN1 knockdown on HU or cisplatin sensitivity in lung adenocarcinoma.\u003c/strong\u003eA: Cell viability assays showing the effects of cisplatin and hydroxycarbamide (HU) on A549 and PC9 cells with HMGN1 knockdown. C-D: Clonogenic assays of A549 and PC9 cells treated with varying concentrations of HU after HMGN1 knockdown. Surviving fraction percentages calculated relative to untreated cells. \u0026nbsp;*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 (Student’s t test)\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/b5152883234587b95f36384a.jpg"},{"id":56488178,"identity":"4d26d187-d5a9-457c-8018-3c4176139019","added_by":"auto","created_at":"2024-05-14 21:29:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1219864,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/dae8813a-c93f-4b79-a9b9-903b378059c7.pdf"},{"id":47454315,"identity":"b8438901-0ec4-4a82-9f2e-84558c82ea28","added_by":"auto","created_at":"2023-12-01 17:30:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":474824,"visible":true,"origin":"","legend":"","description":"","filename":"table.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/734b0ead3b6ded5ac081259b.pdf"},{"id":47452650,"identity":"a8b91341-0b57-4713-bfa9-f3402b11eee6","added_by":"auto","created_at":"2023-12-01 17:22:42","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5689051,"visible":true,"origin":"","legend":"","description":"","filename":"S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/54aceb58d11babe162f11b79.pdf"},{"id":47452647,"identity":"3c9baba0-247b-4e6c-9a36-80f3b459ec3a","added_by":"auto","created_at":"2023-12-01 17:22:42","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":983654,"visible":true,"origin":"","legend":"","description":"","filename":"S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/7349d853b6b444c6cb84df0e.pdf"},{"id":47454316,"identity":"6aeb5a3b-07e4-4bb9-ad47-d5b99d591cf2","added_by":"auto","created_at":"2023-12-01 17:30:42","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1200899,"visible":true,"origin":"","legend":"","description":"","filename":"S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3669863/v1/7c8b74781b3195d296288eaa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"HMGN1 is a potential chemotherapeutic target for lung adenocarcinoma.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer, recognized as the leading cause of cancer-related deaths globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], is primarily categorized into non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC), with NSCLC further subdivided into lung adenocarcinoma (LUAD), squamous cell carcinoma, and large cell carcinoma based on pathological classification [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, LUAD is the most prevalent subtype, accounting for 40% of all new lung cancer cases diagnosed annually worldwide. Despite considerable advancements in therapeutic strategies for LUAD in recent decades, the long-term prognosis for patients remains a significant clinical challenge [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, the development of early diagnostic methods and the implementation of effective targeted therapies are imperative for this devastating disease.\u003c/p\u003e \u003cp\u003eDNA, the carrier of storing and transmitting genetic information, is organized into chromatin in eukaryotic cells through stably interactions with histone and non-histone proteins. The chromatin structure is a dynamic entity, continuously modified by nuclear factors, which compact the genome for efficient accommodation within the nucleus [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Critical DNA-dependent events, including transcription, replication, and DNA repair, are intricately linked to the chromatin, necessitating precise spatial and temporal regulation of its structure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. High mobility group N (HMGN), a group of chromatin-binding architectural proteins, binds specifically to nucleosomes without preference for any DNA sequence [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The association of HMGN with nucleosomes impedes histone H1 binding, thereby influencing chromatin accessibility and remodeling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To date, five HMGN family members have been discovered in human genome. All of them possess a positively charged nucleosome binding domain, a bipartite nuclear localization signal (NLS), and an acidic C-terminal chromatin regulatory domain [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. HMGN1 and HMGN2 are ubiquitously expressed in mammalian cells, while HMGN3, 4, and 5 have more restricted expression patterns [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The interaction of HMGN proteins with nucleosomes destabilizes the high-order chromatin structure, modulates the pattern of histone post-translational modification, and enhances the accessibility of non-histone proteins such as transcription factors, thereby significantly influencing cellular phenotypes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreasing evidence has demonstrated that HMGN genes are implicated in development, immunological processes, and the etiology of disease, including cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For example, appropriate expression levels of HMGN family proteins are crucial in both maintenance of the pluripotent identity of stem cells and facilitating cellular differentiation during embryogenesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. HMGN1, acting as an alarmin, fosters antitumor immunity by inducing maturation in human dendritic cells via Toll-like receptor 4 (TLR-4) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, high HMGN1 levels correlate with increased peri-tumor infiltration of lymphocytes in Her2-positive breast cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, recent studies in head and neck carcinoma have linked elevated cytoplasmic HMGN1 levels with increasing tumor-infiltrating lymphocytes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], indicating potential immunotherapeutic applications. HMGN2 promotes breast cancer progression by facilitating STAT5 access to the promoter region of its targets [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Elevated HMGN4 expression is associated with high grade tumors and poor outcomes in hepatocellular carcinoma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite growing evidence of aberrant HMGN family gene expression across multiple tumor lineages, their specific roles in lung adenocarcinoma, the predominant type of lung cancer, remain largely unexplored.\u003c/p\u003e \u003cp\u003eGiven the crucial role of HMGN proteins in chromatin de-condensation and posttranslational modifications in histone tails, which are integral to DNA damage repair, it is plausible that HMGN family protein may be involved in DNA damage response. Indeed, studies show that HMGN1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and cells are hypersensitive to UV radiation due to impaired accessibility to UV-damaged sites in chromatin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Loss of HMGN1 also impairs ionizing radiation (IR)-induced ATM auto-phosphorylation and the activation of several ATM targets in mouse embryonic fibroblasts [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the mechanisms by which HMGN proteins modulate DNA repair processes are yet to be fully understood, warranting systematic analysis and exploration.\u003c/p\u003e \u003cp\u003ePrior research has indicated aberrant expressions of HMGNs and their prognostic value in some members of HMGN family [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nevertheless, the role of distinct HMGN family members in the development and progression of lung adenocarcinoma have not been clearly defined. Through comprehensive analyses of multi-level omics data, this study investigated the expression and prognostic significance of HMGNs in lung adenocarcinoma. We discovered that HMGN1 was upregulated in LUAD, and its expression was associated with clinical outcomes. Function enrichment analysis further revealed a strong association between HMGN1 expression six DNA damage repair pathways. Finally, the roles of HMGN1 in the ATR-ChK1 and homologous recombination repair pathways was manifested in vitro in lung adenocarcinoma cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e1. Data collection\u003c/p\u003e\n\u003cp\u003e1.1 LUAD cohor\u003c/p\u003e\n\u003cp\u003eClinical information and RNA-seq data of LUAD patients were sourced from the TCGA database [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov\u003c/span\u003e\u003c/span\u003e). Additionally, gene chips numbered GSE11969, GSE10072, GSE13213, GSE19804, and GSE116959 were selected from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Transcriptome data of the 75 LUAD cell lines were obtained from the CCLE database [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e1.2. Human protein atlas\u003c/p\u003e\n\u003cp\u003eHuman Protein Atlas database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.proteinatlas.org/\u003c/span\u003e\u003c/span\u003e) were utilized to analyze HMGNs expression in LUAD tissues compared to adjacent normal tissues at the protein level [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e1.3. Ualcan dataset\u003c/p\u003e\n\u003cp\u003eUalcan dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu\u003c/span\u003e\u003c/span\u003e) was employed to examine the protein expression of HMGNs in LUAD tissues versus normal tissues from Clinical Proteomic Tumor Analysis Consortium (CPTAC). Additionally, this dataset provided DNA promoter methylation profiles of HMGN family in LUAD from TCGA [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e1.4. cBioportal\u003c/p\u003e\n\u003cp\u003eMutation profiles of HMGN family genes were analyzed by a standard processing pipeline in the cBioPortal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org\u003c/span\u003e\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e2. Weighted gene co-expression network analysis (WGCNA)\u003c/p\u003e\n\u003cp\u003eWGCNA was used to analysis gene association patterns in LUAD patients from TCGA. Initially, a correlation coefficient was calculated between two genes based on their expression patterns, followed by the construction of a gene network based on these coefficients. Optimal soft thresholding was determined using the \u0026apos;pick soft threshold\u0026apos; function to categorize genes with high correlation into modules. The module containing HMGN1 was identified as \u0026apos;brown\u0026apos;, and genes in this module were subjected to GO analysis using the DAVID dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/summary.jsp\u003c/span\u003e\u003c/span\u003e). The top 8 channels with P value less than 0.05 were selected [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e3. Gene Set Enrichment Analysis(GSEA)\u003c/p\u003e\n\u003cp\u003eGSEA was conducted using the clusterProfiler (version 4.4.4) and enrichplot (version 1.16.1) R packages. RNA-Seq data from 516 lung adenocarcinoma specimens and pre-defined gene sets based on the Human MSigDB v2022.1 were used to identify signatures associated with HMGN1 expression in LUAD [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e4. Cell culture\u003c/p\u003e\n\u003cp\u003eLUAD cell lines A549 and PC9 were maintained in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.Regular testing for bacterial and mycoplasma contamination was conducted.\u003c/p\u003e\n\u003cp\u003e5. RNA interference and stable cell line construction\u003c/p\u003e\n\u003cp\u003eHMGN1 siRNAs sequences, referenced from relevant literature [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] and synthesized by General Biosystems (Hefei, China). Cells at 50% density confluency in 6-well plates were transfected with HMGN1 siRNAs or NC-siRNA using the Effectine transfection agent, following the manufacturer\u0026rsquo;s instruction. After 48 hours of culture, cells were harvested for further analysis. For stable cell line construction, the short hairpin RNA (shRNA) oligonucleotides of HMGN1 were synthesized based on siRNA sequence and cloned into the pLKO.1 vector (Sigma). Resulting constructs were packaged into lentivirus for transduction into LUAD cancer cells. Stable cell lines were established by puromycin selection over one week.\u003c/p\u003e\n\u003cp\u003e6. RNA preparation and quantitative real-time PCR\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cultured cells using the Trizol-up plus RNA kit (Thermo Scientific), following the manufacturer\u0026rsquo;s protocol. cDNA synthesis was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time quantitative PCR (RT-qPCR) was conducted using ChamQ SYBR qPCR Master Mix (Vazyme) in a Roche LC96 Real-Time PCR System. mRNA expression levels were calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. The primers used were as follows:\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003cp\u003eHMGN1-F: 5\u0026rsquo;-CGGGAAAGGATAAAGCATCA-3\u0026rsquo;\u003c/p\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003cp\u003eHMGN1-R: 5\u0026rsquo;-TGGACTCTGGTTTTCCGTCT-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e\u0026beta;-actin-F: 5\u0026rsquo;-CATGTACGTTGCTATCCAGGC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e\u0026beta;-actin-R: 5\u0026rsquo;-CTCCTTAATGTCACGCACGA-3\u0026rsquo;\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003cp\u003eRAD51-F: 5\u0026rsquo;-CAACCCATTTCACGGTTAGAGC-3\u0026rsquo;\u003c/p\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003cp\u003eRAD51-R: 5\u0026rsquo;-TTCTTTGGCGCATAGGCAACA-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e7. Western blot\u003c/p\u003e\n \u003cp\u003eStandard Western blotting protocols were followed as previously described [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Primary antibodies used included RAD51 (1:1000, Abcam ab133534), \u0026gamma;H2AX (1:1000, CST 2577), p-CHK1 (1:1000, Ser317, CST 12302), CHK1 (1:1000, CST 2G1D5), \u0026beta;-actin (1:1000, TransGen HC201-02), p-RPA2 (1:1000,Ser4, Ser8, NBP1-23017), RPA2 (1:1000, Abcam ab2175), HMGN1 (1:1000,proteintech 11695-1-AP). Secondary antibodies were Rabbit IgG (1:3000, CST 7074) and mouse IgG (1:3000, CST 7076).\u003c/p\u003e\n \u003cp\u003e8. Colony formation assay\u003c/p\u003e\n \u003cp\u003eStable knockdown cells were cultured in six-well plates until colonies reached appropriate size (approximate 7\u0026ndash;10 days). Cells were fixed with methanol for 5\u0026ndash;10 minutes, then stained with 0.1% crystal violet solution for 15 min. After rinsing and drying, colonies were photographed.\u003c/p\u003e\n \u003cp\u003e9. Statistical analysis\u003c/p\u003e\n \u003cp\u003eAll in vitro analyses were replicated at least three times. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Data were analyzed using two-tailed unpaired Student\u0026rsquo;s t tests with GraphPad Prism software.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The datasets generated during and/or analyzed during the current study are available in the TCGA repository, https://tcgadata.nci.nih.gov/tcga; UALCAN repository, http://ualcan.path.uab.edu; Human Protein Atlas repository, https://www.proteinatlas.org; cBioPortal repository, https://www.cbioportal.org; KMplot repository, https://.kmplot.com .\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eExpression levels of HMGNs in LUAD.\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTo investigate the deregulation of HMGNs in lung adenocarcinoma (LUAD), the RNA-sequencing data of five HMGN members in LUAD and normal lung tissues were extracted from the TCGA database and two GSE data sets and analyzed. Volcano plots analysis revealed a significant upregulation of HMGN1 (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The up-regulation of HMGN1 was confirmed in the GSE19804 data set (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and the GSE10072 (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) data sets, while HMGN3 and HMGN5 did not exhibit a similar trend. Analysis of gene expression of LUAD and matched adjacent non-tumor tissues from three cohorts showed consistent upregulation of HMGN1 in LUAD across all cohorts, with HMGN5 downregulated in two of them (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). Finally, pan-cancer analysis across various cancers, including breast, colon, liver, and stomach cancers, also indicated high expression of HMGN1 (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eProtein expression of HMGNs in LUAD was then assessed using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) mass-spectrometry-based proteomics dataset. The results were in line with mRNA expression data, showing increased protein levels of HMGN1 and decreased levels of HMGN4/5 in LUAD (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Additionally, the Human Protein Atlas was used to corroborate these findings at the protein level (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In short, these findings indicate that abnormal expression of HMGN family members is associated with patients with LUAD.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2. Genetic and epigenetic alterations of HMGNs in LUAD\u003c/h2\u003e \u003cp\u003eThe aberrant expression of HMGNs might be due to genetic alterations that occur in coding regions and dysregulation of epigenetics at the promoter region. Therefore, we characterized the mutation patterns of HMGNs in LUAD using the cBioPortal online tool. In general, the mutation rates of HMGNs are relatively low. HMGN genes were altered in 25 of 507 specimens (5%). Two alterations were simultaneously detected in almost 1/20 of the samples (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Since genomic structural variations are the most genetic alterations in LUAD, the MEXPRESS online tools were applied to assess the transcriptional consequences of HMGN copy number variation (CNV). As shown in Figure. 2B, a significantly greater percentage of copy number gain and losses of HMGN1/2/3/4/5 in LUAD was observed. Increasing evidence has shown that DNA methylation level at promoter region is a critical factor determining transcription. Thereby the promoter methylation of HMGNs in LUAD was analyzed using the UALCAN portal. As shown in Figure.2C, reduced methylation levels of HMGN1/2/4 were found in primary lung tumors. And promoter methylation of HMGN3/5 were upregulated in tumor. These results implied that HMGNs gene were mutated infrequently, and the deregulation of HMGNs might be at least partly due to copy number variation and abnormal promoter methylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3. Potential of HMGN1 as a biomarker and prognosis in LUAD\u003c/h2\u003e \u003cp\u003eWe selected HMGN1 for subsequent assessment of prognostic values and function enrichment analysis in LUAD since HMGN1 displays most prominent changes in expression, copy number variation promoter methylation. First, the relationship between HMGN1 mRNA levels and clinicopathological subgroups of LUAD patients was evaluated in the TCGA lung cancer and GSE11969 cohorts. As shown in Figure.3A, the expression of HMGN1 in all tumor stages (I-IV) was significantly higher than that in normal group. In addition, the expression of HMGN1 in different subtypes of LUAD was significantly higher than that in normal group (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u0026minus;\u0026thinsp;3C). Furthermore, HMGN1 expression in four major subtypes of lung cancer was significantly higher than normal control (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, no difference was seen between different tumor stages/subtypes (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eTo further seek the possibility of HMGN1 as a biomarker, we analyzed the diagnostic efficiency of HMGN1 expression in discriminating LUAD patients from healthy individuals using receiver operating characteristic (ROC) curves from TCGA and GEO datasets. The data showed that HMGN1 had a very high area under the curve (AUC\u0026thinsp;=\u0026thinsp;0.91) value. Similar high AUC values were observed in other three lung cancer cohorts (AUC\u0026thinsp;=\u0026thinsp;0.89 in GSE10072, AUC\u0026thinsp;=\u0026thinsp;0.98 in GSE11969, and AUC\u0026thinsp;=\u0026thinsp;0.87 in GSE19804) (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). And we analyzed the diagnostic efficiency of HMGN1 expression in discriminating LUAD patients from other lung cancer using ROC curves from GEO dataset. The data showed that HMGN1 had no high area under the curve value (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To date, the potential prognostic value of HMGN family members remains unclear. Therefore, the prognostic significance of HMGN1 mRNA expression were evaluated by using three public available datasets. Kaplan\u0026ndash;Meier (KM) curve analysis showed that HMGN1 expression correlated significantly with overall survival (OS, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and disease-specific survival (DSS, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) in LUAD patients in the TCGA cohort. Moreover, the association of HMGN1 mRNA expression with worse survival in LUAD was also seen in two other cohorts (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK \u0026minus;\u0026thinsp;3L). However, the association of HMGN1 mRNA expression with worse survival in LUAD was also seen no significantly in TCGA cohort (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). And we analyzed data from the KM ploter website and found similar results in three survival analyses for OS, FPS, and PPS (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, the univariate and multivariate Cox analyses were performed to investigate the relationship between HMGN1 expression and OS or DSS. As indicated in Tables\u0026nbsp;1 and 2, the analysis shown that T stage, N stage, HMGN1 expression were associated with OS and DSS in LUAD patients. These results indicated that the expression of HMGN1 might be helpful in the prognosis of LUAD patients.\u003c/p\u003e \u003cp\u003e4. \u003cb\u003eFunctional enrichment analysis of the HMGN1 in LUAD\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the function of HMGN1 in lung adenocarcinoma, we first explored the potential co-expression genes of HMGN1 using the weighted correlation network analysis (WGCNA) followed by GO analysis. The network constructed on 1157 genes led to the identification of 100 modules comprising genes with similar expression patterns (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). GO analysis further pointed out that cell division, protein ubiquitination, intracellular protein, cell cycle, and DNA repair are the top five modules which were related to the expression of HMGN1 in LUAD (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To further verify the physiological functions of HMGN1, Gene Set Enrichment Analysis (GSEA) analysis was then undertaken to investigate hallmarks associated with HMGN1 expression. The results confirmed the association of HMGN1 with DNA repair pathways (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additionally, DNA repair were the top two gene sets with significant association with HMGN1 in multiple LUAD cohorts (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F andsupplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, GSEA analyses of HMGN1 in several other cancer types have also emphasized the essential roles of HMGN1 in DNA repair (shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). The enrichment degree of specific pathways related to DNA repair is shown in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF. And as displayed in supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, the changes of marker genes in DNA repair pathway were analyzed according to different expressions of HMGN1.DNA repair pathways primarily consist of six modalities: base excision repair (BER), nucleotide excision repair (NER), crypto-chrome/photolyase family (CPF), Fanconi Anemia DNA Repair Pathway (FA), non-homologous end joining (NHEJ), and homologous recombination repair (HRR). Correlation analysis indicated that HMGN1 expression was linked to all of these six DNA damage repair pathways, especially HRR (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-L).\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eHMGN1 loss reduces the DNA repair response\u003c/b\u003e.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe mRNA expression of RAD51, a key recombinase in homologous recombination repair, showed positive correlation with HMGN1 in the TCGA LUAD cohorts (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Since HMGN1 function in HRR remains largely unknown, this data and above analysis prompted us to test whether HMGN1 is involved in homologous recombination repair in lung adenocarcinoma cells. Efficient depletion HMGN1 expression using siRNA was confirmed at the RNA and protein level in A549 and PC9 cells (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We then assessed the effect of HMGN1 knockdown on Rad51 expression. The results showed that RAD51 expression was remarkably decreased at the mRNA and protein level (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E). Importantly, p-CHK1, an essential component of the ATR-ChK1 pathway, was markedly reduced. At the same time, p-RPA2, a marker monitoring the activity of DNA end resection that is prerequisite for homologous recombination, was remarkably accumulated after knocking down HMGN1. In agreement with the western results for Rad51, p-CHK1, and p-RPA2, HMGN1 depletion led to robust accumulation of γH2AX, a classic DSB marker (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Similar phenomenon was also seen in HMGN1 stably knockdown A549 and PC9 cells (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In sum, these data suggest that HMGN1 inhibition attenuates the ATR-ChK1 pathway and subsequent Rad51-mediated recombination, thereby impairing homologous recombination capacity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e6. HMGN1 depleted cells are more sensitive to HU treatment\u003c/h3\u003e\n\u003cp\u003eThe indispensable role of HMGN1 in HRR prompted us to test the potential of HMGN1 inhibition to sensitize lung adenocarcinoma cells to DNA damage agents, which is frequently applied in clinical setting. First, we investigated the effect of HMGN1 knockdown on the sensitivity of hydroxyurea (HU). As shown in Figure.6A, HMGN1-deficent A549 and PC9 cells were more much more sensitive to HU treatment, which was consistent with the role of HMGN1 in the ATR-ChK1 pathway. Moreover, long-term colony formation assays demonstrated that HMGN1 could protect the cells from HU-induced toxicity (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). To explore the chemosensitizing potential of targeting HMGN1, we treated A549 and PC9 cells with a range of concentrations of cisplatin to assess cell survival in the absence of HMGN1. As displayed in Figure.6C, HMGN1 silencing could enhance the cytotoxic activity of cisplatin, a commonly used chemotherapeutic drug in the clinic.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLung cancer, as the most common cause of cancer-related deaths globally, with lung adenocarcinoma (LUAD) being its most common histological subtype, continue to present significant challenges. Despite advances in diagnosis and treatment in the past decades, there remains a critical need for novel prognostic markers and therapeutic strategies for LUAD, which is characterized by an extremely poor 5-year survival rate. Aberrant epigenetic regulation, alongside genetic alterations, has been implicated in every step of lung cancer. Among epigenetic factors, the High Mobility Group Nucleosome Binding (HMGN) family, known for its specific interaction with nucleosomes in chromatin, particularly in enhancers and promoters, is crucial in establishing cell-type-specific gene expression programs. Although HMGNs' involvement in various cancers has been noted, their roles in LUAD remain unclear.\u003c/p\u003e \u003cp\u003eThis study utilized publicly available datasets and bioinformatics approaches to comprehensively analyze the expression and prognostic value of HMGNs in LUAD. We focused on HMGN1 due to its significant differential gene expression. Our results suggest HMGN1's potential as a prognostic marker for distinguishing LUAD patients from healthy individuals. Functional enrichment analysis and cellular experiments emphasized HMGN1's critical role in the DNA damage response, particularly in Homologous Recombination (HR).\u003c/p\u003e \u003cp\u003eWe observed that HMGN1 were highly expressed in LUAD tissues compared with normal tissues at the RNA and protein level, while HMGN3/5 were expressed at a lower level in the tumor tissues compared to normal controls. Mutation profile and promoter methylation analysis indicate that both copy number variation and promoter methylation might contribute the dysregulation of HMGNs, though the underlying mechanisms lead to these alterations remain under investigation. Interestingly, HMGNs show a relatively low mutation rate in lung adenocarcinoma, this phenomenon has been observed for a number of epigenetic factors. We speculated that HMGN1 might be too critical to be mutated due to its importance in modulation of chromatin structure and histone modifications.\u003c/p\u003e \u003cp\u003eTo identify potential signaling pathways regulated by HMGN1, we constructed a module-centric co-expression network based on weighted correlation network analysis (WGCNA) and analyzed the possible functions of the correlated gene set by GO analysis. The results highlighted that cell division, protein ubiquitination, intracellular protein, cell cycle, and DNA repair were significantly associated with HMGN1 expression in LUAD. Moreover, functional enrichment analysis by GSEA confirmed that HMGN1 was highly involved in the regulation of the DNA repair signaling, which is in line with the previous findings in UV and irradiation-treated cells. Since the roles of HMGN1 in specific DNA repair pathways are not well characterized. We set to correlate the specific DNA repair pathways with HMGN1 expression and found that HRR and NER were the top two pathways among the six classic DNA repair pathways. Subsequent in vitro experiments using PC9 and A549 cells confirmed that silencing HMGN1 attenuated HR repair capacity.\u003c/p\u003e \u003cp\u003eIndeed, several studies suggest that HMGN1 manifests anti-tumor effects by promoting DNA repair and genome stability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, high HMGN1 expression correlates with poor prognosis in LUAD, despite its role in promoting DNA repair. Two recent studies have demonstrated thatHMGN1 acts chromatin architectural protein to compete with histone H1 for nucleosome binding sites [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. HMGN1 supports repair of DNA lesions, it would be expected that histone H1 will have an opposite effect on DNA damage repair. Indeed, a number of studies suggest that histone H1 has an inhibitory effect on repair of DNA lesions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Another investigation indicates that histone H1 suppresses repair of DSB in vitro when present in high concentrations [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The above observations suggest that the balance between the relative amounts of HMGN1 and histone H1, as well as their chromatin binding abilities, may be essential for the regulation of the cellular response to DNA damage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, how HMGN1 ultimately participates in the progression of lung adenocarcinoma remains to be explored.\u003c/p\u003e \u003cp\u003eHowever, there are some limitations to this study. First, although our experimental data showed the functional connection between HMGN1 and the DNA damage response pathway, the underlying mechanisms leading to the down-regulation of RAD51 and CHK1 phosphorylation remain to be determined. Perturbation of HMGN expression might interfere with the expression of some DNA damage response genes including RAD51 on the basis of the preferential association of HMGN proteins with chromatin regulatory sites including enhancers and promoters. Second, only one pathway of DNA repair has been verified, and other enrichment pathways need to be further addressed.\u003c/p\u003e \u003cp\u003eIn summary, our systematic analysis positions HMGN1 as a diagnostic and prognostic marker for lung adenocarcinoma. Moreover, HMGN1 holds great potential as an epigenetic therapeutic target given that HMGN1 plays critical roles in the DNA damage response, especially in HRR. Given the pivotal role of HMGN1 in peri-tumor infiltration of lymphocytes, combining HMGN1 targeting with immunotherapy might achieve better therapeutic outcomes for lung adenocarcinoma.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLUAD：Lung adenocarcinoma\u003c/p\u003e\n\u003cp\u003eHMGN: The high mobility group nucleosome-binding protein\u003c/p\u003e\n\u003cp\u003eCNV: DNA copy number\u0026nbsp;variation\u003c/p\u003e\n\u003cp\u003eROC: The receiver operating characteristic curve\u003c/p\u003e\n\u003cp\u003eAUC: Area under the curve\u003c/p\u003e\n\u003cp\u003eHR: Hazard ratio\u003c/p\u003e\n\u003cp\u003eOS: Overall survival\u003c/p\u003e\n\u003cp\u003ePFS: Progression survival\u003c/p\u003e\n\u003cp\u003eDFS: Disease-free survival\u003c/p\u003e\n\u003cp\u003eDSS: Disease-specific survival\u003c/p\u003e\n\u003cp\u003eHU: Hydroxyurea\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interests and do not exist any commercial or financial relationships, and acknowledgment that all authors have contributed significantly, and that all authors are in agree with the content of the manuscript. And The manuscript has not been published nor submitted for publication elsewhere except as a brief abstract in the proceedings of a scientific meeting or symposium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Source:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (81972191 and 81672647), Science and Technology Major Project of Anhui Province (18030801140), and the 100-Talent Program of Chinese Academy of Sciences. A portion of this work was supported by the High Magnetic Field Laboratory of Anhui Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conceptualization and design: WX, CG, FJ, LW; Development of methodology and investigation, WX, CG; Writing, review, and/or revision of the manuscript: WX, CG, FJ, LW. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the TCGA repository, https://tcgadata.nci.nih.gov/tcga; MEXPRESS repository, https://mexpress.be; UALCAN repository,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehttp://ualcan.path.uab.edu; Human Protein Atlas repository, https://www.proteinatlas.org; cBioPortal repository, https://www.cbioportal.org;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKMplot repository, https://.kmplot.com\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. 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Biochim Biophys Acta. 2010 Jan-Feb;1799(1\u0026ndash;2):80\u0026thinsp;\u0026ndash;\u0026thinsp;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbagrm.2009.10.007\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagrm.2009.10.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LUAD, HMGN1, biomarker, DNA repair","lastPublishedDoi":"10.21203/rs.3.rs-3669863/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3669863/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe high mobility group nucleosome binding (HMGN) family, constitutes a large family of non-histone protein family known to bind the acidic patch of the nucleosomes with various key cellular functions. Several studies have highlighted the pivotal roles of HMGNs in the pathogenic process of various cancer types. However, the roles of HMGN family in lung adenocarcinoma (LUAD) have not been fully elucidated. Herein, integrative analyses of multiple-omics data revealed that HMGNs frequently exhibit dysregulation in LUAD. Subsequent analysis of the clinical relevance of HMGN1 demonstrated its association with poor prognosis in LUAD and its potential as a diagnostic marker to differentiate LUAD from heathy controls. Additionally, functional enrichment analysis suggested that HMGN1 was mainly involved in DNA repair. To corroborate these findings, cellular experiments were conducted, confirming HMGN1\u0026rsquo;s crucial involvement in homologous recombination repair and its potential to enhance the sensitivity of LUAD cells to standard chemotherapeutic drugs. This study proposes HMGN1 as a novel prognostic biomarker and a promising target for chemotherapy in lung adenocarcinoma.\u003c/p\u003e","manuscriptTitle":"HMGN1 is a potential chemotherapeutic target for lung adenocarcinoma.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-01 17:22:36","doi":"10.21203/rs.3.rs-3669863/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-25T04:41:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-30T03:26:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f17b50d4-c1ee-4257-ad2d-38f7dff64c72","date":"2023-12-20T22:33:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d7f6e4f2-d8b5-4a83-991c-0a0f39f83cd4","date":"2023-12-20T00:10:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-17T19:43:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-11T16:50:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-11-28T07:10:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-28T07:03:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-11-27T02:23:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5dfa1273-2a2c-462d-aa37-bb641b5d1903","owner":[],"postedDate":"December 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":26800474,"name":"Biological sciences/Cancer/Lung cancer"},{"id":26800475,"name":"Biological sciences/Cancer/Tumour biomarkers"},{"id":26800476,"name":"Biological sciences/Cancer"},{"id":26800477,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":26800478,"name":"Biological sciences/Molecular biology"},{"id":26800479,"name":"Health sciences/Biomarkers"},{"id":26800480,"name":"Health sciences/Molecular medicine"},{"id":26800481,"name":"Health sciences/Oncology"}],"tags":[{"value":"featured","date":"2023-12-01 18:52:29"}],"updatedAt":"2024-05-14T21:23:21+00:00","versionOfRecord":{"articleIdentity":"rs-3669863","link":"https://doi.org/10.1038/s41598-024-60352-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-06 21:18:04","publishedOnDateReadable":"May 6th, 2024"},"versionCreatedAt":"2023-12-01 17:22:36","video":"","vorDoi":"10.1038/s41598-024-60352-8","vorDoiUrl":"https://doi.org/10.1038/s41598-024-60352-8","workflowStages":[]},"version":"v1","identity":"rs-3669863","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3669863","identity":"rs-3669863","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}