SPATS2 is correlated with cell cycle progression and immune cells infiltration in hepatocellular carcinoma

SPATS2 is correlated with cell cycle progression and immune cells infiltration in hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SPATS2 is correlated with cell cycle progression and immune cells infiltration in hepatocellular carcinoma Jia Yan, Jing Lin, Xiu ling Deng, Changshan Wang, Hai sheng Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2106467/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jan, 2023 Read the published version in BMC Gastroenterology → Version 1 posted 9 You are reading this latest preprint version Abstract The spermatogenesis associated serine rich 2 (SPATS2) is a member of RNA-binding protein in which the abnormal expression is linked with carcinogenesis in serval types of cancer. However, there is no systematic study on the differential expression, prognostic significance, epigenetic regulation, immune infiltration of SPATS2 in HCC. In the present study, we investigated the expression, prognosis, epigenetic regulation, and immune cell infiltration of SPATS2 in HCC. We found that the elevated expression of SPATS2 was unfavorably associated with the clinical pathological stage and prognosis. Functional enrichment analysis revealed that SPATS2 associated with cell cycle, apoptosis and cancer cell metastasis processes in HCC. Our results confirmed that knockdown of SPATS2 will affect cell cycle, apoptosis and invasion of HCC cell lines. Moreover, the expression of SPATS2 is upregulated by epigenetic regulation, including DNA methylation, m6A and histone modification in HCC. In addition, SPATS2 expression was positively correlated with immune cell or gene markers of immune infiltration in HCC. Taken together, our data demonstrated that SPATS2 was associated with progression and immune infiltration, and could serve as a prognostic biomarker for HCC. In conclusion, these results highlight the potential of SPATS2 to be used as a therapeutic target for HCC. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The spermatogenesis associated serine rich 2 (SPATS2) is a cytoplasmic RNA-binding protein, which is mainly expressed in adult testis and slightly expressed in liver and other tissues [ 1 , 2 ]. It has been reported that SPATS2 serves a tumorigenic role in several cancers, such as esophageal squamous cell carcinoma, colorectal cancer, prostate cancer, and hepatocellular carcinoma (HCC) [ 1 – 4 ]. SPATS2 is identified as a novel diagnostic biomarker in squamous cell carcinoma [ 5 ]. Moreover, SPATS2 promotes lncRNA SNHG5-mediated survival of colorectal cancer cells through pro-proliferative and anti-apoptotic effect [ 3 ]. Recently, SPATS2 is reported to be a diagnostic and prognostic biomarker in liver cancer [ 6 ]. SPATS2 negatively regulates by miR-145-5p and results in promoting hepatocellular carcinoma progression through regulating cell cycle [ 1 ]. Moreover, it is still involved in the proliferation and invasion of HCC cells through TRIM44-STAT3 signaling pathway [ 7 ]. Therefore, SPATS2 may be a potential liver cancer marker. However, the function of SPATS2 in HCC needs to be further clarified. Extensive studies have illustrated that the interplay between cancer cells and the tumor microenvironment (TME) plays a significant role in ineffective treatment and a poor prognosis of cancer [ 8 ]. The main cellular components in the HCC TME include immune cells, fibroblasts, macrophages, and cancer stem cells [ 9 ]. The levels of these cells and related molecules are crucial for the tumor cell survival, growth, proliferation, epithelial–mesenchymal transition, metastasis and tumor immune escape [ 10 ]. Therefore, finding and understanding the function of TME-related molecules are essential for the effective management and precision anticancer therapies [ 10 ]. In recent years, immunotherapy has brought beneficial effects in a variety of solid tumors. In hepatocellular carcinoma (HCC) patients, it was only a subgroup of HCC patients responded to immunotherapy [ 11 , 12 ]. It is important to explore new prognostic biomarkers and potential predictors of immunotherapeutic response for HCC. In this study, we assessed the diagnostic and prognostic values of SPATS2 in HCC. Moreover, the increased mRNA level and reduced methylation level of SPATS2 were associated with poor survival of patients with HCC. It is a prognostic biomarker and involved in cell cycle, apoptosis, and metastasis of HCC progressions. In addition, SPATS2 expression and its methylation were associated with the immune infiltration levels of different immune cell subtypes in HCC. Therefore, SPATS2 is likely a potential prognostic and diagnostic biomarker related to immune infiltration in HCC microenvironment. This will be benefit to improve the prognostic prediction and personalized treatment management of immunotherapy in HCC. Materials And Methods Gene expression analysis For differential expression analysis, GEPIA2 was used to detect the expression of SPATS2 based on the TCGA and GTEx (Gene Tissue Expression) databases. The expression of SPATS2 in different tumor stages and grades was confirmed using UALCAN database based on the TCGA _LIHC (Liver Hepatocellular Carcinoma) data. In addition, the protein expression level of SPATS2 was further verified by immunohistochemical staining in tumor tissues from patients with HCC using the HPA (Human Protein Atlas) database. Kaplan–Meier survival analysis The survival data of patients with liver cancer was derived from the TCGA database. Kaplan-Meier survival analysis was performed to determine the predictive value of SPATS2 in LIHC, including overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), and progression-free survival (PFS). Moreover, Kaplan-Meier survival analysis was completed to confirm the OS of patients with HCC based on the SPATS2 expression and the infiltration levels of different immune cell subtypes. Analysis hub genes of SPATS2 co-expressed in HCC We obtained the differentially expressed genes related to SPATS2 in LIHC using the LinkedOmics database. These SPATS2 related genes were annotated using Gene Ontology (GO) analysis. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed via the gene set enrichment analysis (GSEA) model in LIHC. Additionally, The GSCA database was used to analyze the pathway enrichment of the co-expressed gene set of SPATS2. The Cytoscape software was used to determine its hub genes in HCC base on the proteins interaction networks from String database. Cell culture and transfection The normal liver cell line (LO 2 ) and HCC cell lines (HepG2 and MHCC97-H) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% FBS (Gibco, USA), 10 U/ml penicillin, and 10 mg/ml streptomycin (Sigma, USA). The cells were grown in a sterile incubator with a humidified atmosphere containing 5% CO 2 at 37°C. The specific siRNA targeted to SPATS2 was synthesized by GenePharma (Shanghai, China). Empty vector was utilized as negative control. Lipofectamine 2000 (ThermoFisher Scientific, Waltham, USA) was used for the transfection of all these vectors and reagents into cells. All transfected cells were collected for subsequent use after 48h later. Cell apoptosis assay We collected 1×10 6 transfected HCC cells and treated with buffering from FITC-Annexin V apoptosis kit (Sungenebiotech, China), which includes 5 µl Annexin V-FITC and 5 µl PI for 20 minutes at room temperature in a dark environment. Then, the rate of cell apoptosis was obtained from flow cytometry (Beckman Coulter, Inc., Brea, USA). Cell cycle assay The transfected HCC cells were fixed with 70% pre-chilled ethanol overnight. They were washed with PBS and stained with 20 µL Propidium iodide (Sigma, USA). Then, the rate of different cell cycle was analyzed by flow cytometry (Beckman Coulter, Inc., Brea, USA). Cell invasion assay The transfected HCC cells (1×10 5 ) were suspended in 200 µl of serum-free medium and seeded into the upper chamber, while 600 µl of medium containing 10% FBS was added to the lower chamber. After incubation for 48 h at 37°C, the remaining cells on the upper surface were removed with cotton swabs. The membranes were fixed in methanol and stained with 0.5% crystal violet. Cells on the lower surface of the membrane were counted in randomly selected fields. Epigenetic analysis DNA methylation of SPATS2 at TSS1500 sites and the prognostic value of this site in HCC were confirmed by MethSurv database. The methylation level of the SPATS2 promoter region was determined via UALCAN database based on the TCGA_LIHC data. Immune infiltration analysis The tumor immune estimation resource (TIMER) database and the gene set cancer analysis (GSCA) was used to calculate the abundance of tumor infiltrating immune cells (TIICs) in tumor tissues of LIHC [ 13 , 14 ]. TIMER was used to analyze the mRNA expression-related infiltration of six types of immune cells in liver cancer. The relationships between SPATS2 mRNA levels and the infiltration of different immune cell subtypes were performed using the immune module of GSCA database. The correlations of SPATS2 expression with the critical immunomodulators in liver cancer were evaluated using TISIDB, which is an integrated repository portal for tumor-immune system interactions [ 15 ]. Statistical analysis All the experiment was independently repeated three times. Data were summarized as the mean ± standard deviation (SD). Partial results were analyzed using GraphPad Prism 8.0 software. The Student’s t -test was used to analyzed the different between two groups, while one-way ANOVA wase performed to evaluate the statistical significance among multiple groups. The results were considered to be statistically significant when the value of p was < 0.05. Results Relationships Between The Clinicopathological And Prognostic Features Of Spats2 In Hcc To further evaluate the functions of SPATS2, we assessed the profiles of SPATS2 expression across various types of cancers based on the results in TCGA databases. We found that SPATS2 was upregulated in LIHC (Fig. 1 A). GSCA result showed that SPATS2 expression was significantly increased in tumor tissues of LIHC than that in normal tissues (Fig. 1 B). Moreover, SPATS2 protein level also was upregulated in LIHC based on the protein expression results of HPA database in liver cancer (Fig. 1 C). Following, the relationships of SPATS2 expression with the clinicopathological parameters of patients with HCC were assessed by UALCAN database. As shown in Fig. 1 D, a significant correlation was found between SPATS2 expression and tumor grades of patients with HCC. Moreover, it is associated with pathologic stage of LIHC, especially in the stage III (Fig. 1 E, F). Additionally, SPATS2 was elevated in the nodal of metastasis status in LIHC (Fig. 1 G). Taken together, SPATS2 mRNA level was significantly correlated with clinicopathological parameters of patients with HCC. To investigate the association of SPATS2 expression with prognosis, the survival association analysis was performed in pan cancer. As shown in Fig. 2 A, overexpression of SPATS2 was related to poor prognosis in most cases, including LIHC. Moreover, Kaplan–Meier plotter tool showed that the elevated SPATS2 was drastically associated with a shorter OS, PFS, DSS, and DFI in patients with HCC (Fig. 2 B-E). These results suggested that SPATS2 has a good prognostic evaluation value in the whole processes of liver cancer development. Functional Enrichment Analysis Of Spats2 And Its Co-expressed Genes In Hcc To further explore the biological function of SPATS2 in HCC, we performed KEGG pathway analysis based on the TCGA-LIHC data. As shown in Fig. 3 A, the top three significantly enriched pathways of SPATS2 included cell cycle, spliceosome, and MicoRNAs in HCC cancer. Furthermore, pathways analysis via GSCA indicated that SPATS2 expression was markedly correlated with apoptosis, and EMT; whereases negatively associated with hormone, RASMAPK and RTK pathways (Fig. 3 B). GSEA results also showed that SPATS2 was positively associated with cell cycle, EMT and apoptosis (Fig. 3 C). Therefore, SPATS2 plays an important role in cell cycle, cell apoptosis, and cancer cell metastasis processes in HCC. Next, we obtained the SPATS2 mRNA expression-associated genes in liver cancer. In total, 401 positively (R > 0.5) and 159 negatively (R<-0.5) correlated gene were found, and the top 50 related genes were displayed on the heat map (Figure S1). These genes were significantly enriched for cancer-promoting terms, such as cell cycle and DNA replication (Fig. 3 D). Furthermore, GSCA pathways enrichment analysis results showed that these genes were also positively correlated with apoptosis, cell cycle and EMT pathways in LIHC (Fig. 3 E). Subsequently, these pathways related genes were obtained from GSEA database and PPI networks were constructed. In total 550 SPATS2 significantly related genes, we only found that SPATS2 directly interact with DLGAP5. GSEA result indicated that DLGAP5 was also enriched in the oxidative phosphorylation, cell cycle, and DNA repair pathways (Fig. 3 F). In addition, the interacted proteins of SPATS2 were obtained based on the GeneMANIA database (Fig. 3 G). YEATS2, AACS and PRDM4 were co-localization proteins of SPATS2. These genes were up-regulated in LIHC (Fig. 3 H). These results indicated that SPATS2 maybe regulate these genes expression to affect cell apoptosis, cell cycle, and invasion processes in HCC progression. Knockdown Of Spats2 Affects Cell Cycle, Apoptosis And Invasion Of Hcc Cells To further investigate the functions of SPATS2 in HCC progression, the siRNAs was transfected in HepG2 and MHCC97H cells to silence SPATS2 mRNA expression. Hereafter, we examined cell cycle and apoptosis progression in HCC cell lines by flow cytometry. The results showed that knockdown significantly increased the percentage of cells in the G0/G1 phase and decreased the G2/M phase, suggesting that SPATS2 may regulate the cell cycle in liver cells (Fig. 4 A-D). Moreover, the downregulation of SPATS2 reduced apoptosis of HCC cells ((Fig. 4 E-F). In addition, knockdown of SPATS2 dramatically inhibited the invasion ability of HCC cells (Fig. 4 I-M). Therefore, SPATS2 plays an important role in HCC progression. The Expression Of Spats2 Is Upregulated By Epigenetic Modification In Hcc It is well known that epigenetic regulation plays an important role in gene mRNA expression. DNA methylation level of SPATS2 was assessed in HCC. We found that SPATS2 was similarly unmethylated in HCC samples (Fig. 5 A). MSP assay indicated that the methylation level was decreased in HepG2 cells (Fig. 5 B). Moreover, high risk was observed in low methylation group (Fig. 5 C). The expression of SPATS2 was negatively associated with its methylation level of SPATS2 in HCC (Fig. 5 D). Considering that m6A methylation in tumorigenesis and development. Then, we explored the relationship between the expression of SPATS2 mRNA and m6A methylation in LIHC. The heatmap indicated that SPATS2 mRNA was positively correlated with most m6A methylation regulatory factors (Fig. 5 E). In addition, we also investigated the histone modification in SPATS2 promoter region. As shown in Fig. 5 F, H3K4me1, H3K4me3, H3K27ac, and H3K36me3 modifications that promote gene expression were significantly enriched in the promoter region of SPATS2 gene (Fig. 5 G). Additionally, Multiple acetylation and methylation sites were found in SPATS2 (Fig. 5 H). Taken together, these data suggested that epigenetic alterations play an important role in regulating the abnormal expression of SPATS2 in LIHC. Correlations Between Spats2 Expression And Immune Infiltration In Hcc To further reveal the functions of SPATS2 in the whole processes of liver cancer development, the relationship between the SPATS2 expression and immune cell in tumor microenvironment was investigated in LIHC. TIMER results indicated that there was a statistically positive correlation between SPATS2 mRNA expression and most of immune cells, such as B cells, CD4 + T cells, Macrophage, Neutrophil, and Dendritic cells (Fig. 6 A). Additionally, GSCA database results indicated that SPATS2 expression was positively associated with B cell and nTreg (cor > 0.4, p < 0.05) (Fig. 6 B). Interestingly, methylation level of SPATS2 was significantly negatively associated with most of immune cells based on the GSVA results (Fig. 6 C). In total, 15 of 24 immune cells were found that have a relationship with SPATS2 methylation. Specifically, the methylation of SPATS2 has a significant association (Cor<-0.4, p < 0.05) with immune cell CD8_T, Tfh, and Th1cells. To further investigate whether the expressions of SPATS2 affected prognosis of patients with LIHC is partly attributed to immune cells infiltration, a prognosis analysis was performed via the Kaplan Meier plotter based on the mRNA level of SPATS2 and the immune cells infiltration levels in HCC. The results showed that the high expression of SPATS2 with low CD4 + T and memory resting cells or neutrophil cells infiltrating indicated a worse prognosis in patients with HCC (Fig. 6 D, E). However, SPATS2 overexpression with high level of macrophages cell infiltration is associated with a worse prognosis in patients with HCC, especially macrophages M2 cell (Fig. 6 F, G). Taken together, SPATS2 overexpression may affect prognoses of patients with HCC in part due to immune cells infiltration. Expression Of Spats2 In Immune Cells Based On The Hcc Single-cell Sequencing Data In order to further elucidate the function of SPATS2 in immune infiltration, single-cell analysis was performed to assess the expression of SPATS2 in immune cells of HCC patients. As shown in Fig. 7 A, the tSNE maps demonstrated that 38 immune cell types were annotated. By comparing tumor with adjacent tissue, we found that SPATS2 level was widely expressed in B plasma, DC cell, and macrophages cells (Fig. 7 B, C). Moreover, cell interaction results showed that B plasma has a correlation with DC, NK and macrophages cells in HCC patients (Fig. 7 D). It is consistent with result that SPATS2 is positively associated with B cells, DC, and macrophage cells. Therefore, SPATS2 likely plays an important role in tumor immune response. Relationship Between Spats2 And Immune-related Genes In Hcc As the expression of SPATS2 showed a significant association with immune cell infiltration, we evaluated the correlations between the expression of SPATS2 and immunomodulators and the infiltration level in HCC. As shown in Fig. 7 E, SPATS2 expression was significantly correlated with immune-related inhibitory and stimulatory. Moreover, thirteen chemokine-related genes, nine MHC-related genes and receptor-related genes, were associated with SPATS2 expression (Fig. 7 E). Furthermore, the relationships between the SPATS2 expression and the most common immune checkpoints were analyzed. The expression of SPATS2 was positively correlated with most immune checkpoints in LIHC (Fig. 7 F). These findings further supported the result that SPATS2 is positively important in immune regulation of HCC. Discussion In recent years, SPATS2 has been reported to contribute to the tumorigenesis of multiple malignancies, including liver cancer [ 1 , 7 ]. However, the potential role of SPATS2 in HCC is yet to be elucidated, especially in tumor immune microenvironment. To further validate the function of SPATS2 in HCC, we comprehensively analyzed gene expression, prognosis, epigenetic regulation, and tumor immune cells infiltration of SPATS2 in HCC. In the present study, SPATS2 was determined to be upregulated in HCC tissues and cells. Furthermore, high SPATS2 expression was indicative of an unfavorable clinicopathological feature and poor prognosis in patients with HCC. Moreover, we found that SPATS2 dramatically promotes cells proliferation and invasion in HCC. Consistent with previous results, SPATS2 expression acts as an oncogene, which could be served as a diagnostic and prognostic biomarker in liver cancer. In addition, biological pathway and functional enrichment analysis in our present study illustrated that SPATS2 likely regulates cell cycle, apoptosis, and EMT in HCC. We further explored that SPATS2 co-expressed genes were also enriched for cell cycle, DNA replication, apoptosis, and EMT in HCC. Functionally, our results indicated that knockdown of SPATS2 likely dampened HCC development and metastasis by regulating cell cycle and cell apoptosis. Previous study has been reported that tumor immune cell infiltration can affect the effects of chemotherapy, radiotherapy, and immunotherapy in multiple cancer. Tumor immunotherapy is a relatively new treatment option for liver cancer [ 16 , 17 ]. In our study, SPATS2 was found for the first time to be highly positively correlated with activated immune cell subtypes infiltration, including macrophage, neutrophil, DC cell, B cell, and CD4 + T cell. Previous study has reported that macrophages is innate immune cell that plays a key role in early neoplastic transformation and metastatic progression [ 18 ]. It has been indicated that M2 macrophages can promote angiogenesis, metastasis, and, most significantly, immunosuppression of hepatocellular carcinoma cells [ 19 ]. Consistence with that, our study showed that SPATS2 overexpression with high macrophage cell infiltration patients had a worse overall survival, especially macrophage M2 cell. This result indicated that SPATS2 may be involved in maintaining the recruitment of M2 macrophages to affect macrophage cells infiltration in HCC. Additionally, a low neutrophil count in blood was favorable for prognosis in multiple cancer types. The infiltration of neutrophils into the tumor is an unfavorable prognostic marker in pancreatic carcinoma and renal cell carcinoma [ 20 ]. We found that the overexpression of SPATS2 with low level of neutrophil immune cell infiltration led to poor prognosis in HCC. Taken together, these results indicated that SPATS2 plays an important role in the recruitment and regulation of some immune infiltrating cells in liver cancer. SPATS2 may be served as a target gene for immunotherapy due to its positive correlation with immune checkpoint inhibitors. Epigenetic regulation was involved in the development and progression of a variety of tumors. We found that the expression of SPATS2 was significantly negatively correlated with the average methylation level of the promoter, suggesting that DNA methylation may be one of the mechanisms for its up-regulation. Moreover, our results showed that DNA methylation of SPATS2 was significantly negatively correlated with the infiltration of immune cells. SPATS2 was significantly negatively associated with the CD8 T, Th1, and Thf cells, positively correlated with the neutrophil and Th17 cells in HCC. It has been confirmed that the major reasons for tumor escape in the immune system include the dysfunction of CD8 + T cells and the presence of excessive suppressor T cells [ 21 ]. More research results have established the function of CD8 + T cells in the formation and progression of HCC, including diagnosis/treatment/prognosis [ 22 ]. Moreover, targeting CD8 + T cells is the main direction of immunotherapy for HCC [ 23 ]. The neutrophils cells are significantly associated with cancer progression and metastasis [ 24 ]. It also mainly suppresses antitumor immunity by inducing apoptosis of CD8 + T cells through nitric oxide production-mediated TNF-α [ 25 ]. Furthermore, we found that SPATS2 are positively correlated with the expression of multiple immune checkpoints. Therefore, these studies suggest that SPATS2 plays an important role in occurrence and development of tumors and contribute to the development of immunotherapy therapies. However, its exact mechanism needs to be further confirmed. In conclusion, our present study explored that SPATS2 was significantly correlated with cancer progression, poor survival, epigenetic regulation and immune infiltration in patients with HCC. Moreover, SPATS2 was associated with increased immune cellular infiltration and the expression of immune checkpoints. Therefore, SPATS2 may be an important factor in the progression of HCC. The present study helps us to elucidate the significance of SPATS2 in HCC progression, especially in tumor immune microenvironment. These results will provide a new theoretical basis for targeted therapy in HCC. Declarations Ethics approval and consent to participate TCGA and GEO belong to public databases. The patients involved in the database have obtained ethical approval. Users can download relevant data for free for research and publish relevant articles. Our study is based on open-source data, so there are no ethical issues and other conflicts of interest. Consent for publication Not applicable. Availability of data and materials The datasets used in this article are publicly available as described in Materials and Methods. All data generated during this study are included in this published article [and its supplementary information files]. The data available in TCGA (https://portal.gdc.cancer.gov/) databases. The datasets analyzed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no conflict of interest. Funding This work was supported by National Natural Science Foundation of China (CN) (grant Nos. 81660024), the Natural Science Foundation of Inner Mongolia (Grant Nos. 2020MS08096). Inner Mongolia Autonomous Region Science and Technology Innovation Guidance Project (KCBJ2018021) Authors' contributions Jia Yan designed the study and wrote the manuscript. All data analysis was performed by Jia Yan, Jing Lin and Xiu ling Deng. Hai sheng Wang and Chang Shan Wang provided reagents and participated in the preparation of the manuscript. All authors provided critical feedback on the manuscript. All authors read and approved the final manuscript. Acknowledgements No References Dong G, Zhang S, Shen S, Sun L, Wang X, Wang H, Wu J, Liu T, Wang C, Wang H, Lu T, Rao B, Ren Z. SPATS2, negatively regulated by miR-145-5p, promotes hepatocellular carcinoma progression through regulating cell cycle. Cell Death Dis. 2020;11(10):837. doi: 10.1038/s41419-020-03039-y. Senoo M, Hoshino S, Mochida N, Matsumura Y, Habu S. Identification of a novel protein p59(scr), which is expressed at specific stages of mouse spermatogenesis. Biochem Biophys Res Commun. 2002; 292:992–998. doi: 10.1006/bbrc.2002.6769. Damas ND, Marcatti M, Come C, Christensen LL, Nielsen MM, Baumgartner R, Gylling HM, Maglieri G, Rundsten CF, Seemann SE, et al. SNHG5 promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization. Liu YP, Cao Q, Li L, Zhang M. High expression of spermatogenesis associated serine rich 2 promotes tumorigenicity in esophageal squamous cell carcinoma cells and is associated with poor patient prognosis. Exp Ther Med. 2021 Jul;22(1):698. doi: 10.3892/etm.2021.10130. Takamochi K, et al. Novel biomarkers that assist in accurate discrimination of squamous cell carcinoma from adenocarcinoma of the lung. BMC Cancer. 2016; 16:760. doi: 10.1186/s12885-016-2792-1. Xing J, Tian Y, Ji W, Wang X. Comprehensive evaluation of SPATS2 expression and its prognostic potential in liver cancer. Medicine (Baltimore). 2020 Feb;99(9): e19230. doi: 10.1097/MD.0000000000019230. Chen L, Yi C, Li W, Tseng Y, Zhang J, Liu J. Inhibition of SPATS2 Suppresses Proliferation and Invasion of Hepatocellular Carcinoma Cells through TRIM44-STAT3 Signaling Pathway. J Cancer. 2021 Jan 1;12(1):89-98. doi: 10.7150/jca.47526. eCollection 2021. Wu ZH, Yang DL, Wang L, Liu J. Epigenetic and Immune-Cell Infiltration Changes in the Tumor Microenvironment in Hepatocellular Carcinoma. Front Immunol. 2021 Dec 2; 12:793343. doi: 10.3389/fimmu.2021.793343. eCollection 2021. Hernandez-Gea V, Toffanin S, Friedman SL, Llovet JM. Role of the Microenvironment in the Pathogenesis and Treatment of Hepatocellular Carcinoma. Gastroenterology (2013) 144(3):512–27. doi: 10.1053/j.gastro.2013.01.002 Miao D, Margolis CA, Gao W, Voss MH, Li W, Martini DJ, Norton C, Bossé D, Wankowicz SM, Cullen D, et al. Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma. Science (New York, NY). 2018;359(6377):801–6. El-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, Kim TY, Choo SP, Trojan J, Welling TH Rd, Meyer T, Kang YK, Yeo W, Chopra A, Anderson J, Dela Cruz C, Lang L, Neely J, Tang H, Dastani HB, Melero I. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet. 2017 Jun 24; 389(10088):2492-2502. Macek Jilkovaa Z, Ghelfi J, Decaens T. Immunomodulation for hepatocellular carcinoma therapy: current challenges. Curr Opin Oncol. 2021 Dec 17. doi: 10.1097/CCO.0000000000000812. Online ahead of print. Li T, Fan J, Wang B, Traugh N, Chen Q, Liu JS, Li B, Liu XS. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells. Cancer Research. 2017. 77(21): e108–e110. DOI 10.1158/0008-5472.CAN-17-0307. Liu CJ, Hu FF, Xia MX, Han L, Zhang Q, Guo AY. GSCALite: a web server for gene set cancer analysis. Bioinformatics. 2018. 34(21):3771-3772 DOI 10.1093/bioinformatics/bty411. Ru B, Wong CN, Tong Y, Zhong JY, Zhong SSW, Wu WC, Chu KC, Wong CY, Lau CY, Chen I, Chan NW, Zhang J. TISIDB: an integrated repository portal for tumor-immune system interactions. Bioinformatics. 2019. 35(20):4200–4202 DOI 10.1093/bioinformatics/btz210. Hilmi M, Vienot A, Rousseau B, et al. Immune therapy for liver cancers. Cancers (Basel) 2019; 12:77. 10.3390/cancers12010077. Nishida N, Kudo M. Immune phenotype and immune checkpoint inhibitors for the treatment of human hepatocellular carcinoma. Cancers (Basel) 2020; 12:1274. 10.3390/cancers12051274. Bourayou E, Golub R. Signaling Pathways Tuning Innate Lymphoid Cell Response to Hepatocellular Carcinoma. Front Immunol. 2022 Feb 23; 13:846923. doi: 10.3389/fimmu.2022.846923. Xu W, Cheng Y, Guo Y, Yao W, Qian H. Targeting tumor associated macrophages in hepatocellular carcinoma. Biochem Pharmacol. 2022 Mar 11:114990. doi: 10.1016/j.bcp.2022.114990. Moghaddam MZ, Ansariniya H, Seifati SM, Zare F, Fesahat F. Immunopathogenesis of endometriosis: An overview of the role of innate and adaptive immune cells and their mediators. Am J Reprod Immunol. 2022 Mar 9: e13537. doi: 10.1111/aji.13537. Zheng C, Zheng L, Yoo JK, Guo H, Zhang Y, Guo X, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell. 2017; 169:1342–56. doi: 10.1016/j.cell.2017.05.035. Xiaopei Hao, Guangshun Sun, Yao Zhang, Xiangyi Kong, Dawei Rong, Jinhua Song, Weiwei Tang, Xuehao Wang. Targeting Immune Cells in the Tumor Microenvironment of HCC: New Opportunities and Challenges. Front Cell Dev Biol. 2021; 9: 775462. Published online 2021 Nov 12. doi: 10.3389/fcell.2021.775462. Ma J, Zheng B, Goswami S, Meng L, Zhang D, Cao C, et al. PD1(Hi) CD8(+) T cells correlate with exhausted signature and poor clinical outcome in hepatocellular carcinoma. J Immunother Cancer. 2019; 7:331. doi: 10.1186/s40425-019-0814-7. Wu L., Saxena S., Awaji M., Singh R.K. Tumor-Associated neutrophils in cancer: Going pro. Cancers. 2019; 11:564. doi: 10.3390/cancers11040564. Michaeli J., Shaul M.E., Mishalian I., Hovav A.H., Levy L., Zolotriov L., Granot Z., Fridlender Z.G. Tumor-Associated neutrophils induce apoptosis of non-activated CD8 T-cells in a TNFalpha and NO-dependent mechanism, promoting a tumor-supportive environment. Oncoimmunology. 2017;6: e1356965. doi: 10.1080/2162402X.2017.1356965. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 11 Jan, 2023 Read the published version in BMC Gastroenterology → Version 1 posted Editorial decision: Major revision 22 Nov, 2022 Reviews received at journal 20 Nov, 2022 Reviews received at journal 19 Oct, 2022 Reviewers agreed at journal 01 Oct, 2022 Reviewers invited by journal 01 Oct, 2022 Editor assigned by journal 01 Oct, 2022 Editor invited by journal 30 Sep, 2022 Submission checks completed at journal 30 Sep, 2022 First submitted to journal 26 Sep, 2022 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-2106467","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":140881090,"identity":"a5faae43-6a3f-401c-98ee-469882c3ede8","order_by":0,"name":"Jia Yan","email":"","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Yan","suffix":""},{"id":140881091,"identity":"5699f46f-00e3-48cd-a04f-b1a2e22db2b7","order_by":1,"name":"Jing Lin","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Lin","suffix":""},{"id":140881092,"identity":"2dd686d7-6806-4c42-baa5-43581bbe094e","order_by":2,"name":"Xiu ling Deng","email":"","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"ling","lastName":"Deng","suffix":""},{"id":140881093,"identity":"7d4798d0-c01c-43da-aa3f-5c250864b20e","order_by":3,"name":"Changshan Wang","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changshan","middleName":"","lastName":"Wang","suffix":""},{"id":140881094,"identity":"544c40e5-e04a-404d-ba8d-ce2994620eb5","order_by":4,"name":"Hai sheng Wang","email":"data:image/png;base64,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","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hai","middleName":"sheng","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-09-27 01:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2106467/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2106467/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12876-022-02633-y","type":"published","date":"2023-01-11T18:17:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27388978,"identity":"7419616d-f822-41ac-a5cb-f694091a7ae7","added_by":"auto","created_at":"2022-10-05 18:25:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1017447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical implication of SPATS2 in LIHC. \u003c/strong\u003e(A) The expression of SPATS2 in pan cancer based on the TCGA results. (B) The expression of SPATS2 in LIHC and paired normal tissues by box plot via GSCA database. (C) Immunohistochemical analysis of SPATS2 protein expression in HCC tissues based on the protein expression results of HPA database. (D-G) Correlation of SPATS2 with clinical information of HCC. (D) SPATS2 is correlated with tumor grades of LIHC. (E, F) SPATS2 is associated with pathologic stage of LIHC. It is upregulated in stage III of patients with HCC. (G) Expression of SPATS2 is higher in metastasis status of LIHC. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/3bb12321ed0fe0253c237dba.png"},{"id":27389241,"identity":"74f427c2-6d8c-4609-897c-c6fff1c470ee","added_by":"auto","created_at":"2022-10-05 18:30:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1828251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrognostic value of SPATS2 in patients with HCC. \u003c/strong\u003e(A) Survival map of SPATS2 gene with significant associations in pan cancer. (B-E) The high expression group of SPATS2 has significantly worse OS (B), PFS (C), DSS (D), and DFI (E) than the low expression group in HCC. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/4d225df9126d19acb3c5fd07.png"},{"id":27388982,"identity":"c6ac18e9-4f29-4c5a-b9b4-b5a72a8a4261","added_by":"auto","created_at":"2022-10-05 18:25:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1526648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnrichment analysis of SPATS2 and its co-expressed genes in HCC. \u003c/strong\u003e(A) The enrichment analysis based on TCGA_LIHC data. Representative GO terms of SPATS2 in LIHC. (B) The enrichment analysis of SPATS2-related pathways in LIHC. Red represents positive correlation; blue represents negative correlation. (C) GSEA analysis the enrichment of signature genes involved in E2F target, G2M checkpoint, EMT, and apoptosis in SPATS2 high or low expression group. (D) The KEGG function enrichment analysis of SPATS2-related genes in LIHC. (E) The biological pathways of SPATS2 co-expressed genes in HCC. Red represents positive correlation; blue represents negative correlation. * indicates \u003cem\u003eP \u003c/em\u003evalue ≤ 0.05; # represents FDR ≤ 0.05.\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant. (F) GSEA analysis the enrichment of signature genes involved in E2F target, G2M checkpoint, Mitotic spindle, and DNA repair in DLGAP5 high or low expression group. (G) SPATS2 interacting proteins are predicted through GeneMANIA database. (H) The expression of SPATS2 interacting proteins (DLGAP5, YEATS2, AACS and PRDM4) in HCC.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/d5ca853b9f8004fbbd4e6b04.png"},{"id":27389244,"identity":"77460635-b7b8-40a2-a130-6bd512cdcb8b","added_by":"auto","created_at":"2022-10-05 18:30:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5792693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SPATS2 affects cell cycle, apoptosis and cell invasion in HCC cells. \u003c/strong\u003e(A-D) The flow cytometry was performed to analyze the cell cycle distribution of HepG2 and MHCC97-H cells after SPATS2 down-regulation. (E-H) The flow cytometry was performed to analyze the cell apoptosis of HepG2 and MHCC97-H cells after SPATS2 down-regulation. (I-M) Transwell assays were performed to detect the migration of HepG2 and MHCC97-H cells after SPATS2 down-regulation. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/ceb3de0734f5c001ab612f79.png"},{"id":27389679,"identity":"ea98f0fc-c856-417b-b9b0-69d13b8d6f47","added_by":"auto","created_at":"2022-10-05 18:40:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1055977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of epigenetic alterations associated with SPATS2 in HCC.\u003c/strong\u003e (A-D) Correlation between SPATS2 mRNA expression and its methylation levels. (A\u003cstrong\u003e) \u003c/strong\u003ePromoter methylation level of SPATS2 is lower in primary tumor than normal tissue. (B) Promoter methylation level of SPATS2 is decreased in HepG2 cells. (C) The overall survival was compared between patients with high or low methylation of SPATS2. (D) The correlation between SPATS2 expression and DNA methylation level. (E) The correlation between the expression of SPATS2 mRNA and m6A methylation regulatory factors in HCC. Correlations are depicted with Spearman’s rho values and statistical significance. (F-H) Histones epigenetic alterations in SPATS2 promoter. (F) Histones epigenetic regulatory potential in SPATS2 mRNA expression. (G) H3K4me1, H3K4me3, H3K27ac, H3K9ac were found enrichment in SPATS2 promoter in HepG2 cell. (H) Methylation and acetylation sites in SPATS2 protein.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/2b68b877f9ae2064b2b4f461.png"},{"id":27389316,"identity":"749d47a4-82e2-47e7-bd32-5b942fff498d","added_by":"auto","created_at":"2022-10-05 18:35:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1247896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between immune cell infiltration and SPATS2 level in HCC.\u003c/strong\u003e (A) Correlation of SPATS2 expression with immune cells infiltration levels in HCC. (B, C) Correlations of SPATS2 expression (B) and methylation (C) with infiltration levels of different immune cell subtypes in HCC. Red represents positive correlation; blue represents negative correlation. (D-F) Comparison of Kaplan-Meier survival curves of the high and low expression of SPATS2 in HCC based on immune cell subgroups, including CD4+ T and memory resting cell (D), neutrophil cell (E), macrophages cell (F), and macrophages M2 cell (G).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/eca2f4040d6f3ef854c4dba5.png"},{"id":27388984,"identity":"409cd4cc-25eb-4378-bc04-09734424a6db","added_by":"auto","created_at":"2022-10-05 18:25:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2897969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA-seq analysis of SPATS2 in the tumor tissue of HCC patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The t-SNE map depicting clusters of immune cells in the tumor tissues and adjacent tissues of five HCC patients. (B) Expression t-SNE maps for the SPATS2 in the tumor tissues and adjacent tissues of HCC. (C) The iolin plots showing the distribution of SPATS2 in various immune cell clusters. (D) Circle plot illustrating the Interaction between B plasma and other immune cells. (E) Heat maps show that the expression of SPATS2 has a significantly positive correlation with immune related genes, including inhibitory and stimulatory, immune chemokine, receptor, MHC. (F). Heat maps display a correlation between SPATS2 expression and immune checkpoints. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/65c5e9cf56068e271480b52a.png"},{"id":44716306,"identity":"5e16d7a2-ae1b-49d4-b773-26e449433723","added_by":"auto","created_at":"2023-10-16 18:25:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3672543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/e1991638-94ed-4c88-9c0a-4d197e662ff0.pdf"},{"id":27388979,"identity":"7b427064-a576-4ad9-93ad-8adb7d4393e7","added_by":"auto","created_at":"2022-10-05 18:25:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2632244,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2106467/v1/0adcd6c2220c0c4075d59442.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SPATS2 is correlated with cell cycle progression and immune cells infiltration in hepatocellular carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe spermatogenesis associated serine rich 2 (SPATS2) is a cytoplasmic RNA-binding protein, which is mainly expressed in adult testis and slightly expressed in liver and other tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It has been reported that SPATS2 serves a tumorigenic role in several cancers, such as esophageal squamous cell carcinoma, colorectal cancer, prostate cancer, and hepatocellular carcinoma (HCC) [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. SPATS2 is identified as a novel diagnostic biomarker in squamous cell carcinoma [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, SPATS2 promotes lncRNA SNHG5-mediated survival of colorectal cancer cells through pro-proliferative and anti-apoptotic effect [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recently, SPATS2 is reported to be a diagnostic and prognostic biomarker in liver cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. SPATS2 negatively regulates by miR-145-5p and results in promoting hepatocellular carcinoma progression through regulating cell cycle [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Moreover, it is still involved in the proliferation and invasion of HCC cells through TRIM44-STAT3 signaling pathway [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, SPATS2 may be a potential liver cancer marker. However, the function of SPATS2 in HCC needs to be further clarified.\u003c/p\u003e \u003cp\u003eExtensive studies have illustrated that the interplay between cancer cells and the tumor microenvironment (TME) plays a significant role in ineffective treatment and a poor prognosis of cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The main cellular components in the HCC TME include immune cells, fibroblasts, macrophages, and cancer stem cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The levels of these cells and related molecules are crucial for the tumor cell survival, growth, proliferation, epithelial\u0026ndash;mesenchymal transition, metastasis and tumor immune escape [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, finding and understanding the function of TME-related molecules are essential for the effective management and precision anticancer therapies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In recent years, immunotherapy has brought beneficial effects in a variety of solid tumors. In hepatocellular carcinoma (HCC) patients, it was only a subgroup of HCC patients responded to immunotherapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is important to explore new prognostic biomarkers and potential predictors of immunotherapeutic response for HCC.\u003c/p\u003e \u003cp\u003eIn this study, we assessed the diagnostic and prognostic values of SPATS2 in HCC. Moreover, the increased mRNA level and reduced methylation level of SPATS2 were associated with poor survival of patients with HCC. It is a prognostic biomarker and involved in cell cycle, apoptosis, and metastasis of HCC progressions. In addition, SPATS2 expression and its methylation were associated with the immune infiltration levels of different immune cell subtypes in HCC. Therefore, SPATS2 is likely a potential prognostic and diagnostic biomarker related to immune infiltration in HCC microenvironment. This will be benefit to improve the prognostic prediction and personalized treatment management of immunotherapy in HCC.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis\u003c/h2\u003e \u003cp\u003eFor differential expression analysis, GEPIA2 was used to detect the expression of SPATS2 based on the TCGA and GTEx (Gene Tissue Expression) databases. The expression of SPATS2 in different tumor stages and grades was confirmed using UALCAN database based on the TCGA _LIHC (Liver Hepatocellular Carcinoma) data. In addition, the protein expression level of SPATS2 was further verified by immunohistochemical staining in tumor tissues from patients with HCC using the HPA (Human Protein Atlas) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKaplan\u0026ndash;Meier survival analysis\u003c/h2\u003e \u003cp\u003eThe survival data of patients with liver cancer was derived from the TCGA database. Kaplan-Meier survival analysis was performed to determine the predictive value of SPATS2 in LIHC, including overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), and progression-free survival (PFS). Moreover, Kaplan-Meier survival analysis was completed to confirm the OS of patients with HCC based on the SPATS2 expression and the infiltration levels of different immune cell subtypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis hub genes of SPATS2 co-expressed in HCC\u003c/h2\u003e \u003cp\u003eWe obtained the differentially expressed genes related to SPATS2 in LIHC using the LinkedOmics database. These SPATS2 related genes were annotated using Gene Ontology (GO) analysis. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed via the gene set enrichment analysis (GSEA) model in LIHC. Additionally, The GSCA database was used to analyze the pathway enrichment of the co-expressed gene set of SPATS2. The Cytoscape software was used to determine its hub genes in HCC base on the proteins interaction networks from String database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eThe normal liver cell line (LO\u003csub\u003e2\u003c/sub\u003e) and HCC cell lines (HepG2 and MHCC97-H) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, USA) containing 10% FBS (Gibco, USA), 10 U/ml penicillin, and 10 mg/ml streptomycin (Sigma, USA). The cells were grown in a sterile incubator with a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe specific siRNA targeted to \u003cem\u003eSPATS2\u003c/em\u003e was synthesized by GenePharma (Shanghai, China). Empty vector was utilized as negative control. Lipofectamine 2000 (ThermoFisher Scientific, Waltham, USA) was used for the transfection of all these vectors and reagents into cells. All transfected cells were collected for subsequent use after 48h later.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assay\u003c/h2\u003e \u003cp\u003eWe collected 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e transfected HCC cells and treated with buffering from FITC-Annexin V apoptosis kit (Sungenebiotech, China), which includes 5 \u0026micro;l Annexin V-FITC and 5 \u0026micro;l PI for 20 minutes at room temperature in a dark environment. Then, the rate of cell apoptosis was obtained from flow cytometry (Beckman Coulter, Inc., Brea, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle assay\u003c/h2\u003e \u003cp\u003eThe transfected HCC cells were fixed with 70% pre-chilled ethanol overnight. They were washed with PBS and stained with 20 \u0026micro;L Propidium iodide (Sigma, USA). Then, the rate of different cell cycle was analyzed by flow cytometry (Beckman Coulter, Inc., Brea, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion assay\u003c/h2\u003e \u003cp\u003eThe transfected HCC cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were suspended in 200 \u0026micro;l of serum-free medium and seeded into the upper chamber, while 600 \u0026micro;l of medium containing 10% FBS was added to the lower chamber. After incubation for 48 h at 37\u0026deg;C, the remaining cells on the upper surface were removed with cotton swabs. The membranes were fixed in methanol and stained with 0.5% crystal violet. Cells on the lower surface of the membrane were counted in randomly selected fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEpigenetic analysis\u003c/h2\u003e \u003cp\u003eDNA methylation of SPATS2 at TSS1500 sites and the prognostic value of this site in HCC were confirmed by MethSurv database. The methylation level of the SPATS2 promoter region was determined via UALCAN database based on the TCGA_LIHC data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmune infiltration analysis\u003c/h2\u003e \u003cp\u003eThe tumor immune estimation resource (TIMER) database and the gene set cancer analysis (GSCA) was used to calculate the abundance of tumor infiltrating immune cells (TIICs) in tumor tissues of LIHC [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TIMER was used to analyze the mRNA expression-related infiltration of six types of immune cells in liver cancer. The relationships between SPATS2 mRNA levels and the infiltration of different immune cell subtypes were performed using the immune module of GSCA database. The correlations of SPATS2 expression with the critical immunomodulators in liver cancer were evaluated using TISIDB, which is an integrated repository portal for tumor-immune system interactions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiment was independently repeated three times. Data were summarized as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Partial results were analyzed using GraphPad Prism 8.0 software. The Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to analyzed the different between two groups, while one-way ANOVA wase performed to evaluate the statistical significance among multiple groups. The results were considered to be statistically significant when the value of \u003cem\u003ep\u003c/em\u003e was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\n\u003ch3\u003eRelationships Between The Clinicopathological And Prognostic Features Of Spats2 In Hcc\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the functions of SPATS2, we assessed the profiles of SPATS2 expression across various types of cancers based on the results in TCGA databases. We found that SPATS2 was upregulated in LIHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). GSCA result showed that SPATS2 expression was significantly increased in tumor tissues of LIHC than that in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, SPATS2 protein level also was upregulated in LIHC based on the protein expression results of HPA database in liver cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Following, the relationships of SPATS2 expression with the clinicopathological parameters of patients with HCC were assessed by UALCAN database. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, a significant correlation was found between SPATS2 expression and tumor grades of patients with HCC. Moreover, it is associated with pathologic stage of LIHC, especially in the stage III (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). Additionally, SPATS2 was elevated in the nodal of metastasis status in LIHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Taken together, SPATS2 mRNA level was significantly correlated with clinicopathological parameters of patients with HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the association of SPATS2 expression with prognosis, the survival association analysis was performed in pan cancer. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, overexpression of SPATS2 was related to poor prognosis in most cases, including LIHC. Moreover, Kaplan\u0026ndash;Meier plotter tool showed that the elevated SPATS2 was drastically associated with a shorter OS, PFS, DSS, and DFI in patients with HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). These results suggested that SPATS2 has a good prognostic evaluation value in the whole processes of liver cancer development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFunctional Enrichment Analysis Of Spats2 And Its Co-expressed Genes In Hcc\u003c/h3\u003e\n\u003cp\u003eTo further explore the biological function of SPATS2 in HCC, we performed KEGG pathway analysis based on the TCGA-LIHC data. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the top three significantly enriched pathways of SPATS2 included cell cycle, spliceosome, and MicoRNAs in HCC cancer. Furthermore, pathways analysis via GSCA indicated that SPATS2 expression was markedly correlated with apoptosis, and EMT; whereases negatively associated with hormone, RASMAPK and RTK pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). GSEA results also showed that SPATS2 was positively associated with cell cycle, EMT and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Therefore, SPATS2 plays an important role in cell cycle, cell apoptosis, and cancer cell metastasis processes in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we obtained the SPATS2 mRNA expression-associated genes in liver cancer. In total, 401 positively (R\u0026thinsp;\u0026gt;\u0026thinsp;0.5) and 159 negatively (R\u0026lt;-0.5) correlated gene were found, and the top 50 related genes were displayed on the heat map (Figure S1). These genes were significantly enriched for cancer-promoting terms, such as cell cycle and DNA replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, GSCA pathways enrichment analysis results showed that these genes were also positively correlated with apoptosis, cell cycle and EMT pathways in LIHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eSubsequently, these pathways related genes were obtained from GSEA database and PPI networks were constructed. In total 550 SPATS2 significantly related genes, we only found that SPATS2 directly interact with DLGAP5. GSEA result indicated that DLGAP5 was also enriched in the oxidative phosphorylation, cell cycle, and DNA repair pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In addition, the interacted proteins of SPATS2 were obtained based on the GeneMANIA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). YEATS2, AACS and PRDM4 were co-localization proteins of SPATS2. These genes were up-regulated in LIHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These results indicated that SPATS2 maybe regulate these genes expression to affect cell apoptosis, cell cycle, and invasion processes in HCC progression.\u003c/p\u003e\n\u003ch3\u003eKnockdown Of Spats2 Affects Cell Cycle, Apoptosis And Invasion Of Hcc Cells\u003c/h3\u003e\n\u003cp\u003eTo further investigate the functions of SPATS2 in HCC progression, the siRNAs was transfected in HepG2 and MHCC97H cells to silence SPATS2 mRNA expression. Hereafter, we examined cell cycle and apoptosis progression in HCC cell lines by flow cytometry. The results showed that knockdown significantly increased the percentage of cells in the G0/G1 phase and decreased the G2/M phase, suggesting that SPATS2 may regulate the cell cycle in liver cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Moreover, the downregulation of SPATS2 reduced apoptosis of HCC cells ((Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). In addition, knockdown of SPATS2 dramatically inhibited the invasion ability of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-M). Therefore, SPATS2 plays an important role in HCC progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe Expression Of Spats2 Is Upregulated By Epigenetic Modification In Hcc\u003c/h3\u003e\n\u003cp\u003eIt is well known that epigenetic regulation plays an important role in gene mRNA expression. DNA methylation level of SPATS2 was assessed in HCC. We found that SPATS2 was similarly unmethylated in HCC samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). MSP assay indicated that the methylation level was decreased in HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, high risk was observed in low methylation group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The expression of SPATS2 was negatively associated with its methylation level of SPATS2 in HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering that m6A methylation in tumorigenesis and development. Then, we explored the relationship between the expression of SPATS2 mRNA and m6A methylation in LIHC. The heatmap indicated that SPATS2 mRNA was positively correlated with most m6A methylation regulatory factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In addition, we also investigated the histone modification in SPATS2 promoter region. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, H3K4me1, H3K4me3, H3K27ac, and H3K36me3 modifications that promote gene expression were significantly enriched in the promoter region of SPATS2 gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Additionally, Multiple acetylation and methylation sites were found in SPATS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Taken together, these data suggested that epigenetic alterations play an important role in regulating the abnormal expression of SPATS2 in LIHC.\u003c/p\u003e\n\u003ch3\u003eCorrelations Between Spats2 Expression And Immune Infiltration In Hcc\u003c/h3\u003e\n\u003cp\u003eTo further reveal the functions of SPATS2 in the whole processes of liver cancer development, the relationship between the SPATS2 expression and immune cell in tumor microenvironment was investigated in LIHC. TIMER results indicated that there was a statistically positive correlation between SPATS2 mRNA expression and most of immune cells, such as B cells, CD4\u0026thinsp;+\u0026thinsp;T cells, Macrophage, Neutrophil, and Dendritic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, GSCA database results indicated that SPATS2 expression was positively associated with B cell and nTreg (cor\u0026thinsp;\u0026gt;\u0026thinsp;0.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Interestingly, methylation level of SPATS2 was significantly negatively associated with most of immune cells based on the GSVA results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In total, 15 of 24 immune cells were found that have a relationship with SPATS2 methylation. Specifically, the methylation of SPATS2 has a significant association (Cor\u0026lt;-0.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with immune cell CD8_T, Tfh, and Th1cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate whether the expressions of SPATS2 affected prognosis of patients with LIHC is partly attributed to immune cells infiltration, a prognosis analysis was performed via the Kaplan Meier plotter based on the mRNA level of SPATS2 and the immune cells infiltration levels in HCC. The results showed that the high expression of SPATS2 with low CD4\u0026thinsp;+\u0026thinsp;T and memory resting cells or neutrophil cells infiltrating indicated a worse prognosis in patients with HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). However, SPATS2 overexpression with high level of macrophages cell infiltration is associated with a worse prognosis in patients with HCC, especially macrophages M2 cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). Taken together, SPATS2 overexpression may affect prognoses of patients with HCC in part due to immune cells infiltration.\u003c/p\u003e\n\u003ch3\u003eExpression Of Spats2 In Immune Cells Based On The Hcc Single-cell Sequencing Data\u003c/h3\u003e\n\u003cp\u003eIn order to further elucidate the function of SPATS2 in immune infiltration, single-cell analysis was performed to assess the expression of SPATS2 in immune cells of HCC patients. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the tSNE maps demonstrated that 38 immune cell types were annotated. By comparing tumor with adjacent tissue, we found that SPATS2 level was widely expressed in B plasma, DC cell, and macrophages cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). Moreover, cell interaction results showed that B plasma has a correlation with DC, NK and macrophages cells in HCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). It is consistent with result that SPATS2 is positively associated with B cells, DC, and macrophage cells. Therefore, SPATS2 likely plays an important role in tumor immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRelationship Between Spats2 And Immune-related Genes In Hcc\u003c/h3\u003e\n\u003cp\u003eAs the expression of SPATS2 showed a significant association with immune cell infiltration, we evaluated the correlations between the expression of SPATS2 and immunomodulators and the infiltration level in HCC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, SPATS2 expression was significantly correlated with immune-related inhibitory and stimulatory. Moreover, thirteen chemokine-related genes, nine MHC-related genes and receptor-related genes, were associated with SPATS2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Furthermore, the relationships between the SPATS2 expression and the most common immune checkpoints were analyzed. The expression of SPATS2 was positively correlated with most immune checkpoints in LIHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These findings further supported the result that SPATS2 is positively important in immune regulation of HCC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, SPATS2 has been reported to contribute to the tumorigenesis of multiple malignancies, including liver cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the potential role of SPATS2 in HCC is yet to be elucidated, especially in tumor immune microenvironment. To further validate the function of SPATS2 in HCC, we comprehensively analyzed gene expression, prognosis, epigenetic regulation, and tumor immune cells infiltration of SPATS2 in HCC. In the present study, SPATS2 was determined to be upregulated in HCC tissues and cells. Furthermore, high SPATS2 expression was indicative of an unfavorable clinicopathological feature and poor prognosis in patients with HCC. Moreover, we found that SPATS2 dramatically promotes cells proliferation and invasion in HCC. Consistent with previous results, SPATS2 expression acts as an oncogene, which could be served as a diagnostic and prognostic biomarker in liver cancer. In addition, biological pathway and functional enrichment analysis in our present study illustrated that SPATS2 likely regulates cell cycle, apoptosis, and EMT in HCC. We further explored that SPATS2 co-expressed genes were also enriched for cell cycle, DNA replication, apoptosis, and EMT in HCC. Functionally, our results indicated that knockdown of SPATS2 likely dampened HCC development and metastasis by regulating cell cycle and cell apoptosis.\u003c/p\u003e \u003cp\u003ePrevious study has been reported that tumor immune cell infiltration can affect the effects of chemotherapy, radiotherapy, and immunotherapy in multiple cancer. Tumor immunotherapy is a relatively new treatment option for liver cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our study, SPATS2 was found for the first time to be highly positively correlated with activated immune cell subtypes infiltration, including macrophage, neutrophil, DC cell, B cell, and CD4\u0026thinsp;+\u0026thinsp;T cell. Previous study has reported that macrophages is innate immune cell that plays a key role in early neoplastic transformation and metastatic progression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has been indicated that M2 macrophages can promote angiogenesis, metastasis, and, most significantly, immunosuppression of hepatocellular carcinoma cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consistence with that, our study showed that SPATS2 overexpression with high macrophage cell infiltration patients had a worse overall survival, especially macrophage M2 cell. This result indicated that SPATS2 may be involved in maintaining the recruitment of M2 macrophages to affect macrophage cells infiltration in HCC. Additionally, a low neutrophil count in blood was favorable for prognosis in multiple cancer types. The infiltration of neutrophils into the tumor is an unfavorable prognostic marker in pancreatic carcinoma and renal cell carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We found that the overexpression of SPATS2 with low level of neutrophil immune cell infiltration led to poor prognosis in HCC. Taken together, these results indicated that SPATS2 plays an important role in the recruitment and regulation of some immune infiltrating cells in liver cancer. SPATS2 may be served as a target gene for immunotherapy due to its positive correlation with immune checkpoint inhibitors.\u003c/p\u003e \u003cp\u003eEpigenetic regulation was involved in the development and progression of a variety of tumors. We found that the expression of SPATS2 was significantly negatively correlated with the average methylation level of the promoter, suggesting that DNA methylation may be one of the mechanisms for its up-regulation. Moreover, our results showed that DNA methylation of SPATS2 was significantly negatively correlated with the infiltration of immune cells. SPATS2 was significantly negatively associated with the CD8 T, Th1, and Thf cells, positively correlated with the neutrophil and Th17 cells in HCC. It has been confirmed that the major reasons for tumor escape in the immune system include the dysfunction of CD8\u0026thinsp;+\u0026thinsp;T cells and the presence of excessive suppressor T cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. More research results have established the function of CD8\u0026thinsp;+\u0026thinsp;T cells in the formation and progression of HCC, including diagnosis/treatment/prognosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, targeting CD8\u0026thinsp;+\u0026thinsp;T cells is the main direction of immunotherapy for HCC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The neutrophils cells are significantly associated with cancer progression and metastasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It also mainly suppresses antitumor immunity by inducing apoptosis of CD8\u0026thinsp;+\u0026thinsp;T cells through nitric oxide production-mediated TNF-α [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, we found that SPATS2 are positively correlated with the expression of multiple immune checkpoints. Therefore, these studies suggest that SPATS2 plays an important role in occurrence and development of tumors and contribute to the development of immunotherapy therapies. However, its exact mechanism needs to be further confirmed.\u003c/p\u003e \u003cp\u003eIn conclusion, our present study explored that SPATS2 was significantly correlated with cancer progression, poor survival, epigenetic regulation and immune infiltration in patients with HCC. Moreover, SPATS2 was associated with increased immune cellular infiltration and the expression of immune checkpoints. Therefore, SPATS2 may be an important factor in the progression of HCC. The present study helps us to elucidate the significance of SPATS2 in HCC progression, especially in tumor immune microenvironment. These results will provide a new theoretical basis for targeted therapy in HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTCGA and GEO belong to public databases. The patients involved in the database have obtained ethical approval. Users can download relevant data for free for research and publish relevant articles. Our study is based on open-source data, so there are no ethical issues and other conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this article are publicly available as described in Materials and Methods. All data generated during this study are included in this published article [and its supplementary information files]. The data available in TCGA (https://portal.gdc.cancer.gov/) databases. The datasets analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (CN) (grant Nos. 81660024), the Natural Science Foundation of Inner Mongolia (Grant Nos. 2020MS08096). Inner Mongolia Autonomous Region Science and Technology Innovation Guidance Project (KCBJ2018021)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJia Yan designed the study and wrote the manuscript. All data analysis was performed by Jia Yan, Jing Lin and Xiu ling Deng. Hai sheng Wang and Chang Shan Wang provided reagents and participated in the preparation of the manuscript. All authors provided critical feedback on the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n\u003cli\u003eDong G, Zhang S, Shen S, Sun L, Wang X, Wang H, Wu J, Liu T, Wang C, Wang H, Lu T, Rao B, Ren Z. SPATS2, negatively regulated by miR-145-5p, promotes hepatocellular carcinoma progression through regulating cell cycle. Cell Death Dis. 2020;11(10):837. doi: 10.1038/s41419-020-03039-y.\u003c/li\u003e\n\u003cli\u003eSenoo M, Hoshino S, Mochida N, Matsumura Y, Habu S. Identification of a novel protein p59(scr), which is expressed at specific stages of mouse spermatogenesis. Biochem Biophys Res Commun. 2002; 292:992\u0026ndash;998. doi: 10.1006/bbrc.2002.6769.\u003c/li\u003e\n\u003cli\u003eDamas ND, Marcatti M, Come C, Christensen LL, Nielsen MM, Baumgartner R, Gylling HM, Maglieri G, Rundsten CF, Seemann SE, et al. SNHG5 promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization.\u003c/li\u003e\n\u003cli\u003eLiu YP, Cao Q, Li L, Zhang M. High expression of spermatogenesis associated serine rich 2 promotes tumorigenicity in esophageal squamous cell carcinoma cells and is associated with poor patient prognosis. Exp Ther Med. 2021 Jul;22(1):698. doi: 10.3892/etm.2021.10130.\u003c/li\u003e\n\u003cli\u003eTakamochi K, et al. Novel biomarkers that assist in accurate discrimination of squamous cell carcinoma from adenocarcinoma of the lung. BMC Cancer. 2016; 16:760. doi: 10.1186/s12885-016-2792-1. \u003c/li\u003e\n\u003cli\u003eXing J, Tian Y, Ji W, Wang X. Comprehensive evaluation of SPATS2 expression and its prognostic potential in liver cancer. Medicine (Baltimore). 2020 Feb;99(9): e19230. doi: 10.1097/MD.0000000000019230.\u003c/li\u003e\n\u003cli\u003eChen L, Yi C, Li W, Tseng Y, Zhang J, Liu J. Inhibition of SPATS2 Suppresses Proliferation and Invasion of Hepatocellular Carcinoma Cells through TRIM44-STAT3 Signaling Pathway. J Cancer. 2021 Jan 1;12(1):89-98. doi: 10.7150/jca.47526. eCollection 2021.\u003c/li\u003e\n\u003cli\u003eWu ZH, Yang DL, Wang L, Liu J. Epigenetic and Immune-Cell Infiltration Changes in the Tumor Microenvironment in Hepatocellular Carcinoma. Front Immunol. 2021 Dec 2; 12:793343. doi: 10.3389/fimmu.2021.793343. eCollection 2021.\u003c/li\u003e\n\u003cli\u003eHernandez-Gea V, Toffanin S, Friedman SL, Llovet JM. Role of the Microenvironment in the Pathogenesis and Treatment of Hepatocellular Carcinoma. Gastroenterology (2013) 144(3):512\u0026ndash;27. doi: 10.1053/j.gastro.2013.01.002\u003c/li\u003e\n\u003cli\u003eMiao D, Margolis CA, Gao W, Voss MH, Li W, Martini DJ, Norton C, Boss\u0026eacute; D, Wankowicz SM, Cullen D, et al. Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma. Science (New York, NY). 2018;359(6377):801\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eEl-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, Kim TY, Choo SP, Trojan J, Welling TH Rd, Meyer T, Kang YK, Yeo W, Chopra A, Anderson J, Dela Cruz C, Lang L, Neely J, Tang H, Dastani HB, Melero I. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet. 2017 Jun 24; 389(10088):2492-2502.\u003c/li\u003e\n\u003cli\u003eMacek Jilkovaa Z, Ghelfi J, Decaens T. Immunomodulation for hepatocellular carcinoma therapy: current challenges. Curr Opin Oncol. 2021 Dec 17. doi: 10.1097/CCO.0000000000000812. Online ahead of print.\u003c/li\u003e\n\u003cli\u003eLi T, Fan J, Wang B, Traugh N, Chen Q, Liu JS, Li B, Liu XS. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells. Cancer Research. 2017. 77(21): e108\u0026ndash;e110. DOI 10.1158/0008-5472.CAN-17-0307.\u003c/li\u003e\n\u003cli\u003eLiu CJ, Hu FF, Xia MX, Han L, Zhang Q, Guo AY. GSCALite: a web server for gene set cancer analysis. Bioinformatics. 2018. 34(21):3771-3772 DOI 10.1093/bioinformatics/bty411.\u003c/li\u003e\n\u003cli\u003eRu B, Wong CN, Tong Y, Zhong JY, Zhong SSW, Wu WC, Chu KC, Wong CY, Lau CY, Chen I, Chan NW, Zhang J. TISIDB: an integrated repository portal for tumor-immune system interactions. Bioinformatics. 2019. 35(20):4200\u0026ndash;4202 DOI 10.1093/bioinformatics/btz210.\u003c/li\u003e\n\u003cli\u003eHilmi M, Vienot A, Rousseau B, et al. Immune therapy for liver cancers. Cancers (Basel) 2019; 12:77. 10.3390/cancers12010077. \u003c/li\u003e\n\u003cli\u003eNishida N, Kudo M. Immune phenotype and immune checkpoint inhibitors for the treatment of human hepatocellular carcinoma. Cancers (Basel) 2020; 12:1274. 10.3390/cancers12051274.\u003c/li\u003e\n\u003cli\u003eBourayou E, Golub R. Signaling Pathways Tuning Innate Lymphoid Cell Response to Hepatocellular Carcinoma. Front Immunol. 2022 Feb 23; 13:846923. doi: 10.3389/fimmu.2022.846923.\u003c/li\u003e\n\u003cli\u003eXu W, Cheng Y, Guo Y, Yao W, Qian H. Targeting tumor associated macrophages in hepatocellular carcinoma. Biochem Pharmacol. 2022 Mar 11:114990. doi: 10.1016/j.bcp.2022.114990.\u003c/li\u003e\n\u003cli\u003eMoghaddam MZ, Ansariniya H, Seifati SM, Zare F, Fesahat F. Immunopathogenesis of endometriosis: An overview of the role of innate and adaptive immune cells and their mediators. Am J Reprod Immunol. 2022 Mar 9: e13537. doi: 10.1111/aji.13537.\u003c/li\u003e\n\u003cli\u003eZheng C, Zheng L, Yoo JK, Guo H, Zhang Y, Guo X, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell. 2017; 169:1342\u0026ndash;56. doi: 10.1016/j.cell.2017.05.035.\u003c/li\u003e\n\u003cli\u003eXiaopei Hao, Guangshun Sun, Yao Zhang, Xiangyi Kong, Dawei Rong, Jinhua Song, Weiwei Tang, Xuehao Wang. Targeting Immune Cells in the Tumor Microenvironment of HCC: New Opportunities and Challenges. Front Cell Dev Biol. 2021; 9: 775462. Published online 2021 Nov 12. doi: 10.3389/fcell.2021.775462.\u003c/li\u003e\n\u003cli\u003eMa J, Zheng B, Goswami S, Meng L, Zhang D, Cao C, et al. PD1(Hi) CD8(+) T cells correlate with exhausted signature and poor clinical outcome in hepatocellular carcinoma. J Immunother Cancer. 2019; 7:331. doi: 10.1186/s40425-019-0814-7.\u003c/li\u003e\n\u003cli\u003eWu L., Saxena S., Awaji M., Singh R.K. Tumor-Associated neutrophils in cancer: Going pro. Cancers. 2019; 11:564. doi: 10.3390/cancers11040564.\u003c/li\u003e\n\u003cli\u003eMichaeli J., Shaul M.E., Mishalian I., Hovav A.H., Levy L., Zolotriov L., Granot Z., Fridlender Z.G. Tumor-Associated neutrophils induce apoptosis of non-activated CD8 T-cells in a TNFalpha and NO-dependent mechanism, promoting a tumor-supportive environment. Oncoimmunology. 2017;6: e1356965. doi: 10.1080/2162402X.2017.1356965.\u003c/li\u003e\n\u003c/ol\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":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2106467/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2106467/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe spermatogenesis associated serine rich 2 (SPATS2) is a member of RNA-binding protein in which the abnormal expression is linked with carcinogenesis in serval types of cancer. However, there is no systematic study on the differential expression, prognostic significance, epigenetic regulation, immune infiltration of SPATS2 in HCC. In the present study, we investigated the expression, prognosis, epigenetic regulation, and immune cell infiltration of SPATS2 in HCC. We found that the elevated expression of SPATS2 was unfavorably associated with the clinical pathological stage and prognosis. Functional enrichment analysis revealed that SPATS2 associated with cell cycle, apoptosis and cancer cell metastasis processes in HCC. Our results confirmed that knockdown of SPATS2 will affect cell cycle, apoptosis and invasion of HCC cell lines. Moreover, the expression of SPATS2 is upregulated by epigenetic regulation, including DNA methylation, m6A and histone modification in HCC. In addition, SPATS2 expression was positively correlated with immune cell or gene markers of immune infiltration in HCC. Taken together, our data demonstrated that SPATS2 was associated with progression and immune infiltration, and could serve as a prognostic biomarker for HCC. In conclusion, these results highlight the potential of SPATS2 to be used as a therapeutic target for HCC.\u003c/p\u003e","manuscriptTitle":"SPATS2 is correlated with cell cycle progression and immune cells infiltration in hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-05 18:25:15","doi":"10.21203/rs.3.rs-2106467/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-11-22T12:20:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-11-20T17:02:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-19T07:41:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46d8245e-7c81-4eb9-96ea-1b33e004ddc5","date":"2022-10-01T05:09:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-01T04:53:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-01T04:47:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-09-30T12:20:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-09-30T12:17:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Gastroenterology","date":"2022-09-27T01:44:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bea2752e-872f-404b-a7a9-feddb4fb5cfb","owner":[],"postedDate":"October 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:21:40+00:00","versionOfRecord":{"articleIdentity":"rs-2106467","link":"https://doi.org/10.1186/s12876-022-02633-y","journal":{"identity":"bmc-gastroenterology","isVorOnly":false,"title":"BMC Gastroenterology"},"publishedOn":"2023-01-11 18:17:20","publishedOnDateReadable":"January 11th, 2023"},"versionCreatedAt":"2022-10-05 18:25:15","video":"","vorDoi":"10.1186/s12876-022-02633-y","vorDoiUrl":"https://doi.org/10.1186/s12876-022-02633-y","workflowStages":[]},"version":"v1","identity":"rs-2106467","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2106467","identity":"rs-2106467","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}