Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy [
1,
2]. It is the sixth most commonly diagnosed malignancy and the third leading cause of cancer-related mortality worldwide [
2,
3]. In the past few decades, new therapeutic strategy, such as molecular targeted therapies and immunotherapy, have been developed and clinically evaluated with interesting results in HCC [
4]. Remarkably, the combination of immune checkpoint inhibitors and VEGF inhibitors (Atezolizumab plus Bevacizumab) have been tested and approved for the treatment of advanced HCC [
5]. Although many new therapeutic strategies have been widely used and improved, the long-term survival rate of HCC is still unsatisfactory [
5]. Therefore, it is critical to reveal the carcinogenesis mechanisms and explore new potential therapeutic targets to improve survival of HCC patients.
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 [
6,
7]. It has been reported that SPATS2 serves a tumorigenic role in several cancers, such as esophageal squamous cell carcinoma, colorectal cancer, prostate cancer, and HCC [
6‐
9]. SPATS2 is identified as a novel diagnostic biomarker in squamous cell carcinoma [
10]. Moreover, SPATS2 promotes lncRNA SNHG5-mediated survival of colorectal cancer cells through pro-proliferative and anti-apoptotic effect [
8]. Recently, SPATS2 is reported to be a diagnostic and prognostic biomarker in liver cancer [
11]. SPATS2 negatively regulates by miR-145-5p and results in promoting hepatocellular carcinoma progression through regulating cell cycle [
6]. Moreover, it is still involved in the proliferation and invasion of HCC cells through TRIM44-STAT3 signaling pathway [
12]. 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 [
13]. The main cellular components in the HCC TME include immune cells, fibroblasts, macrophages, and cancer stem cells [
14]. 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 [
15]. Therefore, finding and understanding the function of TME-related molecules are essential for the effective management and precision anticancer therapies [
15]. 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 [
16,
17]. 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 (The Cancer Genome Atlas) and GTEx (Gene Tissue Expression) databases. The expression of SPATS2 in different tumor stages and grades was confirmed using UALCAN (The University of Alabama at Birmingham Cancer data analysis Portal) 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. The results of p < 0.05 were considered to be statistically significant.
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 (Gene Set Cancer Analysis) 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 (LO2) 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% CO2 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 48 h later.
Cell apoptosis assay
We collected 1 × 106 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 min 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 × 105) 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, a web tool to perform multivariable survival analysis using DNA methylation data. The methylation level of the SPATS2 promoter region was determined via UALCAN database based on the TCGA_LIHC data.
Immune infiltration analysis
The TIMER (Tumor Immune Estimation Resource) database and the GSCA was used to calculate the abundance of tumor infiltrating immune cells (TIICs) in tumor tissues of LIHC [
18,
19]. TIMER was used to analyze the mRNA expression-related infiltration of different 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 [
20]. The
p < 0.05 results were considered to be statistically significant.
Statistical analysis
All the experiment was independently repeated three times. Data were summarized as the mean ± SD (Standard Deviation). 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.
Discussion
In recent years, SPATS2 has been reported to contribute to the tumorigenesis of multiple malignancies, including liver cancer [
6,
12]. 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. Furthermore, high expression of SPATS2 was indicative of an unfavorable clinicopathological feature and poor prognosis in patients with HCC. Moreover, we found that SPATS2 dramatically promotes proliferation and invasion of HCC cells. 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. Furthermore, we 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 apoptosis.
It is well known that the immune system plays an important role in controlling cancer progression. Recent year, immunotherapy has developed as the new first-line treatment option for advanced HCC [
4,
21,
22]. Previous study indicated that tumor immune cell infiltration generates an immunosuppressive TME and presences a generally correlations with a worse prognosis [
23,
24]. In tumor infiltrated immune cells, DCs are a unique class of immune cells that act as a bridge between innate and adaptive immunity, which plays a critical role in generating anti-tumor CD8 T cell immunity [
25]. Treg cells has been reported that the they could inhibit the killing ability of CD8+T cells and results in a poor prognosis in HCC [
24,
26]. In our study, SPATS2 was found for the first time to be highly positively correlated with the tumor infiltration of Tregs, B cell, macrophage, and DC cells in HCC. Moreover, the single cell data of HCC showed that SPATS2 was specifically expressed in B cells, macrophage, and DC cells in HCC. Thus, SPATS2 plays an important role in the recruitment and regulation of some immune infiltrating cells in liver cancer. Furthermore, we also confirmed that SPATS2 combined with CD4+ , Tregs, or macrophage infiltration level was associated with prognosis in HCC. Taken together, we hypothesized that SPATS2 expression could lead to HCC progression and poor prognosis by affecting the infiltration of above immune cells.
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 [
26]. More research results have established the function of CD8+T cells in the formation and progression of HCC, including diagnosis/treatment/prognosis [
27]. Moreover, targeting CD8+T cells is the main direction of immunotherapy for HCC [
28]. The neutrophils cells are significantly associated with cancer progression and metastasis [
29]. It also mainly suppresses antitumor immunity by inducing apoptosis of CD8+T cells through nitric oxide production-mediated TNF-α [
30]. 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. SPATS2 may be a novel target gene for immunotherapy in HCC. 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.
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