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. 1A). GSCA result showed that SPATS2 expression was significantly increased in tumor tissues of LIHC than that in normal tissues (Fig. 1B). Moreover, SPATS2 protein level also was upregulated in LIHC based on the protein expression results of HPA database in liver cancer (Fig. 1C). Following, the relationships of SPATS2 expression with the clinicopathological parameters of patients with HCC were assessed by UALCAN database. As shown in Fig. 1D, 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. 1E, F). Additionally, SPATS2 was elevated in the nodal of metastasis status in LIHC (Fig. 1G). 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. 2A, 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. 2B-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. 3A, 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. 3B). GSEA results also showed that SPATS2 was positively associated with cell cycle, EMT and apoptosis (Fig. 3C). 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. 3D). Furthermore, GSCA pathways enrichment analysis results showed that these genes were also positively correlated with apoptosis, cell cycle and EMT pathways in LIHC (Fig. 3E).
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. 3F). In addition, the interacted proteins of SPATS2 were obtained based on the GeneMANIA database (Fig. 3G). YEATS2, AACS and PRDM4 were co-localization proteins of SPATS2. These genes were up-regulated in LIHC (Fig. 3H). 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. 4A-D). Moreover, the downregulation of SPATS2 reduced apoptosis of HCC cells ((Fig. 4E-F). In addition, knockdown of SPATS2 dramatically inhibited the invasion ability of HCC cells (Fig. 4I-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. 5A). MSP assay indicated that the methylation level was decreased in HepG2 cells (Fig. 5B). Moreover, high risk was observed in low methylation group (Fig. 5C). The expression of SPATS2 was negatively associated with its methylation level of SPATS2 in HCC (Fig. 5D).
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. 5E). In addition, we also investigated the histone modification in SPATS2 promoter region. As shown in Fig. 5F, H3K4me1, H3K4me3, H3K27ac, and H3K36me3 modifications that promote gene expression were significantly enriched in the promoter region of SPATS2 gene (Fig. 5G). Additionally, Multiple acetylation and methylation sites were found in SPATS2 (Fig. 5H). 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. 6A). Additionally, GSCA database results indicated that SPATS2 expression was positively associated with B cell and nTreg (cor > 0.4, p < 0.05) (Fig. 6B). Interestingly, methylation level of SPATS2 was significantly negatively associated with most of immune cells based on the GSVA results (Fig. 6C). 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. 6D, 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. 6F, 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. 7A, 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. 7B, C). Moreover, cell interaction results showed that B plasma has a correlation with DC, NK and macrophages cells in HCC patients (Fig. 7D). 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. 7E, 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. 7E). 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. 7F). These findings further supported the result that SPATS2 is positively important in immune regulation of HCC.