3.1 The expression of potential drug target RPS6KB2 in various normal and tumor tissue.
We first investigated the expression of a potential target, RPS6KB2, based on the GTEx and CCLE databases. The results showed that the mRNA expression of RPS6KB2 was comparable in all organs (Fig. 1A, B). Using TCGA data alone, variations were found in 16 of the 20 tumor types except for KIRP, LGG, PAAD, and THCA, whereas when TCGA and GTEx data were combined, variations were found in 21 of the 27 tumors, except for BRCA, CESC, GBM, KIRP, LIHC, and PAAD (Fig. 1C, D).
3.2 The expression of RPS6KB2 correlates with the pathological tumor stage.
The "Pathological Stage Plot" module of GEPIA2 was used to investigate the association between RPS6KB2 expression and the pathological stages of tumors. A significant positive correlation was observed between RPS6KB2 expression and pathological tumor stage (Fig. 2).
3.3 RBS6KB2 is a potential prognostic biomarker of tumors.
To investigate the association between RPS6KB2 expression in different tumor types and the survival of tumor patients, we performed univariate Cox regression analysis based on data from the TCGA database. Patients' overall survival (OS) was significantly poorer in those with high RPS6KB2 expression than those with low expression (HR = 1.3, Fig. 3A). According to the following survival studies, patients with high RPS6KB2 expression had worse overall survival (OS) in the ACC, KICH, KIRC, LGG, SKCM, and THYM (Fig. 3B). Significant RBS6KB2 hazard ratios were shown by ACC, KICH, KIRC, LGG, and SKCM, with KICH having the greatest risk impact (Fig. 3C). Similar results demonstrated that patients in the ACC, KICH, KIRC, KIRP, LGG, and SKCM had worse DSS due to higher RPS6KB2 expression. (Fig. 3D).
Univariate Cox regression analysis was then used to examine the association between RBS6KB2 expression and PFS. Due to a lack of pertinent data, it is impossible to calculate the hazard ratio of RPS6KB2 in LAML. The ACC, KICH, LIHC, TGCT, and UCS hazard ratios for RBS6KB2 were all statistically significant (Fig. 4A). According to the following survival studies, which used patient data dichotomized by the ideal cut-off value for each tumor type, patients with high RPS6KB2 expression had worse PFS in ACC, KICH, LIHC, TGCT, and UCS (Fig. 4B).
3.4 RPS6KB2 is involved in the activation and proliferation of immune cells
We investigated the association between RPS6KB2 expression and immune cell infiltration to determine whether RPS6KB2 expression affected the tumor immunological environment. Using the infiltration scores of six immune cell types (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) based on the Tumor Immune Estimation Resource (TIMER) database, we did find a strong correlation in numerous malignancies.
BRCA, COAD, and THYM were the three tumor cohorts with the highest rankings. The corresponding linear regression diagrams revealed a correlation between high RPS6KB2 expression and increased immune cell infiltration. High RPS6KB2 expression was associated with increased immune cell infiltration, based on THYM's linear regression graphs produced by THYM. Importantly, dendritic cells in THYM had a rather high coefficient (Fig. 5A).
The top three tumors that had the strongest correlations with RPS6KB2 expression were PRAD, LUAD, and BRCA (StromalScore), THCA, GBM, and LUAD (ImmuneScore), and PRAD, THCA, and LUAD (ESTIMATEScore) (Fig. 5B). The results demonstrated a strong correlation between RPS6KB2 expression and immune infiltration in malignancies.
3.5 RPS6KB2 regulated immune processes in tumors.
The TCGA dataset was used to investigate whether there is an association between the expression of RPS6KB2 and immune checkpoint genes and the underlying mechanism of RPS6KB2 involving tumor immunity. RPS6KB2 was significantly linked with TNFRSF14, CD276, TNFRSF25, TNFRSF18, CD40, and TNFSF15, according to a correlation study of the expression of RPS6KB2 and checkpoint genes in different tumor types. These findings, particularly for the immune checkpoint molecules LAML, LGG, LIHC, PAAD, and PCPG, where RPS6KB2 expression was positively correlated with most of them, suggesting that RPS6KB2 was involved in the regulation of tumor immune responses through modulating immune checkpoint activity. Similarly, but not significantly, the expression of RPS6KB2 was inversely correlated with most immune checkpoint molecules in COAD, ESCA, GBM, HNSC, MESO, SARC, SKCM, and THYM (Fig. 6A). Therefore, in LUAD, BRCA, UCEC, and STAD, RPS6KB2 expression was significantly associated with neoantigen count (Fig. 6B).
In some tumors, RPS6KB2 was associated with TMB and MSI. RPS6KB2 expression was positively correlated with TMB in UCEC, STAD, PAAD, LUAD, LUSC, LGG, KICH, and BRCA, but negatively correlated with TMB in THYM and THCA (Fig. 6C). RPS6KB2 expression was inversely correlated with MSI in THCA, SKCM, SARC, PRAD, LGG, and DLBC but positively correlated in COAD, STAD, and UCEC (Fig. 6D).
3.6 RPS6KB2 expression strongly correlated to MMR defects in different tumors and might affect methylation.
The association between RPS6KB2 expression and carcinogenesis, particularly with MMR defects and the methylation of particular tumor suppressor genes, needed further investigation after the correlation between RPS6KB2 expression and mutation indicators TMB and MSI had been established. Therefore, the expression of some known MMR genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) and their correlation with RPS6KB2 were investigated. The expression of RPS6KB2 was strongly correlated with the MMR gene in most tumor types (33 kinds, omitting CESO, CHOL, COAD, DLBC, ESCA, LAML, LUSC, MESO, SARC, SKCM, UCS, and UWM), while MLH1, MSH2, and MSH6 were all positively correlated with RPS6KB2. RPS6KB2 expression was remarkably negatively associated with all five MMR genes in colon tumor types (READ and COAD), suggesting that MMR may play a role in colon carcinogenesis (Fig. 7A). As for the correlation between RPS6KB2 and 4 methyltransferases (DNMT1, DNMT2, DNMT3A, DNMT3b), the results showed that the two were co-expressed in many specific tumor types (ACC, BRCA, COAD, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, OV, PAAD, PCPG, SKCM, STAD, TGCT, THCA, THYM, UCEC) (Fig. 7B).
3.7 Enrichment analysis of RPS6KB2 in tumor therapy.
We conducted a pathway enrichment analysis of the gene RPS6KB2 to investigate its role in metabolism. The strong relationship between the up-regulation of RPS6KB2 and the neurotrophin and Wnt signaling pathways was discovered by KEGG analysis. HALLMARK study revealed a strong link between the up-regulation of RPS6KB2 and the Wnt/-catenin signaling pathway, the p53 pathway, and fatty acid metabolism. Furthermore, the down-regulation of RPS6KB2 was significantly correlated with epithelial-mesenchymal transition (Fig. 8).
3.8 The potential drug target RPS6KB2 was upregulated in HCC tissues.
We examined the expression of RPS6KB2 in HCC tissues and surrounding tissues to confirm the high expression of RPS6KB2 in tumor tissues. Western blot revealed that HCC tissues expressed more RPS6KB2 than neighboring tissues (Fig. 9).
3.9 RPS6KB2 promotes the invasion and migration of HCC cells.
Since the expression of RPS6KB2 positively correlates with the occurrence and development of tumors, we investigated whether RPS6KB2 functionally enhanced tumor occurrence and development. We performed transwell and wound healing assays to examine how RPS6KB2 contributes to HCC's invasive and migratory capacities. As a result, RPS6KB2 knockdown reduced cell invasion and migration based on wound healing and transwells data (Fig. 10A, B). Further investigation revealed that RPS6KB2 silencing inhibited the capacity of cells to invade and migrate (Fig. 10C), which was supported by the lower expression of MMP-2 and MMP-9. Thus, we can conclude that RPS6KB2 may promote the invasion and motility of HCC cells in vitro.
3.10 RPS6KB2 regulated apoptosis and cell proliferation in HCC cells.
We carried out EDU staining and Western blot to investigate the function of RPS6KB2 in cell growth and apoptosis. EdU staining showed that RPS6KB2 knockdown significantly reduced cell proliferation in cells (Fig. 11A). Western blot revealed that when RPS6KB2 was silenced in HCC cells, BCL2-associated X protein (Bax) expression increased while B-cell lymphoma-2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were downregulated (Fig. 11B). These findings show that RPS6KB2 suppresses apoptosis and boosts cell proliferation.
3.11 RPS6KB2 was involved in the secretion of inflammatory cytokines In the HCC
Western blot was applied to analyze the expression of three inflammatory cytokines, IL-1, IL-6, and TNF-α, and determine if RPS6KB2 affects inflammatory cytokine release. The expression of IL-1, IL-6, and TNF-α are downregulated when RPS6KB2 is expressed at a low level, while the expression of IL-1, IL-6, and TNF-α are upregulated when RPS6KB2 is expressed at a high level (Fig. 12), demonstrating the close relationship between RPS6KB expression and inflammatory cytokines. These inflammatory factors may be closely related to the tumor microenvironment and tumor occurrence, which may also be a possible mechanism for RPS6KB2 to regulate immunity and even play an antitumor role in inflammation, immunity, and tumors.