1. Introduction
RNA binding motif, single stranded interacting protein 2 (RBMS2), a gene commonly linked with tumor-suppressive properties, is the focus of our study. Positioned on chromosome 12q13.3 [Reference Gross1], RBMS2 plays a critical role in the regulation of transcription and alternative splicing, both key cellular processes related to cell proliferation and programmed cell death or apoptosis. Alterations or disruptions in the normal functioning of RBMS2 have been found to be associated with the development of various types of cancers, underlining its crucial role in maintaining the delicate balance of cellular homeostasis [Reference Sun, Hu and Wu2].
Investigations into different types of cancers such as lung cancer, gastric cancer, and hepatocellular carcinoma have reported a downregulation of RBMS2. This diminished expression of RBMS2 is seen to contribute to the initiation and progression of these cancers, thereby establishing a link between RBMS2 downregulation and oncogenesis [Reference Xu, Xia and Xu3]. However, the role of RBMS2 in kidney renal clear cell carcinoma (ccRCC), which is the most prevalent subtype of kidney cancer, has not been thoroughly explored.
The primary objective of this study is to illuminate the role of RBMS2 in ccRCC and to establish its clinical and cellular implications. This is achieved by investigating the relationship between the expression of RBMS2 and the clinico-pathological characteristics of patients suffering from ccRCC. We delve into the prognostic potential of RBMS2 and its impact on the growth of ccRCC cells. An additional layer of complexity is added by the tumor microenvironment, which plays a significant role in the progression of the disease. In this context, we analyze the relationship between RBMS2 expression and immune infiltration in the tumor microenvironment of ccRCC.
The tumor microenvironment, a complex and dynamic entity, has a profound impact on the development and progression of cancer [Reference Anderson and Simon4]. It consists of various immune cells, stromal cells, and extracellular matrix components and is known to be involved in immune surveillance, tumor progression, and therapeutic response [Reference Hinshaw and Shevde5]. Recent studies have started to unravel the complex interplay between cancer cells and their microenvironment, revealing that cancer cells can manipulate their surroundings to promote tumor growth, invasion, and resistance to therapy [Reference Jin and Jin6]. In the context of ccRCC, the tumor microenvironment has been found to be highly immunogenic, with a high degree of immune cell infiltration [Reference Joyce7]. However, despite the immunogenic nature of ccRCC, immune checkpoint inhibitors, which are designed to reactivate the antitumor immune response, have only shown limited efficacy in this cancer type. This suggests that there are still unknown mechanisms through which ccRCC can evade the immune system and highlights the need for further research in this area.
In this study, we also shed light on these issues by investigating the role of RBMS2 in ccRCC and its relationship with the tumor microenvironment. We hope that our findings will not only contribute to the understanding of ccRCC pathogenesis but also open new avenues for the development of more effective therapeutic strategies for this devastating disease.
2. Materials and Methods
2.1 Online Database
We harnessed the potential of the Cancer Genome Atlas (TCGA) database, a comprehensive resource housing genomic and clinical data from thousands of cancer patients [Reference Tomczak, Czerwińska and Wiznerowicz8]. The ccRCC dataset from TCGA, which includes gene expression data and associated clinico-pathological information, served as the foundation for our investigation into the role of RBMS2 in ccRCC.
2.2 Immunohistochemical (IHC) Staining
IHC was utilized in this study to evaluate the protein expression of RBMS2 in ccRCC and adjacent nontumorous tissue samples. The process commenced with the preparation of 4 μm thick tissue sections, which underwent a sequence of treatments involving deparaffinization and rehydration utilizing successive xylene and ethanol immersions. Subsequently, heat-induced epitope retrieval was conducted by subjecting the samples to antigen retrieval, employing citrate buffer (pH 6.0) within a microwave oven. To inhibit any inherent peroxidase activity, a 3% hydrogen peroxide solution was applied for a duration of 10 minutes, followed by incubation with bovine serum albumin (BSA). The tissue sections were subsequently subjected to an overnight incubation at 4°C, employing a primary antibody specific to RBMS2 at a 1 : 200 dilution. Afterward, PBS was used to thoroughly rinse the sections, and they were then exposed to a secondary antibody, conjugated with horseradish peroxidase (HRP), for an hour at room temperature. Detection of the antigen-antibody complex was facilitated by employing a 3,3′-diaminobenzidine (DAB) substrate solution, yielding a brown-colored precipitate indicative of antigen localization. Hematoxylin counterstaining was subsequently applied to facilitate the visualization of cell nuclei. Negative controls were integrated into the procedure by omitting the primary antibody.
Following the staining process, the tissue sections underwent dehydration via a series of ethanol washes and clearing with xylene. Ultimately, the slides were mounted with a coverslip utilizing mounting medium. The stained tissue sections were subsequently examined to evaluate the extent of RBMS2 protein expression.
2.3 Cell Culture and Transfection
Cellular experiments were performed to validate the findings from the database analysis. We used two ccRCC cell lines, 786-O and Caki-2, for these experiments. Transient transfection techniques were employed to overexpress pcDNA3.1-vector or pcDNA3.1-RBMS2 in these cell lines by Lipo3000 reagent, allowing us to observe the impact of RBMS2 on ccRCC cell growth [Reference Zhang, Xiao and Paredes9]. The overexpression plasmids were synthesized in GenePharma (Shanghai, China). Western blotting was used to assess the transfection efficacy.
2.4 Proliferation Assay
We utilized the CCK-8 assay to assess the proliferation capacity of the ccRCC cell lines following RBMS2 overexpression based on the manufacturer’s instructions as previously reported.
2.5 Migration Assay by Transwell
The transwell migration assay was used to evaluate the impact of RBMS2 overexpression on the migration ability of the ccRCC cell lines. Cells (2 × 105) were suspended in 100 μl serum-free medium at a density of 105/ml and seeded into the upper chamber. The bottom wells were filled with 600 μl of 10% FBS medium. Cells that had migrated or invaded to the lower surface of the membrane were fixed with methanol and glacial acetic acid and then stained with 20% Giemsaand. Five random fields were chosen to count migrated cell numbers.
3. Statistics
Statistical analyses were performed using R software. All data are presented as mean ± SD. The correlation between RBMS2 expression and clinico-pathological characteristics was assessed using the chi-square test. Survival analysis was conducted using the Kaplan–Meier method and the Cox regression model. Cellular experimental data were tested by Student’s t-test. P < 0.05 was considered statistically significant.
4. Ethics Statement
This study was conducted in accordance with the Declaration of Helsinki. Since the study used publicly available data and in vitro experiments, it did not involve human participants or animals and thus did not require ethical approval.
5. Results
5.1 Interplay between RBMS2 Expression and Clinico-Pathological Traits in ccRCC Patients
Figure 1 and Table 1 showcase a significant downregulation of RBMS2 expression in ccRCC patients presenting with severe disease markers, such as advanced age (P = 0.035), increased serum calcium level (P = 0.002), advanced T stage (P < 0.001), distant metastasis (P < 0.001), advanced TNM stage (P < 0.001), and poorer differentiation grade (P < 0.001).
Figure 1 Associations between RBMS2 and clinico-pathological traits of ccRCC patients. RBMS2 mRNA levels were found to be significantly diminished in ccRCC patients presenting with elder age (a), elevated serum calcium level (b), advanced T stage (c), distant metastasis (d), advanced TNM stage (e), and poorer differentiation grade (f).
Table 1 ccRCC patients’ characteristics and correlations with RBMS2 expression level.
To further validate the expression difference of RBMS2 in ccRCC and adjacent nontumorous kidney tissues, we next collected 13 ccRCC specimens together with their paired nontumorous tissues. RT-qPCR data revealed that 10 of the 13 cases showed higher RBMS2 mRNA level in adjacent tissues than that in ccRCC tissues, and the mean fold change in each tissue pair is exhibited in Figure 2(a). Consistently, IHC immunostaining also reflected higher protein level of RBMS2 in nontumorous kidney tissue than that in ccRCC tissue (Figures 2(b) and 2(c)).
Figure 2 The mRNA and protein levels of RBMS2 in ccRCC specimens compared to adjacent nontumorous tissues. (a) RT-qPCR data reflected a decreased mRNA level of RBMS2 in ccRCC specimens compared to paired adjacent nontumorous tissues (n = 13, P = 0.0004). Data were compared by paired Student’s t-test. (b) Representative IHC staining of RBMS2 protein in ccRCC tissues. (c) Representative IHC staining of RBMS2 protein in adjacent nontumorous tissues.
5.2 The Prognostic Relevance of RBMS2 in ccRCC Patients
The prognostic potential of RBMS2 in the ccRCC cohort from the TCGA dataset is depicted in Figure 3. Downregulated RBMS2 is significantly correlated with worse overall survival (Figure 3(a)), disease-specific survival (Figure 3(b)), and progression-free survival (Figure 3(c)).
Figure 3 Prognostic relevance of RBMS2 in the ccRCC cohort. Higher RBMS2 levels correlated to improved overall survival (a), disease-specific survival (b), and progression-free survival (c), as depicted by the Kaplan–Meier analysis of the cohort from the TCGA dataset.
In the multivariate analysis for overall survival (Table 2), age over 60 years (hazard ratio = 1.728, P = 0.033), advanced T stage (hazard ratio = 2.207, P = 0.003), and the presence of metastasis (hazard ratio = 5.154, P < 0.001) were identified as factors significantly linked with worse survival. High RBMS2 expression was found to be a significant factor in the overall survival multivariate analysis although it did not reach statistical significance (hazard ratio = 0.719, P = 0.195). Similar trends were observed in the multivariate analysis for disease-specific survival (Table 3) and progression-free survival (Table 4), with advanced T stage and metastasis significantly associated with worse survival. Of note, high RBMS2 expression was significantly associated with better disease-specific survival (hazard ratio = 0.387, P = 0.004) and progression-free survival (hazard ratio = 0.527, P = 0.02).
Table 2 ccRCC overall survival analysis.
Bold values indicate P<0.05.
Table 3 ccRCC disease-free survival analysis.
Bold values indicate P<0.05.
Table 4 ccRCC progression-free survival.
Bold values indicate P<0.05.
5.3 Survival Nomograms for ccRCC Patients
Based on the multivariate analyses, we next formulated disease-specific survival and progression-free survival nomograms to predict the 1-, 3-, and 5-year survival rates of ccRCC patients (Figures 4(a) and 4(b)). These nomograms incorporate various factors, including gender, age, serum calcium level, histological grade, T stage, N stage, M stage, and RBMS2 levels.
Figure 4 Survival nomograms for ccRCC patients. Disease-specific survival (a) and progression-free survival (b) nomograms were established to predict the 1-, 3-, and 5-year survival rates using the ccRCC cohort from TCGA dataset. The variables included are gender, age, serum calcium level, histological grade, T stage, N stage, M stage, and RBMS2 levels.
5.4 Cellular Experiments on the Tumor-Suppressing Role of RBMS2 in ccRCC
Cellular experiments were undertaken to examine the tumor-suppressing role of RBMS2 in ccRCC. We conducted overexpression and knockdown of RBMS2 in two different ccRCC cell lines, 786-O and Caki-2. The CCK-8 assay results demonstrated that overexpressing RBMS2 significantly inhibited the proliferation capacity of both 786-O and Caki-2 ccRCC cell lines (Figures 5(a) and 5(b)). In addition, the transwell migration assay results showed that overexpressing RBMS2 significantly reduced the migration capacity of these ccRCC cell lines (Figures 5(c) and 5(d)). In contrast, compared with the scrambled control group, siRNA-induced knockdown of RBMS2 significantly downregulates the proliferation and migration of the two cell lines (Figures 5(e)–5(h)).
Figure 5 Cellular experiments examining the tumor-suppressing role of RBMS2 in ccRCC. (a, b) The CCK-8 assay revealed that overexpressing RBMS2 can significantly inhibit the proliferation capacity of 786-O and Caki-2 ccRCC cell lines. (c, d) The transwell migration assay demonstrated that overexpressing RBMS2 can significantly reduce the migration capacity of 786-O and Caki-2 ccRCC cell lines. (e, f) The CCK-8 assay revealed that RBMS2 knockdown can significantly enhance the proliferation capacity of 786-O and Caki-2 ccRCC cell lines. (g, h) The transwell migration assay demonstrated that RBMS2 knockdown can significantly upregulate the migration capacity of 786-O and Caki-2 ccRCC cell lines.
5.5 Associations between RBMS2 Expression and Immune Infiltrations of ccRCC
We also presented the correlations between RBMS2 expression and immune infiltrations in ccRCC. Figure 6(a) shows different enrichments of 24 immune cell types in samples with high or low RBMS2 levels. Notably, RBMS2 expression showed a positive correlation with Tcm cells and a negative correlation with CD56bright cells in ccRCC samples (Figures 6(b) and 6(c)).
Figure 6 Correlations between RBMS2 expression and immune infiltrations in ccRCC. The enrichment of 24 immune cell types in samples with high or low RBMS2 levels was examined (a). For example, RBMS2 expression was positively correlated with Tcm cells (b) and negatively correlated with CD56bright cells (c) in ccRCC samples.
6. Discussion
Our research delves into the implications of RNA binding motif, single stranded interacting protein 2 (RBMS2) in kidney renal clear cell carcinoma (ccRCC), contributing to the growing body of evidence that underlines the tumor-suppressive role of RBMS2. Our findings reveal that RBMS2 expression is significantly downregulated in ccRCC patients with more severe disease characteristics. Moreover, RBMS2 has demonstrated potential as a prognostic indicator for survival outcomes in ccRCC.
Our findings are in line with previous research that has underscored the tumor-suppressive role of RBMS2 in various malignancies [Reference Sun, Hu and Wu2, Reference Fang, Chen and Ma10]. RBMS2 is known to inhibit the activation of c-Myc, an oncogene that plays a pivotal role in cell proliferation and growth [Reference Sun, Hu and Wu2]. Downregulation of RBMS2 has been implicated in the pathogenesis and progression of various cancers, such as lung cancer and breast cancer. Our research extends these findings to ccRCC, suggesting a broad implication of RBMS2 in cancer biology. Furthermore, our in vitro experiments provide biological evidence for the tumor-suppressive role of RBMS2 in ccRCC. We have demonstrated that overexpression of RBMS2 can significantly inhibit the proliferation and migration of ccRCC cell lines. These findings highlight the therapeutic potential of RBMS2 in ccRCC. Indeed, RBMS2 was reported to chemosensitize breast cancer cells to doxorubicin, implying its therapeutic potential [Reference Xu, Xia and Xu3].
Additionally, our study has shed light on the interaction between RBMS2 expression and immune infiltration in the ccRCC tumor microenvironment, hinting at a role for RBMS2 in modulating the tumor immune microenvironment. The precise mechanisms through which RBMS2 influences immune infiltrations in ccRCC warrant further investigation.
While our study provides valuable insights, it does come with a few limitations. First, our findings are based on a retrospective analysis of a public database, and thus prospective studies are needed to validate these findings. Second, while our in vitro experiments have demonstrated the tumor-suppressive role of RBMS2 in ccRCC, further in vivo experiments and mechanistic studies are required to comprehensively understand the biological function of RBMS2 in ccRCC. Moving forward, our study opens several avenues for future research. Unraveling the molecular mechanisms underpinning the tumor-suppressive role of RBMS2 in ccRCC could provide novel insights into the pathogenesis of ccRCC. Additionally, as our study suggests a potential role of RBMS2 in modulating the tumor immune microenvironment, it would be worthwhile to investigate whether RBMS2 could serve as a target for immunotherapy.
7. Conclusions
Our research delves deep into the role of RBMS2 in ccRCC and reveals that RBMS2 expression is significantly diminished in patients displaying severe disease characteristics. Our findings suggest that higher RBMS2 expression levels correlate with improved survival outcomes, underscoring its potential as a prognostic biomarker. In vitro experiments further reinforce the tumor-inhibitory role of RBMS2 in ccRCC. Importantly, we have also uncovered correlations between RBMS2 expression and immune infiltration in ccRCC, suggesting a potential role of RBMS2 in modulating the tumor immune microenvironment. In conclusion, our study positions RBMS2 as a promising prognostic biomarker and a potential therapeutic target in ccRCC.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
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