The Clinical and Cellular Impact of RBMS2 on the Progression and Prognosis of Kidney Renal Clear Cell Carcinoma

This research delves into the implications of the RNA binding motif, single stranded interacting protein 2 (RBMS2)—a gene associated with tumor-suppressing functions—in the context of kidney renal clear cell carcinoma (ccRCC). Through meticulous exploration of online databases, we have identified a negative association between RBMS2 expression and adverse clinico-pathological features, such as advanced TNM stage. Furthermore, our findings indicate that RBMS2 acts as a prognostic predictor for clinical outcomes in ccRCC, evidenced by both univariate and multivariate analyses. Cellular assays have corroborated these findings, revealing that an overexpression of RBMS2 curtails ccRCC cell proliferation and migration. Additionally, our research has unearthed links between RBMS2 and immune infiltration within the ccRCC tumor microenvironment. Collectively, our results underscore the tumor-inhibiting role of RBMS2 in ccRCC and spotlight its potential as a prognostic marker and therapeutic intervention target.


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[1], 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 [2].
Investigations into diferent types of cancers such as lung cancer, gastric cancer, and hepatocellular carcinoma have reported a downregulation of RBMS2.Tis 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 [3].
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.
Te primary objective of this study is to illuminate the role of RBMS2 in ccRCC and to establish its clinical and cellular implications.Tis is achieved by investigating the relationship between the expression of RBMS2 and the clinico-pathological characteristics of patients sufering 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 signifcant role in the progression of the disease.In this context, we analyze the relationship between RBMS2 expression and immune infltration in the tumor microenvironment of ccRCC.
Te tumor microenvironment, a complex and dynamic entity, has a profound impact on the development and progression of cancer [4].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 [5].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 [6].In the context of ccRCC, the tumor microenvironment has been found to be highly immunogenic, with a high degree of immune cell infltration [7].However, despite the immunogenic nature of ccRCC, immune checkpoint inhibitors, which are designed to reactivate the antitumor immune response, have only shown limited efcacy in this cancer type.Tis 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 fndings will not only contribute to the understanding of ccRCC pathogenesis but also open new avenues for the development of more efective therapeutic strategies for this devastating disease.

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 [8].Te ccRCC dataset from TCGA, which includes gene expression data and associated clinicopathological information, served as the foundation for our investigation into the role of RBMS2 in ccRCC.

Immunohistochemical (IHC) Staining.
IHC was utilized in this study to evaluate the protein expression of RBMS2 in ccRCC and adjacent nontumorous tissue samples.Te process commenced with the preparation of 4 μm thick tissue sections, which underwent a sequence of treatments involving deparafnization and rehydration utilizing successive xylene and ethanol immersions.Subsequently, heat-induced epitope retrieval was conducted by subjecting the samples to antigen retrieval, employing citrate bufer (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).Te tissue sections were subsequently subjected to an overnight incubation at 4 °C, employing a primary antibody specifc 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.Te stained tissue sections were subsequently examined to evaluate the extent of RBMS2 protein expression.

Cell Culture and Transfection.
Cellular experiments were performed to validate the fndings 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-vectoror pcDNA3.1-RBMS2 in these cell lines by Lipo3000 reagent, allowing us to observe the impact of RBMS2 on ccRCC cell growth [9].Te overexpression plasmids were synthesized in GenePharma (Shanghai, China).Western blotting was used to assess the transfection efcacy.

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.

Migration Assay by
Transwell.Te transwell migration assay was used to evaluate the impact of RBMS2 overexpression on the migration ability of the ccRCC cell lines.Cells (2 × 10 5 ) were suspended in 100 μl serum-free medium at a density of 10 5 /ml and seeded into the upper chamber.Te bottom wells were flled with 600 μl of 10% FBS medium.Cells that had migrated or invaded to the lower surface of the membrane were fxed with methanol and glacial acetic acid and then stained with 20% Giemsaand.Five random felds were chosen to count migrated cell numbers.

Statistics
Statistical analyses were performed using R software.All data are presented as mean ± SD.Te 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 signifcant.

Ethics Statement
Tis 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.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 diferentiation grade (P < 0.001).

Interplay between RBMS2 Expression and Clinico-Pathological Traits in ccRCC Patients
To further validate the expression diference 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 refected higher protein level of RBMS2 in nontumorous kidney tissue than that in ccRCC tissue (Figures 2(b) and 2(c)).

Te Prognostic Relevance of RBMS2 in ccRCC Patients.
Te prognostic potential of RBMS2 in the ccRCC cohort from the TCGA dataset is depicted in Figure 3. Downregulated RBMS2 is signifcantly correlated with worse overall survival (Figure 3(a)), disease-specifc survival (Figure 3(b)), and progression-free survival (Figure 3(c)).
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 identifed as factors signifcantly linked with worse survival.High RBMS2 expression was found to be a signifcant factor in the overall survival multivariate analysis although it did not reach statistical signifcance (hazard ratio � 0.719, P � 0.195).Similar trends were observed in the multivariate analysis for disease-specifc survival (Table 3) and progression-free survival (Table 4), with advanced T stage and metastasis signifcantly associated with worse survival.Of note, high RBMS2 expression was signifcantly associated with better disease-specifc survival (hazard ratio � 0.387, P � 0.004) and progression-free survival (hazard ratio � 0.527, P � 0.02).

Survival Nomograms for ccRCC Patients.
Based on the multivariate analyses, we next formulated disease-specifc 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)).Tese nomograms incorporate various factors, including gender, age, serum calcium level, histological grade, T stage, N stage, M stage, and RBMS2 levels.

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 diferent ccRCC cell lines, 786-O and Caki-2.Te CCK-8 assay results demonstrated that overexpressing RBMS2

Associations between RBMS2 Expression and Immune
Infltrations of ccRCC.We also presented the correlations between RBMS2 expression and immune infltrations in ccRCC.Figure 6(a) shows diferent 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)).

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 tumorsuppressive role of RBMS2.Our fndings reveal that RBMS2 expression is signifcantly downregulated in ccRCC patients with more severe disease characteristics.Moreover, RBMS2 has demonstrated potential as a prognostic indicator for survival outcomes in ccRCC.
Our fndings are in line with previous research that has underscored the tumor-suppressive role of RBMS2 in various malignancies [2,10].RBMS2 is known to inhibit the activation of c-Myc, an oncogene that plays a pivotal role in cell proliferation and growth [2].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 fndings to ccRCC, suggesting a broad  Genetics Research      Genetics Research RBMS2 in ccRCC.Indeed, RBMS2 was reported to chemosensitize breast cancer cells to doxorubicin, implying its therapeutic potential [3].Additionally, our study has shed light on the interaction between RBMS2 expression and immune infltration in the ccRCC tumor microenvironment, hinting at a role for RBMS2 in modulating the tumor immune microenvironment.Te precise mechanisms through which RBMS2 infuences immune infltrations in ccRCC warrant further investigation.

Genetics Research
While our study provides valuable insights, it does come with a few limitations.First, our fndings are based on a retrospective analysis of a public database, and thus prospective studies are needed to validate these fndings.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.

Conclusions
Our research delves deep into the role of RBMS2 in ccRCC and reveals that RBMS2 expression is signifcantly diminished in patients displaying severe disease characteristics.Our fndings 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 infltration 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.

Figure 1 Figure 1 :
Figure 1: Associations between RBMS2 and clinico-pathological traits of ccRCC patients.RBMS2 mRNA levels were found to be signifcantly 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 diferentiation grade (f ).
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 signifcantly 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 signifcantly downregulates the proliferation and migration of the two cell lines (Figures 5(e)-5(h)).

Figure 2 :Figure 3 :
Figure 2: Te mRNA and protein levels of RBMS2 in ccRCC specimens compared to adjacent nontumorous tissues.(a) RT-qPCR data refected 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.

Figure 4 :Figure 5 :
Figure 4: Survival nomograms for ccRCC patients.Disease-specifc 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.Te variables included are gender, age, serum calcium level, histological grade, T stage, N stage, M stage, and RBMS2 levels.

Figure 5 :
Figure 5: Cellular experiments examining the tumor-suppressing role of RBMS2 in ccRCC.(a, b) Te CCK-8 assay revealed that overexpressing RBMS2 can signifcantly inhibit the proliferation capacity of 786-O and Caki-2 ccRCC cell lines.(c, d) Te transwell migration assay demonstrated that overexpressing RBMS2 can signifcantly reduce the migration capacity of 786-O and Caki-2 ccRCC cell lines.(e, f ) Te CCK-8 assay revealed that RBMS2 knockdown can signifcantly enhance the proliferation capacity of 786-O and Caki-2 ccRCC cell lines.(g, h) Te transwell migration assay demonstrated that RBMS2 knockdown can signifcantly upregulate the migration capacity of 786-O and Caki-2 ccRCC cell lines.

Table 1 :
ccRCC patients' characteristics and correlations with RBMS2 expression level.