RARRES1 identified by comprehensive bioinformatic analysis and experimental validation as a promising biomarker in Skin Cutaneous Melanoma

Skin cutaneous melanoma (SKCM) is a highly malignant form of skin cancer, known for its unfavorable prognosis and elevated mortality rate. RARRES1, a gene responsive to retinoic acid receptors, displays varied functions in various cancer types. However, the specific role and underlying mechanisms of RARRES1 in SKCM are still unclear. GSE15605 was utilized to analyze the expression of RARRES1 in SKCM. Subsequently, the TCGA and GEO databases were employed to investigate the relationships between RARRES1 and clinicopathological parameters, as well as the prognostic implications and diagnostic efficacy of RARRES1 in SKCM. GO, KEGG, and GSEA analyses were conducted to explore the potential functions of RARRES1. Furthermore, the associations between RARRES1 and immune infiltration were examined. Genomic alterations and promoter methylation levels of RARRES1 in SKCM were assessed using cBioPortal, UALCAN, and the GEO database. Finally, RARRES1 expression in SKCM was validated through immunohistochemistry, and its functional role in SKCM progression was elucidated via in vivo and in vitro experiments. We found that RARRES1 was downregulated in SKCM compared with normal tissues, and this low expression was associated with worse clinicopathological features and poor prognosis of SKCM. The diagnostic efficacy of RARRES1, as determined by ROC analysis, was 0.732. Through GO, KEGG, and GSEA enrichment analysis, we identified 30 correlated genes and pathways that were mainly enriched in the tumor immune microenvironment, proliferation, apoptosis, and autophagy. Additionally, RARRES1 expression was found to be positively related to the infiltration of various immune cells in SKCM, particularly macrophages and T helper cells, among others. Analysis of genomic alterations and promoter methylation revealed that shallow deletion and hypermethylation of the RARRES1 promoter could lead to reduced RARRES1 expression. IHC validation confirmed the downregulation of RARRES1 in SKCM. Moreover, overexpression of RARRES1 inhibited the proliferation and migration of A375 cells, promoted apoptosis, and inhibited autophagic flux. In the mouse xenograft model, RARRES1 overexpression also suppressed SKCM tumor growth. Collectively, these findings suggest that RARRES1 may function as a suppressor and could potentially serve as a prognostic biomarker and therapeutic target for SKCM.

was used to create a co-expression heatmap.Then, we selected 900 genes which were significantly correlated with RARRES1 (P < 0.05, |R|> 0.5) to perform GO and KEGG analysis (Table S1).GO analysis is a widely accepted approach for performing comprehensive functional enrichment studies on a large scale, encompassing biological processes (BP), molecular functions (MF), and cellular components (CC) 13 .KEGG serves as a highly utilized database for archiving data related to genomes, biological pathways, diseases, and pharmaceuticals 14 .GO annotation analysis and KEGG pathway enrichment analysis of correlated genes were performed using R package clusterProfiler.P < 0.05 was considered statistically significant.Results of enrichment analysis were visualized using ggplot2, igraph, and ggraph.

GSEA analysis
Gene Set Enrichment Analysis (GSEA) is a powerful analytical technique designed to assess whether a predefined group of genes exhibits statistically significant and coherent variations between two distinct phenotypes 15 .The RNAseq data of 470 SKCM patients was analyzed by DESeq2 and edgeR.After obtaining gene names and their corresponding log fold changes (logFC), we performed GSEA using the clusterProfiler.The objective was to unveil substantial disparities in functional and pathway profiles between the high-and low-RARRES1 groups.h.all.v2022.1.Hs.entrez.gmt[Hallmarks] in MSigDB Collections (https:// www.gsea-msigdb.org/ gsea/ msigdb/ colle ctions.jsp) was used as the reference gene collection.An adjusted P < 0.05 and False discovery rate (FDR) < 0.25 were considered as significant enrichment.

Correlation analysis between RARRES1 expression and immune infiltration
For the information of 470 SKCM patients in TCGA database, immune infiltration analysis was performed using the ssGSEA algorithm based on that provided in the R package GSVA [1.46.0], using markers of 24 immune cells to calculate the immune infiltration corresponding to the cloud-based data 16,17 .The data were processed using R (4.2.1) and correlation analysis was performed between the primary variables and immune infiltration matrix data, and the results were displayed as lollipop plots and analyzed through Spearman correlation analysis.Data was visualized using ggplot2.

Analysis of gene mutations and promoter methylation analysis
cBioPortal database (http:// cbiop ortal.org) was used to analyze the profile of genetic alterations in SKCM, amino acid changes in proteins, and the effect of different copy number variants (CNV) on RARRES1 mRNA expression 18 .Furthermore, UALCAN database (https:// ualcan.path.uab.edu/) was utilized to assess RARRES1 promoter methylation in SKCM 19 .Finally, we downloaded GSE120878 dataset from the GEO database.This dataset consists of DNA methylation data for 89 SKCM and 73 nevi.Furthermore, EWAS database (https:// ngdc.cncb.ac.cn/ ewas/) was used to retrieve methylation sites specifically associated with RARRES1 in SKCM.

Clinical samples
Paraffin-embedded skin samples were taken from 20 patients (11 male and 9 female, aged 57-74 years) diagnosed with SKCM and 11 healthy controls (6 male and 5 female, aged 60-78 years) diagnosed with intradermal nevus.These samples were sourced from the tissue bank of the First Affiliated Hospital of Xi'an Jiaotong University.All diagnoses were confirmed by two experienced pathologists.Written informed consent was diligently obtained from all patients before sample collection and analysis.Ethics approval for the study was acquired from the Institutional Ethics Committee of Xi'an Jiaotong University.

Cell culture
The A375 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium containing with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37℃ in a humidified atmosphere with 5% CO 2 .

Plasmid construction and transient transfection
The RARRES1 expression plasmid was synthesized by MiaoLing Plasmid (Wuhan, China).A375 cells were transfected with 2 μg of the RARRES1 expression plasmid (pCMV-RARRES1(human)-3×FLAG) or the control pCDNA vector using PEI for 48 h.The transfection procedures were conducted according to the standard protocols.

Transwell assay
Transwell assays were conducted using Transwell chambers (3422; Corning).The lower chamber was filled with 800 μL of complete culture.In the upper chamber, 3 × 10 4 cells were seeded using 200 μL of fetal bovine serumfree medium.After a 24-h incubation, the migrating cells were fixed and then stained with 0.1% aqueous crystal violet.Microscopic images were captured, and the cell count in each field was documented.

Apoptosis assays
Cells transfected by pcDNA3.4-RARRES1/Vector,were stained with FITC Annexin V Apoptosis Detection Kit according to the manufacturer's instructions.Cells apoptosis was analyzed by flow cytometry (NovoCyte flow cytometry, ACEA Biosciences).

ROS determination
ROS levels were measured with DHE (Biyotime, #S0063) dyes.A375 cells were first seeded into 12-well plates overnight and transfected with pcDNA3.4-Vectoror pCDNA3.4-RARRES1for 48 h.Then, cells were incubated with DHE solution dissolved in FBS-free medium for 45 min and then washed with PBS.Nexcope NIB620 inverted microscope (Nexcope, Ningbo, China) was used to take the fluorescent images.Image J software was usd to analyze DHE relative fluorescence intensity.(bar length = 50 um).

LysoSensor green DND-189 staining
Lysosomal staining with LysoSensor Green DND-189, was performed according to the manufacturer (Yeasen 40767ES50, Shanghai, China).After transfection of pCDNA3.4,Lysosensor green was added to the medium and incubated for 10 min at 37℃, with 5% CO 2. Then cells were directly transferred to a fluorescent microscope for imaging.

Xenograft tumor model
A total of 4 × 10 6 cells transfected with pCDH-RARRES1/Vector were subcutaneously injected into4-week-old female BALB/c-nude mice on their right flanks (6 mice/group).Tumor volume was calculated every 3 days.On day 10, the mice were sacrificed and subcutaneous tumors were collected.Tumor volumes were calculated using the formula V = 1/2 × L × W 2 , where "L" represents tumor length and "W" represents tumor width.All animal experiment were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

Ethics approval and consent to participate
Ethics approval for the study was acquired from the Institutional Ethics Committee of Xi'an Jiaotong University, and the use of animals in our experiments was consistent with ethical requirements.

Expression, clinicopathological correlation, prognostic and diagnostic potential of RARRES1 in SKCM
The expression of RARRES1 in SKCM (n = 58) and normal tissues (n = 16) was analyzed using GSE15065 dataset.
As shown in Fig. 1A, RARRES1 expression in SKCM was significantly down-regulated compared to the normal tissues.Subsequently, TCGA database was used to investigate the correlation between RARRES1 expression and clinical characteristics of SKCM.We found that RARRES1 was significantly reduced in SKCM patients of T4 stage, pathological stage II, age over 60, with melanoma ulcer, higher Clark level, higher Breslow thickness and without radiotherapy (Fig. 1B-H), indicating decreased RARRES1 expression was associated with poor clinical features of SKCM.We then assessed the correlation between RARRES1 expression and survival of SKCM patients.The Kaplan-Meier survival analysis indicated that low expression of RARRES1 was associated with unfavorable overall survival (OS) or disease specific survival (DSS) (Fig. 1I-J).Besides, RARRES1 expression significantly affected the OS of SKCM patients undergoing immunotherapy, showing an overall correlation with poor OS after immunotherapy (HR = 0.6 (0.43-0.83), logrank P = 0. 0021 for all anti-PD-1 treatment; HR = 0.049 (0.29-0.83), logrank P = 0.0068 for anti-CTLA-4 treatment) (Fig. 1K-L).We futher evaluated the factors correlated with OS in SKCM, as shown in Table 1.Univariate Cox regression analysis showed that tumor stage (T, N, M), patient age, ulceration, Clark level, Breslow depth, and RARRES1 expression were associated with survival.Multivariate Cox regression analysis demonstrated that only pathological N stage, Breslow depth and RARRES1 are independent prognostic factors for survival.For further verification, we constructed a nomogram of OS that incorporated RARRES1, pathological N stage and Breslow depth, enabling us to predict the 1-, 3-, 5-year survival probability in SKCM (Fig. 1M).Based on the expression data of RARRES1 in GSE15605, the diagnostic value of RARRES1 in SKCM was calculated, the area under the curve (AUC) was 0.732 (P = 0.0048), which illustrated that RARRES1 had a good diagnostic performance in SKCM 21 (Fig. 1N).

Gene correlation and functional enrichment analysis of RARRES1 expression in SKCM
We proceeded to employ bioinformatics analysis in order to examine the biological function of RARRES1.Initially, we conducted correlation analysis between RARRES1 and other genes in the TCGA database.Subsequently, we assigned ranks to genes that exhibited a significant correlation with RARRES1 expression (P < 0.05).Consequently, we identified the top 15 genes that displayed a positive correlation and the top 15 genes that displayed a negative correlation with RARRES1, as visually depicted in the heatmap (Fig. 2A).The GO terms were classified into BP, CC and MF.Within the BP category, the enriched terms included leukocyte cell-cell adhesion, regulation of T cell activation, and positive regulation of leukocyte.In the CC category, the enriched terms included external side of the plasma membrane, T cell receptor complex, and plasma membrane signaling receptor complex.Lastly, in the MF category, the enriched terms included immune receptor activity, cytokine receptor activity, and MHC protein complex binding.In terms of KEGG pathway enrichment, significant enrichments were observed in hematopoietic cell lineage, cell adhesion molecules, and cytokines-cytokine receptor interaction (Fig. 2B-C).We also performed a GSEA analysis to identify the key pathways related to RARRES1.GSEA analysis found that 480 data sets satisfied the criteria of an FDR < 0.25 and a P < 0.05, as shown in Table S2.

Correlation between RARRES1 expression and immune cell infiltration in SKCM
Functional enrichment analysis of RARRES1 revealed that numerous pathways were closely associated with immune processes.Furthermore, tumor-associated immune cells play important roles in determining tumor progression, and the infiltration of immune cells is strongly related to the outcome of the tumor 22 .Therefore, we assessed the correlation between the RARRES1 expression and the extent of immune cell infiltration in SKCM.
As shown in Fig. 3A, RARRES1 expression in SKCM was positively correlated with the infiltration of various immune cells, indicating the low expression of RARRES1 might be unfavorable for immune cell infiltration in SKCM.We further compare the difference in immune cell infiltration between RARRES1 high and low expression groups.The results demonstrated that SKCM with low expression of RARRES1 had less immune cell infiltration (Fig. 3B-U).

Mutation and promoter methylation of RARRES1 in SKCM
Previous studies have demonstrated that the abnormal expression of RARRES1 arises due to hypermethylation of the CpG island associated with the RARRES1 promoter 23 .Thus, we analyzed the genomic alteration and promoter methylation of RARRES1 via cBioPortal, UALCAN and GEO database.In a total of 443 SKCM samples, www.nature.com/scientificreports/ the mutation rate of RARRES1 was found to be 1.8% (Fig. 4A).This implies that there were 8 instances of gene alteration, comprising 6 cases of mutation and 2 cases of deep deletion (Fig. 4B).More specifically, the missense mutation of RARRES1 in SKCM led to changes in the amino acids that constitute the protein (Table 2).All 6 mutated nucleotide positions were visually represented in Fig. 4C.Copy number variations (CNV) involve the amplification or deletion of chromosomal segments resulting from genomic rearrangements.CNVs play a crucial role in influencing gene expression 24 .We then investigated the association between the RARRES1 expression and CNVs.As shown in Fig. 4D, shallow deletion maybe responsible for the downregulated of RARRES1.In addition, data from UALCAN database showed that the promoter methylation of metastatic melanoma was significantly higher than that of the normal group, as shown in Fig. 4E.However, due to the limited sample size of the normal group, we were unable to identify any significant differences in the promoter methylation levels between normal and SKCM tissues.In order to conduct a more comprehensive analysis of the promoter methylation of RARRES1 in SKCM, we identified 15 promoter methylation sites of RARRES1 based on the GSE120878 dataset.Among these sites, 10 exhibited significantly higher levels of methylation in SKCM compared to nevus (Fig. 5A-J).These findings suggest a strong correlation between the downregulation of RARRES1 expression in melanoma and the presence of mutations as well as elevated promoter methylation.

Decreased RARRES1 expression was confirmed in SKCM by immunohistochemistry
To validate the expression of RARRES1, a total of 20 skin samples from patients with SKCM and 11 skin samples from individuals with intradermal nevus were collected for immunohistochemistry staining.As depicted in Fig. 6A, RARRES1 was found to be weakly expressed in the tumor tissues of SKCM.In contrast, a noticeable increase in the expression of RARRES1 was observed in intradermal nevi (Fig. 6B).Furthermore, a semiquantitative analysis demonstrated a significant downregulation of RARRES1 in SKCM (Fig. 6C).

RARRES1 overexpression inhibits A375 cell proliferation and migration
Based on the above results, we suppose that Rarres1 is a tumor suppressor in SKCM.Furthermore, we conducted gain-of-function assays in A375 cells by transfected pCDNA-RARRES1/Vector and lentiviruses-RARRES1/Vector.The overexpressing efficiency is shown in Fig. 7A.Results from MTT assay (Fig. 7B) showed that RARRES1 overexpression inhibited A375 cell proliferation.Consistently, crystal violet staining (Fig. 7C-D) and soft agar assays (Fig. 7E-F) showed an injured ability of colony formation on RARRES1 upregulated A375 cells.Flow cytometry analysis was subsequently conducted to assess the impact of RARRES1 on the cell cycle distribution of A375 cells.As demonstrated in Fig. 7G-H, RARRES1 overexpression resulted in an increased cell population in the G0/G1 phase and a concurrent decrease in the S phase.In addition, we detected the expression of cell cycle related protein.Western blotting results revealed that RARRES1 overexpression led to a reduction in the expression levels of Cyclin D1 and Cyclin E1, as depicted in Fig 7 I and J.As the pathway related to migration and invasion is visible in the enrichment analysis, we next analyzed the role of RARES1 on the migration or invasion of A375 cells.We directly studied in vitro migration in 24-well plates inserted with 8.0-mm-pore transwells.Significant decrease in migration was observed in RARRES1 overexpression cells compared with vector group (Fig. 7K-L).Consistently, an increased expression of E-cadherin was observed upon RARRES1 overexpression (Fig. 7M-N).Overall, these findings indicate that RARRES1 plays an important role in cell proliferation, colony formation, cell cycle and migration, and its overexpression significantly suppresses A375 cell proliferation and migration.

Overexpression of RARRES1 promotes apoptosis and inhibits autophagic flux in A375 cell line
We then investigated the role of RARRES1 in the regulation of cell apoptosis.A375 cells transfected with the pCDNA-vector showed significantly low baseline apoptosis rates, whereas overexpression of RARRES1 noticeably increased the rate of cell apoptosis (Fig. 8A-B).RARRES1 has been reported to activate p53, thereby promoting apoptosis.We subsequently investigated the impact of RARRES1 on p53 in A375 cells, and the results showed that RARRES1 overexpression significantly increased the protein levels of p53 and its target gene BAX.Additionally, we observed an increase in the expression of BAK, another key executor of mitochondrial membrane permeabilization in the mitochondrial pathway apoptosis (Fig. 8C-D).Since ROS plays a crucial role in various  www.nature.com/scientificreports/signaling pathways, such as cell cycle regulation and apoptosis, we further explored the effect of RARRES1 on ROS and observed a significant increase in ROS levels with RARRES1 overexpression (Fig. 8E-F).As ROS elevation also promotes autophagy 25 , we next accessed whether Rarres1 over-expression impacts autophagy.As shown in Fig. 8G-H, an augmented LC3 conversion from LC3-I to LC3-II in cells overexpressing RARRES1 was discovered through immunoblotting assays.These findings indicate that the overexpression of RARRES1 led to the accumulation of autophagosomes in A375 cells.This could be attributed to either an enhancement in autophagy induction or a suppression in autophagosome degradation.To elucidate the underlying factor contributing to the autophagosome accumulation, we initially assessed the expression levels of key autophagy-related proteins.Our results demonstrated that RARRES1 overexpression significantly upregulated the mRNA expression of ATG5, ATG7, and ATG10 (Fig. 8I), demonstrating that RARRES1 overexpression induced autophagic activation.Next, we investigated whether RARRES1 overexpression hinders downstream processes in autophagic flux.We analyzed the expression of the SQSTM1/P62 protein, which acts as a selective autophagy receptor and is degraded alongside ubiquitinated substrates during autolysosome degradation.As demonstrated in Fig. 8J-K, we observed an accumulation of p62 upon Rarres1 overexpression.This finding aligned with the inhibition of autophagic flux during the later stages.Subsequently, we hypothesized that the blockage of autophagic flux could be attributed to lysosomal dysfunction.Since a low lysosomal pH is essential for maintaining proper lysosomal function, we utilized Lysosensor DND-189, a pH-dependent probe that exhibits increased fluorescence upon acidification, to assess lysosomal pH levels.As presented in Fig. 8L-M, Rarres1 overexpression led to a reduction in fluorescence, indicating a decrease in lysosomal acidification and thus contributing to the impairment of autophagic flux.Overall, these findings suggest that Rarres1 overexpression stimulated autophagic initiation while simultaneously impeding the degradation of autophagic cargo during the later stages.In summary, our findings suggest that RARRES1 overexpression inhibits SKCM cell proliferation by inducing ROS-mediated cell cycle arrest, apoptosis induction, and autophagy blockade.Furthermore, RARRES1 overexpression also suppresses the migration of SKCM cells (Fig. 8N).

Overexpression of RARRES1 suppresses tumor growth in vivo
Xenograft tumor experiments were conducted to assess the in vivo effects of RARRES1.The nude mice were administered a subcutaneous injection of stable RARRES1-overexpressing cells.The tumors of RARRES1-overexpressing cells exhibited smaller volume and reduced weight (Fig. 9A-D).The stable overexpression of RARRES1 is depicted in Fig. 9E.Immunohistochemical staining (IHC) demonstrated a significant reduction in the expression of the cell proliferation marker Ki67 in the RARRES1 overexpression group (Fig. 9F-G).These results confirmed that RARRES1 overexpression inhibits tumor growth in vivo, consistent with the in vitro experimental findings.

Discussion
RARRES1, also referred to as Tazarotene-induced gene 1 (TIG1), plays a crucial role in the regulation of various tumorigenic processes.Studies have shown a significant downregulation of RARRES1 expression in several types of cancers, including prostate, colorectal, nasopharyngeal, gastric, endometrial, and testicular cancers.Furthermore, it has been observed that the expression level of RARRES1 is inversely correlated with patient survival in cases of prostate and colorectal cancers 9,10,[26][27][28][29] .Nonetheless, RARRES1 might have a promotive effect on cancer development in specific tumor types.For example, Wang X et al. demonstrated that enhanced RARRES1 expression in inflammatory breast cancer facilitated the growth of tumors 30 .
To elucidate the expression and functional roles of RARRES1 in SKCM, we obtained data from both the GEO and TCGA databases, followed by comprehensive bioinformatic analyses.Our findings revealed a significant downregulation of RARRES1 expression in SKCM.Furthermore, we observed that low RARRES1 expression was associated with unfavorable clinical features and prognostic outcomes, regardless of whether patients received immunotherapy.Consequently, RARRES1 emerged as an independent prognostic factor for SKCM.Moreover, it held promise as a valuable diagnostic marker.These results substantiated the suppressive role of RARRES1 in the progression of SKCM, supporting previous studies exploring RARRES1 in other tumor types 9,10 .A recent study has unveiled that the coexistence of tumor-associated CD8 + T cells and CD20 + B cells is correlated with enhanced survival in metastatic melanoma 31 .Srour et al. performed immunohistochemical staining on samples obtained from melanoma patients receiving immune checkpoint inhibitor.The results of their study revealed a significant association between immune infiltration and the therapeutic efficacy of the treatment 32 .In our study, we conducted GO, KEGG, and GSEA enrichment analysis, which revealed a significant association between RARRES1 expression and the tumor immune microenvironment, cell cycle regulation, migration, apoptosis, autophagy, and oxidative damage.Furthermore, our findings indicated a positive correlation between RARRES1 expression in SKCM and the infiltration of various immune cells, including macrophages, CD8 + T cells, B cells, and T helper cells.These results suggest that RARRES1 may enhance its anti-tumor effect in SKCM by promoting immune cell infiltration.Consistent with our findings, a previous study demonstrated that RARRES1 induces M1 macrophage activation to exert antitumor effects in renal clear cell carcinoma 11 .
We also investigated the underlying mechanism behind the reduced expression of RARRES1 in SKCM.Prior research has established a correlation between the transcriptional silencing of RARRES1 in esophageal, gastric, endometrial, and prostate cancers and epigenetic inactivation resulting from promoter hypermethylation 23,28,29,33 .In accordance with the aforementioned studies, our findings demonstrated a notably increased level of RARRES1 promoter methylation in metastatic melanoma when compared to the control group.Moreover, we identified 10 promoter sites that exhibit higher levels of methylation in SKCM as compared to nevus.Additionally, we  observed that various copy number mutations, particularly shallow deletion, have also been associated with decreased expression of RARRES1 in SKCM.
Based on the findings of bioinformatic analysis, we proceeded with experimental validation in order to corroborate the expression and function of RARRES1 in SKCM.IHC staining showed a significant downregulation of RARRES1 in SKCM compared to intradermal nevus.Both in vitro and in vivo experiments consistently demonstrated that increased expression of RARRES1 suppressed the proliferation of A375 cells, which aligns with the observations made in a prior study conducted on colorectal cancer cells 9 .Through flow cytometry analysis, we discovered that RARRES1 exerts inhibitory effects on cell proliferation by suppressing the G1/S transition, as evidenced by the downregulation of Cyclin D1 and Cyclin E1 expression.According to the results of the enrichment analysis, it was found that RARRES1 is strongly correlated with the migration and invasion of melanoma.Therefore, a thorough examination was conducted to investigate the influence of RARRES1 on cell migration.Consistent with the initial hypothesis, the outcomes of the transwell assay demonstrated that overexpression of RARRES1 effectively impeded the migration of A375 cells, which was accompanied by a notable elevation in the protein levels of E-cadherin.www.nature.com/scientificreports/As previously mentioned, the upregulation of RARRES1 in glomerular disease resulted in the upregulation of genes related to the cell death pathway.Moreover, the overexpression of RARRES1 in podocytes induced the activation of P53, leading to apoptosis and promoting the advancement of glomerular disease.These discoveries implyed that RARRES1 might have a significant involvement in the process of apoptosis 34 .As expected, our results revealed that RARRES1 overexpression induced apoptosis in A375 cells through upregulating the expression of pro-apoptotic factors P53, BAK and BAX.In addition, studies have demonstrated that ROS plays a key role in apoptosis 25,35 .Consistently, we also found that RARRES1 overexpression promoted ROS production, providing another plausible explanation for RARRES1's role in facilitating cell apoptosis.
On the other hand, we found autophagy flux in A375 cell was inhibited by RARRES1 overexpression.Autophagy is an extensively conserved lysosomal degradation pathway that plays a crucial role in maintaining cellular homeostasis by clearing protein aggregates and damaged organelles 36 .Previous studies have shown that RARRES1 activates autophagy to inhibit tumor progression in prostate cancer and cervical cell carcinoma 7,37 .In our study, we discovered that the overexpression of RARRES1 led to an increase in the expression of ATG5/7/10 and LCII, suggesting the activation of autophagy during the early stages.However, we also observed the accumulation of P62, a biomarker associated with the late stages of autophagy.This accumulation indicated that the overexpression of RARRES1 inhibited the progression of autophagy towards the later stages.Recent research has highlighted that the inhibition of autophagy at later stages can lead to the excessive buildup of undegraded materials within autophagic vacuoles, ultimately resulting in an accelerated process of cell death 38 .Therefore, it was hypothesized that the inhibition of autophagic flux is significantly correlated with the heightened prevalence of cellular apoptosis.

Conclusion
In conclusion, the expression of RARRES1 in SKCM was found to be diminished as a result of copy number mutations and high promoter methylation.Additionally, it was observed that low RARRES1 expression correlated with a poorer overall survival.Enrichment analysis suggested that RARRES1 may function as a tumorsuppressive factor by regulating various aspects of immune cell infiltration, proliferation, migration, apoptosis, and autophagy.Experimental validation further confirmed the functional roles of RARRES1 in proliferation, migration, apoptosis, and autophagy.This study emphasizes the significance of RARRES1 as a valuable prognostic biomarker for SKCM and highlights its potential as a promising therapeutic target.

Figure 2 .Figure 3 .
Figure 2. Gene correlation and functional enrichment analysis of RARRES1 expression in SKCM.(A) Top 30 genes correlated with RARRES1 expression.(B) The GO and KEGG enrichment analysis of RARRES1.(C) The network results of GO and KEGG pathways.(D-K) GSEA analysis on (D) mitotic G1 phase and G1/S transition, (E) caspase cascade, (F) oxidative damage response, (G) autophagy, (H) cancer immunotherapy by PD1 blockade, (I) cancer immunotherapy by CTLA4 blockade, (J) modulators of TCR signaling and T cell activation, (K) natural killer cell mediated cytotoxicity.
T-test was used to analyze the differential expression genes between SKCM and normal.Kruskal-Wallis Test, Welch t' test and Wilcoxon rank sum test were used to assess the differences in RARRES1 expression among samples with varying clinical features.Logrank test was used to compare the prognosis between the high and low expression groups of RARRES1.Sperman's correlation was carried out to evaluate enrichment scores of 20 immune cells and RARRES1 expression separately.Experimental data were expressed as the mean ± SEM.Statistical graphs and analyses were conducted using GraphPad Prism software.To compare two unpaired groups, Student's t-test was employed, and P < 0.05 was deemed as statistically significant.

Table 1 .
The univariate and multivariate analyses of overall survival.