Abstract

   This study aims to investigate the role of MSH6 in the diagnosis and
   prognosis of bladder cancer and its association with immunity. Various
   analyses were conducted on The Cancer Genome Atlas (TCGA) data, and the
   results were validated using the Gene Expression Omnibus (GEO)
   database. The potential mechanism of MSH6 in bladder cancer was
   revealed using R tools, Gene Set Enrichment Analysis (GSEA), and Gene
   Ontology (GO) analysis. Bladder cancer patients admitted to the
   Affiliated Hospital of Inner Mongolia Medical University from June 2023
   to December 2023 were enrolled. Pathological specimens of bladder
   cancer and adjacent tissues were obtained. Immunohistochemical results
   showed the expression of MSH6 in bladder cancer tissues was higher than
   in adjacent tissues. High expression of MSH6 was identified as an
   independent prognostic factor for bladder cancer. The occurrence of
   bladder cancer was also influenced by age, pathological T stage, and
   pathological stage. According to the Kaplan–Meier survival curve, the
   overall survival rate of the MSH6 high-expression group was lower
   (P = 0.004). After MSH6 was knocked down in cell experiments, the
   proliferation, colony formation, and migration abilities of bladder
   cancer cells were significantly inhibited. MSH6 is significantly
   associated with the diagnosis and prognosis of bladder cancer,
   suggesting its potential utility as a biomarker for these purposes.
   MSH6 is closely related to the tumor immune microenvironment, implying
   a significant role in immunotherapy.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-09644-1.

   Keywords: Bladder cancer, Bladder urothelial carcinoma, MSH6,
   Biomarkers, Immunization

   Subject terms: Cancer, Immunology

Introduction

   Bladder cancer remains the most common malignant tumor of the urinary
   system. In 2018, 549,393 individuals were diagnosed with bladder
   cancer, and 199,922 deaths were recorded worldwide^[38]1. The most
   common pathological type of bladder cancer is urothelial carcinoma,
   accounting for 90% of all cases. Other types, including squamous cell
   carcinoma and adenocarcinoma, account for approximately 10%^[39]2. At
   diagnosis, 75% of bladder cancers are confined to the mucosal
   urothelium and are classified as non-muscle-invasive diseases. In the
   remaining 25–30% of patients, bladder cancer invade the deep layer of
   the bladder wall or metastasize^[40]3. Therefore, more effective
   biomarkers need to be identified for early diagnosis of bladder cancer.
   The human MSH6 gene is a mismatch repair (MMR) gene located on
   chromosome 2. This gene has a length of 23,806 bp and includes 10 exons
   and 83 untranslated regions. This gene encodes the MSH6 protein, which
   plays an important role in DNA repair^[41]4. Current research indicates
   that individuals carrying pathogenic MMR variants have a high risk of
   developing early-stage cancers, particularly colorectal and endometrial
   cancers^[42]5,[43]6. Moreover, abnormal expression of MSH6 is
   associated with an increased risk of ovarian, gastric, hepatobiliary,
   small bowel, pancreatic, breast, prostate, and adrenocortical cancers,
   and glioblastoma^[44]7–[45]14. However, the role of MSH6 in bladder
   cancer remains unclear.

   The Cancer Genome Atlas (TCGA) data were used to analyze MSH6
   expression in bladder cancer samples, and the Human Protein Atlas (HPA)
   was used to validate protein expression. The relationship between MSH6
   expression and prognostic and clinical parameters was investigated
   using R (version 4.2.1). To clarify the regulatory mechanism of MSH6 in
   bladder cancer, GSEA and GO analyses were performed. Additionally, R
   software was utilized to examine associations between MSH6 and the
   tumor microenvironment and immune system. A nomogram was developed to
   predict overall survival (OS) in bladder cancer patients. Calibration
   curves and receiver operating characteristic (ROC) curves were
   constructed to evaluate the accuracy of the nomogram. Finally, we
   explored the genetic alterations in MSH6 in bladder cancer samples.

   After an in-depth analysis of the association between MSH6 expression
   and bladder cancer, it was found that elevated levels of MSH6 were
   associated with poor overall survival. GSEA indicated that enriched
   pathways were closely related to the immune and central nervous
   systems. In addition, we found a negative correlation between MSH6 and
   NK cells, CD56bright T cells, plasmacytoid dendritic cells, immature
   dendritic cells, mast cells, and T helper cells 17. Thus, potential
   mechanisms and the role of MSH6 in bladder cancer were partly
   elucidated. The work design and flow chart of this study are shown in
   Figure [46]S1.

Materials and methods

Data collection and differential expression analysis

   Data on differentially expressed MSH6 from TCGA cancerous tissues were
   collected and analyzed using the UCSC Xena database
   ([47]https://xenabrowser.net/datapages/).

   MSH6 expression levels and associated clinical data were retrieved from
   the TCGA bladder cancer database. To verify MSH6 mRNA expression in
   bladder cancer patients, the original gene profile ([48]GSE3167) was
   obtained from the Gene Expression Omnibus (GEO) database. Visual
   differential expression analysis was performed using the “ggplot2” R
   package (version 4.2.1; [49]https://www.r-project.org/). The HPA
   ([50]http://www.proteinatlas.org) database contains human protein
   expression and location information for various tissues and organs.
   Online immunohistochemical staining data from the HPA were used to
   assess differences in MSH6 protein expression between normal and
   bladder cancer tissues. A clinicopathological scatter plot was created
   to visualize the relationship between MSH6 expression and
   clinicopathological parameters. Logistic regression analysis was
   conducted with R software to investigate associations between clinical
   characteristics and MSH6 expression.

Survival analysis

   To determine the independent prognostic factors for MSH6, univariate
   and multivariate Cox regression analyses were performed using the R
   package survival, and the results were visualized as forest plots using
   ggplot2. Risk factor maps were drawn based on risk scores and survival
   status obtained from Cox regression analysis. The predictive power of
   MSH6 in bladder cancer was evaluated by ROC curve analysis using pROC
   and visualized with ggplot2. Kaplan–Meier survival analysis was
   conducted using the R package survminer to assess the relationship
   between MSH6 expression and patient survival. P < 0.05 was considered
   to indicate statistical significance.

   For overall survival prediction, a Cox regression-based nomogram was
   constructed using the rms and survival ROC packages in R. The area
   under the curve (AUC) values were calculated to evaluate the model’s
   performance. To evaluate the performance of the constructed nomogram,
   calibration curves were also used.

Functional enrichment analysis

   Differential expression analysis between high and low MSH6 expression
   groups in bladder cancer tissues from TCGA was performed using the
   DESeq2 R package. P < 0.05 was considered to indicate statistical
   significance. GO enrichment analysis, including biological processes
   (BP), molecular functions (MF), and cellular components (CC), was
   conducted using the clusterProfiler package. P < 0.05 was considered to
   indicate statistical significance. In addition, GSEA of standardized
   MSH6 RNA-Seq data from the TCGA cohort was performed with cluster
   Profiler.

Immune correlation analysis

   The correlation between MSH6 expression and immune infiltration was
   analyzed using the GSVA R package and visualized as a lollipop plot.
   The TCGA data were divided into high- and low-expression groups based
   on the median MSH6 expression to investigate differences in immune
   infiltration between the groups. The TIMER database
   ([51]https://cistrome.shinyapps.io/timer/) was used to evaluate the
   relationship of MSH6 with a variety of immune cells.

Genetic alteration analysis through cBioPortal

   MSH6 genetic alterations, including alteration frequency, copy number
   alteration (CNA), structural variations, and mutation types, were
   explored using the cBioPortal database ([52]http://cbioportal.org).
   Kaplan–Meier plots were generated to illustrate the relationship
   between MSH6 gene alterations and survival outcomes in bladder cancer
   patients.

Protein–protein interaction (PPI), tumor mutational burden (TMB), and
microsatellite instability (MSI)

   Using the online STRING ([53]https://string-db.org/) database, an
   analysis of the PPI network was also carried out to find potential
   relationships between MSH6 and other genes in bladder cancer by
   querying MSH6 gene in Homo sapiens. In order to explore the
   relationship between MSH6 gene expression and TMB or MSI, respectively,
   correlation analyses were conducted by querying MSH6 gene in
   single-gene pan-cancer analysis tool of Sangerbox.

Immunohistochemical analysis

   Paraffin-embedded sections from patients admitted to the Affiliated
   Hospital of Inner Mongolia Medical University from June 2023 to
   December 2023, who underwent surgical treatment and were pathologically
   confirmed to have bladder cancer after surgery, were collected. These
   sections included both cancer tissues and adjacent normal tissues. This
   study strictly adhered to the principles of medical ethics and was
   authorized and supervised by the Ethics Committee of Inner Mongolia
   Medical University (No. YKD202401093).

   Immunohistochemical staining was conducted, and the percentage of
   positive cells was calculated. Immunohistochemical staining was
   performed according to the manufacturer’s instructions, and the
   MaxVision two-step immunohistochemical staining was also used in this
   study. The staining scores were determined by evaluating membrane
   staining in tumor cells. According to the 2018 ASCO/CAP criteria, the
   IHC scores were classified as negative (score of 0 or 1 +), equivocal
   (score of 2 +), or positive (score of 3 +)^[54]15.

   Results were observed under a laser scanning confocal microscope, and
   images were acquired using an image analysis system. Tissue staining
   data were obtained using ImageJ 1.8 software, and average optical
   density (AOD) was calculated. Data analysis was performed with GraphPad
   Prism 10.3.1, and results were presented as the mean of each group. A
   paired T test was applied for comparisons between groups, and
   differences were considered statistically significant at P < 0.05.

Cell experiments

   Human bladder cancer cell lines (5637, J82, and SV-HUC-1) were
   purchased from Haixing Biology (Suzhou, China). Cells were cultured in
   specialized medium provided by Haixing at 37 °C with 5% CO[2]. MSH6
   expression was silenced using small interfering RNA (siRNA)
   transfection technology. RNA extraction, reverse transcription, and
   real-time quantitative PCR were performed on the three cell lines.
   Subsequently, cell scratch assays, CCK-8 cell proliferation assays, and
   colony formation assays were conducted. Data were analyzed using
   Graphpad Prism 10.3.1 and expressed as Inline graphic  ± s for each
   group. One-way ANOVA was used for multiple comparisons among groups,
   and T tests were performed for comparisons between two normally
   distributed groups.

Results

MSH6 expression analysis

   We analyzed pancancer data from the TCGA and GTEx data to assess MSH6
   expression. MSH6 expression was elevated in 22 cancer types and reduced
   in 5 compared to normal tissues (Fig. [55]1a). Based on TCGA-BLCA data,
   MSH6 expression levels in 19 paired samples, 19 normal tissues, and 412
   bladder cancer samples were plotted (Fig. [56]1b, d). The analysis
   showed significantly increased MSH6 expression in bladder cancer
   tissues (P < 0.05). These findings were validated using microarray data
   from GEO database [57]GSE3167, confirming higher MSH6 expression in
   bladder cancer compared to normal tissues (Fig. [58]1c).
   Immunohistochemistry data from the HPA database further confirmed these
   results (Fig. [59]1e–h).

Fig. 1.

   [60]Fig. 1

   [61]Fig. 1
   [62]Open in a new tab

   MSH6 expression analysis. (a) MSH6 expression in normal and tumor
   tissues in the TCGA and GTEx pancancer datasets (***P < 0.001;
   **P < 0.01; *P < 0.05). (b) Expression of MSH6 in an unpaired bladder
   cancer sample. c. MSH6 expression in the [63]GSE3167 database. (d)
   Expression of MSH6 in paired bladder cancer samples. (e, f)
   Representative image of an immunohistochemical image showing the
   expression of MSH6 in bladder cancer tissues. (g, h) Representative
   image of the immunohistochemical analysis showing MSH6 expression in
   normal bladder tissue.

Relationships between MSH6 expression and clinicopathological parameters

   Various clinicopathological parameters of bladder cancer patients were
   compared with MSH6 expression. Results showed that MSH6 expression was
   significantly associated with race (P < 0.01; Fig. [64]2a), subtype
   (P < 0.001; Fig. [65]2b), and histological grade (P < 0.001;
   Fig. [66]2c). Logistic regression analysis indicated that MSH6
   expression was significantly correlated with primary therapy outcomes
   (PR&CR vs. PD&SD, P = 0.013) and histologic grade (low grade vs. high
   grade, P = 0.003) (Table [67]1).

Fig. 2.

   [68]Fig. 2
   [69]Open in a new tab

   Differential expression of MSH6 according to various
   clinicopathological parameters. MSH6 expression was correlated with (a)
   race, (b) subtype, and (c) histological grade.

Table 1.

   Association between MSH6 expression and clinicopathological
   characteristics using logistic regression.
   Characteristics Total (N) OR (95% CI) P value
   Age (> 70 vs. <  = 70) 412 1.000 (0.677–1.476) 1.000
   Gender (Male vs. Female) 412 0.739 (0.476–1.149) 0.180
   BMI (> 25 vs. <  = 25) 362 1.141 (0.751–1.733) 0.537
   Race (Black or African American vs. Asian &White) 395 1.372
   (0.587–3.207) 0.466
   Primary therapy outcome (PR&CR vs. PD&SD) 355 0.553 (0.346–0.884) 0.013
   Histologic grade (Low grade vs. High grade) 409 0.045 (0.006–0.339)
   0.003
   Lymphovascular invasion (Yes vs. No) 281 0.709 (0.442–1.136) 0.153
   Pathologic stage (Stage III& Stage IV vs. Stage I& Stage II) 410 1.336
   (0.883–2.024) 0.171
   [70]Open in a new tab

   Significant values are in bold.

Survival analysis

   As shown in Table [71]2, we explored the relationship between MSH6
   expression and overall survival in patients with bladder cancer using
   Cox regression analysis. Univariate Cox regression analysis showed that
   age (HR = 1.424, P = 0.018), pathological T stage (HR = 2.157,
   P < 0.001), pathological stage (HR = 1.677, P = 0.016), and MSH6
   expression (HR = 1.551, P = 0.004) were significantly associated with
   OS. Multivariate Cox regression analysis (Fig. [72]3a, forest plot)
   revealed MSH6 expression (P = 0.012) as an independent prognostic
   factor. The distribution of MSH6 expression, patient survival status,
   and predicted risk scores based on the MSH6 Cox model are shown in
   Fig. [73]3b. In addition, according to the K‒M survival curve, patients
   in the MSH6 high-expression group had a lower overall survival rate
   (P = 0.004; Fig. [74]3c). The ROC curve indicated that MSH6 had good
   prognostic capability (AUC = 0.615) (Fig. [75]3d).

Table 2.

   Correlations between overall survival and multivariate characteristics
   in TCGA patients according to Cox regression analysis.
   Characteristics Total(N) Univariate analysis Multivariate analysis
   Hazard ratio (95% CI) P value Hazard ratio (95% CI) P
   Age 411 0.018
    <  = 70 231 Reference Reference
    > 70 180 1.424 (1.064–1.906) 0.018 1.145 (0.743–1.765) 0.539
   Gender 411 0.395
   Female 108 Reference
   Male 303 0.868 (0.629–1.198) 0.390
   Pathologic T stage 377  < 0.001
   T1&T2 123 Reference Reference
   T3&T4 254 2.157 (1.485–3.132)  < 0.001 2.038 (0.269–15.418) 0.490
   Pathologic stage 275 0.014
   Stage I& Stage II 133 Reference Reference
   Stage III 142 1.677 (1.102–2.553) 0.016 0.897 (0.121–6.647) 0.916
   Histologic grade 408 0.066
   High grade 387 Reference Reference
   Low grade 21 0.338 (0.084–1.365) 0.128 0.811 (0.191–3.440) 0.776
   Race 394 0.403
   Asian& White 371 Reference
   Black or African American 23 1.284 (0.729–2.260) 0.387
   MSH6 411 0.003
   Low 206 Reference Reference
   High 205 1.551 (1.153–2.086) 0.004 1.774 (1.136–2.771) 0.012
   [76]Open in a new tab

Fig. 3.

   [77]Fig. 3
   [78]Open in a new tab

   Survival analysis of patients stratified by MSH6 expression. (a)
   Multivariate Cox regression analysis of MSH6 expression and other
   clinicopathological variables. (b) MSH6 expression distribution and
   survival status (0 = survival; 1 = death). c. MSH6 mRNA expression
   levels and overall survival. d. ROC curve for MSH6.

   Using this nomogram, survival rates for bladder cancer patients at 1,
   3, and 5 years could be predicted with increased accuracy
   (Fig. [79]4a). The AUC values of the nomogram were 0.583, 0.555, and
   0.516, indicating moderate prediction accuracy (Fig. [80]4b). Our
   nomogram calibration curves were plotted at 1, 3, and 5 years
   (Fig. [81]4c–e).

Fig. 4.

   [82]Fig. 4
   [83]Open in a new tab

   Construction and evaluation of the nomogram. (a) Nomogram constructed
   based on MSH6 and clinicopathological variables. (b) OC curve of MSH6.
   (c) Calibration curve at 1 year. d. Calibration curves for 3 years. (e)
   5-year calibration curve.

MSH6 enrichment analysis

   GO and GSEA were used to explore the potential biological functions of
   MSH6. As shown in Fig. [84]5a, GO terms related to BP, MF, and CC
   closely associated with MSH6 included axon development, cell fate,
   neurons, intermediate filaments, keratin fibers, serine hydrolase
   activity, serine-type peptidase activity, and serine-type endopeptidase
   activity. As shown in Fig. [85]5b, GSEA results indicated that genes
   within KEGG and Reactome pathways were downregulated^[86]16–[87]18.

Fig. 5.

   [88]Fig. 5
   [89]Open in a new tab

   Functional and pathway enrichment analysis of MSH6 in bladder cancer.
   (a) Important Gene Ontology terms associated with MSH6, including
   biological process (BP), cellular component (CC), and molecular
   function (MF)([90]www.kegg.jp/kegg/kegg1.html). (b) Significant GSEA
   results associated with MSH6, including KEGG pathway and REACTOME
   pathway enrichment analyses ([91]www.kegg.jp/kegg/kegg1.html).

The relationship between MSH6 expression and immune system and tumor
microenvironment

   We investigated the different immune microenvironment in patients with
   bladder cancer with different tumor MSH6 levels. As shown in
   Fig. [92]6a, compared to the low-expression group, the proportions of T
   helper 2 cells, helper T cells, central memory T cells, T helper 1
   cells, aDCs, macrophages (P < 0.001), Tgd (P = 0.0131), regulatory T
   cells (P = 0.0003), effector memory T cells (P = 0.0022), NK cells
   (CD56dim subsets) (P = 0.0032), T cells(P = 0.00213), and T follicular
   helper cells (P = 0.00213) were increased in the high-expression group.
   As shown in Fig. [93]6b, MSH6 expression levels correlated with the
   activation of dendritic cells, macrophages, helper T cells, central
   memory T cells, TGDS cells, T helper 1 cells, T helper 2 cells
   (P < 0.001), NK cells, the CD56dim subset (P = 0.0048), effector memory
   T cells (P = 0.0011), regulatory T cells (P = 0.0006), NK cells, the
   CD56bright subset, plasmacytoid dendritic cells (P < 0.001), immature
   dendritic cells (P = 0.0118), mast cells (P = 0.0016) and T helper cell
   17 (P = 0.0206). Based on these results, MSH6 may play a key role in
   the immune infiltration of bladder cancer. To investigate the
   association between MSH6 expression and immune cell infiltration in
   bladder cancer, correlation analyses were performed using the TIMER
   database. We found that the expression of MSH6 was positively
   correlated with the infiltration of CD4^+ T cells, CD8^+ T cells,
   neutrophils, macrophages, dendritic cells, and B cells (P < 0.001)
   (Fig. [94]6c).

Fig. 6.

   [95]Fig. 6
   [96]Open in a new tab

   MSH6 expression is correlated with the immune system and tumor
   microenvironment. (a) The varied proportions of 24 subtypes of immune
   cells in high and low MSH6 expression groups in tumor samples. (b)
   Correlation between MSH6 expression and the level of immune
   infiltration. (c) Correlation between the expression of MSH6 and immune
   cell infiltration.

Genetic variant analysis

   Mutation characteristics of MSH6 in bladder cancer patients from TCGA
   were explored using the cBioPortal database, showing a genetic
   alteration frequency of 6.08% (Fig. [97]7a). As shown in Fig. [98]7b,
   alterations in the MSH6 gene were not significantly associated with
   patient survival outcomes. Figure [99]7c illustrates the mutation sites
   of MSH6 in bladder cancer.

Fig. 7.

   [100]Fig. 7
   [101]Open in a new tab

   Mutational signature of MSH6 in patients with bladder cancer in the
   TCGA cohort determined using the cBioPortal tool. (a) Type of MSH6 in
   patients with bladder cancer from the TCGA cohort and the frequency of
   mutation changes. (b) K‒M survival analysis of OS with or without MSH6
   alterations. (c) MSH6 mutation sites in bladder cancer.

Relationships between MSH6 and PPI, MSI and TMB in bladder cancer

   With the help of the online STRING database, MSH6 was analyzed to find
   possible relationships with other genes in bladder cancer using PPI
   network analysis (Fig. [102]8a). According to Fig. [103]8b,c, our
   results revealed that MSH6 expression was not related to MSI and TMB.

Fig. 8.

   [104]Fig. 8
   [105]Open in a new tab

   Relationships between MSH6 and PPI, MSI, and TMB in bladder cancer. (a)
   PPI network. (b) Relationships between MSH6 and MSI. c. Relationships
   between MSH6 and TMB.

Detection of MSH6 expression level in paired tissues by immunohistochemical
staining

   Bladder cancer tissues collected at the Affiliated Hospital of Inner
   Mongolia Medical University were further verified by detecting MSH6
   expression level through immunohistochemical staining. A total of 8
   pairs of bladder cancer tissues and adjacent tissues were collected
   (Fig. [106]9a). Quantitative analysis (Fig. [107]9b) showed that the
   expression level of MSH6 in bladder cancer tissues was significantly
   higher than in adjacent tissues (0.36 ± 0.03 vs. 0.49 ± 0.1, P < 0.01).

Fig. 9.

   [108]Fig. 9
   [109]Open in a new tab

   Immunohistochemical images and quantitative analysis results of MSH6.
   (a) Representative images of MSH6 immunohistochemistry obtained by
   laser scanning confocal microscope. (b) Results of immunohistochemical
   quantitative analysis of MSH6.

The biological function of MSH6 in bladder cancer cells

   We determined MSH6 expression levels in cells using real-time
   fluorescence quantitative PCR.As shown in Fig. [110]10a, MSH6
   expression in bladder cancer cell lines was significantly higher than
   in normal urothelial cells. After knockdown of MSH6 using siRNA,
   proliferation, colony formation, and migration of bladder cancer cells
   were significantly inhibited. Figure [111]10b,c further confirmed the
   crucial role of MSH6 in bladder cancer progression. As shown in
   Fig. [112]10d–g, Cell proliferation and colony formation abilities
   decreased significantly following MSH6 knockdown. Scratch assay results
   (Fig. [113]10d–k) demonstrated a significant reduction in the migration
   ability of bladder cancer cells after MSH6 knockdown.

Fig. 10.

   [114]Fig. 10
   [115]Open in a new tab

   The biological function of MSH6 in bladder cancer cells. (a) The
   expression level of MSH6 mRNA in normal bladder epithelial cell lines,
   J82 and 5637 cell lines (*P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001). (b) qRT-PCR detection of MSH6 mRNA expression in 5637
   cell lines after knockdown (*P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001). (c) qRT-PCR detection of MSH6 mRNA expression in J82
   cell lines after knockdown (*P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001). (d) CCK-8 assay to detect the proliferation ability of
   5637 cells after si-NC and si-MSH6 treatment. (e) CCK-8 assay to detect
   the proliferation ability of J82 cells after si-NC and si-MSH6
   treatment. (f–g) Clonogenic assay of 5637 cells. (h–i) Scratch assay to
   detect the migration ability of 5637 cells after si-NC and si-MSH6
   treatment (*P < 0.05; **P < 0.01). (j–k) Scratch assay to detect the
   migration ability of J82 cells after si-NC and si-MSH6 treatment
   (*P < 0.05; **P < 0.01).

Discussion

   MSH6 is a very attractive biomarker in cancer diagnosis; for example,
   the MSH6 protein exhibits low expression in glioblastoma^[116]19 and
   endometrial cancer^[117]20 but high expression in prostate
   cancer^[118]21. According to our results, MSH6 expression was lower in
   five types of cancer tissues and higher in 22 types, including bladder
   cancer, compared to normal tissues.

   In this article, we analyzed the differential expression of MSH6 in
   bladder cancer using multiple public databases. To further verify MSH6
   expression in bladder cancer, we collected eight pathological
   specimens, including adjacent tissues. Immunohistochemical analysis
   revealed high expression of MSH6 in bladder cancer tissues. MSH6
   expression is associated with various tumor characteristics, such as
   race, subtype, and histological grade. Nomograms have been widely used
   to aid in clinical decision-making. GO enrichment and GSEA analyses
   indicated that MSH6 upregulation was closely associated with signaling
   pathways, the immune system, and the central nervous system,
   particularly the Wnt signaling pathway. The Wnt signaling pathway is
   activated in various cancers and contributes to tumorigenesis^[119]22.

   We further explored the potential association between MSH6 and the
   immune system, primarily regarding immune infiltration and the tumor
   microenvironment. The tumor microenvironment is a highly complex
   system^[120]23,[121]24. The tumor microenvironment can facilitate tumor
   growth, metastasis, and resistance to chemotherapy and
   immunotherapy^[122]25,[123]26. Th1, Th2 and Th17 cells are mostly
   differentiated from naive CD4^+ T cells^[124]27. Th1 and Th2 cells
   participate in cellular immunity and humoral immunity, respectively, by
   secreting various specific cytokines^[125]28. CD8^+ T cells mediate
   cytotoxic activity mainly by inducing tumor cell apoptosis^[126]29.

   Human NK cells are typically classified into CD56brightCD16^- and
   CD56dimCD16^+ subsets^[127]30, with CD56dimCD16^+ NK cells exhibiting
   strong cytotoxic activity upon stimulation by target cells^[128]31. In
   the TCGA cohort, the frequency of genetic alterations in MSH6 was
   6.08%. However, no association was observed between genetic alterations
   of MSH6 and bladder cancer prognosis. We utilized the online STRING
   database to conduct PPI network analysis on MSH6 in the context of
   bladder cancer. This approach allowed exploration of potential
   relationships between MSH6 and other genes. These mutations can hinder
   tumor evolution by triggering an immune response to the tumor^[129]32.
   Cancer immunotherapy involves using drugs to activate or enhance the
   immune system’s natural mechanisms to attack cancer
   cells^[130]33,[131]34. Therefore, immunotherapy is considered a
   promising strategy for treating or even curing certain types of cancer.

   In our MSH6 expression analysis, significant differences were
   identified between bladder cancer cell lines (5637 and J82) and normal
   urothelial cells (SV-HUC-1). These findings suggest that abnormal MSH6
   expression may be closely related to bladder cancer occurrence.
   Biological function experiments further confirmed the critical role of
   MSH6 in bladder cancer. The CCK-8 proliferation assay, colony formation
   assay, and scratch assay showed significant inhibition of
   proliferation, colony formation, migration, and invasion abilities in
   bladder cancer cells (5637 and J82) following MSH6 knockdown by siRNA.
   These findings indicate that MSH6 plays a crucial role in promoting the
   proliferation and self-renewal of bladder cancer cells.

   However, this study has limitations. First, animal model experiments
   were not conducted to further verify biological effects in vivo.
   Second, immune-related experiments were limited, and the relationship
   between MSH6 and the immune microenvironment will be explored in future
   studies. Additionally, only selected bladder cancer cell lines were
   analyzed; therefore, the expression and functional differences of MSH6
   in other cell lines across different clinical stages and pathological
   types require further investigation.

Conclusions

   In summary, our findings indicate that MSH6 may have significant roles
   in the immune system and bladder cancer prognosis. MSH6 significantly
   promotes the proliferation, self-renewal, migration, and invasion of
   bladder cancer cells, suggesting its potential as a therapeutic target.
   Ultimately, we anticipate that our results will provide new insights
   into bladder cancer immunotherapy for future research.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [132]Supplementary Material 1^ (172.7KB, docx)

Abbreviations

   TCGA
          The Cancer Genome Atlas

   BLCA
          Bladder cancer

   GEO
          Gene Expression Omnibus

   GSEA
          Gene Set Enrichment Analysis

   GO
          Gene Ontology

   HPA
          Human Protein Atlas

   OS
          Overall survival

   ROC
          Receiver operating characteristic

   AUC
          Area under the curve

   AOD
          Average optical density

   BP
          Biological process

   MF
          Molecular function

   CC
          Cellular component

   BP
          Biological process

   MF
          Molecular function

   CAN
          Copy number alteration

   K‒M
          Kaplan–Meier survival curve

   siRNA
          Small interfering RNA

   Treg
          Regulatory T cells

   Th1 cells
          Type 1 Th cells

   aDC
          Activated dendritic cells

   Th2 cells
          Type 2 Th cells

   Tfh
          T follicular helper

   Tem
          T effector memory

   pDC
          Plasmacytoid dendritic cell

   NK cells
          Natural killer cells

   Th17 cells
          Type 17 Th cells

Author contributions

   Ning He analyzed the data and wrote the manuscript. Yan Meng designed
   the experiments. Lu-Ri Bao was responsible for data curation.
   Peng-cheng Ma conducted the cell experiments and immunohistochemical
   experiments. Yan Meng reviewed and approved the manuscript. Zhen-Li Fu,
   Li-Gang Chen, Jing-Yuan Wang, Wu-niri Gao were responsible for the
   project administration. Yan Meng supervised the experiments.

Funding

   This work was supported by the General Project of Inner Mongolia
   Natural Science Foundation (No. 2022MS08063), the Key Research and
   Development and Achievement Transformation Project in the Social
   Welfare Field of the 14th Five-Year Plan in the Inner Mongolia
   Autonomous Region (No. 2022YFSH0087), Science and Technology Program of
   the Joint Fund of Scientific Research for the Public Hospitals of Inner
   Mongolia Academy of Medical Sciences (2024GLLH0337), Inner Mongolia
   Medical University Affiliated Hospital Talent Training Project—Sailing
   Series, the Inner Mongolia Health Science and Technology Project in
   2022 (No. 202201293), the National Natural Science Foundation of China
   (No. 81960143), the Inner Mongolia Grassland Talents Program Young
   Innovative Talent Project (No. Q2022082), the Shanghai Key Laboratory
   of Kidney and Blood Purification (No. 14DZ226022, 20DZ2271600), the
   Science and Technology Commission of Shanghai, and the Trinity College
   Students Innovation and Entrepreneurship Cultivation Project of Inner
   Mongolia Medical University (No.SWYT2020008).

Data availability

   Data is provided within the manuscript or supplementary information
   files.

Declarations

Ethics statement

   The studies involving human participants were reviewed and approved by
   the Ethics Committee of Inner Mongolia Medical University (No.
   YKD202401093). The patients/participants provided their written
   informed consent to participate in this study.

Competing interests

   The authors declare no competing interests.

Footnotes

   Publisher’s note

   Springer Nature remains neutral with regard to jurisdictional claims in
   published maps and institutional affiliations.

   Ning He, Lu-ri Bao and Peng-cheng Ma contributed equally to this
   article.

References