Effect of High‑Fat-Diet and Semaglutide on Bladder Cancer in Mice

doi:10.21203/rs.3.rs-4899171/v1

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Research Article

Effect of High‑Fat-Diet and Semaglutide on Bladder Cancer in Mice

https://doi.org/10.21203/rs.3.rs-4899171/v1

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Objective

The aim of this study is to examine the proteomics of adipose tissue in mice with obesity induced by a high-fat diet, in order to investigate how obesity affects the protein expression profile of adipose tissue. Additionally, we seek to establish a foundation for understanding the mechanism through which semaglutide may impact bladder cancer (BLCA) associated with obesity.

Methods

Thirty-six male C57BL/6J mice, all in good health, were chosen and divided into three groups: a group fed a normal diet (referred to as the NCD group), a group fed a high-fat diet (known as the HFD group), and a group fed a high-fat diet along with semaglutide treatment (referred to as the Sema group). We examined how obesity affects serum markers and how semaglutide influences these markers. Additionally, we investigated changes in protein expression within BLCA using proteomics techniques. By employing bioinformatics methods, we identified differentially expressed proteins that may be associated with the hypothesized mechanism of semaglutide's potential for reducing bladder cancer risk.

Results

Our findings indicate that semaglutide has the potential to decrease body weight, enhance glucose metabolism, and improve blood lipid levels. The alterations observed in the expression of Lama2 ( laminin subunit alpha-2), Lama4 (laminin subunit alpha 4), Lamc1 (laminin subunit gamma 1), Thbs2 (thrombospondin 2) genes across the normal group, high fat group, and semaglutide group primarily involve the extracellular matrix (ECM) pathway. Following intervention with semaglutide, a significant reduction in the expression of various proteins was observed in BLCA. These results suggest that by modulating genes such as Lama2, Lama4, Lamc1, Thbs2 and others, semaglutide may potentially mitigate the risk associated with BLCA.

Conclusion

Semaglutide exhibits potential in mitigating obesity induced by a high-fat diet and delaying the onset and progression of bladder cancer. The activation of Lama2, Lama4, Lamc1, Thbs2, and their involvement in the ECM pathway may underlie the mechanism through which semaglutide exerts its effects on bladder cancer.

bladder cancer

semaglutide

GLP-1

Obesity

BLCA

Obesity is a condition characterized by an excessive accumulation of body fat, particularly triglycerides. It is generally considered a lifestyle disorder and is typically assessed through the evaluation of body mass index (BMI) ≥ 30 kg/m²(1). Obesity is often associated with insulin resistance, hyperinsulinemia, impaired glucose tolerance, dyslipidemia (elevated triglyceride levels, increased low-density lipoprotein ratio, decreased high-density lipoprotein cholesterol levels), and inflammation(2, 3). As living standards improve, the prevalence of obesity has increased due to high-fat diets, leading to a rise in obesity-related diseases, including cancer(4, 5). Research has found that obesity is considered a result of inflammation and insulin resistance, and it can increase the risk of cancer.

Glucagon-like peptide-1 (GLP-1) is an important intestinal incretin hormone secreted by enteroendocrine cells or L cells. It plays a crucial role in glucose regulation in the human body through various mechanisms, including increasing insulin secretion, improving βcell function, and inhibiting glucagon release(6, 7). Additionally, GLP-1 slows down gastric emptying, reduces appetite, and promotes satiety, aiding in weight control. Furthermore, GLP-1 has protective effects on pancreaticβcells, reduces insulin resistance, and inhibits fatty acid synthesis, positively impacting insulin sensitivity and lipid metabolism. As a result, GLP-1-based medications are widely used in the treatment of type 2 diabetes and obesity(8). Semaglutide is a long-acting GLP-1 receptor agonist that shares 94% structural similarity with natural GLP-1. It can be safely used in adult and elderly patients with kidney, liver, or cardiovascular diseases(9). Research has found that semaglutide can reduce high blood lipid levels, body weight, and hepatic steatosis, while improving cognitive abilities in neurodegenerative diseases(10). Drug repurposing strategies are effective methods for exploring different therapeutic opportunities, helping to reduce the cost and time required to bring drugs with known safety and pharmacokinetic profiles to the market. Semaglutide is a medication that has been clinically approved for the treatment of type 2 diabetes and obesity, and it has the potential to be repurposed as a multifunctional therapeutic agent.

Biological therapy for cancer, known as targeted cancer therapy, employs therapeutic drugs like therapeutic antibodies to disrupt specific molecular targets necessary for the growth of cancer cells(11). Consequently, there is an immediate requirement for novel prognostic biomarkers that can accurately forecast the prognosis of BLCA patients and offer innovative treatment strategies.

BLCA is the most common cancer of the urinary system and ranks among the top ten causes of cancer-related deaths worldwide(12). The primary treatment options for BLCA include surgery, radiation therapy, chemotherapy, and immunotherapy. Transurethral resection of bladder tumor is the preferred treatment for BLCA(13). However, these methods are invasive, expensive, and have low sensitivity. In recent years, researchers have been actively searching for new prognostic biomarkers for BLCA, aiming to improve early diagnosis, progression monitoring, risk prediction, and treatment optimization for this disease(14). Targeted cancer therapy is a form of biological treatment that utilizes drugs, such as therapeutic antibodies, to disrupt specific molecular targets necessary for cancer cell growth, thereby destroying cancer cells. Hence, there is an urgent need for novel prognostic biomarkers to predict the prognosis of BLCA patients and offer innovative treatment approaches.

2.1 Ethical Approval

All animal testing conducted in this study received approval from the Animal Ethics Committee of Hebei Provincial People's Hospital and adhered to the guidelines outlined in the Regulations on Laboratory Animal Management.

2.2 Animal experiments

Male C57BL/6 mice (6 weeks old) were purchased from Hebei Avivo Biotechnology Co., Ltd. (Shijiazhuang, China). All mice were housed in a specific pathogen-free (SPF) facility with a controlled temperature of 24 ± 2°C and a relative humidity of 60 ± 10%. They were maintained on a 12-hour light-dark cycle. After one week of acclimatization, the mice were randomly divided into two groups: the normal diet group (4% fat, 20% carbohydrates, 20% protein) and the high-fat diet group (60% fat, 20% carbohydrates, 20% protein). After 12 weeks, the mice in the HFD group were further divided into two subgroups: the HFD control group and the Semaglutide group (30 nmol/kg/day, Novo Nordisk, Bagsværd, Denmark)(15). The mice in the NCD and HFD groups received an equal volume of phosphate-buffered saline (PBS) and were intraperitoneally injected with 1% pentobarbital sodium (60 mg/kg) to induce anesthesia. Blood was collected from the retro-orbital sinus and placed in sterile tubes containing 1mm EDTA. Subsequently, the mice were euthanized, and the interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) were weighed. Tissue samples were either stained with hematoxylin and eosin (H&E) or rapidly frozen in liquid nitrogen and stored at -80°C until further analysis.

2.3 Preparation of serum samples

The mice were orally administered a glucose load (2g/kg dose) and their fasting tail blood glucose levels were measured at each time point (0, 15, 30, 60, 90, and 120 minutes) using a blood glucose meter and test strips (AccuCHEK, USA) to assess glucose tolerance. The mice were fasted for 6 hours prior to the experiment. First, the blood samples were allowed to stand at room temperature for 30 minutes. They were then centrifuged at 3500 rpm for 10 minutes at 4°C. The supernatant was collected and stored at -80°C. Serum levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG), oxidative stress marker superoxide dismutase (SOD), and inflammatory factors were measured using enzyme-linked immunosorbent assay (ELISA) kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.4 Histopathological analysis

The bilateral epididymal white adipose tissue and interscapular brown adipose tissue were fixed in 4% paraformaldehyde for 48 hours. After fixation, the tissues were embedded in paraffin and sectioned into 4 µm thick slices using a microtome. After deparaffinization, the tissue sections were stained with H&E. The target regions of the tissue were imaged at 200x magnification using the Nikon Eclipse Ci-L optical microscope (Nikon Eclipse E100). After imaging, data analysis was performed using Image-Pro Plus 6.0 software with Nikon DS-U3 analysis software.

2.5 Protein Extraction and Digestion

To prepare the samples for analysis, we pulverized nine adipose tissues from the epididymis using liquid nitrogen and then subjected them to lysis by incubating in a buffer solution containing SDT (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH7.6).The digestive peptides from each sample underwent desalination using a C18 column (EmporeTM SPE column C18 with standard density), followed by enrichment through vacuum centrifugation and reconstitution in 40 μl of 0.1% formic acid (v/v).The level of protein purity was assessed using the SDS-PAGE method, and subsequently, each sample's peptide mixture was labeled with iTRAQ or TMT reagent (Thermo Scientific).

2.6 LC-MS/M LC-MS/MS Analysis

LC-MS/MS analysis was performed using Q Exactive mass spectrometer analysis (Thermo Scientific, Santa Clara, CA, USA), coupled with Easy nLC, and then labeled peptides were transferred to an inverse trap column (Thermo Fisher Scientific) and C18 inverse analysis column (Thermo Scientific) for linear gradient separation in buffer A and buffer B at a flow rate of 300 nl/min. The MASCOT engine (Matrix Science, UK) integrated within Proteome Discoverer 1.4 software was utilized to extract the MS raw data for qualitative and quantitative analysis of each sample. This facilitated the identification of differentially expressed proteins (DEPs) by filtering out irrelevant data. The differential expression analysis was performed using fold change (FC) and P value as screening criteria. Proteins with FC > 1.2 and P < 0.05 were considered up-regulated, while proteins with FC < 0.83 and P < 0.05 were considered down-regulated.

2.7 Bioinformatics analysis

The protein subcellular localization prediction was conducted using CELLO (http://cello.life.nctu.edu.tw/), a system based on multi-class SVM classification(16). Using the Xiantao database (http://www.xiantao.com), we selected differentially expressed proteins and aligned them to homologous sequences. Subsequently, we performed gene ontology (GO) term localization and functional annotation(http://geneontology.org/)(17). The proteins under study were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp/) to retrieve their KEGGs and then mapped to KEGG pathways. Enrichment analysis was conducted using Fisher's exact test. Additionally, the protein-protein interaction (PPI) network was constructed to demonstrate the interconnections among DEPs, utilizing the string database as a resource.

2.8 Statistical analysis

The statistical analysis was conducted using the software Graphpad Prism 9.5.0. In cases where all samples exhibited normal distribution and homogeneous variation, the analysis of variance was employed for statistical evaluation. Conversely, if any samples deviated from normal distribution or displayed heterogeneous variance, the nonparametric rank sum test was utilized instead. A significance level of p<0.05 was considered to indicate statistically significant differences.

3.1 Mouse Weight Change

The HFD mouse model was effectively established through the implementation of a high-fat diet approach. Figure 1 shows the changes in body weight of mice before high-fat diet feeding, after high-fat diet feeding, and after intervention with semaglutide. We can observe that there was not much change in body weight in the NCD group. After intervention with a high-fat diet, the HFD group of mice showed a significant increase in body weight compared to the NCD group (P < 0.01) (Figure 1B) After intervention with semaglutide, there was a significant decrease in body weight of mice (P < 0.01) (Figure 1C).

3.2 Serum indicators and changes in SOD

The findings from the experiment revealed that obesity could potentially contribute to dyslipidemia and elevated blood glucose levels, as evidenced by the significant increase in LDL-C, HDL-C, TC, TG, and blood glucose levels observed in the HFD group when compared to the NCD group. After administering semaglutide treatment, the Sema group of mice exhibited significantly decreased serum TC and TG levels compared to the HFD group (P < 0.05), suggesting that semaglutide possesses potential in lowering lipid and glucose levels. However, there were no statistically significant disparities observed in the levels of HDL-C and LDL-C between the two groups (P > 0.05) (Figure 2A-D). In addition, our findings indicate a significant reduction in the SOD level among individuals in the HFD group compared to those in the NCD group (P < 0.05). The presence of obesity may contribute to a diminished capacity for combating oxidative stress and elevate susceptibility to oxidative stress-related damage. However, the administration of semaglutide resulted in a notable elevation in the SOD level of mice in the Sema group (P<0.05), suggesting that semaglutide possesses the potential to enhance resistance against oxidative stress (Figure 2E).

3.3 Changes in glucose tolerance of mice

The glucose tolerance of mice was examined, revealing a significant increase in blood glucose levels among the mice in the HFD group. In comparison to the NCD and Sema groups, the HFD group exhibited significantly higher blood glucose values at 15, 30, 60, 90, and 120 minutes with a gradual decline thereafter. However, the administration of semaglutide resulted in significant reductions in glucose levels at 15, 30, 60, 90 and 120 minutes. This suggests that semaglutide effectively mitigated the fluctuations in glucose caused by the high-fat diet and enhanced both glucose metabolism and insulin sensitivity(Figure 2F).

3.4 Changes in Adipose Tissue

The changes in adipocytes were observed, and the results showed that the diameter and area of adipocytes in HFD group were significantly increased compared with those in NCD group, while after the intervention of semaglutide, the diameter and area of adipocytes were significantly reduced (Figure 3).

3.5 Differential protein qualitative and quantitative analysis.

The mass spectrometry analysis yielded a total of 1,038,748 secondary spectra, out of which 130,933 were successfully retrieved from proteomics databases as valid secondary spectra. Through spectral analysis, 53911 peptide segments were successfully identified, of which 48914 were specific. In addition, 7590 types of proteins were detected in this study, with 7553 types available for quantitative analysis (Figure 4).

3.6 Identification of differentially expressed proteins

Firstly, significant differences among different groups can be obviously observed in the principal component analysis score chart, indicating high consistency within each group and good discrimination between groups (Figure 5A-C). Each trial was conducted thrice, and the FC and P values were computed based on the acquired data. In the NCD/HFD group, a total of 683 proteins with differential expression were identified, comprising 342 up-regulated and 341 down-regulated proteins. The HFD/Sema group exhibited 640 differentially expressed proteins, including 292 up-regulated and 348 down-regulated proteins. Similarly, the NCD/Sema group displayed a total of 772 differentially expressed proteins, with 446 up-regulated and 326 down-regulated proteins. To effectively visualize the distinct protein expression patterns among the NCD group, HFD group, and Sema group, we employed a volcano plot to demonstrate their significant differences in distribution (Figure 5D-F).

3.7 Subcellular localization

The CELLO database was utilized to analyze the subcellular localization of all DEPs. The results revealed that in the NCD/HFD group, DEPs were predominantly distributed within various cellular compartments including the cytoplasm, cell membrane, nucleus, mitochondria, extracellular space, endoplasmic reticulum, peroxidase bodies, cytoskeleton and lysosomes (Figure 6A). In the HFD/Sema group, DEPs were mainly concentrated in the cytoplasm, cell membrane, nucleus, endoplasmic reticulum and mitochondria, and also appeared on lysosomes and Golgi complex structures (Figure 6B). Regarding the NCD/Sema group, DEPs were predominantly detected within the cytoplasm, nucleus, and mitochondria. Additionally, they were observed on the endoplasmic reticulum and peroxidase bodies, as well as lysosomes and Golgi complex structures (Figure 6C).

3.8 Enrichment analysis

In order to deeply explore the functions of DEPs, we conducted functional enrichment analysis, including biological process (BP), molecular function (MF) and cellular component (CC). In the NCD/HFD group, BP mainly concentrates on fatty acid metabolism, regulation of external stimulus response, regulation of immune process, activation of immune response and response to oxidative stress. The enrichment of CC is predominantly observed in the inner membrane, sheath, microstructure domain of the mitochondria, as well as in the mitochondrial matrix. On the other hand, MF shows a higher abundance in actin proteins, transmembrane transport proteins, and phospholipid binding (Figure 6D). In the HFD/Sema group, BP exhibits significant enrichment in processes related to positive regulation of immune response, metabolism of fatty acids, regulation of inflammatory response, modulation of immune effects, and responsiveness to external stimuli. CC is primarily found in the extracellular matrix that contains collagen, as well as in the nuclear envelope, mitochondrial inner membrane, and membrane wall. On the other hand, MF is predominantly present in proteins that bind to phospholipids, actin, and GTP (Figure 6E). In the NCD/Sema group, BP primarily exhibits enrichment in immune effect process regulation, positive modulation of external stimulus response, fatty acid metabolic processes, inflammatory response regulation, and oxidative stress responsiveness. CC is primarily found in the extracellular matrix that contains collagen, as well as in the microstructure domain of membranes, cell cortex, and membrane scaffold. On the other hand, MF shows a higher enrichment in activities such as binding to phospholipids, actin proteins, and GTP-binding proteins (Figure 6F).

3.9 Key protein screening and expression changes

KEGG pathway analysis was conducted on the proteins that were up-regulated in the NCD/HFD group and down-regulated in the HFD/Sema group. The findings of this analysis revealed a significant enrichment of ECM pathways closely associated with BLCA in our experimental samples. In the ECM pathway, we performed a screening of four essential proteins, namely Lama2, Lama4, Lamc1 and Thbs2. Comparatively, the HFD group demonstrated significantly increased levels of expression for Lama2, Lama4, Lamc1 and Thbs2 when compared to the NCD group (Figure 7). This implies that the ECM pathway's functionality may be influenced by a high-fat diet, which in turn affects the expression of these four proteins. Consequently, this impacts the onset and progression of BLCA. To further confirm the presence of these proteins in BLCA, we conducted verification through KEGG's official website, as depicted in Figure 8.

3.10 Survival analysis of protein in BLCA

Compared to the group of patients with high expression levels of Lama2, Lama4, Lamc1, and Thbs2 proteins, those with low expression levels exhibited significantly extended overall survival (P < 0.05). This suggests that individuals with elevated expression of these four proteins experience comparatively shorter overall survival in comparison to those with lower expression levels (Figure 9).

The correlation between obesity and BLCA has become a subject of immense interest in scientific investigations(18). Due to the shift in contemporary lifestyle patterns, obesity has progressively emerged as a global health concern, with its prevalence escalating annually(19). Simultaneously, BLCA, being a highly prevalent and recurrent tumor within the urinary system, has exhibited an ongoing upward trajectory. While semaglutide is not commonly used in cancer treatment, there is a growing interest in its potential for preventing cancer. Hence, this article aims to investigate the mechanism behind obesity-related BLCA induced by a high-fat diet and assess the impact of semaglutide on BLCA(10). This research will contribute novel insights into prevention strategies and identify new targets for diagnosing and treating BLCA.

Obesity poses a worldwide health concern, elevating the likelihood of developing at least 13 different forms of cancer(20). BLCA remains a prevalent and fatal malignancy affecting the urinary system, despite significant advancements in its diagnosis and treatment over the past few decades(18). These advancements encompass early identification, surgical interventions, and pharmaceutical approaches. This is influenced not only by the severity of tumors, but also by various factors including the lifestyle choices of patients(19). Over the years, lifestyle choices have gained greater significance. For instance, an unhealthy diet, being overweight, and having metabolic disorders can elevate the chances of developing BLCA(21). As per Koebnick's findings, individuals with obesity face a 28 percent higher likelihood of developing this form of cancer compared to those who maintain a normal weight(22). GLP-1 plays a crucial role as an incretin within the human body, effectively enhancing glucose metabolism and promoting weight loss through its ability to stimulate insulin production, suppress glucagon release, and decelerate gastric emptying(23). Semaglutide, a GLP-1 receptor agonist, has demonstrated notable efficacy in promoting weight reduction, enhancing glucose and lipid metabolism abnormalities, safeguarding cardiovascular health and exhibiting potential associations with cancer(24). While semaglutide has yet to be utilized in the direct treatment of cancer, an increasing number of research studies have begun to focus on its potential as a preventive measure against certain forms of cancer. Hence, it is crucial to investigate the role that semaglutide may play in addressing obesity-related BLCA.

Given that obesity is the primary contributor to BLCA, we employed a high-fat diet approach to establish a mouse model for this experiment. The findings revealed a significant disparity in weight between the NCD group mice and those on the HFD, thereby confirming the successful establishment of our HFD mouse model. After the implementation of a high-fat intervention, there was a significant increase in weight observed among the mice on the HFD, while noticeable differences were found in the weight of NCD mice. These findings suggest that the high-fat diet led to evident obesity development in the mice. After a period of 12 weeks, the mice in the Sema group that were administered semaglutide exhibited significant reduction in weight, which was notably different from the HFD group. This suggests that semaglutide has potential to counteract weight gain caused by high-fat feeding and display favorable effects on weight loss. Numerous studies have established obesity as an autonomous risk factor for various cancers, primarily associated with amplified fat cell count and size among individuals affected by obesity(21). The experiments conducted on animals demonstrated that the introduction of a high-fat diet led to a notable increase in both the diameter and area of adipocytes in mice. However, following the administration of semaglutide, there was a significant reduction observed in the diameter and area of adipocytes among obese mice. These findings align with Chromecki's assertion regarding the positive association between obesity and BLCA(25).

Research has indicated that conditions closely associated with obesity, such as disturbances in glucose metabolism, irregular lipid metabolism, and oxidative stress, could potentially exert detrimental impacts on bladder tissue and heighten the susceptibility to cancer. The findings from this study revealed that the mice in the HFD group exhibited significantly elevated blood glucose levels compared to those in the NCD group, suggesting that obese mice induced by a high-fat diet experienced severe impairments in glucose metabolism when compared to their normal counterparts. These results are consistent with a previous study conducted by Wang Y(26). However, following the administration of semaglutide, there was a significant reduction in blood glucose levels observed in the Sema group. This suggests that semaglutide has the potential to improve glucose metabolism disorders caused by a high-fat diet in obese mice. Furthermore, the findings from this study indicate that obesity may contribute to abnormal lipid metabolism as evidenced by significantly increased levels of LDL-C, HDL-C, TC, and TG compared to the NCD group. Following semaglutide treatment, the Sema group of mice exhibited significantly reduced serum levels of TC and TG compared to those in the HFD group(27). This suggests that semaglutide may have a positive impact on dyslipidemia resulting from obesity. They also discovered that the HFD group exhibited significantly reduced SOD levels compared to the NCD group, implying that obesity might contribute to a decline in antioxidant capacity and an elevated susceptibility to oxidative stress injury. Nevertheless, following semaglutide treatment, the Sema group experienced a significant increase in SOD levels, indicating that semaglutide has the potential to enhance antioxidant capacity.

An increasing number of research studies have indicated that the activation of the ECM pathway plays a crucial role in stabilizing the tumor microenvironment and providing structural support for cells(28). Additionally, it is involved in significant biological processes such as intercellular communication, migration, and differentiation. Furthermore, the activation of the ECM pathway is a crucial factor in promoting tumor proliferation, invasion, immune evasion and resistance to therapeutic drugs(29). Therefore, it is imperative to conduct an extensive investigation into the specific mechanisms underlying ECM pathway activity for effective cancer prevention and treatment. In this research, it was observed that the adipose tissue of obese mice exhibited elevated expression levels of ECM pathway proteins such as Lama2, Lama4, Lamc1 and Thbs2(30, 31). However, following the administration of semaglutide intervention, a significant decrease in the expression of these proteins was noted. These genes play a crucial role in the development, progression, and prognosis of BLCA by activating the ECM pathway(32). Therefore, it is essential to utilize these ECM pathway-related genes for predicting the occurrence, tumor stage, and prognosis of BLCA associated with obesity.

It has been documented that BLCA is closely associated with members of the laminin family, namely Lama2, Lama4, and Lamc1(33). These proteins are known to have a significant impact on the ECM pathway by facilitating cell adhesion, migration, and differentiation(34). In addition, the interaction between cancer cells and matrix facilitated by the protein pair could potentially contribute to the proliferation and metastasis of tumors(35). In the context of BLCA, Lama2, Lama4, and Lamc1 facilitate the activation of the ECM pathway through their interaction with integrin and other receptors(36). This interaction subsequently influences tumor cell behavior and promotes increased production of collagen fibers and elastic fibers by BLCA cells(37). As a result, there is an induction of ECM sclerosis and remodeling, creating a conducive environment for BLCA cell invasion and metastasis.

Lama2 has been identified in multiple types of cancer, including liver cancer, breast cancer, ovarian cancer, pancreatic cancer, and colorectal cancer(38, 39). Its increased expression has the potential to enhance cellular migration and invasion. Additionally, the level of Lama2 expression exhibits a strong correlation with tumor grading, staging, and prognosis(37). However, the direct correlation between Lama2 overexpression and BLCA progression has been largely overlooked by researchers(40). The regulation of adipocyte function through cell receptor signals is directly influenced by Lama4, which in turn indirectly affects adipocyte behavior by modulating the local cellular microenvironment and interacting with other components of the ECM(41). These interactions have a significant impact on the initiation and advancement of BLCA. In addition, the significance of Lama4 in adipose tissue expansion and function has been observed in various studies(42). The attention towards the role of Lama4 in cancer has grown significantly over the past few decades(43). Lama4 exhibits high expression levels within hepatocellular carcinoma, breast cancer, and renal cell carcinoma cells, thereby being closely associated with the development and progression of cancer(44). In terms of functionality, Lamc1 facilitates the growth and movement of cancer cells through its involvement in multiple signaling pathways, interaction with ECM pathways, and initiation of downstream effectors within cellular systems(45). An association has been observed between elevated levels of Lamc1 expression and the malignancy as well as prognosis of BLCA(46). Furthermore, gastric cancer, prostate cancer, hepatocellular carcinoma, endometrial cancer, and various other cancers have demonstrated a significant upregulation of Lamc1(47). Thbs2 is a crucial protein involved in the regulation of platelet function, primarily contributing to the modulation of ECM, cellular adhesion, and angiogenesis(37). Thbs2 belongs to the glycoprotein family, which also exists in the ECM pathway and is closely related to the tumor microenvironment(48). It can stimulate the malignant invasion of cancer(49). Its abnormal expression is associated with the malignant invasion and poor prognosis of BLCA.

In our investigation, it was observed that the adipose tissue of obese mice exhibited elevated expression levels of Lama2, Lama4, Lamc1 and Thbs2. However, following intervention with semaglutide, a significant down-regulation in gene expression was noted. To further investigate the involvement of the protein in BLCA, a prognostic analysis was conducted on these four proteins. The findings indicated that individuals exhibiting elevated levels of Lama2, Lama4, Lamc1, and Thbs2 experienced reduced survival duration compared to those with regular expression. It can be inferred that the upregulation of Lama2, Lama4, Lamc1, and Thbs2 contributes to the advancement of BLCA, while suppressing their expression may impede the progression of BLCA.

Based on the limited existing research regarding the involvement of Lama2, Lama4, Lamc1, and Thbs2 in bladder function and the impact of semaglutide on obesity-related BLCA, this study has discovered that these proteins play a role in promoting the progression of BLCA through the ECM pathway. Additionally, it suggests that semaglutide may potentially influence BLCA by modulating the ECM pathway via Lama2, Lama4, Lamc1, and Thbs2 proteins. Furthermore, it proposes that semaglutide could potentially alleviate obesity-related BLCA resulting from a high-fat diet. Therefore, gaining a comprehensive understanding of the significance of the ECM pathway and its associated proteins in BLCA is crucial for advancing new diagnostic techniques, treatment alternatives, and prognostic assessments. Additionally, targeting the ECM pathway as a potential pharmacological intervention could be explored, with semaglutide serving as a regulator to offer diverse possibilities for managing obesity-related BLCA.

This research showcased the significant impact of obesity on BLCA by examining the influence of body weight, glucose metabolism, lipid metabolism, oxidative stress, and ECM pathway in mice fed a high-fat diet with regards to BLCA associated with obesity. In addition, the regulation of body weight, glucose metabolism, lipid metabolism, oxidative stress and inhibition of ECM pathway in mice by semaglutide can potentially delay the occurrence and progression of BLCA. This finding offers novel insights for identifying new therapeutic targets for BLCA treatment and suggests innovative approaches for utilizing semaglutide in clinical settings. At the moment, there is a need for further investigation into the comprehensive understanding of semaglutide's mechanism of action in BLCA, as well as a lack of sufficient clinical experience in this area.

Abbreviation	Full name
BLCA	bladder cancer
Lama2	laminin subunit alpha-2
Lama4	laminin subunit alpha-4
Lamc1	laminin subunit gamma 1
Thbs2	thrombospondin 2
ECM	extracellular matrix
BMI	body mass index
GLP-1	Glucagon-like peptide-1
PBS	phosphate-buffered saline
iBAT	interscapular brown adipose tissue
eWAT	epididymal white adipose tissue
H&E	hematoxylin and eosin
LDL-C	low-density lipoprotein cholesterol
HDL-C	high-density lipoprotein cholesterol
TC	total cholesterol
TG	triglycerides
SOD	oxidative stress marker superoxide dismutase
ELISA	enzyme-linked immunosorbent
DEPs	differentially expressed proteins
FC	fold change
GO	gene ontology
KEGG	Kyoto Encyclopedia of Genes and Genomes
BP	biological process
MF	molecular function
CC	cellular component

Acknowledgments

This is a short text to acknowledge the contributions of specific colleagues, institutions, or agencies that aided the efforts of the authors.

Author’s contributions

YL and SC conceived and designed the study. JB, XZ and XP provided materials and samples. ZJ and LY collected and assembled the data. All authors contributed to the article and approved the submitted version.

Funding

There is no financial support for this project.

Availability of data and materials

The datasets supporting the conclusions of this article is included within the article.

Consent to Publication

This study complies with the Declaration of Helsinki. The participants gave informed consent. The ethics committee approved the study.

Conflict of interest

This study has no conflict of interest as defined by BMC.

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Effect of High‑Fat-Diet and Semaglutide on Bladder Cancer in Mice

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Ethical Approval

2.2 Animal experiments

2.3 Preparation of serum samples

2.4 Histopathological analysis

2.5 Protein Extraction and Digestion

2.6 LC-MS/M LC-MS/MS Analysis

2.7 Bioinformatics analysis

2.8 Statistical analysis

3 Results

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1