miR-1323 inhibits bladder cancer growth by targeting PMEPA1

doi:10.21203/rs.3.rs-4928041/v1

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Research Article

miR-1323 inhibits bladder cancer growth by targeting PMEPA1

https://doi.org/10.21203/rs.3.rs-4928041/v1

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Background

Bladder cancer is one of the common malignant tumors in the urinary system, and its incidence has been increasing year by year in recent years. However, effective treatment options for advanced bladder cancer are still lacking at present. The molecular mechanisms underlying the occurrence and development of bladder cancer have not been fully elucidated. Previous studies have found that various genes are abnormally regulated in bladder cancer tumor tissues, and the abnormal expression of microRNAs also affects protein expression.

Methods

The analysis of the TCGA-BLCA dataset was used to identify differentially expressed genes in bladder cancer. RT-qPCR and Western blot were used to detect the expression levels of PMEPA1, miR-1323, and SMAD/p-SMAD proteins. The CCK-8 assay and wound healing assay were used to evaluate cell proliferation and migration. Flow cytometry with AnnexinⅤ-FITC/PI double labeling was used to detect apoptosis. The interaction between miR-1323 and PMEPA1 was verified by Dual Luciferase Reporter Assay.

Results:

The results indicate that PMEPA1 is upregulated in bladder cancer tissues and cells. High expression of PMEPA1 stimulates bladder cancer cell proliferation and migration while inhibiting apoptosis. Databases such as TargetScan_8.0 predicted and Dual Luciferase Reporter Assay and Western blot experiments verified the interaction between miR-1323 and PMEPA1. Knockdown of miR-1323 promotes bladder cancer cell proliferation and migration, inhibits apoptosis, and reduces the expression of SMAD2/SMAD3 and p-SMAD2/p-SMAD3 proteins, while simultaneous knockdown of PMEPA1 can counteract these changes induced by miR-1323 knockdown.

Conclusions:

In summary, miR-1323 can inhibit bladder cancer progression by targeting PMEPA1, potentially through suppression of the SMAD-dependent TGF-β signaling pathway. Both miR-1323 and PMEPA1 may serve as potential therapeutic targets for bladder cancer.

Pmepa1

mir-1323

bladder cancer

TGF-β

Bladder cancer is the fourth most common malignant tumor in men and one of the most common malignant tumors of the urinary system^[^1]

It is estimated that in 2020 alone, there were approximately 573,278 new cases of bladder cancer worldwide, and about 212,536 people died from bladder cancer[^2]. About 60% of bladder cancers are non-muscle invasive bladder cancer (NMIBC) at the time of diagnosis, while muscle-invasive bladder cancer (MIBC) accounts for about 20%[^3]. Although non-muscle invasive bladder cancer has a better prognosis, it is highly prone to recurrence [^4]. Compared to non-muscle invasive bladder cancer, muscle-invasive bladder cancer has a shorter survival period and a worse prognosis [^5]. Therefore, finding new biomarkers and potential therapeutic targets is of great clinical significance for patients with bladder cancer.

Prostate transmembrane protein, androgen induced 1 (PMEPA1), is an androgen-responsive gene located on chromosome 20q13, initially discovered in the hormone-responsive prostate cancer cell line LNCaP [^6]. PMEPA1 includes a type Ib transmembrane domain at its N-terminus and two PY motifs (PPxY) that interact with the WW domains of the E3 ubiquitin ligase Nedd4. PMEPA1 plays a dual role in prostate cancer. It can inhibit the proliferation of androgen receptor-positive prostate cancer cell lines [^7]. However, in androgen receptor-negative prostate cancer cell lines, PMEPA1 promotes cell proliferation by inhibiting the Smad3/4–c-Myc–p21 signaling pathway[^8]. Moreover, PMEPA1 is closely related to the bone metastasis of prostate cancer[^9]. PMEPA1 also promotes the progression of various solid tumors, such as pancreatic cancer [^10], colorectal cancer [^11], and lung cancer[^12]. Previous studies have shown that high expression of PMEPA1 in bladder cancer can promote the growth, migration, and invasion of bladder cancer cells and is associated with poorer prognosis, more immune infiltration, and higher levels of TME inflammation[^13]. However, the potential mechanisms of action of PMEPA1 have not been thoroughly investigated.

MicroRNAs, typically 20-23 nucleotides in length, are a class of abundant non-coding RNAs[^14]. The first microRNA, lin-4, was discovered by Ambros and colleagues in the model organism C. elegans [^15]. Currently, 2578 mature miRNA sequences have been identified according to miRBase (http://ww^ew.mirbase.org). However, the functions of many miRNAs have not yet been fully studied. It has been found that miRNAs can be significantly dysregulated during the onset of cancer [^16]. In recent years, the role of miR-1323 in tumors has garnered attention. miR-1323 promotes the migration of lung adenocarcinoma cells by targeting Cbl-b and serves as an early prognostic biomarker for lung adenocarcinoma [^17]. The expression of miR-1323, when suppressed by β-elemene, can inhibit the metastasis of multidrug-resistant gastric cancer cells [^18]. Moreover, miR-1323 can also affect the migration and invasion of breast cancer cells by targeting the tumor protein D52 [^19]. Various pieces of evidence suggest that miR-1323 is closely associated with the development and progression of tumors.

Previous studies have shown that signaling via the transforming growth factor β (TGF-β) superfamily extensively participates in the regulation of cell growth, differentiation, and development, involving both the classical SMAD pathway and non-classical, non-SMAD pathways [^20]. TGF-β signaling plays a significant role in the onset and progression of human cancers, displaying a dual function in tumor regulation [^21]. In the early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis[^22]. Given the critical role of TGF-β and its downstream molecules in cancer progression, targeting TGF-β signaling appears to be a promising strategy for therapy.

Here, we demonstrate that knocking down PMEPA1 promotes proliferation and migration while inhibiting apoptosis in bladder cancer cells. Further experimental validation shows that hsa-mir-1323 can directly target the 3'-UTR region of PMEPA1, thereby affecting PMEPA1 expression. mir-1323 activates the Smads-dependent TGF-β signaling pathway by targeting PMEPA1, which enhances the growth of bladder cancer cells.

Database analysis

Analysis was performed using the BLCA cohort from The Cancer Genome Atlas Program (TCGA) database, with data processed using R. The DESeq2 R package was utilized to analyze differential gene expression, while the clusterProfiler R package was employed for GO/KEGG enrichment analysis. Survival data within the BLCA cohort were analyzed using the Survminer package.

Cells and Cell Culture

The human bladder cancer cell line 5637 cells and 293T cells were purchased from Wuhan Servicebio company. The cells were cultured in a humidified environment at 37°C with 95% air and 5% carbon dioxide. RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (100 U/mL), and penicillin (100 U/mL) was used for cultivation.

Transient Transfection

Cells were seeded into 6-well culture plates at approximately 30% confluence and incubated overnight in a carbon dioxide (CO2) incubator. INTFERin transfection reagent was used according to the manufacturer's recommendations: RNA and transfection reagent were mixed in Opti-MEM medium at the specified ratio. After incubating the mixture for 10 minutes, the transfection mix was added to the culture plates. Cells were further incubated in the CO2 incubator for 48 hours.

RNA Isolation and Quantitative Real-Time PCR

Total RNA was isolated from the cells using Trizol. The cDNA library was constructed using HiScript® III RT SuperMix for qPCR (+gDNA wiper) (Nanjing, Novozymes). qPCR was performed using AceQ qPCR SYBR Green Master Mix (Nanjing, Novozymes), following the manufacturer's recommendations. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The relative expression levels were assessed using the 2^-ΔΔCt method. The primer sequences were as follows: PMEPA1 (F: 5’- TGTCAGGCAACGGAATCCC-3’; R: 5’-CAGGTACGGATAGGTGGGC-3’), GAPDH (F: 5’- AGAAGGCTGGGGCTCATTTG-3’; R: 5’-AGGGGCCATCCACAGTCTTC-3’).

Western Blot

Total protein was isolated from the cells using RIPA buffer. The extracted protein samples (20 µl per lane) were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking with 3% BSA for 2 hours, the membrane was incubated with the primary antibody overnight at 4°C. The membrane was then incubated with the corresponding secondary antibody at room temperature for 1 hour. Protein blots were detected using an enhanced chemiluminescence (ECL) detection reagent.

Wound Healing Assay

To assess cell migration, transfected 5637 cells (1 × 10^5) were seeded in a 6-well plate and incubated overnight at 37°C with 2ml of 1640 medium containing 10% fetal bovine serum until confluence was reached. Afterward, the medium was replaced with serum-free medium. A scratch was made perpendicular to the lines on the 6-well plate using a 200µl pipette tip. Subsequent photographs were taken at fixed intervals using an inverted optical microscope (Olympus, Tokyo, Japan), and further analysis was conducted using ImageJ software.

Dual Luciferase Reporter Assay

The PMEPA1 3’UTR fragment was amplified by PCR and subcloned into a promoter vector containing luciferase. Mutants with altered miR-1323 binding sites were also amplified and subcloned into the vector. Using Lipofectamine 2000 (Life Technologies Corporation), 293T cells were co-transfected with PMEPA1-WT or PMEPA1-MUT and miR-1323. After 48 hours, luciferase activity was assessed using a Dual-Luciferase Reporter Assay System (Beyotime), following the manufacturer's instructions.

Flow cytometry

Collect the cell pellet in a 1.5 ml centrifuge tube, resuspend in 500 μl binding buffer, and incubate with 5 μl Annexin V-FITC and 5 μl PI at room temperature in the dark for 30 minutes. Immediately analyze using flow cytometry. Annexin V-FITC labels early apoptotic cells, while PI labels late apoptotic or necrotic cells. Based on the fluorescence signals, live cells, early apoptotic cells, late apoptotic cells, and necrotic cells can be distinguished.

Statistical Analysis and Graphing

All data were derived from at least three independent replicates and are presented as mean ± standard deviation. Statistical significance was determined using the unpaired two-tailed Student’s t-test for comparisons between two sets of data, and the ANOVA was used to assess the differences between the groups, with P < 0.05 considered statistically significant. GraphPad Prism software was used for analysis and graphical representation.

2.1 PMEPA1 is highly expressed in bladder cancer and correlates with disease progression

Analysis of publicly available RNA-seq data from TCGA-BLCA (Bladder Urothelial Carcinoma) using thresholds of |Log2FC| > 1 and adjusted p-value < 0.05 reveals that 3825 genes are significantly upregulated and 2495 genes are downregulated in tumor tissues compared to adjacent non-cancerous tissues (Figure 1A). Specifically, PMEPA1 is significantly upregulated in tumor tissues compared to adjacent non-cancerous tissues, both in paired and unpaired samples (Figure 1B, C). Analysis of PMEPA1 expression across different clinical stages shows a significant increase from T2 to T3 stages, while other stages exhibit an increasing trend without statistical significance (Figure 1D). Furthermore, high expression of PMEPA1 is associated with poor prognosis in bladder cancer (Figure 1E).

Previous studies have shown that PMEPA1 can promote the progression of various tumors. To further elucidate the expression of PMEPA1 in bladder cancer, we conducted RT-qPCR to measure the mRNA levels of PMEPA1 in cell lines. Compared to SV-HUC-1 cells, PMEPA1 is significantly upregulated in 5637 cells (Figure 2A).

2.2 PMEPA1 Promotes Proliferation, Migration, and Inhibits Apoptosis of Bladder Cancer Cells

Given the significant impact of PMEPA1 on the prognosis of bladder cancer patients, we aimed to understand its involvement in regulating biological behaviors and its potential as a novel target for clinical therapy. To investigate the effect of PMEPA1 on bladder cancer cells, we transfected 5637 cells with siRNA targeting PMEPA1 and measured PMEPA1 expression levels after 24 hours.The RT-qPCR results show that compared to the NC group, siPMEPA1 significantly reduces the mRNA expression levels of the PMEPA1 gene (Figure 2B). Western blot results indicate that compared to the NC group, the expression level of PMEPA1 protein is also reduced in the siPMEPA1 group (**P < 0.01) (Figure 2C, D).Cell viability was assessed using the CCK-8 assay, and the results indicate that knocking down pmepa1 significantly inhibits the growth of 5637 cells compared to the siNC group (Figure 2E).In wound healing experiments, it was observed that knocking down pmepa1 significantly reduces the migration ability of 5637 cells (Figure 2F, G).Additionally, we used flow cytometry to assess the impact of knocking down PMEPA1 on apoptosis in 5637 cells. Compared to the NC group, knocking down PMEPA1 significantly increases apoptosis in 5637 cells (Figure 2H, I).

2.3 hsa-mir-1323 targets PMEPA1 in bladder cancer.

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene expression post-transcriptionally by binding to the 3' untranslated region (UTR) of target genes. They are important regulators of gene expression^[23].To study the upstream regulation of PMEPA1 by microRNAs, we utilized a miRNA binding prediction database established using four different methods. This approach identified 8 potential microRNAs that may interact with PMEPA1 (Figure 3A).

We conducted correlation analysis of PMEPA1 expression and the expression of 8 potential interacting microRNAs in bladder cancer samples from the TCGA-BLCA dataset. Among them, miR-421, miR-1323, and miR-760 showed a significant negative correlation with PMEPA1 expression (p < 0.001) (Figure 3B, Supplementary Figure 1). Considering the expression levels of miRNAs in tumor tissue and the context++ score from the TargetScan_8.0 database, we selected hsa-miR-1323 for further investigation (Supplementary Table 1).

To confirm whether hsa-miR-1323 directly targets PMEPA1 through its 3' untranslated region (UTR) to regulate cell activity, we conducted dual-luciferase reporter assays in 293T cells. After co-transfection with hsa-mir-1323 mimics, the luciferase activity in cells containing PMEPA1-3'UTR-WT was reduced by 33% compared to the control group (P < 0.01). When the PMEPA1-3'UTR region was mutated (PMEPA1-3'UTR-MUT), the luciferase activity in these cells increased by 56.7% compared to PMEPA1-3'UTR-WT (P < 0.01) (Figure 3C). These results demonstrate that miR-1323 directly targets PMEPA1 through its 3'UTR.

Additionally, transfection of miR-1323 mimics into human bladder cancer cells 5637 resulted in significantly downregulated protein expression of PMEPA1 compared to the control group and NC (negative control) group. This further demonstrates that miR-1323 can influence the protein expression levels of PMEPA1 in bladder cancer (Figure 3D-E).

2.4 hsa-miR-1323 inhibits bladder cancer cell proliferation and migration, and promotes apoptosis by targeting PMEPA1.

Cell viability was assessed using the CCK-8 assay, and the results indicate that compared to the control group, inhibition of hsa-miR-1323 significantly enhances the activity of 5637 cells (Figure 4A). Concurrently inhibiting hsa-miR-1323 while knocking down PMEPA1 reduces the enhanced cell activity caused by hsa-miR-1323 inhibition.

In wound healing experiments, it was observed that inhibition of hsa-miR-1323 significantly enhances the migration ability of 5637 cells compared to the control group (Figure 4B). Concurrently inhibiting hsa-miR-1323 while knocking down PMEPA1 reduces the enhanced cell migration ability caused by hsa-miR-1323 inhibition (Figure 4C).

Additionally, we used flow cytometry to assess the effect of inhibiting hsa-miR-1323 on apoptosis in 5637 cells. As shown in the figures, compared to the NC group, inhibition of hsa-miR-1323 significantly reduces apoptosis in 5637 cells (Figure 4D-E). Concurrently inhibiting hsa-miR-1323 while knocking down PMEPA1 partially rescues the apoptosis induced by hsa-miR-1323 inhibition (Figure 4D-E).

2.5 hsa-miR-1323 activates the Smads-dependent TGF-β signaling pathway by inhibiting PMEPA1

To further explore the specific signaling pathways through which PMEPA1 functions in bladder cancer, we conducted differential gene enrichment analysis using KEGG and GO terms on TCGA-BLCA data. The results indicate that the TGF-β signaling pathway is the most likely pathway PMEPA1 is involved in (Figure 5A-B). Protein-protein interaction (PPI) network analysis using the STRING database reveals interactions between PMEPA1 and SMAD2, SMAD3 (Figure 5C). Additionally, Gene Set Enrichment Analysis (GSEA) indicates that the pathway "PATHWAY RESTRICTED SMAD PROTEIN PHOSPHORYLATION" is upregulated in bladder cancer patients with high PMEPA1 expression, showing significant differences (P < 0.01) (Figure 5D).

Additionally, previous studies have shown that PMEPA1 can promote bone metastasis in prostate cancer by blocking TGF-β signaling [^24]. PMEPA1 can also interfere with the phosphorylation of R-Smad proteins to inhibit activation of the TGF-β signaling pathway[^25].

Combining the above information, PMEPA1 likely functions in bladder cancer by regulating the TGF-β signaling pathway.

In our experiments, we explored how hsa-miR-1323 and PMEPA1 influence the TGF-β signaling pathway. Upon inhibiting hsa-miR-1323, we observed a significant decrease in the protein expression levels of Smad2, Smad3, p-Smad2, and p-Smad3 compared to the control group. Conversely, when simultaneously inhibiting hsa-miR-1323 and knocking down PMEPA1, the protein expression levels of Smad2, Smad3, p-Smad2, and p-Smad3 were significantly elevated compared to the control group (Figure 5E-F).

In summary, in bladder cancer, PMEPA1 exerts an inhibitory effect on the Smad-dependent TGF-β signaling pathway, while hsa-miR-1323 can relieve this inhibition by affecting the expression of PMEPA1.

Bladder cancer is the 10th most common cancer type, with approximately 573,000 new cases diagnosed in 2020 [^26]. Bladder cancer, at initial diagnosis, can be classified as Muscle-Invasive Bladder Cancer (MIBC) or Non-Muscle-Invasive Bladder Cancer (NMIBC), which is limited to the mucosa or submucosal connective tissue. MIBC accounts for about 25% and NMIBC for approximately 75% of newly diagnosed bladder cancer cases[^27]. Apart from surgical procedures, advances in molecular biology of bladder cancer have led to the widespread use of various treatments such as immune checkpoint inhibitors and targeted therapies, benefiting patients significantly [^28]. Further research into the molecular mechanisms of bladder cancer can help us better tackle the disease. Previous studies indicate that PMEPA1 is closely related to various tumors, such as breast cancer, prostate cancer, and glioblastoma[^29][30][31]. In this study, we observed that PMEPA1 is highly expressed in bladder cancer patients and is associated with poor disease prognosis. In the bladder cancer cell line 5637, PMEPA1 promotes cell proliferation and migration while inhibiting apoptosis.

PMEPA1, a transmembrane TGF-β-inducible protein, is a direct target gene of the TGF-β signaling pathway. PMEPA1 exerts its negative effect on TGF-β/Smad signaling by interfering with R-Smad phosphorylation, playing a role in various diseases[^32]. Previous research has indicated that PMEPA1 is a prognostic biomarker associated with malignancy and the tumor microenvironment in bladder cancer cells. However, the regulatory mechanisms of PMEPA1 in bladder cancer have not been thoroughly explored[^33]. This study predicts the biological significance of PMEPA1 in the development and progression of bladder cancer by analyzing its expression levels and survival data in bladder cancer patient samples. After knocking down PMEPA1 in the human bladder cancer cell line 5637, various cell phenotypes related to disease progression, including proliferation, migration, and apoptosis, were affected.

To investigate the upstream regulation of PMEPA1 by microRNAs, potential microRNAs that might bind to PMEPA1 were predicted using four databases. Dual-luciferase reporter assays validated that hsa-miR-1323 directly targets the 3’ UTR region of PMEPA1. Previous studies indicate that PMEPA1 can regulate the duration and intensity of TGF-β signaling through various mechanisms [^34][35]. In this study, we confirmed that hsa-miR-1323 affects PMEPA1 protein expression in bladder cancer, which in turn influences the protein levels of TGF-β signaling pathway components, specifically SMAD2/3 and p-SMAD2/3.

In summary, our research reveals the direct targeting effect of hsa-miR-1323 on PMEPA1 and its impact on the proliferation, migration, and apoptosis of bladder cancer cells 5637 through targeting PMEPA1, providing a new potential target for BLCA treatment. Additionally, the experiment uncovered the impact of abnormal PMEPA1 expression on the SMAD-dependent classical TGF-β signaling pathway in BLCA. However, the mechanism by which PMEPA1 suppresses the TGF-β signaling pathway in BLCA remains unclear. Our team will continue to delve deeper into this based on the existing results.

Data availability

The data analyzed during the current study are available in the TCGA database repository,[https://cancergenome.nih.gov/]. All data generated or analysed during this study are included in this published article.

Acknowledgements

Not applicable.

Funding

No funding, grants or other support was received during the writing of this manuscript.

Author information

Department of Urology Surgery, The First Affiliated Hospital of USTC, Hefei, Anhui, China

Haoran Yang，Jing Wang, Tao Haung, Yu Li

Contributions

Yu Li contributed to the study conception and design. Haoran Yang contributed to the Material preparation and Data collection. Jing Wang contributed to Data analysis and Software. The first draft of the manuscript was written by Tao Haung. All authors commented on previous versions of the manuscript, read and approved the final manuscript.

Corresponding author

Correspondence to Yu Li

Ethics declarations

Data from the TCGA database and other experimental materialswere obtained in a public manner, no ethical approval is required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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miR-1323 inhibits bladder cancer growth by targeting PMEPA1

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