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. 2026 Jan 20;26:87. doi: 10.1186/s12935-026-04167-9

Urinary miR-191-5p levels are significantly reduced after radical prostatectomy in patients with prostate cancer

Fatima Domenica Elisa De Palma 1,2,✉, Vincent Carbonnier 3, Gustavo Cernera 1,2, Monica Gelzo 1,2, Carmela Nardelli 1,2, Savio Domenico Pandolfo 2,4,5, Achille Aveta 2,6, Sisto Perdonà 6, Ciro Imbimbo 4, Giuseppe Castaldo 1,2
PMCID: PMC12905937  PMID: 41559725

Abstract

Background

MicroRNAs (miRNA) detection in urine samples may be a scarce invasive approach for diagnosis and monitoring prostate cancer (PCa). The role of miR-191-5p in prostate oncogenesis, as well as its function as a potential diagnostic and prognostic biomarker has been described in many cancers. However, its role as a diagnostic non-invasive indicator in PCa is still under investigated.

Methods

We performed an extracellular vesicle (EV)-based miRNA-sequencing of urine samples (n = 12/group) collected from PCa patients before (T0) and three months after (T1) radical prostatectomy, and healthy individuals. An independent cohort of PCa paired urine samples (n = 25/group) and controls (n = 22) was used to validate our sequencing data by RT-qPCR. Furthermore, we conducted comprehensive in silico analyses on urine and tissue samples extracted from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, respectively. Receiver operator curves analysis was employed to assess the diagnostic value of miR-191-5p. To explore the role(s) of miR-191-5p in prostate carcinogenesis, we predicted miR-191-5p potential targets using four miRNA/target-gene pair databases (i.e., miRDB, miRTarBase, TarBase, and TargetScan). Finally, we corroborated the potential correlation between miR-191-5p and its targets by in silico investigation using TCGA dataset.

Results

Differential expression analysis revealed a significant upregulation of miR-191-5p in PCa patients compared to healthy individuals. Remarkably, urinary miR-191-5p expression levels significantly decreased after surgery in both our discovery and validation cohorts of urine samples by miRNA-seq and RT-qPCR, respectively. Bioinformatics analyses further confirmed high levels of miR-191-5p in urine and tissue samples from PCa patients compared to controls, particularly in patients with high Gleason score. Moreover, miR-191-5p showed a strong diagnostic value in urine and tissue samples. Finally, target prediction analysis identified the genes Satb1 and Ctdsp2 as potential targets of miR-191-5p. This was supported by a significant inverse correlation between the expression of miR-191-5p and these genes in TCGA PCa tissues. Moreover, both Satb1 and Ctdsp2 were downregulated in PCa tissues, and especially in high Gleason score tumors compared to normal and low Gleason score samples.

Conclusions

MiR-191-5p is highly expressed in urine and tissue samples from patients with prostate cancer. Our findings, although preliminary, support miR-191-5p as a promising minimally invasive biomarker for diagnosis of PCa patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04167-9.

Keywords: MicroRNA, Biomarker, Prostate cancer, MiRNome

Background

Prostate cancer (PCa) is the leading type of cancer in men [1, 2]. PCa detection is currently based on the combination of the digital rectal examination, measurement of the glycoprotein prostate-specific antigen (PSA) in blood, and prostate needle biopsy [1]. However, many factors can contribute to inaccurate results with consequent useless invasive prostate biopsy. The diagnostic management of PCa was streamlined in 2012 by the introduction of a novel urinary test, namely PROGENSA [3]. This assay is more specific and sensitive than PSA for PCa detection [3, 4]. It measures the abundance of the long non-coding RNA (lncRNA) PCA3 in urine samples of PCa patients [5]. More recently, Tosoian and collaborators presented a new 18-genes urinary test for PCa patients with high PSA [6]. Indeed, urine is a polyhedric source of biomolecules, including genes, lncRNAs and microRNAs (miRNAs).

MiRNAs circulate stably in biofluids, either as cell-free miRNAs or, with greater stability, encapsulated into extracellular vesicles [7, 8]. Altered expression of onco-/tumor suppressor- miRNAs has been associated with prostate carcinogenesis and disease progression [9–11]. Numerous studies identified single specific miRNA or signatures of miRNAs as potential diagnostic, prognostic, and predictive biomarkers in PCa, with a particular focus on tissue and blood, but less on urine specimens [12–14].

In this framework, we aimed at identifying novel prostate-specific miRNAs as minimally-invasive diagnostic indicators. Thus, we investigated the extracellular vesicles (EVs)-derived miRNome profile in urine samples from PCa patients before and three months after radical prostatectomy, as well as from cancer-free man volunteers. As results, miR-191-5p was overexpressed in PCa patients compared to healthy individuals. Importantly, we observed a significant reduction of urinary miR-191-5p expression levels in PCa patients after prostatectomy with respect to both post-surgery samples and controls. Moreover, miR-191-5p was highly expressed in urine samples and tissue PCa biopsies compared to normal specimens, extracting data from the gene expression omnibus (GEO) and the cancer genome atlas (TCGA) databases, respectively. Furthermore, in silico exploration of miR-191-5p function(s) contributing to prostate carcinogenesis, identified the genes Satb1 and Ctdsp2 as its potential targets. Thus, the altered expression of urinary EVs-derived miR-191-5p, and the consequent deregulation of Satb1 and/or Ctdsp2, might play a critical role in prostate malignant transformation. Altogether, these results evidenced the strong potential of miR-191-5p as a non-invasive diagnostic indicator of PCa.

Methods

Study subjects

Investigational (n = 24) and validation (n = 50) cohorts of urine samples of PCa patients before (T0) and 3 months after (T1) radical prostatectomy, as well as urine samples from male healthy volunteers (controls; investigational cohort, n = 12; validation cohort, n = 22) were obtained from the Department of Neuroscience of University Federico II of Naples, Italy. Urine samples, collected without prior prostate stimulation or digital rectal examination, were aliquoted and immediately stored ad −80 °C until use, as described previously [15]. Individuals with known comorbidities, including inflammatory disorders (e.g., urinary tract infections), were excluded from this study. The main characteristics of the study population are shown in Supplementary Table S1. This study was approved by the local institutional review board of University of Naples, and was conducted in accordance with the Declaration of Helsinki.

EVs isolation and RNA extraction

Extracellular vesicles (EVs) were isolated from human urine samples with exoRNeasy kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Briefly, urine samples were centrifuged at 3000 × g for 15 min at 4 °C to remove cells and debris, and the supernatants collected. Then, 4 mL of prefiltered (0.45 μm syringe filter, Millipore, USA) urine sample was mixed with binding XBP buffer and added to the exoEasy membrane affinity spin column. Subsequently, after washing with XWP buffer, isolated EVs were lysed on the column using QIAzol to extract and purify RNA. Finally, RNA quantity and quality was assessed by Nanodrop.

microRNA-sequencing

Total RNA extracted from urine EVs was used for miRNA-sequencing. Library preparation was carried out using QIAseq miRNA library kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All samples have been sequenced in 75SE on the Illumina NextSeq500. Library preparation and sequencing was performed by Personal Genomics.

RT-qPCR

Ten ng of RNA was reverse transcribed using miR-specific RT primers (hsa-miR-191-5p: #002299; U6 snRNA: #001973; all from Thermo Fisher Scientific) and the TaqMan™ MicroRNA Reverse Transcription kit (#4366596, Thermo Fisher Scientific), according to the manufacturer’s instruction. Then, the qPCR reaction was carried out using the Fast Advanced Master Mix in a total volume of 10 µl on a 384-well QuantStudio 6 Real-time PCR system (ThermoFisher Scientific).

Bioinformatics and statistical analysis

Analysis of miRNA sequencing data from our cohort. The quality control of raw sequencing reads was performed using FastQC (version 0.11.5). MiRNA counts were calculated using the Qiagen portal: Analysis workflow version: 1.2.1, using as reference: miRBase_v22, Homo sapiens (GRCh38.103). MiRNAs expression counts were imported into DESeq2, an R package for normalization and differential expression analysis. Differentially expressed miRNAs for each comparison were selected using absolute log2 Fold Change (log2FC) threshold of 1, p-value ≤ 0.05 and baseMean ≥ 10. Wilcoxon-Mann-Whitney U test was used to evaluate the differential expression of miR-191-5p in normal and PCa samples. Grubbs’s test was used to identify outlier values. Heatmap, Boxplots and Volcano plots were generated using R packages ComplexHeatmap (v2.24.1), GGplot2 (v3.5.2) and EnhancedVolcano (v1.26.0), respectively.

Analysis of public datasets. Concerning Gene Expression Omnibus (GEO) data analysis, GSE45604 (normal, n = 10; PCa, n = 50) and GSE39314 (benign prostate hyperplasia (BPH), n = 8; PCa, n = 9) datasets were retrieved from GEO database (https://www.ncbi.nlm.nih.gov/geo/) [16, 17]. Wilcoxon-Mann-Whitney U test was used to evaluate the differential expression of miR-191-5p in normal/BPH and PCa tissue samples. Grubbs’s test was used to identify outlier values.

Concerning the cancer genome atlas (TCGA) data analysis, miRNA-sequencing and RNA-sequencing data of PCa (PRAD dataset) were downloaded from Genomic Data Commons (https://portal.gdc.cancer.gov) using TCGAbiolinks (v2.36.0) R packages. Wilcoxon-Mann-Whitney U test was used to evaluate the differential expression of miR-191-5p, Satb1 and Ctdsp2 in normal and PCa tissue samples.

All correlation analyses were performed with the “cor.test” R function. Pearson’s correlation was used for continuous variables, and Spearman’s correlation was used in all other cases. All analyses were performed with R (v4.5.0).

The association between miR-191-5p expression and the clinicopathological parameters (e.g., tumor status, lymph node involvement, Gleason score and Prostate Specific Antigen (PSA) level), when such features were available, was calculated using Fisher’s exact test in: (i) our cohort of sequenced PCa patients’ urine samples (n = 12); (ii) GSE45604 cohort from PCa patients’ urine samples (n = 50); and (iii) TCGA cohort from PCa tissue samples (n = 505). For each dataset ROC curve analysis was used to identify the best threshold value to discriminate between samples with a high versus low miR-191-5p expression.

Receiver operating characteristic (ROC) curve analysis. ROC curves were generated and area under the ROC curve (AUC) was calculated using “pROC” R packages [18, 19].

Target prediction analysis. Four different miRNA/target-gene pair databases were used to identify predicted targets of miR-191-5p, including miRDB (mirdb.org), miRTarBase (mirtarbase.cuhk.edu.cn), TarBase (dianalab.e-ce.uth.gr/tarbasev9) and TargetScan (targetscan.org) [20–23]. Targets in TarBase were identified by selecting as filters: “microT prediction score > 0.8”, “high confidence miRNA only”, and “experimental type = direct”. MiR-191-5p binding sites in the 3’UTR of Satb1 and Ctdsp2 were predicted interrogating TargetScan database (targetscan.org). Venn diagram was realized using the R package ggVennDiagram (v1.5.4).

RT-qPCR statistical analysis. Relative expression was calculated according to the 2−∆∆Ct method. U6 snRNA (#001973, ThermoFisher) was used as endogenous control. Significance was assessed using paired or unpaired Student’s test. Outlier values were identified using the ROUT test. Samples and their matched paired samples (in paired analysis) with “undetermined” raw threshold cycle (Ct) values were excluded from the analysis. A p-value ≤ 0.05 was considered statistically significant.

Results

Expression profile of extracellular vesiscles-derived miRNAs in urine samples of prostate cancer patients

Firstly, we investigated the expression profile of EVs-derived miRNAs from urine samples of PCa patients (n = 12) before (T0) and 3 months after (T1) radical prostatectomy. Out of the 33 differentially expressed (DE) miRNAs (baseMean > 10) identified, ten miRNAs were significantly deregulated (p < 0.05, |log2FC| ≥ 1, baseMean > 10) in T0 versus T1 urine samples (Supplementary Table S2, Supplementary Figure S1). Specifically, eight miRNAs (i.e., miR-191-5p, miR-125a-5p, miR-99b-5p, miR-10b-5p, let7i-5p, miR-27b-3p, miR-125b-5p and let-7e-5p) were downregulated, and two miRNAs (i.e., miR-4497 and miR-4488) were upregulated in T1 when compared to T0 samples (Supplementary Table S2, Supplementary Figure S1). Next, we screened for the most significantly deregulated (absolute log2FC ≥ 1.5, adjusted p < 0.05, baseMean > 10) miRNAs. Among the miRNA candidates, urinary miR-191-5p exhibited the largest delta of expression (Supplementary Table S2). More precisely, miR-191-5p showed the most significant deregulated expression (log2FC = − 1.9, adjusted p = 0.039, and p = 0.003) after prostatectomy (Supplementary Table S2, Supplementary Figure S1). Based on these findings, we focused on miR-191-5p for further investigation.

miR-191-5p expression is reduced in urine samples of prostate cancer patients after prostatectomy

In our discovery cohort, urinary miR-191-5p levels were elevated in patients with PCa (T0) when compared to healthy controls (Fig. 1A). Interestingly, urinary levels of miR-191-5p significantly (p = 0.0086) decreased three months after prostatectomy compared to pre-operative levels (Fig. 1A). Of note, miR-191-5p expression was not significantly different (p = 0.58) between controls and T1 samples, indicating a return to physiological level after prostatectomy (Fig. 1A). The decrease in miR-191-5p levels from pre- to post-surgery was also significant (p = 0.027) when assessed in paired urine samples (Fig. 1B). Elevation of urinary miR-191-5p levels in PCa patients with respect to controls was confirmed in the validation cohort (Fig. 1C), as well as its reduction after prostatectomy in paired samples (Fig. 1D).

Fig. 1.

Fig. 1

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Expression level of urinary miR-191-5p in prostate cancer. (A, B) Boxplots illustrating the individual level of expression of extracellular vesicles-derived miR-191-5p in our discovery cohort of whole urine samples from cancer-free male individuals (ctrl) and PCa patients before (T0) and after (T1) radical prostatectomy (A), and of paired urine samples from T0 and T1 patients (B), via miRNA-sequencing. (C) Boxplot representing the individual values of miR-191-5p expression in our validation cohort of whole urine samples from controls and PCa patients (T0), via RT-qPCR. (D) Urinary miR-191-5p expression levels in paired T0 and T1 samples by RT-qPCR. (C, D) miRNA expression was measured by RT-qPCR and calculated according to the 2−∆∆Ct method. u6snRNA was used as endogenous control. PCa, prostate cancer

To further corroborate our findings, we evaluated miR-191-5p levels in an independent cohort of urine samples from healthy subjects and PCa patients using GEO database (GSE45604, n = 60). In line with our data, miR-191-5p was significantly (p = 0.032) elevated in urine samples from PCa patients with respect to controls (Fig. 2A, B). Analysis of an additional GEO dataset (GSE39314, n = 17), comprising urine samples from patients with benign prostatic hyperplasia (BPH) and PCa, revealed a similar pattern of miR-191-5p expression (Supplementary Figure S2). Although the absence of statistical significance (p = 0.21), data showed a trend of miR-191-5p overexpression in PCa compared to BPH controls (Supplementary Figure S2).

Fig. 2.

Fig. 2

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Urine and tissue expression level of miR-191-5p in prostate datasets. (A-D) Boxplots illustrating the individual levels of miR-191-5p in urine samples (A, B) and tissue samples (C, D) from controls (ctrl) and PCa samples (A, C), and from ctrls and prostate tumors based on Gleason score (B, D), extracted from GEO (GSE45604) (A, B) and TCGA (C, D) databases. (D) See Supplementary Table S3 for corresponding data on statistical analysis. (E, F) Diagnostic value of miR-191-5p expression levels in urine (E) and tissue (F) prostate samples through ROC curve analysis from GEO (E) and TCGA (F) database. AUC area under the curve; CPM, count per million; Ctrl, control; GEO, gene expression omnibus; PCa, prostate cancer; ROC, receiver operating characteristic; TCGA, the cancer genome atlas

In the next step, we assessed whether urinary levels of miR-191-5p mirrored tissue expression by interrogating TCGA database (n = 557). miR-191-5p was significantly (p = 2.2e-16) upregulated in malignant tissues compared to controls (Fig. 2C). Remarkably, these tissue expression data were consistent with the level of miR-191-5p detected in urine samples (Figs. 1A and 2A). Moreover, miR-191-5p expression was particularly elevated in PCa patients with high Gleason score (Fig. 2D, Supplementary Table S3).

We also investigated the association between the expression of miR-191-5p and patients’ clinicopathological characteristics (e.g., Gleason score, PSA, and tumor grade) in our cohort, as well as in GEO and TCGA datasets, when such features were available. MiR-191-5p expression did not appear to be associated with the clinicopathological characteristics examined in the datasets (Supplementary Table S4).

Finally, to strength the potential clinical role of miR-191-5p, we assessed its diagnostic potential also by plotting receiver operating characteristic (ROC) curves and calculating area under the curve (AUC) in our discovery cohort, as well as GEO and TCGA datasets. MiR-191-5p appeared to be a good diagnostic indicator in urine samples (our cohort: AUC = 0.792, p = 0.01; GSE45604 cohort: AUC = 0.92, p = 1.71e-23). In addition, tissue miR-191-5p levels (AUC = 0.92, p = 1.71e-23), but not PSA levels (AUC = 0.518, p = 0.676), significantly discriminate PCa patients from control individuals (Fig. 2E, F, and Supplementary Figure S3, S4).

miR-191-5p affects Satb1and Ctdsp2 expression in prostate cancer

To investigate the regulatory role(s) of miR-191-5p in PCa, we looked for its potential targets by exploring four different miRNA/target-gene pair databases, including miRDB, miRTarBase, TarBase and TargetScan (Fig. 3A, Supplementary Table S5).

Fig. 3.

Fig. 3

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Correlation between the expression of Satb1 and Ctdsp2 genes and miR-191-5p level in prostate cancer. (A) Venn diagram displaying the common potential target genes of miR-191-5p in 4 databases, including miRDB, miRTarBase, TargetScan, and TarBase. See Supplementary Table S5 for complementary gene lists. (B, C) Plots showing correlation between the expression of miR-191-5p and Satb1 (B) and Ctdsp2 (C) in PCa tissue samples from TCGA dataset. Correlation coefficient (r) and significance (p) are shown on the graphs. (D-G) Boxplots illustrate the expression of Satb1 (D, F) and Ctdsp2 (E, G) in normal and PCa tissues based on sample type (i.e., control and tumor group) (D, E), and on patient Gleason score (F, G), derived from the TCGA dataset. (F) Complementary data on statistical significance are shown in Supplementary Table S6. CPM, count per million; Ctrl, control group; miRDB, microRNA database; PCa, prostate cancer; TCGA, the cancer genome atlas; TPM, transcript per million

We identified two targets of miR-191-5p, the gene Special AT-rich sequence-binding protein 1 (Satb1) and the gene Carboxy-Terminal Domain Small Phosphatase 2 (Ctdsp2) in the 4 databases (Fig. 3A). MiR-191-5p exhibited strong site-type 8mer and 7mer-m8 interactions with Satb1 and Ctdsp2, respectively, according to TargetScan prediction (Supplementary Figure S5, Supplementary Table S7). Of note, Satb1 and Ctdsp2 implication in different cancer types, including PCa, has been reported [24–27]. The interaction between miR-191-5p and Satb1 and Ctdsp2 was corroborated by investigating their correlation using TCGA dataset. Specifically, in prostate adenocarcinoma samples, miR-191-5p and Satb1, as well as Ctdsp2 showed a significant negative correlation (Fig. 3B, C). In addition, both potential targets demonstrated a significant reduced expression in PCa tissues compared to controls in the TCGA dataset (Fig. 3D, E). More precisely, Satb1 and Ctdsp2 abundance was particularly low in PCa patients with increasing Gleason score (Fig. 3F, G). These results were congruent with miR-191-5p high abundance in PCa patients bearing high Gleasore score (Fig. 2D), corroborating the negative regulation of miR-191-5p on Satb1 and Ctdsp2.

Discussion

Here, we conducted an EVs-derived miRNA-seq profiling on urine samples from PCa patients collected before and three months after radical prostatectomy, and healthy individuals, followed by bioinformatics analyses. Among the deregulated miRNAs, we focused on miR-191-5p because it showed the strongest expression change. Moreover, its contribution to prostate carcinogenesis and its potential as a urinary biomarker in PCa remained poorly investigated. In general terms, miR-191-5p is higher expressed in tumors than in normal tissues [28–32]. Its oncogenic role has been reported in various cancers [28, 30, 31]. Recently, miR-191-5p involvement in prostate carcinogenesis has been demonstrated in vitro [33]. Moreover, miR-191-5p upregulation has been shown to promote cell growth and invasion by targeting the gene Tissue Inhibitor Of Metalloproteinases 3 (TIMP3) in PCa cells [34].

In this study, we observed a significant overexpression of miR-191-5p in PCa patients compared to control subjects. Importantly, miR-191-5p levels decreased significantly after prostatectomy in both our discovery and validation cohorts of urine samples. Of note, low post-surgical levels of miR-191-5p were similar to those of healthy controls. This high expression of urinary miR-191-5p in PCa was also validated using an independent cohort from GEO database. Moreover, a trend in its upregulation in PCa specimens vs. BPH urine samples was detected.

In addition, we showed that miR-191-5p was also deregulated in PCa tissues using TCGA database. Consistent with our findings in urine samples, miR-191-5p was significantly overexpressed in PCa tissues when compared to normal specimens, especially in PCa samples with high Gleason score. Our results are supported by several studies [35–37]. For instance, miR-191-5p exhibited a significant high expression in 146 PCa tissues when compared to normal adjacent samples [37]. Ray and collaborators showed miR-191-5p deregulation in prostate tissues and its function in promoting radiation resistance by interacting with RXRA, both in vitro and in vivo [36].

The diagnostic, predictive and prognostic potential of miR-191-5p as a tissue (mainly) and circulating (e.g., serum, plasma) indicator has been described in several cancers (e.g., breast and lung cancer) [29, 32, 35, 36]. Notably, miR-191-5p has been identified as an early non-invasive (serum) diagnostic biomarker in ovarian clear cell carcinoma [29]. Its role as a non-invasive circulating (plasma) therapeutic biomarker for radiotherapy in esophageal squamous carcinoma patients has been also described [38].

In PCa, a recently published paper reported higher plasma levels of miR-191-5p in PCa patients than controls [33]. Moreover, multivariate analysis showed that miR-191-5p upregulation could serve as an independent prognostic marker of unfavorable overall survival in subjects with PCa [37].

Here, we demonstrated (via ROC curve analysis) that miR-191-5p has a good potential as a diagnostic biomarker for PCa in urine samples from both our cohorts and GEO datasets, as well as in tissues from TCGA dataset. In addition, we showed that tissue miR-191-5p levels presented a better ability than PSA to discriminate between patients with and without PCa. Accordingly, it has been demonstrated that plasma miR-191-5p levels, but not PSA levels, segregate PCa patients from controls, especially in the PSA “grey zone” [33]. Similarly, in another study, miR-191 expression was associated with Gleason score and tumor status, but not with PSA levels in tissue samples [37]. In our bioinformatics analyses, we did not observe a significant association between urinary or tissue miR-191-5p expression and clinicopathological features (e.g., Gleason score, PSA). This could be explained by differences in cohort size, detection methods, statistical analyses, and/or threshold settings to classify samples as high or low miR-191-5p expression.

Hence, it appears plausible the potential of measuring miR-191-5p in liquid instead of tissue biopsies to discriminate healthy individuals from PCa patients. Further investigation involving larger cohorts of paired tissue and urine samples (including normal, BPH and PCa individuals) collected before and at multiple time points after prostatectomy (ranging from 3 months up to 5 years), is necessary to comprehensively assess the diagnostic, prognostic and predictive significance of miR-191-5p. Unfortunately, long-term clinical data on disease recurrence, as well as BPH controls and paired tissues, were not available in our current cohorts, limiting these analyses.

Exploration of four public databases revealed the genes Satb1 and Ctdsp2 as potential targets of miR-191-5p. Both genes were significantly low expressed in PCa tissues when compared to normal specimens, based on our bioinformatic analyses (TCGA database). Ctdsp2 is known to be a tumor suppressor gene in clear renal cell carcinoma and in non-small cell lung cancer [27, 39]. The altered abundance of Satb1, as well as its role as oncogene (mainly) or tumor suppressor gene in many cancers has been already reported [25, 40–44]. Our results showing Satb1 downregulation in PCa are in contrast with many published papers [25, 41, 42, 44]. For instance, one study reported high level of Satb1 in metastatic PCa [41]. However, Satb1 expression was evaluated comparing PCa versus benign prostate hyperplasia, without analyzing normal tissues [41]. Moreover, it has been shown that Satb1 upregulation promotes epithelial-mesenchymal transition (EMT) and metastasis in PCa [44]. EMT is a reversible process of cell de-differentiation [45]. However, despite Satb1 known association with EMT, Satb1 expression has not been evaluated in PCa according to the degree of tissue de-differentiation. In this study, we demonstrated that Satb1 (and Ctdsp2) expression decreases with increasing de-differentiation. Notably, Satb1 level was particularly lower in high Gleason score tissues than those with low Gleason score. This corroborates our data on Satb1 downregulation in PCa. Additionally, we observed a significant negative correlation between miR-191-5p and both Satb1 and Ctdsp2 in PCa tissue samples, further supporting our findings. Indeed, it is well known that miRNAs function as negative regulators of gene expression. Thus, we can speculate that high level of miR-191-5p represses Satb1 and/or Ctdsp2 expression with the consequent initiation and/or progression of prostate cancer. Our hypothesis on miR-191-5p/Satb1 interaction was corroborated by several studies [46–48]. Lena et al., reported the same inverse correlation between the expression of miR-191 and Satb1 in the context of cell senescence [47]. Similarly, the lncRNA ANRIL regulates inflammation through the modulation of the miR-191-5p/Satb1 axis in ulcerative colitis [48]. However, the exact mechanisms by which EVs-derived miR-191-5p in PCa modulates Satb1 and Ctdsp2 still need further investigation. Regulation of miR-191-5p/Satb1 interaction might be a future therapeutic strategy to reduce progression and aggressiveness of PCa.

Conclusions

Here, we identified urinary miR-191-5p as a potential diagnostic non-invasive biomarker of PCa. Our study is limited by the small number of patients and the absence of benign prostate samples. Nevertheless, our findings are supported by comprehensive in silico investigation on larger publicly datasets and corroborated by evidence from the literature. Moreover, functional studies on miR-191-5p interaction with Satb1 and Ctdsp2 are necessary to deepen into miR-191-5p oncogenic role in prostate carcinogenesis. Further investigation will validate the clinical relevance of the miR-191-5p role for the management of this disease. Tracking cancer using liquid biopsy appears to be the ideal, though still challenging, strategy to streamline and improve the clinical management of cancer patients [49, 50]. In PCa patients, urine seems to be the most suitable biofluid for detecting and measuring stable biomolecules, particularly miRNAs encapsulated into extracellular vesiscles, to distinguish healthy from cancer-affected individuals [6, 9, 12].

Supplementary Information

Supplementary Material 1. (506.9KB, pdf)

Abbreviations

microRNA

miRNA

PCa

Prostate cancer

EV

Extracellular vesicle

T0

Time 0, pre-surgery patients

T1

Time 1, post-surgery patients

TCGA

The Cancer Genome Atlas

ROC

Receiver operator curve

AUC

Area under the curve

Satb1

Special AT-rich sequence-binding protein 1 gene

Ctdsp2

Carboxy-Terminal Domain Small Phosphatase 2 gene

PSA

Prostate-specific antigen

lncRNA

Long non-coding RNA

RT

Retrotranscription

qPCR

quantitative PCR

Ct

threshold cycle

FC

Fold change

Ctrl

Control

RXR

Retinoid X receptor alpha

EMT

Epithelial-mesenchymal transition

Author contributions

FDEDP performed most of the experiments, elaborated and interpreted data. VC performed bioinformatics and statistical analyses. MG and GCe (Gustavo Cernera) contributed to RT-qPCR experiments. CN, SDP, AA, SP and CI enrolled and selected patients in the clinical areas and collected biological materials. GC (Giuseppe Castaldo) supervised the whole set of the experiments. GC and FDEDP conceived the study, wrote and revised the manuscript. All authors have contributed, read, and agreed to the published version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the commercial, or not-for-profit sectors.

Data availability

MicroRNA-sequencing and RNA-sequencing data of PCa patients from the TCGA datasets were downloaded from the website Genomic Data Commons (https://portal.gdc.cancer.gov). GEO datasets used in the study are publicly available. In particular, GSE45604 and GSE39314 datasets, were downloaded from GEO database (https:/www.ncbi.nlm.nih.gov/geo) [16, 17]. Data on differential expression analysis and target identification are provided within the manuscript or supplementary information files (**Supplementary Tables S2, S5**). All the other data that support the findings of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethical Committee of University of Naples (protocol number 253/2023 of 05/06/2023, Naples, Italy) and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all the participants.

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (506.9KB, pdf)

Data Availability Statement

MicroRNA-sequencing and RNA-sequencing data of PCa patients from the TCGA datasets were downloaded from the website Genomic Data Commons (https://portal.gdc.cancer.gov). GEO datasets used in the study are publicly available. In particular, GSE45604 and GSE39314 datasets, were downloaded from GEO database (https:/www.ncbi.nlm.nih.gov/geo) [16, 17]. Data on differential expression analysis and target identification are provided within the manuscript or supplementary information files (**Supplementary Tables S2, S5**). All the other data that support the findings of this study are available from the corresponding author upon request.

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