Current landscape of exosomal non-coding RNAs in prostate cancer: Modulators and biomarkers

Abstract

Prostate cancer (PCa) has the highest frequency of diagnosis among solid tumors and ranks second as the primary cause of cancer-related deaths. Non-coding RNAs (ncRNAs), such as microRNAs, long non-coding RNAs and circular RNAs, frequently exhibit dysregulation and substantially impact the biological behavior of PCa. Compared with circulating ncRNAs, ncRNAs loaded into exosomes are more stable because of protection by the lipid bilayer. Furthermore, exosomal ncRNAs facilitate the intercellular transfer of molecules and information. Increasing evidence suggests that exosomal ncRNAs hold promising potential in the progression, diagnosis and prognosis of PCa. This review aims to discuss the functions of exosomal ncRNAs in PCa, evaluate their possible applications as clinical biomarkers and therapeutic targets, and provide a comprehensive overview of the ncRNAs regulatory network in PCa. We also identified ncRNAs that can be utilized as biomarkers for diagnosis, staging, grading and prognosis assessment in PCa. This review offers researchers a fresh perspective on the functions of exosomal ncRNAs in PCa and provides additional options for its diagnosis, progression monitoring, and prognostic prediction.

Graphical abstract

In prostate cancer (PCa), non-coding RNAs (ncRNAs) are transferred from donor cells to recipient cells via exosomes and involved in gene expression through various mechanisms. Due to the regulative effects of exosomal ncRNAs on physiological and pathological processes, they are potential biomarkers and promising therapeutic tools in PCa.

1. Introduction

Prostate cancer (PCa) has the highest diagnostic rate among male malignancies. Moreover, it ranks second among the leading causes of cancer-associated mortality in the United States [[1], [2], [3]]. Globally, approximately 10 million men have been diagnosed with PCa, and the incidence of metastatic PCa is increasing, leading to over 3.8 million deaths annually [[4], [5], [6]]. By 2040, this number is estimated to be at least twice as high as it is today [7,8]. Since the survival rate of PCa is strongly linked to early diagnosis [9], prostate-specific antigen (PSA) is used for mass screening of suspected patients. However, due to the non-cancer specificity, PSA often leads to unnecessary overdiagnosis and treatment [10]. Given the significant burden of PCa, it is crucial to explore its evolutionary mechanisms and identify potential diagnostic biomarkers and therapeutic tools.

After reticular cells were discovered to secrete vesicles containing biomolecules outside the cell [11], exosomes have emerged as the shining stars of liquid biopsies, displaying profound potential in the realm of cancer diagnosis and treatment. Once considered mere cellular waste bins, exosomes are now recognized as vital vehicles for transporting specific molecules between cells [11]. Exosomes comprise diverse types of biomolecules, including nucleic acids, proteins, lipids, sugars and metabolites [12]. Furthermore, non-coding RNAs (ncRNAs) contained within exosomes, are enriched and more stable than circulating ncRNAs because of the protection provided by the lipid bilayer [13,14]. These exosomal ncRNAs have been found to modulate multiple cellular processes in malignancies by regulating gene expression [[15], [16], [17]]. In addition, molecular and cellular biological studies of PCa have emphasized the key players in its progression [18,19]. Owing to their unique advantages and functions, exosomal ncRNAs serve as invaluable resources for gaining novel insights into PCa development and potential guides for its diagnosis and treatment.

In this review, we present a brief overview of exosomes and their components. Moreover, we emphasize the modulatory functions of exosomal ncRNAs in the initiation, growth, progression, and therapeutic resistance of PCa, with attention to their possible role as promising biomarkers. We also explore the clinical utilities of exosomal ncRNAs in PCa.

2. Exosomes

Exosomes are the most extensively investigated subset among extracellular vesicles (EVs), varying in size from 30 to 150 nm. They are produced by both eukaryotic cells and prokaryotic cells and can be found in all body fluids [20,21]. These vesicles are generated via an endosomal degradation pathway and cargo inside, on or outside cell can enter exosomes, supporting the essential function of exosomes in intercellular communication [20,22]. While the selective packaging mechanism of exosomes is still unknown, it has been demonstrated that exosomes from different sources or the same cells under different conditions carry a unique content profile [23]. This profile reflects the constituents and current status of the source and may indicate a modulatory selective packaging. Therefore, exosomes have demonstrated potential as modulators and biomarkers of various diseases.

Exosomes contain diverse components including proteins, lipids, nucleic acids, metabolites and small molecules. Proteins, partly originating from the cell membrane, endosomal membrane and cytoskeletal components [24], have been reported to regulate cancer progression [25,26] and serve as biomarkers for diagnostic and prognostic purposes [27,28]. Exosomal lipids are primarily located in the exosomal membrane and play a major role in exosomal formation and homeostasis in recipient cells [27]. They have also been found to modulate cancer and aid diagnosis because of their unique profiles [[29], [30], [31], [32]]. In addition, exosomes also contain nucleic acids such as DNAs and RNAs. It has been demonstrated that the number of DNAs with more bases is relatively higher in larger exosomes than in smaller exosomes [33]. This may indicate that small nucleotides are the most nucleic acids in exosomes. The ncRNAs consisting of dozens to over 200 nucleotides constitute over 98 % of the genome and are abundant in exosomes due to their short length [34]. They regulate gene expression by interacting with mRNAs, remodeling chromatin and cooperating with other biomolecules, thus constructing multiple regulatory networks that modulate the levels of a variety of fundamental protein effectors [35]. The relative abundance and function of ncRNAs in exosomes make them the principal players in cancer modulation [[15], [16], [17]].

The ncRNAs encompass various types of RNA molecules, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) [36]. MiRNAs, serving as short ncRNAs of roughly 22 nucleotides in length, are also the most common ncRNA type transported by exosomes and function by engaging in interactions with mRNAs [37]. LncRNAs are longer than 200 nucleotides and exhibit extensive functional diversity. They can guide chromatin-modifying complexes to target gene promoters and affect transcriptional regulation, as well as bind to miRNAs to modulate molecular functions [38]. CircRNAs are more than 200 nucleotides in length like lncRNAs and are protected from exonuclease degradation due to the closed circular structures and the absence of polyadenylated tail [39]. Therefore, circRNAs in mammalian cells have a longer half-life that is approximately 2.4 times longer than linear RNAs [40] and exosomes exhibit a higher proportion of circRNAs to linear RNAs, with a ratio that is approximately 6 times higher than in cells [41]. Furthermore, circRNAs act as sponges and scaffolds for specific proteins and can be translated to perform specific functions in cancer [17].

3. Exosomal ncRNAs as modulators in PCa

Exosomes affect initiation, growth, development and treatment of cancer by facilitating cellular communication [42]. There is growing evidence indicating that exosomal ncRNAs are the major contributors to these effects. Exosomal ncRNAs can influence the levels of downstream target mRNAs, genes or proteins, altering various signaling pathways (Table 1). Subsequently, they trigger a range of physiological and pathological processes, including angiogenesis, proliferation, apoptosis, migration, invasion, phenotypic transformation, bone microenvironment regulation, and therapy resistance (Fig. 1). Therefore, comprehending the role of exosomal ncRNAs could offer a valuable perspective on PCa.

Table 1.

Molecular mechanisms of exosomal ncRNAs in modulation of prostate cancer.

Process	Types	ncRNAs	Signaling Pathways and Targets
Initiation	miRNAs	miR-27a-3p [48], miR-21 [[51], [52], [53], [54],121] and let-7b [55]	VEGF [49,56,57], PTEN/AKT [54] and PTEN/ERK [54] pathway
	lncRNAs	/	/
	circRNAs	circRNA HIPK3 [150]	BMI-1 [151]
Growth	miRNAs	miR-183 [65], miR-217 [70], miR-23b-3p [70], miR-143 [141], miR-205 [74], miR‐95 [77], miR-153 [85], miR-99b-5p [61], miR-888 cluster [88], miR-1246 [98], miR-26a [99], miR-424 [104], miR-31 [121] and miR-145 [121]	PI3K/AKT [62,63,78,79], NF-κB [62,64], Rho [75,76], STAT3 [86,87,105], TGF-β and mTOR [100] pathway; EZH2 [89]
	lncRNAs	MYU [135], lncAY927529 [139] and PCSEAT [141]	NF-κB [140] and PI3K/AKT [136] pathway; c-Myc [135]
	circRNAs	circRNA HIPK3 [150], circ_0044516 [154] and circ-XIAP [161]	Wnt/β-catenin pathway [155]; BMI-1 [151] and TPD52 [161]
Progression	miRNAs	miR-27a-3p [117], miR-99b-5p [61], miR-183 [65], miR-217 [70], miR-23b-3p [70], miR-143 [141], miR-205 [74], miR‐95 [77], miR-153 [85], miR-146a-5p [94], miR-26a [99,117], miR-1246 [98], miR-888 cluster [88], miR-125b [101], miR-130b [101], miR-141-3p [108], miR-155 [101], let-7b [55], miR-424 [104], miR-375 [110], miR-940 [113], miR-1275 [116], miR-92a-1-5p [119], miR-31 [121], miR-145 [121] and miR-21 [55,121]	PI3K/AKT [98,103,120,142], NF-κB [118], Rho [114,115], STAT3 [86,87,105,107], TGF-β [120], EGFR [94,98], mTOR [100], Lats2/YAP [101], MAPK [108], MEK/ERK [120] and Wnt/β-catenin [142] pathway; EZH2 [89], p53 [127], PDCD4 [101], RUNX2 [111,116,117] and FAM134A [113]
	lncRNAs	MYU [135], lncAY927529 [139], PCSEAT [141], HOXD-AS1 [143] and NEAT1 [145]	NF-κB [140], PI3K/AKT [136] and Wnt/β-catenin [142] pathways; c-Myc [135], FOXM1 [143] and PTBP2/SFPQ complex [145]
	circRNAs	circRNA HIPK3 [150], circ_0044516 [154], circ_0081234 [157] and circ-XIAP [161]	Wnt/β-catenin [155] and MAPK [158] pathway; BMI-1 [151] and TPD52 [161]
Therapy resistance	miRNAs	miR-31 [121], miR-145 [121], miR-21 [121], miR-27a [127], miR-423-5p [130] and miR-34a [131,132]	STAT3 [129], TGF-β [130], NF-κB [133] and PI3K/AKT [134] pathway
	lncRNAs	LINC01213 [148]	Wnt/β-catenin pathway [148]
	circRNAs	circ-XIAP [150]	TPD52 [161]

Fig. 1.

The modulatory mechanisms of exosomal ncRNAs in PCa. Exosomes derived from diverse cell types transport non-coding RNAs, which in turn modulate the expression of downstream genes, target mRNAs or proteins and alter various signaling pathways. The exosomal ncRNAs trigger a cascade of physiological and pathological events, encompassing angiogenesis, proliferation, apoptosis, migration, invasion, phenotypic transformation, bone microenvironment regulation and therapeutic resistance. Ultimately, these processes affect the occurrence, growth, development and treatment of PCa.

3.1. Exosomal miRNAs in PCa

3.1.1. Exosomal miRNAs in PCa initiation

The first step in cancer development is initiation, in which angiogenesis provides oxygen and nutrients and eliminates metabolic waste and carbon dioxide from tumor cells [43]. Angiogenesis, which is the neovascularization from the original ones, is typically activated transiently in response to various stimuli, including wound healing and the female reproductive cycle [44]. However, the angiogenic switch is always activated in cancer to support the growth of cancer cells. Without the support of angiogenesis, the cancer cells would only reside within 100 μm of capillary blood vessels and could not expand [45]. Angiogenesis is regulated by several growth factors and cytokines, such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α [15]. Tumor-derived exosomes can regulate angiogenesis through proangiogenic biomolecules, which induce or inhibit angiogenesis by modulating angiogenic signaling pathways in endothelial cells [46,47].

Exosomal miRNAs are significant modulators of angiogenesis in PCa, involving multiple signaling pathways dominated by VEGF. Exosomal miR-27a-3p is overexpressed in the PC-3 PCa cell line, which induces angiogenesis by enhancing endothelial tube formation [48]. In the proangiogenic process, miR-27a-3p directly targets tissue factor pathway inhibitor (TFPI), inhibits the expression of TFPIα, reduces the phosphorylation of VEGFR2 at residue K951 [49], and then restrains endothelial cell migration [50]. Therefore, miR-27a-3p may induce angiogenesis through the miR-27a-3p/TFPI/VEGF pathway. Additionally, the HIF-1α/VEGF pathway is another common regulatory target. Exosomal miR-21 is upregulated in patients with PCa and DU145 PCa cell line [[51], [52], [53], [54]]. It targets phosphatase and tensin homolog deleted on chromosome ten (PTEN), activates AKT and extracellular regulated kinases (ERK) 1/2, upregulates HIF-1α and VEGF [54], and promotes angiogenesis via HIF-1α/VEGF pathway. Similarly, exosomal let-7b is overexpressed in PC-3 cell-derived exosomes, increased in recipient cells, and promotes tube-like structure formation, which is an essential step during neoangiogenesis in human umbilical vein endothelial cells (HUVEC) [55]. Moreover, let-7b is a hypoxia-responsive miRNA that modulates the expression of VEGF by targeting argonaute 1 (AGO1) [56,57], indicating that the HIF-1α/let-7b/AGO1/VEGF pathway might be the potential mechanism of angiogenesis in PCa. In conclusion, exosomal miRNAs engage in PCa angiogenesis through various signaling pathways dominated by VEGF.

3.1.2. Exosomal miRNAs in PCa growth

With sustained angiogenesis, the tumor acquires sufficient support for growth by regulating cell proliferation and apoptosis. Cell proliferation refers to the controlled growth and division of cells, leading to the generation of new daughter cells and an overall increase in cell population. Apoptosis is an automatic and orderly cell death program that is crucial for cell consumption. Thus, cell proliferation and apoptosis are essential processes in tumor growth and correlate with a rise in the number of malignant cells [45]. These crucial cellular processes are regulated by various physical and chemical signals. Exosomal miRNAs have been reported to modulate cell proliferation and apoptosis in various cancers, including colorectal [58], gastric [59] and ovarian [60] cancers.

Exosomal miRNAs are involved in modulating cell proliferation as either tumor promoters or suppressors through a series of signaling pathways in PCa. One such tumor suppressor is miR-99b-5p, which is upregulated in exosomes obtained from human bone marrow mesenchymal stem cells (hBMSCs). This elevation significantly inhibited PCa growth in vivo and in vitro by downregulating insulin-like growth factor-1 receptor (IGF1R) [61]. IGF1R mediates the phosphoinositide 3-kinase (PI3K)/AKT [62,63] and nuclear factor-kappaB (NF-κB) pathways [62], promoting cell proliferation in PCa [62,64]. Another miRNA, miR-183, is upregulated in LNCaP and PC-3 cell-derived exosomes and enhances cell proliferation by downregulating tropomyosin1 (TPM1) [65], which acts as a tumor suppressor in PCa [66,67]. The underlying mechanism of TPM1 in PCa requires further investigation, but the TPM1/mitogen-activated protein kinase kinase (MEK)/ERK pathway and TPM1/NF-κB pathway in colorectal cancer [68] and osteosarcoma [69] may offer possible pathways by which TPM1 functions in PCa.

The modulation of apoptosis by exosomal miRNAs in PCa involves multiple pathways. Exosomal miR-217 is significantly upregulated in the serum of patients with PCa and acts as an oncogenic factor by inhibiting apoptosis, whereas miR-23b-3p is significantly downregulated and acts as a pro-apoptotic factor [70]. However, how miR-217 and miR-23b-3p function in PCa remains unexplored. Notably, miR-217 has oncogenic and anti-tumor effects. MiR-217 significantly promotes apoptosis in chemotherapy-treated PC-3 cells by regulating polo-like kinase 1 (PLK1) and protein tyrosine kinase 2 (PTK2) expression [71]. Other studies have suggested several potential mechanisms, including PLK1/androgen receptor (AR) [72] and PTK2/PI3K/AKT [73] pathways. The dual roles of miR-217 on apoptosis were observed in PC-3 cells, which excluded the possibility of cell line diversity and suggested that the effect of chemotherapy may be the underlying reason. After chemotherapy, intracellular molecules in PC-3 cells may alter, resulting in a reduction in the levels of the miR-217 initial target and an elevation of new targets such as PLK1 and PTK2, or the activation of other complicated mechanisms. This suggests that chemotherapy may trigger the conversion of biomolecules from pro-cancer molecules to anti-cancer molecules.

MiRNAs in exosomes derived from other non-cancerous cells also regulate apoptosis. For instance, hBMSCs-derived exosomes overexpressing miR-205 promote apoptosis in vivo and tumor growth in vitro by suppressing rhophilin-2 (RHPN2) [74]. By targeting RHPN2, miR-205 indirectly regulates apoptosis via the Rho pathway [75,76]. MiR-95, which is significantly increased in tumor-associated macrophages (TAMs)-derived exosomes, directly targets JunB, thereby inhibiting PCa cell apoptosis [77]. JunB is functionally relevant for increased proliferation and decreased senescence in PCa through interaction with PI3K/AKT pathway [78,79]. Overall, exosomal miRNAs primarily modulate PCa growth via PI3K/AKT, AR and Rho pathways.

3.1.3. Exosomal miRNAs in PCa progression

PCa undergo certain changes as they progress, where cancer cells become more aggressive, non-cancerous cells change their phenotype, and the tumor microenvironment is remodeled. These characteristics enable tumors to alter their microenvironment and grow in distant organs, thereby promoting their survival and development. Invasion and migration increase cell mobility, allowing tumor cells to penetrate the vascular matrix. Recent studies on the new hallmarks of cancer have shown that phenotypic transformation is a crucial factor in neoplastic development [43]. Additionally, the “seed and soil” theory suggests that the emergence of a bone pre-metastasis niche creates favorable conditions for PCa metastasis [80].

Cumulative evidence has revealed that exosomes have emerged as a novel messaging system in organisms, mediating cell-cell and inter-organ communication in tumor progression. Specifically, biologically functional molecules inside exosomes are transferred to recipient cells, facilitating tumor progression by influencing invasion [81], migration [82], phenotypic transformation [83] and bone microenvironment regulation [84].

Cell invasion and migration are hallmark features of aggressive cancer and are significantly influenced by exosomes, particularly exosomal miRNAs. Exosomes facilitate the detachment of tumor cells in situ and their transport to other sites via the bloodstream or lymphatic system [11]. In PCa, exosomal miRNAs have been identified as oncogenic factors that promote cell invasion and migration through multiple pathways. For instance, in the DU145 and PC3 cell lines with high Gleason score (GS ≥ 7), miR-153 is overexpressed and transferred through exosomes, promoting cell invasion and migration by regulating kruppel-like factor 5 (KLF5) [85]. KLF5 is deleted and plays a suppressor role by inhibiting the IGF1/signal transducer and activator of the transcription 3 (STAT3) pathway in PCa [86,87]. Exosomal miR-888 and its cluster members are enriched in PC3-ML cells. MiR-888 inhibits KLF5, retinoblastoma-like 1 (RBL1), tissue inhibitor of metalloproteinase 2 (TIMP2) and SMAD4 levels and enhances cell migration and invasion in vitro [88]. KLF5 is also a target of exosomal miR-153 [87]. RBL1 interferes with the enhancer of zeste homolog 2 (EZH2) and is involved in oncogenic processes [89]. TIMP2 inhibits cell migration, invasion and cancer cell-mediated tissue remodeling [90], possibly through the PI3K/AKT pathway [91]. SMAD4 regulates cell invasion and motility in PCa [92], likely through the SMAD4/TGF-β pathway [93]. Overall, low levels of KLF5, RBL1, SMAD4, and TIMP2 levels are associated with PCa progression.

Exosomal miRNAs also act as suppressors of cell invasion and migration. Cancer-associated fibroblasts (CAFs) also contribute to PCa progression. CAFs-derived exosomes contain miR-146a-5p, which is taken up by LNCaP and DU145 cells and inhibits epidermal growth factor receptor (EGFR)/ERK pathway [94]. This inhibition results in a decrease in cell migration and invasion of PCa [95]. In addition, EGFR is related to biochemical relapse and high GS in PCa [96]. Therefore, exosomal miRNAs modulate cell invasion and migration in PCa through various pathways, including the IGF/STAT3, RBL1/EZH2, TGF-β, PI3K/AKT and EGFR/ERK pathways.

Moreover, exosomal miRNAs are associated with cellular phenotypic transformations that enhance PCa cell aggressiveness. Epithelial-mesenchymal transition (EMT) is a well-known phenotypic transformation and a conserved developmental program controlled by several signaling pathways. It confers metastatic properties and enhances cancer aggressiveness by regulating cell activity, invasion and apoptosis [97].

Exosomal miRNAs are also involved in PCa EMT. The miR-1246 selectively is secreted into exosomes in PCa. It targets EMT-related genes, inhibits EMT, and regulates other cellular processes via the EGFR and PI3K/AKT pathways [98]. Likewise, Exosomal miR-26a is increased in LNCaP cells and significantly downregulates the expression of EMT-related factors in recipient cells [99] by regulating the la-related proteins 1 (LARP1)/mechanistic target of rapamycin (mTOR) pathway [100].

Other phenotypic transformations such as mesenchymal-epithelial transition (MET) and neoplastic transformations also occur in the tumor microenvironment. Co-culture with C4–2B and PC-3 cell-conditioned media induces phenotypic transformation in stem cells, resulting in the formation of prostate-like neoplastic lesions. This transformation is facilitated by exosomes, which upregulate oncogenic factors (miR-125b, miR-130b, and miR-155) and downregulate tumor suppressors (large tumor suppressor homolog 2 (LATS2) and programmed cell death protein 4 (PDCD4)) [101]. The LATS2/yes associated protein (YAP) pathway is critical for tissue homeostasis in PCa [102], and the PI3K/AKT pathway is associated with the expression of PDCD4 [103]. Additionally, exosomal miRNAs are involved in the acquisition of stem cell-like and tumorigenic properties in prostate epithelial cells. In patients with metastatic PCa, there is a higher frequency of circulating miR-424-positive exosomes compared to those with primary tumors and benign prostatic hyperplasia (BPH). MiR-424-loaded exosomes promote stem cell-like properties and tumor initiation, contributing to tumorigenesis in recipient cells [104]. Meanwhile, miR-424 promotes tumorigenic traits via the miR-424/constitutive photomorphogenic 1 (COP1)/STAT3 axis [105].

Phenotypic transformation is also present in the immune system, not only in the mesenchymal and epithelial cells. Overexpressed let-7b in PC-3 cell-derived exosomes can be transferred to THP-1 monocytes, inducing TAM-like polarization [55], which facilitates tumor cell growth [106]. These effects of let-7b are attributed to the suppressor of cytokine signaling 1 (SOCS1)/STAT3 pathway [107]. To conclude, exosomal miRNAs modulate the transformation of cell phenotypes and enhance malignant activity through the EGFR, PI3K/AKT, LARP1/mTOR, Lats2/YAP, and STAT3 pathways.

PCa frequently results in the development of bone metastasis. Exosomes are essential in the preparation of bone pre-metastatic niches. They contain miRNAs that stimulate the formation of a metastatic microenvironment. Exosomal miR-141-3p is overexpressed in MDA PCa 2b cells. It can enter osteoblasts and stimulate their activity. This can lead to bone metastasis and osteogenic damage in PCa. The underlying mechanism of miR-141-3p involves the inhibition of deletion of liver cancer-1 (DLC1) and the activation of the mitogen-activated protein kinase (MAPK) pathway [108]. DLC1 modulates the Rho pathway and suppresses the invasion of highly metastatic PCa cells [109]. Similarly, exosomal miR-375 is identified to promote osteoblast activity [110] via the miR-375/runt-related transcription factor 2 (RUNX2) pathway [111]. RUNX2 is a major modulator of osteoblast activity and related to the metastatic traits of C4–2B cells [112]. In addition, C4–2B cell-derived exosomal miR-940 enhances osteoblastic differentiation in human mesenchymal stem cells by the modulation of Rho GTPase-activating protein 1 (ARHGAP1) and family with sequence similarity 134 member A (FAM134A) [113]. ARHGAP1 is a potential tumor suppressor that mediates osteoblastic differentiation via the Rho pathway [114,115], whereas FAM134A is an oncogenic factor in tumor metastasis, and its physiological function in osteogenesis remains unclear [113]. The transfer of miR-1275 from PC-3 cells to osteoblasts via exosomes significantly enhances osteoblast proliferation by inhibiting sirtuin 2 (SIRT2) and increasing RUNX2 expression [116]. Collectively, exosomal miRNAs have a critical function in PCa bone metastasis.

In addition to osteoblastic lesions, exosomal ncRNAs have been implicated in osteolytic loss. RM1-BM PCa cell-derived exosomes show increased expression of miR-26a-5p, miR-27a-3p and miR-30e-5p. These miRNAs suppress osteogenesis and osteoblast differentiation [117]. The miR-26a-5p targets bone morphogenetic protein 2 (BMP-2), which was reported to activate NF-κB mediated BMP-2-SMAD signaling cascade [118]. Both miR-26a-5p and miR-27a-3p can rejuvenate the level of t RUNX2 [117]. Exosomes derived from PCa cells regulate bone homeostasis, leading to osteoclastic lesions and the promotion of bone tumor growth. MiR-92a-1-5p is overexpressed in MDA PCa 2b cell exosomes and transferred to osteoclasts, promoting osteoclast differentiation through regulating collagen type I alpha 1 (COL1A1) [119]. COL1A1 might be regulated by TGF-β pathway, EGFR/MEK/ERK pathway and PI3K/AKT pathway [120], which implies the involvement of these pathways in the regulation of osteoclastic lesions.

3.1.4. Exosomal miRNAs in PCa therapy resistance

Therapy resistance inevitably presents a complex challenge in PCa, including resistance to androgen deprivation therapy (ADT) and chemotherapy. ADT is the recommended primary therapy for PCa and can improve patient prognosis to a certain extent. However, almost all androgen-sensitive PCa finally becomes androgen-resistant, leading to castration-resistant PCa (CRPC) that can metastasize to distant organs. Exosomes are pivotal in modulating therapy resistance and promoting PCa progression by conveying anti- and pro-tumor signals [27]. Consequently, exosomes can positively or negatively impact therapy resistance by delivering their cargo, indirectly affecting treatment and prognosis.

Mesenchymal-like PCa cells (Mes-PCa)-derived exosomes are involved in promoting mesenchymal characteristics in recipient cells, resulting in resistance to enzalutamide. Genetic analysis of the recipient cells revealed the downregulation of AR and AR-regulated genes, which may be associated with miR-31, miR-145 and miR-21. Mes-PCa-derived exosomes deliver these miRNAs to recipient cells where they target the AR pathway. The upregulation of these miRNAs is associated with message delivery by Mes-PCa-derived exosomes [121]. MiR-31 and miR-21 act as oncogenic factors in regulating therapeutic resistance [[122], [123], [124]], while miR-145 plays a tumor suppressor role and inhibits chemo-radio-resistance [125,126]. Although these miRNAs are all upregulated in Mes-PCa-derived exosomes, the effect of Mes-PCa-derived exosomes is mainly attributed to miR-31 and miR-21.

Non-cancerous cell-derived exosomes implicate in chemoresistance as well. For example, primary prostate fibroblasts (PSC-27)-derived exosomes carrying miR-27a promote chemoresistance of PC-3 cells by inhibiting p53 [127]. The inhibition of p53 can confer resistance to chemotherapeutic drugs [128], activate the STAT3 pathway, and promote tumor progression [129]. MiR-423-5p is overexpressed in CAF-derived exosomes and modulates drug resistance by targeting gremlin 2 via TGF-β pathway [130].

Moreover, exosomal miRNAs implicate in upregulating chemotherapy sensitivity. For example, exosomes derived from docetaxel-resistant PC-3 and 22Rv1 cells decrease levels of miR-34a, which leads to increased docetaxel resistance by upregulating B-cell lymphoma-2 [131,132]. It is related to NF-κB [133], PI3K/AKT [134] and other pathways. Overall, exosomal miRNAs derived from different cells have a significant impact on regulating therapy resistance via the AR, STAT3, TGF-β, NF-κB and other signaling pathways, contributing to chemoresistance and sensitivity to chemotherapy.

3.2. Exosomal lncRNA in PCa

3.2.1. Exosomal lncRNAs in PCa growth

Exosomal lncRNAs act as miRNA sponges and indirectly regulate PCa growth. They have been shown to exert significant effects on cell proliferation and apoptosis. Exosomal lncRNA MYU complements miR-184, resulting in the upregulation of c-Myc level, which subsequently stimulates the proliferation of PC-3 cells [135]. The c-Myc maintains a high cell proliferation rate and cooperates with the PI3K/AKT pathway [136] to promote PCa cell survival [137,138]. Additionally, the upregulation of exosomal lncAY927529 inhibits apoptosis by positively regulating C-X-C motif chemokine ligand-14 level [139]. It promotes M2 macrophage polarization through the NF-κB pathway and contributes to LNCaP and PC-3 cell proliferation, invasion, colony formation and tumor growth [140].

3.2.2. Exosomal lncRNAs in PCa progression

Exosomal lncRNAs, boasting over 200 nucleotides, possess distinctive nucleotide sequences that allow them to complement and pair with corresponding miRNAs, thereby exerting an indirect influence on gene expression and modulating the progression of PCa. For instance, exosomal lncRNA PCSEAT, which is overexpressed in patients with PCa, is transmitted to promote migration and proliferation of recipient cells via exosomes. PCSEAT modulates EZH2 by interacting with miR-143-3p and miR-24-2-5p [141]. EZH2 plays a crucial role in activating the PI3K/AKT/mTOR and Wnt/β-catenin pathways [142].

Exosomal lncRNAs induce phenotypic transformation in PCa. Exosomal lncRNA HOXD-AS1 is increased in LNCaP-Bic and LNCaP-AI cell-derived exosomes. In recipient cells, HOXD-AS1 competitively binds to miR-361-5p, upregulates forkhead box protein M1 (FOXM1), and induces a metastasis-associated phenotype in vitro and in vivo [143]. The HIF-1α/FOXM1 pathway is mediated by EMT in PCa [133]. Moreover, FOXM1 may also be involved in the modulation of the AR pathway through interaction with the AR [144].

In the bone microenvironment, exosomal lncRNAs regulate osteogenic activity. Exosomal lncRNA nuclear-enriched abundant transcript 1 (NEAT1) is transported to hBMSCs, which upregulates RUNX2 level by interacting with miR-205-5p [145]. Additionally, the overexpression of RUNX2 could be partly attributed to the splicing factor proline- and glutamine-rich (SFPQ)/polypyrimidine tract-binding protein 2 (PTBP2) axis [145]. Additionally, exosomal HOXD-AS1 enhances osteolytic loss and tumor metastasis in the microenvironment via the miR-361-5p/FOXM1 axis [143]. FOXM1 is involved in a metastasis-related gene network, including those related to cellular adhesion and bone microenvironment [146].

3.2.3. Exosomal lncRNAs in PCa therapy resistance

Exosomal lncRNAs modulate androgen sensitivity in PCa, ultimately influencing the efficacy of ADT. Androgens are key drivers of prostate growth and are known to contribute to tumor progression. As androgen sensitivity changes in tumor cells, PCa can progress to CRPC and become androgen-independent [147]. Androgen-independent PCa cell-derived exosomes enhance the acquisition of androgen independence. This effect is mediated in part by overexpression of the LINC01213, which confers androgen deprivation tolerance by activating the Wnt/β-catenin pathway [148]. Activation of this pathway is more commonly observed in CRPC than in treatment-naïve PCa, and its inhibitors can reduce therapy resistance in PCa [149].

3.3. Exosomal circRNA in PCa

3.3.1. Exosomal circRNAs in PCa initiation

Exosomal circRNAs modulate angiogenesis in PCa by regulating miRNAs. For example, circHIPK3 interacts with miR-212 [150] and its overexpression leads to upregulation of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) and inhibition of angiogenesis in PCa [151]. BMI-1 significantly affects the initiation and development of PCa [152]. Moreover, BMI-1 has been shown to promote angiogenesis via the NF-κB pathway in gliomas [153], which may be a potential mechanism of BMI-1 in PCa.

3.3.2. Exosomal circRNAs in PCa growth

Exosomal circRNAs also implicate in modulating tumor growth by acting as miRNA sponges in PCa. Circ_0044516 overexpression in exosomes downregulates miR-29a-3p and enhances the proliferation as an oncogenic factor in PCa [154]. MiR-29a-3p regulates cell proliferation by mediating the classical Wnt/β-catenin pathway in PCa [155]. Exosomal circHIPK3 reduces apoptosis via the circHIPK3/miR-212/BMI-1 axis [150]. The inhibition of BMI-1 impairs apoptosis-related protein expression via the ubiquitination pathway and promotes apoptosis in PCa [156].

3.3.3. Exosomal circRNAs in PCa progression

Exosomal circRNAs modulate PCa progression by acting as miRNA sponges. Exosomes derived from patients with PCa and spinal metastasis (SM) showed higher expression levels of circ_0081234 compared to those without SM. Overexpression of circ_0081234 promoted malignant activity by increasing MAP3K1 levels as a miR-1 sponge [157]. MAP3K1 is part of the MAPK/MEK/ERK pathway, which is a significant signaling pathway in PCa EMT [158]. By the circHIPK3/miR-212/BMI-1 axis, exosomal circHIPK3 enhances cell viability, migration and invasion [150]. BMI-1 also modulates cell migration and invasion in PCa progression [156,159,160].

3.3.4. Exosomal circRNAs in PCa therapy resistance

Exosomal circRNAs implicate in the upregulation of chemoresistance via message delivery. Docetaxel (DTX)-resistant PCa cell-derived exosomes enhance DTX resistance, in which circ-XIAP is overexpressed, directly targets miR-1182, and increases TPD52 level [161]. TPD52 avoids apoptosis in response to androgen deprivation by activating the PI3K/AKT and STAT3 pathways [[162], [163], [164]].

Signaling pathways mediated by miRNAs, lncRNAs and circRNAs in exosome-derived PCa cells are not only involved in a single cellular activity. For example, the PI3K/AKT pathway is associated with cellular proliferation, apoptosis, invasion, migration, phenotypic transformation, regulation of the bone microenvironment and therapy resistance in PCa. Therefore, exosomal ncRNAs are delivered and target the same or correlated signaling pathways, which in turn affect cellular activity. This establishes a complicated modulatory network in PCa and sheds new light on the pathogenesis of PCa. Overall, the regulatory mechanisms of PCa are diverse and every cellular process cannot be viewed unilaterally. Additional exploration is necessary to reveal the underlying mechanisms and crosstalk among the aforementioned pathways.

4. Exosomal ncRNAs as biomarkers in PCa

Due to their capacity to regulate a wide range of cellular processes, exosomal ncRNAs serve as valuable biomarkers for diagnosis, staging, grading and prognosis assessment. The levels of exosomal ncRNAs reflect the status of donor and recipient cells due to the specific roles of exosomal ncRNAs in PCa. Profiling and quantifying exosomal ncRNAs have shown differential expression levels among healthy individuals, patients with BPH, patients with androgen-sensitive PCa and patients with CRPC [12,165,166]. Therefore, exosomal ncRNAs are of the utmost importance in providing precise diagnostic, staging, grading and prognostic biomarkers for PCa (Table 2).

Table 2.

Biomarkers of exosomal ncRNAs in prostate cancer.

Biomarker Type	Role	Source
		Urine	Plasma	Serum
Diagnostic biomarkers	Distinguish PCa from HS	miR-19b [172], miR-21 [51,52], miR-141-5p [52], miR-375 [171], miR-486-5p [171], miR-451a [171], miR-486-3p [171], miR-196a-5p [170], miR-501-3p [170], let-7c [51], miR-574-3p [52] and miR-2909 [174]	miR-125a-5p [175], miR-141-5p [175] and lncAY927529 [139]	miR-141 [52,176,177], miR-212 [150], HIPK3 [150] and circ_0044516 [154]
	Distinguish PCa from BPH	miR-145 [173], miR-1290 [173], miR-2909 [174] and lncRNA-p21 [181]	miR-21-5p [53] and miR-200c-3p [53]	/
	Distinguish PCa from negative prostate biopsy	PCA3 [178] and MALAT1 [178]	/	/
Staging and grading biomarkers	Identify metastatic PCa	miR-375 [171,173] and miR-1290 [171,173]	/	miR-141 [52,176,177] and circ_0081234 [157]
	Distinguish treatment-naïve PCa from CRPC	/	miR-423-3p [183]	/
	Distinguish PCa with different GS	miR-145 [173], PCA3 [178] and MALAT1 [178]	let-7a-5p [53]	/
	Identify metastatic PCa after radical prostatectomy	/	/	miR-141 [52,176,177], miR-375 [177] and miR-1246 [98]
Prognostic biomarkers	Associated with poor overall survival	/	miR-375 [184] and miR-1290 [184]	/

Abbreviation: PCa, prostate cancer; CRPC, castration-resistant prostate cancer; GS, Gleason Score; SM, spine metastasis; HS, healthy subjects; BPH, benign prostatic hyperplasia.

4.1. Exosomal ncRNAs as diagnostic biomarkers

Exosomal ncRNAs represent reliable and precise biomarkers for PCa diagnosis. At present, PSA screening is the first choice for all patients due to its efficiency. However, it also leads to overdiagnosis and overtreatment because it is prostate-specific, not PCa-specific [10]. Other diagnostic methods, such as tissue biopsy, imaging examination and digital rectal examination (DRE), are not routinely recommended for newly admitted patients because of their limitations, such as invasiveness, high cost, and poor diagnostic performance [167,168]. Exosomes have become a research hotspot and provide a promising alternative to liquid biopsy with better overall diagnostic performance [169]. Particularly, ncRNAs in exosomes are important modulators and biomarkers. Therefore, exosomal ncRNAs may offer a new and valuable choice for PCa diagnosis.

The focus of numerous research has been on exosomal miRNAs as biomarkers for diagnosing PCa. For example, the levels of miR-196a-5p [170], miR-501-3p [170], miR-375 [171] and miR-19b [172] are significantly decreased in urinary exosomes from patients with PCa in comparison to those from non-cancerous subjects. Conversely, the expression levels of miR-451a [171], miR-486-3p [171], miR-486-5p [171], miR-21 [51,52], miR-375 [51], let-7c [51], miR-141-5p [52], miR-574-3p [52], miR-145 [173], miR-1290 [173] and miR-2909 [174] are significantly higher in urinary exosomes from patients with PCa in comparison to those from non-cancerous subjects. In exosomes from plasma, miR-200c-3p [53], miR-21-5p [53] and miR-141-5p [175] are significantly overexpressed and utilized to differentiate between patients with PCa and non-cancerous subjects. Additionally, the decline of miR-125a-5p in plasma exosomes is valuable for diagnosing PCa patients and healthy subjects [175]. Similarly, in exosomes from serum, miR-212 is significantly downregulated [150], whereas miR-141 is significantly upregulated [52,176,177], which is meaningful in PCa diagnosis. Moreover, combining multiple biomarkers, including miR-375, miR-451a, miR-486-3p and miR-486-5p [171], miR-21 and miR-375 [51], miR-125a-5p and miR-141-5p [175], PCA3 and MALAT1 [178], or SChLAP1 and SAP30L-AS1 [179], can increase the diagnostic accuracy.

As a part of ncRNAs, exosomal lncRNAs are also valuable diagnostic biomarkers in PCa. Specific exosomal lncRNAs are enriched or decreased and contain miRNA seeds or RNA-binding protein binding (RBP) motifs [180], which make lncRNAs valuable diagnostic biomarkers. In urinary exosomes, PCA3 [178], MALAT1 [178] and lncRNA-p21 [181] are significantly upregulated and serve for PCa diagnosis. The diagnostic performance of exosomal PCA3 and MALAT1 in combination is superior to that of PCA3 or MALAT1 alone. Moreover, the diagnostic performance is further enhanced by combining urinary exosomal PCA3 and MALAT1, PSA, fPSA/tPSA, and a model that includes age, prostate volume, and DRE status [178]. Similarly, serum exosomes from patients with PCa show significant upregulation of SChLAP1 [179] and lncAY927529 [139], compared to those from normal volunteers.

Exosomal circRNAs can also function for PCa diagnosis. Specifically, exosomal circ_0044516 is increased in the serum of patients with PCa and differentiates them from normal subjects [154]. Similarly, exosomal circHIPK3 is also a diagnostic biomarker found in serum and it is meaningfully increased in patients with PCa in comparison to normal subjects [150]. Both circ_0044516 and circHIPK3 act as miRNA sponges and bind to miR-212 and miR-330-5p, respectively [150,182]. This may explain the decline of miR-212 and miR-330-5p in the exosomes.

4.2. Exosomal ncRNAs as staging and grading biomarkers

In addition to diagnosing PCa, exosomal ncRNAs also function as staging and grading biomarkers. Understanding the stage and grade of PCa is essential as it reveals the current status of the disease and the degree of malignancy. This information can help clinicians make informed therapeutic decisions.

Different PCa stages affect the choice of therapy, making it necessary to determine the disease stage. According to studies, patients with metastatic PCa exhibit overexpression of miR-1290 and decreased levels of miR-375 in urinary exosomes as compared to those with localized PCa [171,173]. Furthermore, overexpression of exosomal miR-141 [176] and miR-1246 [98] in serum can distinguish whether PCa metastasizes or not like miR-1290. Additionally, serum exosomal circ_0081234 is meaningfully overexpressed in patients with PCa SM, suggesting its utility for PCa staging [157]. Plasma exosomal miR-423-3p is meaningfully increased in patients with CRPC than in those with non-CRPC [183]. Additionally, after undergoing radical prostatectomy, serum exosomal miR-375 and miR-141 are meaningfully increased in patients with metastatic PCa compared to those without relapse [177].

The GS of PCa is associated with treatment decisions and risk stratification. Exosomal ncRNAs can help determine GS values. For example, overexpression of urinary exosomal miR-145 can be used to significantly determine GS ≥ 8 or GS ≤ 7 in patients with PCa [173]. Conversely, the decline of plasma exosomal let-7a-5p is meaningful when distinguishing GS ≥ 8 or GS ≤ 6 in patients with PCa [53]. PCA3 and MALAT1 are also associated with GS. The levels of these biomarkers are significantly upregulated in urinary exosomes and utilized to distinguish patients with PCa with a high GS (GS ≥ 7) from those with other non-aggressive diseases (PCa with a GS ≤ 6 and benign disease) [178]. By quantifying these ncRNAs, clinicians can determine the stage and grade of patients without invasive biopsies.

4.3. Exosomal ncRNAs as prognostic biomarkers

Exosomal ncRNAs are also useful for predicting patient survival. For example, patients with CRPC after ADT failure show significant upregulation of plasma exosomal miR-1290 and miR-375, indicating their potential for survival prediction. Moreover, the predictive performance of the prognosis is significantly improved by measuring the ratio of miR-1290/miR-375 [184]. Similarly, circ_14736 and circ_17720 form a part of the circRNA signature model. This model, including eight circRNAs, predicted the biochemical recurrence of PCa [185]. These findings suggest that exosomal ncRNAs have prognostic value and can be used to develop effective predictive models for patients with PCa.

5. Clinical application of exosomal ncRNAs

5.1. Exosomal ncRNAs as biomarkers

Given the roles of exosomal ncRNAs as modulators and biomarkers, they hold promise as novel biomarkers in clinical settings. Based on the apparent differences in exosomal ncRNAs between patients with PCa and others, some studies have used them to diagnose. Wei et al. [186] and Wang et al. [187] designed diverse techniques for the ultrasensitive detection of miR-141. Additionally, Kim et al. [188] successfully measured urinary exosomal miR-6090 and miR-3665 levels in patients with PCa, developed a dual amplification approach, and identified its potential in PCa diagnosis. Moreover, by targeting exosomal miR-21 and surface CD63, Cho et al. [189] could determine whether exosomes derived from cancerous prostate cells or not. The sequence between two lncRNAs or circRNAs has some repeat sites, leading to low efficiency in constructing a system to detect specific sequences of lncRNAs or circRNAs. Hence, real-time fluorescence polymerase chain reaction (PCR) and other methods are commonly used to directly measure the expression of lncRNAs or circRNAs.

Although significant progress has been made in detecting specific biomarkers in the plasma, serum or exosomes, the time-consuming procedures and high technical requirements of some methods make biosensing through exosomal ncRNAs non-mainstream. However, the high enrichment of ncRNAs in exosomes makes it easier to collect the same type of ncRNAs and send signals without acquiring a large volume of body liquid. In addition, some researchers have successfully applied their detection assays from basic research to clinical applications with high diagnostic efficiency [187,188], showing that exosomal ncRNAs have excellent detection performance in PCa. These detection systems not only show compatibility with PCR but also have higher specificity than typical PSA tests.

It is critical to standardize the collection and measurement of exosomal ncRNAs before accurately understanding the relationship between ncRNAs and PCa. Several highly sensitive and specific assays have been developed to determine exosomal miRNAs related to PCa. However, the absence of standardization in the collection and measurement of miRNAs can lead to different conclusions. For instance, Foj et al. [51] found that urinary exosomal miR-141 exhibits no apparent difference in diagnosing PCa upon using differential centrifugation, which differs when comparing urinary exosomal miR-141 by lectin-based exosome agglutination method [52]. Additionally, other potential factors should be explored, such as prostate massage, urine volume, urine concentration, renal function and hemodynamic status [14].

5.2. Exosomal ncRNAs as potential therapeutic tools

Besides as biomarkers, exosomal ncRNAs are hopeful targets for cancer therapy. The ncRNAs encased in exosomes are biocompatible and more prone to evade attacks by the immune system. Exosomal ncRNAs are more stable than ncRNAs in bodily fluids and can cross physiological barriers, because of their biocompatibility. Specific miRNAs in endogenous exosomes derived from stem cells can protect adjacent cells [190,191]. Exogenous ncRNAs can be incorporated into synthetic exosomes using methods like electroporation, lipofection, sonication or calcium chloride treatment [192]. Synthetic exosomes can induce gene silencing with loaded ncRNAs, leading to the knockdown of cancer oncogenes [193,194]. Furthermore, exosomal ncRNAs have been used to breast cancer [195], ovarian cancer [196], osteosarcoma [197], colorectal cancer [198] and other cancers (Table 3). Overall, loading ncRNAs into exosomes is promising for PCa.

Table 3.

The therapeutic potential of exosomal ncRNAs.

ncRNAs	Carrier	Target	Model	Role	Reference
let-7a	Modified exosomes	EGFR-expressing breast cancer	In vivo and in vitro	Inhibited tumor growth	[195]
miR-497	Hybrid nanoparticles of liposomes and exosomes	Ovarian cancer	In vivo and in vitro	Led to tumor cell death and overcame drug resistance	[196]
anti-miR-21	Anti-exosome antibody–oligonucleotide complexes	Exosome	In vitro	Inhibited exosomal miR-21 to prohibit cancer cell growth	[199]
lncRNA MEG3	Modified exosomes	Osteosarcoma	In vivo and in vitro	Inhibited proliferation, migration and promoted apoptosis by regulating miR-185-5p	[197]
ciRS-122	Exosome	Colorectal cancer	In vivo and in vitro	Suppressed glycolysis and reversed resistance to oxaliplatin by regulating the ciRS-122-miR-122-PKM2 pathway	[198]

6. Conclusions

According to published studies, exosomal ncRNAs have emerged as key regulators of multiple signaling pathways, critically influencing PCa initiation, growth, progression and therapy resistance. The relationship among exosomal ncRNAs, signaling pathways and cellular activities is complex and requires further exploration. Understanding the involvement of exosomal ncRNAs in tumorigenesis, progression and therapeutic response will usher in a new era in PCa diagnosis and treatment. Given their predictive effects in PCa, exosomal ncRNAs can function as innovative biomarkers and construct a professional system that can identify, classify, monitor PCa, predict patient survival, and assist in medical decision-making. Additionally, new therapeutic methods may target exosomal ncRNAs to intervene in oncogene expression, and exosomes can be designed to transport functional ncRNAs to cancer cells. However, the lack of a recognized protocol for obtaining exosomes remains a challenge and researchers must collaborate to determine the best separation and detection techniques based on stability, accuracy and selectivity to facilitate credible research results. Furthermore, exosomal ncRNA expression in various body fluids of patients with PCa at each stage requires reliable average measurements through multiple assessments. With the combined efforts of researchers, exosomal ncRNAs in PCa will be better explored and successfully applied in clinical practice.

Funding

This study was supported by the National Natural Science Foundation of China (82122042), Chongqing Science Fund for Distinguished Young Scholars (CSTB2022NSCQ-JQX0007), Chongqing Medical Scientific Research Project (2023ZDXM021) and Key Project for Clinical Innovation of Army Medical University (CX2019LC107).

CRediT authorship contribution statement

Yongxing Li: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xiaoqi Tang: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Binpan Wang: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Ming Chen: Writing – review & editing, Supervision. Ji Zheng: Writing – review & editing, Supervision, Funding acquisition. Kai Chang: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

There are no conflicts of interest to declare.