MicroRNAs (miRNAs) are small single-stranded, noncoding RNAs approximately 21–23 nucleotides in length. The first discovered miRNA, Lin-4, was found in Caenorhabditis elegans by Ambros’s group in 1993 [1], followed by the discovery of let-7 in 2000, which further led to similar findings in other species and humans [2]. In human cells, the biogenesis of miRNAs follows events in the nucleus and cytoplasm. After the initial capping and polyadenylation of primary miRNAs by RNA polymerase II (Pol II), pri-miRNAs are formed. The Drosha/DGCR8 heterodimer microprocessor further processes and crops pri-miRNAs into hairpin-structured pre-miRNAs. For the majority of miRNAs, transcription is performed by RNA polymerase II; however, some miRNAs with Alu sequence repeats are transcribed by Pol III [3]. The 364 kDa Drosha microprocessor attaches to the junction between ssRNAs and dsRNAs and two DGCR8 molecules that help to bind and stabilize Drosha [4], followed by the export of the pre-miRNAs to the cytoplasm through exportin 5 (XPO5)/Ran-GTP. In the cytoplasm, Dicer (an RNase III enzyme), along with the cofactors TAR RNA-binding protein 2 (TRBP2) and the protein activator of PKR (PACT), cleaves pre-miRNAs into small miRNA duplexes [5]. Dicer further promotes duplex unwinding through its helicase activity, resulting in one miRNA strand acting as a guide strand that complexes with the RNA-induced silencing complex (RISC). After forming the miRNA-RISC complex, it recognizes the imperfectly matched/complementary target sequences of pre-mRNAs with the help of the Argonaute (Ago) protein [6]. The primary role of these small noncoding RNAs is to recognize Watson–Crick complementary binding sites of the 3′-untranslated regions (3′-UTRs) of mRNAs and regulate their stability. While mature miRNAs are synthesized in the cytoplasmic region, reports have suggested that up to 75% of mature miRNAs can be found in both the cytoplasm and the nucleus. Apart from their main role as post-transcriptional gene regulators, miRNAs are also associated with circular RNA (circRNA), long noncoding RNAs (lncRNAs), and pseudogenes to encourage miRNA suppression or enhance competition for binding sites of miRNAs [7]. A single miRNA can concurrently target multiple mRNAs, affecting various biological processes, such as cell proliferation, cell growth, tissue differentiation, apoptosis, and embryonic development [8]. Changes in the gene expression profiles of miRNAs may influence the expression/regulation of miRNA target genes, leading to alterations in cell homeostasis [7]. The dysregulation/alteration of miRNAs is one of the crucial causes of human diseases, including cancer [9]. Accumulating studies have suggested that the deregulation of miRNAs and miRNA-mediated genes are involved in the pathogenesis of various cancers, including prostate, lung, breast, colon, head and neck cancer, liver, thyroid, endometrial, brain, ovarian, and hematologic cancers ^[10][11][12][10,11,12].

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer deaths among American men [13]. Despite its prevalence, the genetic/epigenetic mechanisms underlying PCa aggressiveness and drug resistance remain elusive. Various clinical criteria, including prostate-specific antigen (PSA) levels, imaging diagnostics, and histopathological scores (such as Gleason scores), are commonly utilized as diagnosis/screening or stratification parameters for PCa [14]. However, given the limitation of these procedures in precision detection and prediction, additional diagnostic and prognostic biomarkers are needed to accurately stratify the PCa disease. In recent years, emerging data have suggested the deregulation of miRNAs as a critical epigenetic mechanism involved in the development and progression of human PCa [15]. To date, more than 50 miRNAs have been implicated in PCa disease [16]. Accumulating studies have provided convincing evidence that deregulated miRNAs are actively involved in the initiation, progression, and metastasis of PCa ^[17][18][19][17,18,19]. Specifically, differential miRNA expression signatures were identified between benign and malignant PCa [20], and deregulated miRNAs are associated with PCa development, invasion, migration, and metastasis ^[18][21][18,21]. Drug resistance is a leading cause of treatment failure and mortality. However, effective therapies for reversing/overcoming PCa drug resistance still remain a clinical challenge. Several mechanisms, including drug efflux, alterations in signaling, epigenetic deregulation, alternative splicing, autophagy, tumor heterogeneity, and the tumor microenvironment, have been associated with PCa drug resistance ^{[22][23][24][25]}[22,23,24,25]. Notably, miRNAs are involved in the regulation of multiple key signaling pathways in PCa progression, such as androgen receptor (AR) signaling ^[26][27][26,27], PTEN/PI3K/AKT signaling [28], p53 signaling [29], Wnt/β-catenin signaling [30], and autophagy [31] pathways. Moreover, miRNA expression profiles are altered in response to chemotherapy, radiotherapy, and androgen deprivation therapy in PCa ^[32][33][32,33]. Therefore, the development of miRNA-based therapies specifically targeting aberrant signaling (AR, PTEN/PI3K/AKT, autophagy signaling, etc.) and/or alternative splicing mechanisms may further lead to novel therapeutics for treatment-resistant PCa.

2. miRNA Deregulation in Prostate Cancer: An Overview

In cancers, aberrantly expressed miRNAs function as either tumor-suppressive or oncogenic miRNAs [5]. The dysregulation of miRNAs, therefore, accounts for one of the mechanisms underlying the development and progression of PCa and other cancers [11]. Let-7, as mentioned above, was first discovered in C. elegans and was found to be downregulated in various cancers, and it acts as a tumor suppressor by targeting several oncogenes [34]. Previous studies have shown that decreased levels of let-7 in lung cancer directly increased the expression of the oncogene RAS. Conversely, restoring let-7 levels in hepatic and colon cancer restricts the growth and proliferation of cancerous cells [35]. The downregulation of let-7a has been demonstrated in several studies. For example, Guanlin et al. showed a negative correlation between the expression levels of CC chemokine receptor 7 (CCR7) and let-7a in PCa. The inhibition of let-7a resulted in the upregulation of CCR7, thereby enhancing CCR7 binding to its ligands (CCRL19 and CCRL21) and triggering the activation of MAPK-mediated EMT signaling in PCa. This hyperactivation of CCR7/MAPK/EMT signaling (due to the downregulation of let-7a) further promotes PCa cell invasion [36]. In castration-resistant prostate cancer (CRPC), let-7c is downregulated. Upon transfection of let-7c, cell growth and clonogenicity were inhibited in PCa [37]. MiR-1 is known to be suppressed in many cancers, including PCa, hepatocarcinoma, chordoma, and esophageal squamous cell carcinoma [38]. It has been shown that miR-1 is downregulated in PCa cells when compared to normal human prostate epithelial cells. MiR-1 is able to reduce cell viability and proliferation by targeting the c-Met/AKT/mTOR signaling pathway in PC-3 cells [38]. The luciferase assay further confirmed MET and KRAS (encoding c-Met and K-Ras, respectively) to be direct targets of miR-1 ^[38][39][38,39]. The inhibition of miR-1 using a miR-1 antagomir promoted cell proliferation in PC-3 cells [38]. In contrast, the overexpression of miR-1 inhibits cell proliferation, invasion, migration, and epithelial–mesenchymal transition (EMT) in PCa cells ^[38][39][38,39] and also suppresses tumorigenesis in xenograft PCa mouse models [39]. Studies have suggested that miR-29b and miR-30c play important roles in regulating cellular functions such as differentiation, proliferation, and apoptosis in cancers. RT-qPCR results have shown that the expression levels of miR-30c/-29b were reduced in PCa tissues compared to non-cancerous tissues. Additionally, a negative correlation was observed between miR-30c/-29b and cancer aggressiveness. Specifically, lower expression levels of miR-30c/-29b are correlated with more lymph node metastasis, more bone metastasis, and a higher Gleason score in PCa [40]. A previous study revealed that miR-30c targets the 3′-UTR of SRSF1 (encoding alternative splicing factor/splicing factor 2, ASF/SF2) and E2F7 (encoding E2F Transcription Factor 7) [41]. MiR-29b targets and negatively regulates MCL1 and AKT2, while AKT2 expression suppresses Bim1. It has been shown that the overexpression of miR-29 inhibits AKT2, thereby upregulating Bim1 and enhancing Bim-1-mediated cell apoptosis in PCa [42]. The downregulation of tumor-suppressive miR-29b was observed in various cancers, such as lung, breast, colon, gastric, and ovarian cancers, medulloblastoma, and multiple myeloma ^[43][44][43,44]. MiR-34a and miR-99b are two important tumor-suppressive miRNAs in cancers, including PCa ^[45][46][47][45,46,47]. MiR-34a belongs to the miR-34 family, along with two other miRNAs: miR-34b and miR-34c [48]. The expression level of miR-34a is higher than that of miR-34b/c in most tissues ^[49][50][49,50]. MiR-34a is a direct target of p53 [51] and is involved in p53-mediated apoptosis, cell cycle arrest, and senescence [52]. Additionally, miR-34a functionally regulates the cell cycle, cell proliferation, senescence, migration, and invasion by targeting multiple genes, such as CDK4/6, E2F3, MET, BCL2, SIRT1, MYC, NOTCH1, and CD44 ^[48][53][48,53]. Previous studies have reported that miR-34a acts as a tumor suppressor and is downregulated in a wide range of cancers, such as PCa, glioma, medulloblastoma, laryngeal cancer, lung, breast cancer, ovarian cancer, bladder cancer, colon cancer, and liver cancer ^{[54][55][56][57]}[54,55,56,57]. The study by Fujita et al. showed that the expression of miR-34a was reduced in PCa specimens and cell lines when compared with their normal controls [58]. Another tumor-suppressive miRNA, miR-99b, belongs to the miR-125a-let-7e cluster. MiR-99b-5p is frequently downregulated and was found to regulate differentiation, proliferation, invasion, and migration in PCa, cervical, breast, esophageal, gastric, and colon cancers ^{[59][60][61][62][63][64]}[59,60,61,62,63,64]. Members of miR-99 were downregulated in advanced PCa compared to the normal prostate epithelium. Through the luciferase reporter assay, three direct target genes were identified: SMARCD1, SMARCA5, and MTOR. In addition, by increasing the levels of miR-99 family members, a reduced level of the androgen receptor (AR) was found in the C4-2 cell line. At both the mRNA and protein levels, miR-99 members reduced PSA expression [60]. TheOur recent studies have also confirmed that miR-99b-5p negatively regulates the expression levels of mTOR ^[45][65][45,65] and AR [65] and blocks the nuclear translocation of AR and mTOR [65]. The overexpression of miR-99b-5p resulted in the inhibition of cell proliferation/viability, the enhancement of cell apoptosis, and the sensitization of PCa, breast, colon, and lung cancer cells to docetaxel [65]. MiR-99b-5p has been shown to be a tumor-suppressive miRNA in various cancers, including PCa [66]. In PCa, a functional link between miR-133a-5p and FUS/AR was revealed by Zheng et al. Specifically, miR-133a-5p directly targets the 3′-UTRs of FUS and AR and inhibits the expression of FUS and AR [67]. In addition, the overexpression of miR-133a resulted in a significant reduction in the cell viability of the PCa cell lines VCaP and LNCaP. Not only FUS and AR but also the downstream targets of AR, such as IGF1R and EGFR, were negatively regulated by miR-133a. Moreover, cell proliferation is enhanced upon the inhibition of miR-133a [67]. In another study by Tang et al., miR-133a-3p was found to directly target EGFR, FGFR1, IGF1R, and MET in PCa cells. Due to the downregulation of miR-133a-3p, EGFR/PI3K/AKT signaling is upregulated in PCa [68]. It has been indicated that miR-134-5p plays a critical role in suppressing human cancers [69]. It affects various oncogenic signaling pathways, such as MAPK/ERK, Notch, and EGFR signaling, by targeting different genes within those pathways. When miR-134 is upregulated, it inhibits the expression of cyclin D/cyclin D2/CDK4, KRAS, EGFR, POGLUT1, and STAT5B proteins, resulting in a decrease in cell proliferation. Furthermore, miR-134 targets and inhibits KRAS, NANON, HNF4A, EGFR, ITGB1, and FOXM1, leading to the inhibition of tumor invasion and metastasis in PCa [70]. In addition, Ngalame et al. demonstrated a negative correlation between miR-134 and RAS oncogenes. The downregulation of miR-134 is associated with the activation of RAS/ERK and PI3K/PTEN/AKT signaling pathways in human prostate epithelial and stem cells [71]. The study by Pelka et al. further revealed the downregulation of miR-134-5p in PCa vs. BPH and demonstrated a negative correlation between miR-134-5p expression levels and Gleason scores [69]. MiR-205 serves a major function as a tumor-suppressive miRNA in cancers. MiR-205 is known to regulate cell proliferation, migration, and invasion by regulating E-cadherin through the targeting/inhibition of ZEB1 and ZEB2 [72]. In PCa, miR-205 is frequently downregulated and functions as a tumor-suppressive miRNA. It inhibits the expression of the androgen receptor (AR) and mitogen-activated protein kinase (MAPK). Similarly, miR-205 is downregulated and also functions as a tumor suppressor in other cancers, such as breast cancer, liver cancer, skin cancer, glioblastoma, pancreatic cancer, colorectal cancer, and renal cancer [73]. MiR-221-5p is a miRNA demonstrating dual functional roles. Kiener et al. explored the tumor-suppressive role of miR-221-5p in PCa. The expression level of miR-221-5p was found to be downregulated in PCa vs. normal prostate tissue, as well as in metastasis vs. primary PCa [74]. The overexpression of miR-221-5p in a PCa cell line decreased cell proliferation, migration, and colony formation. In contrast, the knockdown of miR-221-5p led to the opposite changes [74]. MiR-221-5p has also been reported to target SOCS1 and inhibit its expression at RNA and protein levels, subsequently suppressing Ras/Raf/MEK/ERK signaling cascades in PCa [75]. Moreover, miR-221-5p targets BMI1 and inhibits BMI protein expression, resulting in the inhibition of PCa proliferation [76]. On the other hand, miR-221 has also been found to be upregulated in bone metastatic CRPC, suggesting its role as an oncogenic miRNA. Sun et al. identified HECTD2 and RAB1A as targets of miR-221. The overexpression of miR-221 inhibits HECTD2 and RAB1A expression, promoting androgen independency, AR reprogramming, and CRPC progression [77]. Similar to miR-221-5p, miR-375-3p represents another miRNA that potentially exhibits dual functional roles. MiR-375 was found to be downregulated in PCa. It was found that miR-375 targets and inhibits QKI5 in PCa, inhibiting QKI5-mediated gene expression in PCa [78]. In addition, miR-375 targets/inhibits CD44 and its splice variants. The downregulation of miR-375 was observed in androgen-independent PC-3 and DU-145 [79]. Abramovic and colleagues, however, reported that miR-375-3p is a potent PCa biomarker due to its oncogenic properties. They found that miR-375-3p was highly expressed in the blood plasma of males with PCa compared to benign prostatic hyperplasia (BPH) patients. It was also suggested to be a higher-performance diagnostic marker compared to PSA [80]. MiR-21 has been found to be overexpressed in various types of cancers, including PCa and a wide range of other cancers [81]. With its ability to promote cell proliferation/invasion and metastasis and inhibit apoptosis, miR-21 is known as an oncogenic miRNA [82]. A previous report demonstrated that miR-21 is overexpressed in doxorubicin (DOX)-resistant PC-3 cells compared to the parental PC-3 cells. PTEN was identified as a direct target of miR-21. The suppression of miR-21 resulted in the upregulation of PTEN and a significant reduction in p-glycoprotein (P-gp, an MDR protein), subsequently inhibiting PI3K/AKT signaling, reversing drug resistance, and enhancing apoptosis in DOX-resistant PC-3 cells [83]. Studies have shown that miR-96-5p is significantly upregulated in PCa and various cancers, and it has a functional role linked to the tumor size, metastasis, and malignancy [84]. In PCa, miR-96-5p has been shown to target tumor suppressor genes FOXO1 and MTSS1. The overexpression of miR-96-5p, therefore, promotes cell proliferation, colony formation, and invasion in PCa [85]. It has also been reported that EGFR induces/activates miR-96-5p expression, which in turn targets and inhibits the tumor suppressor gene ETV6 and consequently promotes PCa cell proliferation. In addition, miR-96-5p has been demonstrated to regulate autophagy by targeting MTOR and ATG7 in PCa under hypoxia [69]. MiR-106b functions as an oncogene, and it is upregulated in various cancers, including PCa, breast cancer, lung cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma [86]. MiR-106b target genes include TEN, AKT, CNN1, LARP4B, RUNX3, DAB2, DLC1, FOG2, REST1, FUT6, ALEX1, and BTG3 [86]. In the PCa cell line LNCaP, miR-106b directly targets and inhibits CDKN1A (encoding p21), which leads to a G2/M cell cycle arrest. MiR-106b also regulates the expression of Ki67, MMP2, CD44, and Smad2 proteins in PCa cells [87]. Another miRNA that displays oncogenic properties is miR-125b. It has been reported that PCa tumor growth is significantly increased when miR-125b is overexpressed in either intact or castrated male nude mice [88]. Notably, one of the oncogenic mechanisms underlying PCa progression is mediated through the inhibition of the pro-apoptotic genes TP53, PUMA, and BAK1 and their downstream signaling pathways by miR-125b. In contrast, the inhibition of miR-125b increases cell apoptosis in PCa cells [88]. The oncogenic miRNA miR-141-3p is overexpressed in PCa cell lines and patient samples. It has been shown that the high-level expression of miR-141-3p promotes cell proliferation, 3D spheroid formation, tumorigenesis, stemness, and chemoresistance in PCa cell lines [89]. In addition, miR-141 is involved in cell proliferation by targeting/inhibiting PTEN, BRD3, and UBAP1, enhancing the activation of AKT and Rb/E2F signaling pathways in nasopharyngeal carcinoma [89]. The deregulation of miRNAs is one of the mechanisms conferring drug resistance in cancers, thereby leading to therapeutic failures. For example, the upregulation of miR-145 has been correlated with chemoresistance in PCa and other cancers [90]. A previous study further showed that the upregulation of miR-145 resulted in the downregulation of P-gp (P-glycoprotein) and pRb (retinoblastoma protein) by targeting and inhibiting SP1 (encoding specific protein 1) and CDK6 (encoding cyclin-dependent kinase 6) [90]. MiR-182 was also found to be upregulated in PCa. The overexpression of miR-182 results in increased cell proliferation through the targeting/regulation of the expression of NDRG1, GNA13, and BRCA1 in PCa. The study by Bai et al. showed that miR-182 acts as an oncogene and is upregulated in breast cancer, ovarian cancer, pancreatic cancer, and colorectal cancer. In PCa, miR-182 directly targets the 3′-UTR of ST6GALNAC5 [91]. MiR-200c, belonging to the miR-200 family, is known to play a role in promoting tumor progression in PCa and various cancers [92]. In the study of Lin et al., miR-200c was highly expressed in PCa cell lines compared to normal prostate cells [93]. Similarly, miR-200c is highly expressed in ovarian, endometrial, and esophageal cancers, consequently enhancing tumor growth and promoting chemoresistance [92]. However, various studies have expressed contrasting roles of miR-200c. For instance, it was found that miR-200c negatively regulates the expression of ZEB1 and vimentin, displaying an inhibitory effect on EMT-mediated cell proliferation, invasion, and migration in PCa [94]. Moreover, miR-200c acts as a tumor suppressor by inhibiting FOXF2 and BMI1 expression, consequently inhibiting cancer invasion and migration [95]. It was also found that miR-222 targets and negatively regulates CDKN1B (encoding p27kip1), a negative regulator of cell cycle progression in PCa and many other cancers [96]. The expression levels of miR-222 were found to be negatively correlated with PCa aggressiveness. Additionally, the overexpression of miR-222 resulted in increased cell proliferation in vitro and tumorigenicity in vivo [97]. Similar to PCa, the expression level of miR-222 was significantly associated with the tumor grade in uterine cancer [98] and tamoxifen resistance in breast cancer [99].