Effectiveness of miRNAs as Potential Non-Invasive Liquid Biomarkers for the Diagnosis and Prognosis of Prostate Cancer

Abstract

Background

Prostate cancer (PCa) is one of the most prominent cancers worldwide. However, many limitations in its diagnostic and prognostic protocols lead to severe unreliability. Numerous studies have identified miRNAs as potential non-invasive liquid biomarkers of PCa. This research aims to evaluate the current literature to better understand the effectiveness of miRNAs as potential diagnostic and prognostic non-invasive liquid biomarkers of prostate cancer.

Methods

A systematic review was conducted by thorough searches on the Omni and PubMed database for articles in the past five years. The eligibility criteria, including demographics, study methodology, and sample type, were developed, enabling the final selection of 18 articles that fit these guidelines. Characteristics of the chosen studies varied, as the goal was to include diverse populations and methodologies to encompass all the current literature. Key differentially expressed and statistically significant miRNAs were extracted from the research, including their dysregulation signatures and associated statistical values, and compiled into a Google Doc.

Results

Various single miRNAs and miRNA panels show statistically significant differential expression between healthy controls and PCa patients, displaying potential as biomarkers. MiRNA panels, in conjunction with current diagnostic protocols and clinicopathological factors, display the most promise as a future diagnostic tool for PCa. More specifically, a 2-miRNA and 5-cs-miRPs panel show remarkable potential for future use in prostate cancer screening and diagnosis. These panels displayed significant specificity, sensitivity, and accuracy in the diagnostic power of prostate cancer, even performing better than the prostate-specific antigen test, the current gold-standard liquid biomarker. Some limitations of the supporting evidence include heterogeneity between studies’ methodology and analysis, lack of standardization in the current protocols of miRNA collection and quantification, and the influence of genetic and environmental factors on the expression of these biomarkers.

Conclusion

Future research should validate which miRNAs to include in a panel, how to standardize their storage, collection, and quantification, and how to incorporate them into the current protocols. Clinical applications of miRNAs as non-invasive liquid biomarkers can contribute to early cancer detection and prevention, thus improving outcomes for prostate cancer patients.

Introduction

Prostate cancer (PCa) arises when cancer cells invade and develop in the prostate tissue, with the ability to metastasize to other areas of the body.¹ PCa is an exceedingly prevalent disease, ranking as the second most frequently diagnosed cancer and the fifth leading cause of cancer-related deaths among men worldwide.² According to the Canadian Cancer Society, PCa is the most common cancer among Canadian men.³ Prostate cancer’s heterogeneity between patients leads to immense difficulty in diagnosis and treatment.⁴

Current diagnostic strategies for PCa have been heavily criticized for their uncertainty, leading to frequent overtreatment and false-positive or false-negative outcomes.⁵ The primary screening and diagnostic tools are the prostate-specific antigen (PSA) test, digital rectal exam (DRE), and tissue biopsy.¹

MiRNAs are small, non-coding ribonucleic acids that regulate gene expression through post-transcriptionally modifying messenger RNAs (mRNAs).⁶ MiRNAs bind to their target mRNAs, inhibiting translation or degrading the molecule to regulate their expression.⁷ This regulation of gene expression influences functions such as apoptosis, proliferation, differentiation, metabolism, homeostasis, and cell cycle control. MiRNA biogenesis must be exceptionally precise to ensure that these small molecules have the proper structure and function.

Dysregulation of miRNAs can be a prominent contributor to the development and progression of diseases, such as cancer.⁸ MiRNAs can be upregulated or downregulated, with various effects on cancer pathways. The amplification of oncogenic and/or the loss of tumour-suppressor miRNAs can contribute to cancer tumorigenesis, and the difference in miRNA expression patterns could identify healthy individuals versus cancer patients.⁹ Numerous miRNAs and their dysregulation have been identified in prostate cancer patients. The use of miRNAs as diagnostic and prognostic biomarkers for PCa, such as miR-17-3p and miR-1185-2-3p has shown increasing promise.¹⁰

This research aims to evaluate the current literature to better understand the effectiveness of miRNAs as potential non-invasive liquid biomarkers for detection and prognosis of prostate cancer. In doing so, the following questions will be addressed: How effective are miRNAs as non-invasive liquid biomarkers for the diagnosis and prognosis of prostate cancer? Which specific miRNAs have shown the most promise in the current literature for their potential as diagnostic and prognostic markers of PCa? Given the frequent overtreatment and diagnostic unreliability associated with the current protocols, there is a pressing clinical need for a more specific and reliable biomarker of PCa. MiRNAs, with their ability to stably reflect unique dysregulation signatures in liquid biopsies, offer a promising solution. By thoroughly evaluating the current literature and identifying specific miRNAs, this review seeks to contribute to the development of non-invasive diagnostic tools, ultimately improving early detection and prognosis of prostate cancer. This could lead to more personalized and effective treatment strategies, reducing the burden of this prevalent disease.

The unique dysregulation signatures of specific micro ribonucleic acids (miRNAs) in liquid biopsies can serve as non-invasive biomarkers for early detection and prognosis of prostate cancer. Liquid samples, collected primarily from urine, saliva, serum, or plasma, are extremely rich in analytes with great potential as diagnostic and prognostic markers for prostate cancer.¹¹ The use of liquid samples presents significant advantages as early detection of prostate cancer is crucial for improving patient outcomes and reducing mortality rates. This review aims to explore the potential of miRNAs to enhance early detection and provide reliable prognostic information, ultimately leading to better clinical management of PCa.

Current Diagnosis and Prognosis Protocols of PCa

The current diagnostic and prognostic protocols for prostate cancer (PCa) are continually scrutinized for their limitations. The primary screening and diagnostic procedures consist of a prostate-specific antigen (PSA) test, digital rectal exam, and transrectal biopsy, all with significant limitations. The prostate-specific antigen test measures the level of PSA, which is the current gold-standard liquid biomarker for screening.¹ In prostate cancer, PSA levels are expected to be higher than 4 ng/ml, which is considered the cut-off level to indicate a prostate biopsy.¹¹ However, levels of PSA deemed to be normal (< or = 4.0 ng/mL) can still be detected in individuals with biopsy-confirmed prostate cancer.¹²  Furthermore, PSA levels may also be elevated in men who have benign prostatic hyperplasia (BPH), infection, or inflammation of the prostate. Liu et al. determined a prostate-specific antigen test sensitivity and specificity average of 51% and 68%, respectively.¹⁰ PSA’s limited ability to distinguish the presence and aggressive nature of PCa leads to substantial screening and diagnostic unreliability.¹³

A digital rectal exam (DRE) involves examining the prostate for any abnormalities through the rectal wall. DRE’s are highly subjective to the physician performing the examination; thus, the accuracy may depend on their expertise. DRE’s estimated specificity of 59% and sensitivity of 51% can result in unnecessary biopsies or no further investigation into a severe issue.^11,14

To diagnose PCa, histopathological confirmation is needed through a transrectal biopsy. A piece of tissue is removed from the prostate with a thin needle inserted through the rectum and viewed by a pathologist to identify the presence of cancer cells.¹ The biopsy presents many complications as an invasive procedure, including bleeding, infection, prostatitis, hematospermia, and difficulties urinating.^11,15 Due to its invasive nature, this procedure is not used to continuously monitor the tumour. The current screening and diagnostic protocols for PCa have many limitations, demonstrating the clinical need for a more reliable, less invasive, and specific biomarker for this disease.

PCa’s prognosis is determined by numerous factors, including the cancer grade and stage. The cancer cells’ abnormality and aggression will be given a grade known as the Gleason score, ranging from 6 to 10. A Gleason score of 6 indicates low-grade cancer, medium-grade cancer has a score of 7, and high-grade PCa has a score of 8-10.¹ Furthermore, a combination of factors determines the cancer stage, ranging from stage I to stage IV. PCa staging plays a critical role in patients’ prognosis. Diagnosis and prognosis are heavily connected, as early detection typically results in better patient outcomes.

Liquid Samples

Liquid samples are an emerging non-invasive biopsy procedure for many cancers, including prostate cancer. Samples of serum, plasma, urine, and other bodily fluids can be analyzed for specific analytes to indicate a particular disease. Analytes in liquid samples can include cancer-circulating tumour cells, miRNAs, circulating RNA, cell-free DNA, and extracellular vesicles (exosomes). ¹¹ Dysregulation signatures of specific analytes can be indicative of diseases. Liquid biopsies show significant promise for their benefits and potential use in collecting biomarkers. Samples can be taken and scheduled at any time with significantly fewer complications and are extremely valuable in cancer surveillance.¹¹

The main challenges of liquid biopsies are ensuring quality, reliability, reproducibility, and accuracy due to a lack of standardized methods in collection and analysis.⁴ Additionally, results from serum, plasma, whole blood, or urine should not be compared to one another due to different biomarker signatures.¹³ Current liquid biopsy assays approved by the Food and Drug Administration (FDA) include Oncotype DX AR-V7 Nucleus Detect, Foundation One Liquid, Sangia Total PSA Test, Progensa PCA 3 Assay, and 4 K Score Test; none of which include miRNAs.¹¹ As further research is conducted and a standardized methodology develops, non-invasive liquid biopsies will become more prominent in establishing biomarkers for PCa.

MiRNAs as PCa Biomarkers

The pioneering discovery of the association between miRNAs and human cancer was first reported by Calin et al. in 2002.¹⁶ Since then, researchers have continued to investigate miRNAs' involvement in prostate cancer and their potential use as diagnostic and prognostic biomarkers. Researchers are also improving their understanding of the pathways that miRNAs contribute to prostate cancer. Altered expression of miRNAs can arise from several intrinsic and extrinsic factors. Intrinsic factors include molecular and genetic aspects, such as aberrant miRNA biogenesis and chromosomal modifications.⁷ Extrinsic factors include modified epigenetic regulation from one’s environment, lifestyle, and exposures.⁷ Aberrant miRNAs can then contribute to prostate cancer via various pathways including androgen receptor regulation, p53 repression, PTEN repression, single nucleotide polymorphisms, and copy number changes of miRNAs.^7,17

The stable detection of miRNAs in liquid biopsies as cell-free circulating or exosomal miRNAs can be of practical clinical application.¹³ There are numerous methods to quantify the expression of miRNAs, such as Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), Microarrays, Nanostring’s nCounter and Next Generation Sequencing, which are all currently utilized.¹⁸ Davey et al. utilized qRT-PCR, while Koh et al. utilized Next Generation Sequencing to determine miRNA expression in prostate cancer.^19,20 Expression analysis allows for miRNAs dysregulation signatures to be identified, and their cancer-specific pathways can be elucidated.

Despite significant progress, there are numerous gaps within the current literature, with the lack of a standardized method for miRNA detection, storage, and quantification being the most notable. Also, the role of lifestyle, genetics, and other factors impacting miRNA signatures have not been clearly explored in PCa research. Lastly, the large number of miRNAs presents challenges in deducing which should be utilized as biomarkers. Further research is essential to address these gaps and enhance the clinical utility of miRNAs in PCa diagnosis and prognosis.

Methods

Eligibility Criteria

Case-control and cohort studies published in the past five years, from 2019-2024, were included in this review. Men of all ages of any ethnicity/race with prostate cancer were considered. The goal was to obtain data representative of diverse populations; thus, all ethnicities/races were included. Human studies with liquid samples were only considered; however, exceptions were made for studies including both human and cell culture cohorts. Studies with both liquid and tissue samples were also included. These studies included valuable information, warranting their consideration. Studies must have had a pretreatment collection of liquid samples in either urine, serum, or plasma to properly assess the miRNA dysregulation. Animal studies, cell culture studies, and studies with only tissue samples were excluded due to the focus on human clinical application and the use of liquid biomarkers. The overall outcome of interest was to identify and assess dysregulation signatures of specific miRNAs of clinical and statistical significance.

Information Sources

Articles were collected using both PubMed and Omni databases. For the review, primary articles were found in Omni. Meta-analyses, reviews, and systematic reviews were found using PubMed. These sources were used to conduct a literature review and gain further understanding of the field. Google was used to search for general information and to gain supplementary knowledge outside of the literature, including statistics, definitions, and PCa information. Articles were collected between May 3^rd and 12^th , 2024, with additional research occurring from May 3^rd to July 27^th, 2024.

Search Strategy

Articles and data were collected using both PubMed and Omni databases. Two different sources were used for organizational and accessibility purposes. The Omni database was used to gather primary sources, using the search keywords “miRNAs liquid biomarkers and prostate cancer.” The search was filtered for peer-reviewed articles within the last five years, from 2019-2024, that were written in English. Human samples were only included. Additionally, review articles, duplicate articles, animal trials, and all other articles within the exclusion criteria were filtered out of the search. The PubMed database was used to gather secondary sources using the keywords “miRNA biomarkers and prostate cancer.” Filters were applied for meta-analyses, reviews, and systematic reviews in the last five years that were written in English to be included and evaluated.

Selection Process

Studies were systematically narrowed down based on eligibility criteria and their relevance to the research objectives. Comprehensive searches were conducted on the Omni database, with each article individually assessed. Initial screenings involved reading the abstracts and methodology sections to ensure they met the predefined criteria. Articles meeting these criteria were then downloaded to Zotero for thorough reading and annotation. The final selection was made after a complete evaluation of each article's usefulness and relevance. Articles were excluded during the identification phase if they were textbooks, newspaper articles, or any mediums other than peer-reviewed journal articles. During the screening process, records were excluded based on their relevance to the research topic (Figure 1).

Data Collection Process

Data collection was conducted by the single reviewer. Following the selection of studies that met the eligibility criteria, the focus was placed on the results and discussion sections to gather data. Key differentially expressed and statistically significant miRNAs were extracted from the primary research, including their dysregulation signatures and associated statistical values. The extracted data was systematically compiled into a table using Google Docs and subsequently presented in the results section. Additionally, articles were thoroughly highlighted and annotated within the Zotero application to capture important information from the full texts.

Figure 1: PRISMA Diagram for Study Selection. Flowchart displays the study identification and selection process.²¹

Study Risk of Bias Assessment and Study Quality

A risk of bias and quality assessment were not conducted due to limitations in feasibility, time, and expertise. These constraints prevented the inclusion of such analyses in the current study.

Results

Study Characteristics

A total of 18 studies were reviewed and analyzed. Key characteristics of each study, including methodologies, sample populations, and notable findings were documented to provide comprehensive understanding. The studies collectively investigated the use of miRNA biomarkers in prostate cancer across 16 different countries. The sample sizes varied significantly, ranging from small cohorts of 10 patients to larger groups of over 250 participants. These studies focused on both PCa patients and healthy controls or benign controls to ensure comprehensive comparisons. The primary sample types used were urine, plasma, and serum collected before and after various medical procedures such as biopsies, digital rectal exams, and radical prostatectomies. Different kits, such as the RNeasy Plus Micro Kit and miRNeasy Plasma Kit, were used for RNA extraction and isolation. Techniques such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing were employed to quantify and analyze miRNA expression levels. Some studies used databases like TargetScan, miRTarBase, and Diana microT-CDS to validate miRNA targets. The summarized information is presented in Table 1.

Table 1: Characteristics of the included studies

Dysregulated miRNAs for Prostate Cancer Diagnosis and Prognosis

Numerous differentially expressed and statistically significant miRNAs have been identified, underscoring their potential as biomarkers for PCa. The associated statistical results were also documented. The identified miRNAs were either cell-free circulating or exosomal (extracellular vesicles) in the liquid samples, specifically plasma, serum, or urine. A significant trend of upregulation in many miRNAs associated with PCa was observed across the selected studies, although a few downregulated miRNAs were also identified, contrary to the overall pattern.

Table 2 presents the most significant single miRNA from each study, while Table 3 highlights the most significant miRNA panels. A comprehensive table is included in the Appendix 1.

Among the single miRNAs identified to be differentially expressed with statistical significance, miR-17-3p, miR-1185-3p, miR-21, and miR-18a have demonstrated substantial value as diagnostic markers of prostate cancer (as indicated by the * in Table 2). Both miR-17-3p and miR-1185-2-3p displayed significant area under the curve (AUC), specificity, and sensitivity values in distinguishing PCa patients from healthy individuals.¹⁴ MiR-21 and miR-18a effectively differentiated localized PCa from non-malignant cases with very high statistical significance (p-values of <0.001).³⁴

Furthermore, miR-221, miR-940, and miR-423-3p have shown potential as prognostic biomarkers of PCa (as indicated by the ** in Table 2). MiR-221 was able to distinguish between metastatic and localized PCa with an AUC of 0.982 and a sensitivity of 92.9%.³⁴ MiR-940 was associated with a Gleason score of 7 or greater, aiding in the prediction of cancer aggression with a p-value of 0.0008 and AUC of 0.75.³¹ Lastly, miR-423-3p was shown to differentiate between treatment-naive and castration-resistant PCa, indicating cancer aggression, with statistical significance (p-value of <0.0001).²⁵ These markers exhibit the highest statistical significance among the single miRNAs for potential use as non-invasive diagnostic and prognostic biomarkers.

In addition to the single miRNAs identified in the literature, there are notable panels of miRNAs that have demonstrated greater diagnostic power for PCa (as indicated by the * in Table 3). Liu et al.’s 2-miRNA diagnostic model, comprising of serum miR-17-3p and miR-1185-2-3p, performed exceptionally well across training, test, and validation cohorts, achieving an average accuracy of 92.76%, sensitivity of 88.90%, specificity of 95.65%, and an AUC of 0.9785. Additionally, their 5-class separability miRNA pairs (cs-miRPs) diagnostic model achieved an average accuracy of 99.32%, sensitivity of 99.42%, specificity of 99.67%, and AUC of 0.9959.¹⁰ Similarly, Urabe et al. identified a robust diagnostic model for PCa also consisting of serum miR-17-3p and miR-1185-2-3p, which demonstrated a sensitivity of 91%, specificity of 97%, accuracy of 92%, and an AUC of 0.99.¹⁴

Two separate 3-miRNA models and a 5-miRNA model have shown promise as prognostic biomarkers for PCa (as indicated by ** in Table 3). Gandellini et al.’s 3-miRNA model predicted PCa reclassification with 80% sensitivity, 50% specificity, and an AUC of 0.67.²⁷ Moreover, Ramirez-Garrastacho et al.’s 3-miRNA model distinguished between grade 1 and grade 3 PCa with an AUC of 0.85, while their 5-miRNA model achieved an AUC of 0.93 for the same distinction.²⁸ These miRNA panels exhibit significant clinical potential as non-invasive diagnostic and prognostic biomarkers.

Discussion

This review evaluates the current literature to better understand the effectiveness of miRNAs as potential liquid biomarkers for prostate cancer diagnosis and prognosis. Numerous miRNAs have shown statistically significant dysregulation signatures in PCa, highlighting their potential use as biomarkers. The current gold-standard screening liquid biomarker, the prostate-specific antigen (PSA) test, has demonstrated sensitivity and specificity averages of 51% and 68%, respectively according to Liu et al.¹⁰ Additionally, serum PSA showed an AUC of 0.695, and urinary PSA had an AUC of 0.741 in predicting aggressive PCa.³⁶ Despite some single miRNAs exhibiting greater predictive value than PSA, miRNA panels show the most promise as diagnostic tools.

 Liu et al.’s serum 2-miRNA diagnostic model, comprising of miR-17-3p and miR-1185-2-3p outperforms PSA significantly.¹⁰ This model, also tested by Urabe et al. demonstrated superior specificity, sensitivity, accuracy, and AUC values compared to PSA in predicting PCa.¹⁴ The mechanistic role of miR-17-3p supports these findings, as it was shown to promote tumour cell survival and proliferation, along with heightening tumorigenesis, by targeting TIMP3, p21, and PTEN. TIMP3, p21, and PTEN act as tumour suppressors; thus, their downregulation by an upregulated miR-17-3p may be influential in prostate cancer. ³⁷ Both Yang et al. and Farran et al. have also shown statistically significant upregulation of miR-17-3p, further providing evidence to support their promise as PCa diagnostic biomarkers.^37,38 Additionally, Liu et al.’s 5-class separability miRNA pairs (cs-miRPs) diagnostic model also showed better performance than PSA in all the evaluated metrics.¹⁰ A key component of this panel, miR-1290, has been linked to GLUT4 translocation to the plasma membrane. GLUT4 overexpression seems to promote PCa development and progression.¹⁰ The findings of Huang et al., Wang et al., and Li et al. have all supported miR-1290 upregulation in PCa.^39,40,41 These miRNA panels, with their enhanced performance over the current gold-standard liquid biomarker, warrant serious consideration for their PCa diagnostic capabilities.

In determining prognosis, Ramirez-Garrastacho et al. 's 3-miRNA and 5-miRNA models achieved higher AUCs than both serum and urinary PSA in predicting PCa aggression.²⁸ The 5-miRNA model also includes miR-1290, which was previously noted to have specific pathways in PCa. However, there is insufficient supportive statistical data to make definitive conclusions about the prognostic capabilities of these miRNA panels.

Among the single miRNAs, mir-221 showed the most promise as a prognostic tool, with the highest statistical significance, AUC, and sensitivity for distinguishing between localized and metastatic PCa.³⁴ MiR-221 was shown to promote PCa cell proliferation and migration through SOCS1 downregulation. SOCS1 is a known tumour suppressor gene, and its downregulation may influence prostate cancer tumorigenesis. Furthermore, miR-221 targets the Ras/Raf/MAPK/ERK signaling pathway and regulates Epithelial-mesenchymal transition (EMT), which has a role in cell proliferation and tumour-promoting activity.⁴² Zedan et al. confirmed miR-221 upregulation in PCa, supporting Ibrahim et al.’s findings and the mechanistic roles.^34,35 In contrast, Goto et al. showed downregulation of miR-221 in prostate cancer, indicating the need for further research to validate this specific miRNA.⁴³

Overall, while miRNA panels show the most potential for diagnostic purposes, their role as prognostic markers remains inconclusive. Proper clinical application of these panels could reduce unnecessary transrectal biopsies, overtreatment, and overdiagnosis, potentially improving patient outcomes. Future research and validation of these miRNA panels hold great promise for their non-invasive screening and diagnostic capabilities.

Diagnostic Recommendations

MiRNAs, particularly within multivariable panels, show significant promise as non-invasive liquid biomarkers for screening and diagnosing PCa. Specifically, a 2-miRNA panel of serum miR-17-3p and miR-1185-2-3p and a 5 cs-miRPs panel demonstrate the most potential.^10,14 Utilizing miRNA panels alongside current screening methods and clinicopathological factors could enhance specificity, sensitivity, and overall diagnostic performance for PCa patients. MiRNAs could be particularly useful in the initial diagnostic step, where screening for these biomarkers could provide specific and sensitive detection of PCa, indicating the need for a biopsy. However, there are a few limitations in the use miRNAs as biomarkers, including their necessity to be used in conjunction with transrectal biopsies to confirm pathology, implementing an individualized and preventative approach to establish baseline and dysregulated counts of specific miRNAs, and the availability of financial and medical resources. Addressing these limitations could allow for the emergence of a preventative attitude towards prostate cancer, thereby enhancing the clinical management of this disease.

Limitations

Despite these promising findings, the analyzed studies have several limitations. The heterogeneity among studies, including differences in methodologies, sample populations, and protocols, presents challenges in comparing results and drawing definitive conclusions. The lack of standardized methods for collecting, storage, and quantifying miRNA biomarkers in liquid samples means that different methodologies can yield significantly different miRNA profiles. Furthermore, miRNA signatures in specific populations may not be evident in others due to genetic and environmental variability. Inconsistent data collection, sampling, populations, and analysis methods across studies make it difficult to draw comprehensive conclusions and compare study results.

Conclusion & Future Direction

Prostate cancer is a significant contributor to cancer cases and mortalities worldwide; yet current diagnostic and prognostic tools have numerous limitations. There is a pressing clinical need for a more reliable, accurate, and specific biomarker for PCa to reduce uncertainty in diagnosis and prognosis. MiRNAs have shown great promise as non-invasive liquid biomarkers, especially within multivariable panels, for screening and diagnosing PCa. However, further research is necessary to identify and validate the most predictive miRNAs, standardize the storage, collection, and quantification methods for these biomarkers, and refine their application within multivariable panels. Notably, a model comprising of serum miR-17-3p and miR-1185-2-3p, as well as Liu et al.'s 5 cs-miRPs model, shows exceptional potential and should encourage additional research.^10,14 Future investigations will be pivotal in enhancing the clinical utility of miRNAs as non-invasive liquid biomarkers, thereby improving diagnostic and prognostic outcomes for prostate cancer patients.

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