In this study we aimed to find a new approach to non-invasive prostate cancer screening methods as PSA serum measurements produce high numbers of false positive prostate cancer suspects [19]. We hypothesized that PCa-specific miRNAs measured in saliva exosomes show significant expression level differences between men suffering from prostate cancer and a non-cancer control group enabling a reliable differentiation of both groups in the common preventive screening program. The control group in the present study consisted of men suffering from other diseases of the urogenital tract associated with PSA level elevations. The acquired results confirmed our hypothesis, two out of sixteen examined microRNA sequences (hsa-mir-331, hsa-mir-200b) were significantly reduced in saliva samples from prostate cancer patients compared to the no-cancer control group. These differences correspond with former studies reporting a significant reduction of hsa-mir-200b and hsa-mir-331 in prostate tumor cells compared to healthy prostate tissue samples [20] [21] [22] [23]. Further determination of diagnostic value of both sequences via ROC curve analysis revealed moderate but reliable differentiation strength (AUC of .663 for hsa-mir-200b and .648 for hsa-mir-331) [16]. A recent study from 2017 of Souza et al found similar diagnostic values for hsa-mir-200b with a sensitivity of 67% and specificity of 75% for PCa patients, although examining concentration differences in blood serum samples rather than saliva exosomes [24]. A more clinical and comprehensible assessment of the diagnostic strength is the prognostic value of a test method. Both, hsa-mir-200b and hsa-mir-331 achieved a positive predictive value for prostate cancer of 71%, meaning the saliva test results were consistent with the histologic findings from prostate stance biopsy in 70% of the cases, malign as well as benign diagnose. To our knowledge, this is the first time hsa-mir-200b and hsa-mir-331 were measured in saliva exosomes with the ability to differentiate between men suffering from prostate cancer and a control group.
Lastly, analysis of case numbers and microRNA test results revealed that most of the patients tested positive not only for one but both sequences (i.e., measured expression level indicating prostate cancer according to cut off value), only 15 patients showed a positive test for only one microRNA, either hsa-mir-200b or hsa-mir-331. Significant results for more than one PCa-specific microRNA could indicate the existence of a diagnostic pattern of different microRNA sequences possibly with higher prognostic value for prostate cancer than the diagnostic ability of any microRNA sequence alone. To investigate this hypothesis, we compared sensitivity and specificity of a double positive test result to the diagnostic accuracy of hsa-mir-200b or hsa-mir331 calculated separately and individually. None of those interpretation possibilities showed superiority in discrimination between both groups as sensitivity and specificity were approximately the same. Therefore, we conclude that a microRNA saliva test with a single PCa-specific sequence with high diagnostic value could be more efficient than a combined examination of different microRNAs and development of a diagnostic pattern or panel. Anyhow, for a profound and certain answer to this question, further research on PCa-specific microRNA measured in saliva exosomes is needed.
Although the present study produced promising results indicating a useful augmentation of the standard PCa screening by saliva-based microRNA examination, the overall representative strength of our findings is limited by the composition of the study cohort. The study design included only men classified as prostate cancer suspects due to elevated PSA serum levels admitted to hospital for further verification of a potential cancer threat. Half of the patients were diagnosed with prostate cancer, the others suffered from other diseases of the urogenital tract also accompanied by PSA level elevation. Healthy individuals were not included. Therefore, detected significant group differences do not qualify to assess the identification ability of prostate cancer patients among healthy individuals, which is the purpose of preventive screening programs.
Nevertheless, the study design highlights a problematic aspect of the current PCa screening program to which salivary microRNA tests could provide a solution; that is high numbers of false positive prostate cancer suspects due to low predictive value of PSA serum measurements [25]. Examining the differences between true prostate cancer patients and those falsely suspected to suffer from prostate cancer (control group) after preventive PCa screening, this study revealed a reliable differentiation between both groups via non-invasive measurement of hsa-mir-331 and/or hsa-mir-200b in saliva samples. The current study proved the differentiation ability of those two microRNA sequences via ROC curve analysis and determined cut-off scores with good prognostic value. Augmentation of a conspicuous PSA test result by this method could verify or refute an uncertain cancer suspicion. In consequence, hsa-mir-331 and hsa-mir-200b concentration measurement in saliva samples could reduce numbers of false positive cancer suspects in the preventive screening program and thereby reduce numbers of falsely indicated prostate biopsies. All in all, two out of the 16 examined microRNAs showed prostate cancer specific changes in expression levels when measured in salivary exosomes. Hsa-mir-200b is a member of the mir-200-family whose involvement in carcinogenesis and aberrant cell development is documented for many different cancer types. Gene targets of hsa-mir-200b in prostate cancer development in particular are among others AMACR, BCL2, GOLM1, OR51E2, SIM2 or Bmi-1. Bmi-1 for example is accountable for abnormal cell proliferation, migration and chemosensitivity and is described to be suppressed by hsa-mir-200b [26]. Therefore, loss of or reduction of hsa-mir-200b as measured in PCa patients can induce abnormal cell proliferation in prostate gland tissue. Other research shows, downregulation of hsa-mir-200b can also induce epithelial-mesenchymal transition (EMT) in prostate gland cells. EMT describes the pathological change of epithelial cells from a cobblestone-like, tissue-adherend phenotype to a spindle-like, loosely attached cell character often found in embryonic mesenchymal stem cells leading to more cell motility, invasion of other tissue layers and eventually metastasis to other regions of the body [27] [28]. Recent studies stated, that hsa-mir-200b supports E-cadherin expression by suppressing its transcription repressor ZEB1. Transcriptions factor ZEB1 binds to ZEB-type E-boxes (CACCTG) within E-cadherins promoter region inducing chromatin condensation and gene silencing [29]. E-cadherin is one of the most important members of cell connection molecules like desmosomes or tight junctions and therefore very important to EMT.
A stunning number of studies already revealed important roles of hsa-mir-331 in several cancer types like leukemia, lung, gastric and liver cancer. Some references deal with the role in prostate cancer development and interestingly all of them have suggested a tumor suppressor role for hsa-mir-331 in prostate cancer, unanimously. Shee et al. provided the most recent review of the pathological and physiological roles of the miRNA-331 family in cancer [30]. The findings of Epis et al. (2009) suggest a role for hsa-mir-331 in the development and progression of prostate cancer while focusing on ERBB-2 as a target of this miRNA. They found hsa-mir-331 expression to be decreased in ERBB-2 overexpressing PCa tissue relative to normal adjacent tissue. Furthermore, they have shown that hsa-mir-331 blocked androgen receptor (AR) signaling in PCa cells. A loss of hsa-mir-331 expression could promote the increased ERBB-2 expression and signaling seen in many prostate cancers [21].
Wang et al. (2009) confirmed hsa-mir-331 to play an important role when identifying candidate genes involved in causal pathways of aggressive prostate cancer. They found hsa-mir-331 to be differentially expressed in prostate cancer cell lines and implicated an important part that hsa-mir-331 plays in cell cycle regulation [23].
In a later study, Epis et al. (2011) confirmed the reduced expression of hsa-mir-331 in aggressive prostate cancer. They showed that the RNA-binding protein HuR induces the ERBB-2 expression in prostate cancer by preventing the degradation of ERBB-2 mRNA by hsa-mir-331 [22]. Epis et al. (2011) were also involved in the findings that showed hsa-mir-331 playing an inhibiting part in the growth of prostate cancer cells. hsa-mir-331 seems to repress the expression of the deoxyhypusine hydroxylase (DOHH). This enzyme is an important player in protein modification [22]. When it comes to the aggressiveness of prostate cancer, which can variate widely, Fredsoe et al. (2019) developed a five-miRNA model for predicting prostate cancer aggressiveness using cell-free urine. hsa-mir-331 plays a decisive role in this 5-miRNA orchestra [31].
In epithelial-mesenchymal transition, another target for hsa-mir-331 seem to be Neuropilin 2 and nucleus accumbens-associated protein 1. The findings of Fujii et al. (2016) indicate these targets for hsa-mir-331[32]. White et al. (2012) confirmed hsa-mir-331 to be a tumor suppressor via its regulation of Kallikrein-related peptidases 4 (KLK4) expression [33].
In this study, hsa-mir-331 was one out of two miRNAs to show significantly different results in cancer patients compared with the healthy control group – has-mir-331 was significantly downregulated. When comparing both groups (cancer and comparison group) hsa-mir-331 is decreased by a 2.64-fold change.
When tested for significance, the different expression of hsa-mir-331 shows a significant level with a p-value of 0.031 (with a threshold of p = 0.05).
Decreased levels of hsa-mir-331 concur with former studies that mostly showed decreased levels of hsa-mir-331 in prostate cancer patients [32].
The 4 studies showing results that indicate a downregulation of hsa-mir-331 were studying cell lines rather than body fluids. Epis et al. (2009, 2011) worked with prostate cancer tissue and Wang et al. (2009) focused on cancer cell lines [23] [21] [22]. However, the results oppose the findings of Fujii et al. (2016). Looking closely at the study of Fujii et al. (2016), they found that hsa-mir-331 expression was much higher in prostate cancer cells with higher Gleason scores (7, 8, and 9) [32]. The patient data of the cancer group of this study shows, that 64% of the prostate cancer patients had higher Gleason scores of 7 to 10.
In summary, hsa-mir-331 holds true for the hypothesis to be a potential circulating exosome-derived biomarker in saliva for the detection of prostate cancer.
The other 14 examined circulating microRNA presented no significant group differences between cancer and control group, although being described as PCa-specific in preliminary literature research. Included within this paper were studies identifying potential PCa-microRNA. However, all of them examined expression levels in prostate tissue, cancer cell lines or blood serum samples rather than salivary exosomes. Supplementary research on the influence of saliva filtration processes or active microRNA equipment of salivary exosomes were limited but could provide information on missing disease specific differences of certain circulating microRNA. Therefore, occurrence or absence of PCa-specific microRNA in saliva exosomes need to be part of future investigations. Another reason for inconsistent PCa specificity of the other 14 microRNA may derive from the composition of the control group in this study. In contrast to other studies working with healthy individuals for comparison, the present control collective consisted of patients suffering from other diseases associated with PSA level elevations. Especially diseases of the urogenital tract could be influenced by the same microRNA known for prostate cancer carcinogenesis. Hence, the other 14 examined microRNA might be indicators for general urogenital pathologies rather than only prostate cancer. Future research needs to identify and verify PCa specificity of microRNA in salivary exosomes in comparison to other diseases of the urogenital tract to enable a salivary microRNA test method with high diagnostic accuracy.