The major source of existing discrepancies across studies lies in differing methodological approaches, a lack of suitable housekeeper miRNAs, disease and tumor heterogeneity as well as in the nature of miRNAs themselves.
The methodological approach and single steps of miRNA analysis has a significant impact on the final results (40, 51). For example, northern blotting, microarray-based detection, next generation sequencing and real-time RT-PCR differ in sensitivity and specificity and their random use has led to inconsistent results.
Dahiya et al. describe this issue in their study and literature research (13). MiRs-21, -155 and let-7d were downregulated in tissue and cell lines, whereas miR-100 showed to be upregulated in that microarray-based approach. In addition, they found that only 16 of 192 analyzed miRNAs showed consistent expression patterns across studies (13). The given study demonstrated a downregulation of let-7d in cell culture analyses intra- and extracellularly and found higher expression levels of miR-100 in the intracellular compartment of SK-OV-3 cells only.
Four more studies on OC tissue showed diverging results as well (31, 52, 73, 77). Iorio et al. applied microarray analyses and found different miRNAs to be deregulated (31). MiR-125b1 was downregulated. MiR-100 showed lower expression levels in OC tissues compared to HCs (31). Nam et al. showed an upregulation of miR-20a and miR-21 in ovarian tumor tissue (52, 77). In the presented study, miR-125b showed a similar lower expression in EFO-27 intracellularly, extracellularly and tendentially under acidosis. In SK-OV-3 and OAW-42 cell lines, it showed higher expression levels intracellularly only. This is why it must be hypothesized that miR-125b is subtype specific. Moreover, miR-100 exhibited higher expression levels intracellularly in SK-OV-3 and miR-20a showed higher expression levels in the supernatant of all three analyzed cell lines in our study as well.
Methodologically, Wyman et al. used next generation sequencing and subsequent qRT-PCR (73). They found miR-15a and miR-20a to be upregulated like proven extracellularly in the given study. However, they also showed a downregulation of miR-21 and miR-100 (73).
Regarding the impact of hypoxia and acidosis on miRNA expression levels in OC, results are scarce. We found sporadic alterations of miRNA expression levels as well (see results). Giannakakis et al. demonstrated the involvement of miR-210 in the HIF pathway (23). Compared to our study, hypoxia was methodologically induced using a lower amount of oxygen (1.5% vs. 3%) and a different panel of miRNAs was analyzed (23). Another study on endometrial cancer cell lines also showed sporadic alterations only: miR-15a, miR-20a, miR-20b, miR-21 under hypoxia and let-7a, miR-22 and miR-125b under acidosis (17). These results were inconsistent with comparable studies (41).
We hypothesize that OC patients can be distinguished from HC comparing their urinary miRNA expression levels. However, urine as well as cell culture supernatant are both challenging for cancer detection because of their biochemical conditions. The feasibility of urinary miRNA-based detection of cancer has already been proven in other tumor entities like BC (18, 48, 53). Several studies verified the stability of miRNAs under harsh conditions like extreme pH-values (51). Compared to proteins, miRNAs undergo less degradation through ribonucleases due to their packaging into exosomes and the RISC and also because of their small size (26, 70). However, the amount of total miRNA in urine and cell culture supernatant is small, which emphasizes that miRNA quantity and quality are crucial for the final detection (22, 48).
Targeting the correlation of tumor cell derived and urinary miRNAs, one study on OC tissue and serum of the same patients conducted by Taylor et al. found matching expression patterns of eight miRNAs and therefore hypothesized that miRNAs in the serum derive directly from the tumor itself (64). Nakamura et al. also suggest that miRNAs in biofluids reflect tissue miRNA expression levels accurately (51). Microarray analyses of tissue, ascites and serum of EOC patients also showed uniform alterations (12). However, in serum, additional miRNAs evolved to be regulated (12). In our study we detected differing miRNA signatures in cell culture, in cell culture supernatant and in urine.
Furthermore, OC detection based on urinary miRNAs is hindered by disease heterogeneity. OC is a highly individual disease that differs in histology, stage, metastatic status and molecular tumor-characteristics (47, 51). The studies conducted by Iorio et al. and Calura et al. picture this as well (8, 31). Both showed histotype specific miRNA expression signatures. MiR-222 showed specific alteration in endometrioid and clear cell subtypes whereas miR-21 did in endometrioid subtypes only (8, 31). In our in vitro study, we were also able to detect subtype specific miRNA alterations.
Not only disease but also tumor heterogeneity complicate miRNA-based OC detection. Additionally, the tumor microenvironment consists of different cells and acellular components connected to different miRNA alterations. According to this, the same tumor might be able to exhibit a varying miRNA expression pattern at different states of its existence. This could explain the inconsistency of miRNA alterations across the published studies.
For example, miR-15a as well as let-7d showed significant downregulation in a cell culture study conducted by Zhang et al. (79). Interestingly, the degree of downregulation rose with stage and wasn´t detectable in 23.9% of the examined samples. They finally suggested these two miRNAs to be tumor suppressors (79). MiR-15a was neither down- nor upregulated in the intracellular compartment of the given study but was significantly elevated in the extracellular compartment of all three analyzed cell lines. This suggests that the intracellular downregulation or absence of miR-15a is a possible result of an upregulated trafficking into the tumor microenvironment.
Moreover, the significant downregulation of let-7d in OC cell lines could be proven intracellularly in SK-OV-3 and OAW-42, extracellularly in EFO-27 and OAW-42 and under hypoxia and acidosis in SK-OV-3. Let-7d emerged its role as tumor suppressor by negatively regulating the RAS-pathway in lung cancer (34). In conjunction with tumor heterogeneity, tumor immunology revealed a crucial role in the development and formation of cancer specific miRNA patterns (54).
Resnick et al. identified miR-21 and miR-155 as possible biomarkers (56). While miR-21 was upregulated, miR-155 was downregulated (56). We observed this in our in vitro study as well, but not in the urine of OC patients. This suggests that urinary miRNAs express differently because of the activity of urine-specific enzymes and several cellular mechanisms. Some other blood-based studies emphasize this hypothesis showing aberrantly altered miRNAs compared to the given urine-based study (26, 35, 36, 44, 60, 80). However, it must be considered that the analyzed OC histologic subtypes and samples as well as the applied methods and the investigated miRNA panels varied tremendously across these six studies.
Finally, patient heterogeneity modifies the results of the given study to an unknown extent, given that each individual presents with many exogeneous as well as endogenous confounders. In detail, cardiovascular, rheumatologic, dermatologic, neurologic, renal and many other diseases lead to specific miRNA expression alterations (22, 53, 69).
Zhou et al. performed qRT-PCR to determine exosomal miRNA expression levels (81). Not only OC samples but also benign ovarian tumors and gastric as well as colon carcinomas were analyzed and prove OC specificity of miR-30a-5p upregulation and the downregulation of 37 more miRNAs in OC exclusively (81).
Zavesky et al. performed qRT-PCR on cell free urine samples as well and found miR-100 to be downregulated (78). The strength of this work was prior assessment of RNA quality and quantity, while the inclusion of carcinomas of the fallopian tube into the study group of only 11 patients is questionable (78).