The expression of three members of the miR-200 family was analyzed in plasma, urine and tumor tissue collected from HGSOC patients and patients with benign ovarian tumor. The aim was to assess possible similarities in the expression profile of these commonly de-regulated miRNAs between the different sample types and, whether urine samples could replace or complement blood samples in miRNA biomarker analysis.
MiR-200a, mir-200b and miR-200c levels showed similar trend in all patients based on sample type, suggesting that they are co-expressed at similar levels. The relative levels of the miRNAs in urine and plasma were found to be comparable in the samples obtained from the patients with a malignant tumor, with most patients showing high miRNA expression in plasma also having high miRNA expression in urine. Tissue and plasma miR-200 analysis could distinguish malignant and benign cases. Moreover, this pilot study shows that plasma and urine miR-200 expression levels correlate in HGSOC but not in benign cases. This suggests that the role of urine samples, at least in parallel with plasma samples, should be further investigated in miRNA biomarker detection in HGSOC.
Small-nuclear RNAs (snRNAs) are commonly used as reference genes for miRNA expression normalization. Thus, tumor tissue expression values were normalized against RNU6B. However, several studies have reported differential expression of snRNAs in cancer and the suitability of each snRNA should be assessed for each individual experimental setup [14, 15]. The expression data of plasma and urine samples were normalized against an exogenous spike-in reference miRNA (cel-miR-39) since there is currently no consensus on appropriate endogenous reference to be used for these sample types. The miRNA expression in urine samples showed the overall largest range of variation, with most samples having relatively low expression levels, while samples from three patients with a malignant tumor and two of the controls showing very high relative levels. In future, better normalization strategies and/or absolute quantification PCR can improve the reliability of the results for liquid biopsies.
The relative expression levels in tumor tissues were not comparable with the liquid biopsies. In several cases, however, an opposite expression trend, was observed with patients having high miR-200 levels in both plasma and urine, showing low expression in tissue samples. This could be interpreted as an active secretion of miRNAs from tumor tissue, but such straightforward conclusion cannot be made from the present data taking into account that most of the patients having low expression in liquids had no extensive peritoneal carcinosis in addition to tumors. Exosomes are lipoprotein complexes acting as small membranous vesicles. The factors defining the fate of given miRNA to be secreted in exosomes are still largely unknown. Epithelial ovarian cancer (EOC) neoplastic cells have been shown to have an enhanced exosomal output as compared to normal epithelial cells . Based on in vitro findings with chemoresistant ovarian carcinoma cells, it has been postulated that the release of exosomes may be a mechanism by which neoplastic EOC cells could ‘educate’ each other, thereby enhancing the development of platinum-resistant disease . It will be interesting to test this hypothesis of possible predictive value of high miRNA secretion in liquid biopsies versus tumor tissue in the larger CHEMOVA cohort. We isolated total miRNA from plasma, but from urine the exosomal fraction was used. The high levels of RNase in urinary tract leads to degradation of free miRNAs and practically only exosomal miRNAs remain detectable in urine [18,19,20].
In conclusion, this study aimed to assess the feasibility of liquid biopsies in miRNA expression profiling in HGSOC. The most significant finding is that the three members of the miR-200 family are detectable in urine, plasma and tissue samples obtained from the same ovarian cancer patients. Tumor tissue and plasma analysis could discriminate malignant and benign ovarian samples. Moreover, a correlation was observed between miR-200 expression in urine and plasma of ovarian cancer patients, but not in patients with benign tumor. This pilot study showed that plasma and urine as liquid biopsies could be useful in miRNA biomarker analyses in HGSOC, but more studies are needed to validate the current findings in larger cohorts of patients, including patients with early stage disease.