Αθηνά Λινού-Παπαδημητρίου, Καθηγήτρια, Ιατρική, Ε.Κ.Π.Α.
Γεώργιος Ζωγράφος, Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
Σοφία Τσελένη-Μπαλαφούτα, Καθηγήτρια, Ιατρική Σχολή, Ε.Κ.Π.Α.
Δημήτριος Λινός, Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
Γεώργιος Μαστοράκος, Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
Νικόλαος Αρκαδόπουλος, Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
Νικόλαος Δανιάς, Επίκουρος Καθηγητής, Ιατρική Σχολή, Ε.Κ.Π.Α.
The pathologies of the thyroid gland constitute a common health issue, demanding accurate diagnosis and treatment. As new evidence emerges, new diagnostic methods, prognostic tools and treatment protocols are implemented. The elucidation of molecular mechanisms of diseases is important for the identification of useful prognostic markers in patients, especially in those being at high risk. To date, there is no way to diagnose non-invasively thyroid cancer and to monitor the progression of this disease. MicroRNAs (miRNAs) are small, endogenous, single-stranded, non-coding RNA molecules of approximately 22 nucleotides. They act post-transcriptionally and regulate negatively their targets, depending on the complementarity with their mRNA targets.
The aim of the present PhD thesis was to contribute to the development of new tools for the accurate and non-invasive diagnosis of thyroid cancer.
For this purpose, we collected 35 cancerous tissue samples and adjacent normal thyroid tissue samples from patients with papillyary throid cancer. In 16 out of 35 patients, we also collected preoperative blood serum. Moreover, we collected blood sera from 48 additional patients with papillary thyroid cancer as well as from 72 patients with non-cancerous thyroid (e.g. bening thyroid tumors, hyperparathyroidism etc.). We performed thyroid tissue homogenization and isolated total RNA from these homogenized tissue samples as well as from the aforementioned blood sera. Next, we determined the concentration and purity of the extracted total RNA. After that, we performed miRNA-seq. Moreover, we carried out total RNA polyadenylation and reverse transcription into first-strand cDNA, followed by quantitative real-time PCR (qPCR). Last, we extensive biostatistical analysis was performed on qPCR results.
Among the identified miRNAs, the ten most promising miRNAs in terms of variation of their expression are the following: miR-125a-5p, miR-144-3p, miR-622, miR-361-5p, miR-146a-3p, miR-340-5p, miR-199a-5p, miR-205-5p, miR-335, and miR-129-5p. The levels of these miRNAs were significantly different between cancerous and non-cancerous thyroid tissues. Using the developed qPCR protocols, we validated the expression results regarding these ten miRNAs in the four pairs of cancerous and adjacent normal thyroid tissues, and the we extended quantification of these ten miRNAs in another 31 pairs of thyroid tissues. Eight out of these ten miRNAs (all but miR-125a-5p and miR-205-5p) were differentially expressed at a statistically significant level between cancerous and paired non-cancerous thyroid tissues. We also found that high expression of miR-144-3p, miR-146a-3p and miR-340-5p, as well as low expression of miR-622, miR-361-5p, miR-199a-5p, miR-335-5p, and miR-129-5p is associated with particular clinicopathological features, such as lymph node infiltration, extrathyroidal infiltration, and the existence of multifocal tumors. The levels of five out of eight miRNAs were significantly different in the serum of patients with papillary thyroid cancer, compared to the serum samples of patients with other (non-malignant) thyroid pathologies. Τα miRNAs αυτά ήταν τα εξής: miR-144-3p, miR-146a-3p, miR-340-5p, miR-199a-5p και miR-335. Moreover, biostatistical analysis including construction of ROC curves and univariate logistic regression showed that the expression of these five miRNAs can discriminate between patients with papillary thyroid cancer and other patients or normal population. Thus, these five miRNAs could be combined for the differential diagnosis of papillary thyroid cancer. The levels of the five selected miRNAs (miR-144-3p, miR-146a-3p, miR-340-5p, miR-199a-5p, and miR-335) in blood serum of patients are associated with particular clinicopathological features, at a statically significant level.
In conclusion, according to the results of the present PhD thesis, a molecular signature consisting of miR-144-3p, miR-146a-3p, miR-340-5p, miR-199a-5p and miR-335 is likely to able to diagnose papillary thyroid cancer with high sensitivity and specificity, starting from a blood sample. People with deregulated levels of at least 4 of these 5 miRNAs are very likely to be diagnosed with papillary thyroid cancer, compared to the rest of the population. The differential diagnostic potential of this molecular signature is rathe independent of other factors. Last, the deregulation of the expression levels of these 5 miRNAs is associated with clinicopatholofical features of malignant papillary thyroid tumors.