Objective: Owing to their remarkable stability in the serum, microRNAs are being investigated as circulating cancer biomarkers. Several studies have explored their plasma or serum levels in thyroid cancer in order to differentiate benign from malignant nodules preoperatively. However, very few studies have explored the role of microRNAs to detect recurrent disease during postoperative follow-up and none of them used high through-put technologies. Recombinant human TSH (rhTSH) is used to optimize serum thyroglobulin (Tg) stimulation during follow-up. Our aim was to determine through NGS the circulating miRNA profiles of patients with papillary thyroid carcinoma recurrence before and after rhTSH stimulation.
Material and methods: We collected the serum of thyroid cancer patients after total thyroidectomy and raidioiodide ablation. Illumina small RNA sequencing was performed on 7 patients with recurrent/persistent disease and 4 patients in complete remission, both at basal and rhTSH stimulated time points. Mean Tg basal and rhTSH-stimulated levels were 13 ng/ml and 55 ng/ml respectively for patients with recurrent disease. We used miRPara software tool for novel micro RNA discovery. Sequencing results were validated on 16 patients with recurrence and 14 patients in remission using LNA technology-based qPCR assays, currently the most specific qPCR method for miRNA measurement. Thyroid tumour and adjacent normal tissue were used for evaluating the levels of candidate microRNAs. TPC-1 cell line expressing functional TSH receptor was used to demonstrate microRNA regulation by TSH.
Results: MicroRNA sequencing detected 210 circulating miRs. Our analysis did not show any significant differences in the expression of circulating miRs between recurrent and non-recurrent patients or before and after rhTSH. Validation with qPCR confirmed these results. Interestingly, several sequences designated as putative microRNAs showed significantly different levels in patients with recurrence only after rhTSH stimulation. qPCR analysis confirmed particularly higher levels of one putative microRNA after rhTSH. In addition, this putative miR is upregulated in primary thyroid tumors compared to adjacent normal tissue (P=0.003, paired t-test) and is also expressed in TPC-1 cells and upregulated by TSH (P=0.0208, paired t-test).
Conclusions: Our study shows that currently annotated microRNAs cannot be used as serum markers of recurrence. However, our results point to several putative microRNAs that may be upregulated by rhTSH in serums of patients with recurrent disease. One such microRNA seems promising but further functional studies are being performed to confirm that this TSH-induced small RNA is indeed a novel microRNA with a role in thyroid carcinogenesis.
18 - 21 May 2019
European Society of Endocrinology