In Situ Hybridization Analysis of MiR-146b-5p and MiR-21 Expression in Thyroid Carcinoma and Prognostic Implications

Background: MiR-146b-5p and miR-21 deregulation has been associated with progression and metastasis of thyroid cancers. However the utility of in situ hybridization (ISH) to determine the cellular localization and prognostic signifi cance of miR-146b-5p and miR-21 expression has not been previously analyzed. We examined the expression of miR-146b-5p and miR-21 by ISH and by qRT-PCR in benign thyroid tissues and thyroid carcinomas. Design: Anaplastic thyroid carcinomas (ATC, n=35), poorly differentiated thyroid carcinomas (PDTC, n=21), papillary thyroid carcinomas (PTC, n=58), follicular carcinomas (FTC, n=28), follicular adenomas (FA, n=32), nodular goiters (NG, n=10) and normal thyroids (NT, n=10) were studied by in situ hybridization (ISH) using formalin-fi xed paraffi n-embedded tissue microarrays (TMAs) with probe labeled at both 3’ and 5’ end with digoxigenin. ISH was scored based on intensity of stained cells. The association with miR-146b-5p and miR-21 with clinical feature and prognostic factors were analyzed.MiR-146b-5p and miR-21 expression in thyroid cancers were also analyzed by qRT-PCR. Results: MiR-146b-5p was overexpressed in 37 out of 58 (64%) PTCs. PTC cases with higher expression of miR-146b-5p had signifi cantly poorer disease-free survival rates. MiR-21 was overexpressed in 29 out of 35 (83%) ATCs and 4 out of 21 (19%) PDTCs, while miR-21 was overexpressed in 32 out of 58 (55%) PTCs. PTC cases with positive expression of miR-21 had signifi cantly poorer disease-free survival rates and higher lymph node (LN) metastasis. PDTC cases with positive expression of miR-21 had signifi cantly poorer overall survival rates. Normal thyroid tissues and most of benign goiter were negative for miR-146b-5p and miR-21. QRT-PCR analysis supported the ISH data. Conclusions: MiR-146b-5p is mainly expressed in PTC and is not expressed in most FTC, PDTC and ATC. Thus it may serve as a useful prognostic marker for PTC. MiR-21 is highly expressed in ATC and PTC and serves as a marker of LN metastasis in PTC. ISH is a useful method to analyze microRNA expression in formalin fixed paraffin embedded tissue.