The role of microRNA genes in papillary thyroid carcinoma

Abstract

Apart from alterations in the RET/PTC-RAS-BRAF pathway, comparatively little is known about the genetics of papillary thyroid carcinoma (PTC). We show that numerous microRNAs (miRNAs) are transcriptionally up-regulated in PTC tumors compared with unaffected thyroid tissue. A set of five miRNAs, including the three most up-regulated ones (miR-221, -222, and -146), distinguished unequivocally between PTC and normal thyroid. Additionally, miR-221 was up-regulated in unaffected thyroid tissue in several PTC patients, presumably an early event in carcinogenesis. Tumors in which the up-regulation (11- to 19-fold) of miR-221, -222, and -146 was strongest showed dramatic loss of KIT transcript and Kit protein. In 5 of 10 such cases, this down expression was associated with germline single-nucleotide changes in the two recognition sequences in KIT for these miRNAs. We conclude that up-regulation of several miRs and regulation of KIT are involved in PTC pathogenesis, and that sequence changes in genes targeted by miRNAs can contribute to their regulation.

Fig. 1.

MiRNA expression microarray data confirmation. (A) Semiquantitative RT-PCR of miR-221, miR-222, miR-146b, and CITED1 in the paired PTC samples from nine patients. GAPDH was used as an internal control for RT-PCR; M, DNA marker; (-) negative control; N, normal thyroid tissue; T, tumor tissue. (B) Northern blot showing the overexpression of mature miR-221 and miR-146b in PTC tumors. The same filter was used for both hybridizations after stripping. 5S RNA in an ethidium bromide gel was used as loading control.

Fig. 2.

Overexpression of miR-221 in normal thyroid tissue (N-PTC) adjacent to PTC tumor (T-PTC). (A) Real-time RT-PCR quantification of miR-221 expression in N-PTCs and T-PTCs. The expression level of sample N-Thy4 (thyroid tissue from an individual without thyroid disease) was set as a reference (N-Thy4 expression = 1). (B) Semiquantitative RT-PCR of miR-221 expression in two N-PTC samples and three samples of normal thyroid tissue from individuals without clinical thyroid disease (N-Thy). RT (-) and RT (+), absence vs. presence of reverse transcriptase in the RT-PCR reactions.

Fig. 3.

Down-expression of KIT transcript and Kit protein in PTC tumor tissues. Semiquantitative RT-PCR of KIT expression in five paired PTC samples and two PTC cell lines. Western blot of Kit in protein extract from the same sample set. Bands of ≈140 kDa and ≈120 kDa represent the mature and immature forms of Kit protein, respectively. GAPDH was used as a loading control.

Fig. 4.

Computational modeling of the interaction of miR-221 and miR-146 with the KIT gene. (A) Hybridization of miR-221 (green color) and KIT mRNA (red color); arrows highlight a polymorphic site within the binding domain (SNP rs17084733). mfe, minimum free energy. (B) Hybridization of miR-146b (green color) and KIT mRNA (red color); arrows highlight a polymorphic site within the binding domain (SNP rs3733542).