Inverse Correlation of MiRNA and Cell Cycle-associated Genes Suggests Influence of MiRNA on Benign Thyroid Nodule Tumorigenesis

Context: The molecular etiology of cold and benign thyroid nodules (CBTNs) is largely unknown. Increased thyroid epithelial cell proliferation is a hallmark of CBTNs. MicroRNAs (miRNAs) are prominent regulators of cell proliferation.

Objective: Our objective was to assess the influence of miRNAs on the increased proliferation and thus the molecular etiology of CBTNs.

Design: By using microarrays, we defined the molecular pattern of increased proliferation of CBTNs as a differential expression of cell-cycle-associated genes and miRNAs. In silico integration of differentially expressed miRNAs and mRNAs showed an inverse correlation between the expression of 59 miRNAs and 133 mRNAs. Inverse correlations between cell-cycle-associated genes such as CDKN1C and miR-221, CCND1 and miR-31, GADD45A and miR-130b, or CDKN1A and let-7f suggest a modulation of proliferation in CBTNs by miRNAs. Their expression was validated using quantitative RT-PCR and functionally characterized in cell line models.

Results: Comparative quantitative RT-PCR of 20 samples of CBTNs and their surrounding tissue revealed an 11-fold down-regulation of miR-31 with a 2.6-fold up-regulation of CCND1, and a 2.6-fold up-regulation of miR-130b with a 2.3-fold down-regulation of its target GADD45A. Using HTori and FTC-133 cell lines, we analyzed proliferation, cell cycle, and apoptosis after transfection of miRNA-31 and miRNA-130b mimic and inhibitors. Overexpression of miR-31 and the resultant down-regulation of CCND1 led to an arrest in the cell cycle phase G1. Overexpression of miR-130b led to an increase of apoptosis and necrosis within 72 h.

Conclusion: miR-31 and miR-130b may have an effect on tumorigenesis of CBTNs by regulating proliferation and apoptosis and the cell cycle through cyclin D1.