ABSTRACT
Purpose: It is not established if healthy aging of the thyroid axis is associated with alterations other than changes in hormone secretion.
Methods: The expression of thyroid hormone receptor β gene (THRB) was analyzed in peripheral blood mononuclear cells (PBMC) obtained from young, elderly, and long-lived individuals. The interaction between the 3ʹUTR of TRβ1 mRNA and selected miRNAs was measured using pmirGLO reporter vector. Methylation of the THRB CpG island was analyzed using methylation-sensitive restriction/RT-PCR and bisulfite sequencing methods.
Results: Old age was associated with a significantly lower amount of total TRβ mRNA (p = 0.033) and of TRβ1 mRNA (p = 0.02). Older age was also associated with significantly higher methylation of the THRB promoter (restriction/RT-PCR: p = 0.0023, bisulfite sequencing: p = 0.0004). Higher methylation corresponded to a lower expression of the THRB mRNA, but this correlation did not reach the level of significance. miR-26a interacted with two sites in the 3’UTR of the TRβ1 mRNA leading to the decrease of the reporter protein activity (p < 0.0001 and p = 0.0005), and miR-496 interacted with one of the two putative binding sites which also decreased the reporter protein activity (p < 0.0001). Analysis of the expression of miR-21, miR-26a, miR-146a, miR-181a, miR-221, and miR-496 showed that the expression of miR-26a was significantly decreased in old subjects (p = 0.017), while the levels of other miRNAs were unaffected.
Conclusions: Age-related decrease of THRB expression in PBMC of elderly and long-lived humans might be, in part, a result of the increased methylation of its promoter, but is unrelated to the activity of the miRNAs analyzed here.
Acknowledgments
We thank Dr Jacek Polosak, Mossakowski Medical Research Centre, PAS, for help in preparation of RNA and Paula Dobosz for help in establishing RT-PCR conditions.
Declaration of interest
The authors report no conflict of interest.
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Notes on contributors
Monika Puzianowska-Kuznicka
All authors participated in interpretation of the studies and analysis of the data and review of the manuscript; MPK and EPP conceived and designed the experiments; EPP, MB, ZW, ADS, MO, MG, and MPK conducted the experiments; MRG supplied critical reagents (PBMC and RNA); MPK and EPP wrote the manuscript.