Age-associated increase of thyroid hormone receptor β gene promoter methylation coexists with decreased gene expression

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.

KEYWORDS:

• Aging
• miRNA
• peripheral blood mononuclear cells (PBMC)
• promoter methylation
• thyroid hormone receptor β gene (THRB)

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.

Additional information

Funding

This work was supported by the Polish Ministry of Science and Higher Education [grant NN401037338].

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.