Because miR-146a and miR-155 are significantly elevated in TSH-treated GOFs compared to NOFs and since these miRNAs promote cellular proliferation in other cells, key miR-146a and miR-155 target gene expression was analyzed. GOFs were treated with TSH as described for 48 hours and total RNA was isolated. As shown above, miR-146a and miR-155 levels were induced by TSH (
Fig. 8A).
ZNRF3, a miR-146a target gene that inhibits cell proliferation in osteosarcoma cells,
44 was analyzed by qPCR in untreated and TSH treated GOFs (
Fig. 8B, left panel). Interestingly,
ZNRF3 was dose dependently decreased by TSH in GOFs. Next,
PTEN, a miR-155 target gene and inhibitor of PI3K/Akt induced proliferation, was also analyzed by qPCR (
Fig. 8B, right panel). Like ZNRF3,
PTEN was reduced by TSH in a dose-dependent manner in GOFs. To test if ZNRF3 and
PTEN are targeted by miR-146a and miR-155 in GOFs, control or miRNA mimics were introduced into GOFs and after 48 hours, RNA isolated and analyzed by qPCR (
Fig. 8C). ZNRF3 mRNA levels were significantly reduced by miR-146a. Likewise,
PTEN mRNA levels were attenuated by exogenous expression of miR-155.