A Possible Function for mRNA of PPARγ in Human Embryonic Stem Cell-Derived Neural Differentiation upon Retinoic Acid Treatment.

Objective: PPARμ, a ligand dependent nuclear receptor, play its role as a transcription factor in many of cells especially adipose tissue. In our recent previous studies, although we have shown a significant increase in mRNA level of PPARμ in neural differentiation from human embryonic stem cells (hESCs), a similar pattern in relevant protein level was not detected. To seek additional evidence for how the expression of PPARμ is regulated, we evaluated here two main regulatory systems, proteasome and miRNAs. Materials and Methods: Treated hESCs with noggin and bFGF were induced to differentiate into neuroectodermal cells with retinoic acid for 6 days. Subsequently, cells were collected in TRIzol reagent to extract total RNA for miRNA specific cDNA synthesis, and evaluate miRNA expression. Furthermore, we used MG132 as a proteasome inhibitor to address the question whether this protein complex can involve in regulation of PPARμ expression. To this end, we treated neuroectodermal cells with MG132 in day 6 RA treatment where we had observed the most expression of PPARμ. Results: In the case of miRNA, we measured the expression of three miRNAs, miR20, 27, 133, which had been previously reported as miRNAs specifically targeting PPARμ mRNA. We did not observed significant enhancement related to none of the mentioned miRNAs similar to what we detected regarding PPARμ mRNA. Moreover, we investigated involvement of proteasome in this pathway. Although we inhibited proteasome by MG132, we detected no increase in PPARμ protein compare to untreated group indicating that proteasome could not involve in protein degradation. Conclusion: According to our result, it can be concluded that miRNAs and proteasome don't involve in regulation of PPARμ expression. Possibly, mRNA of PPARμ plays a prominent role in retinoic acid-induced neural differentiation similar to Tpt1 mRNA in mouse embryonic stem cells where there is negative correlation between mRNA and protein level of Tpt1 [ABSTRACT FROM AUTHOR]