Introduction: FGF2 and thrombospondin-1 (THBS1) expression in corpus luteum (CL) exhibited the most divergent profile of induction by prostaglandin F2a (PGF2α). FGF2, a potent angiogenic pro-survival factor was increased in the Day 4 CL. In contrast, the anti-angiogenic, apoptotic factors, THBS1, transforming growth factor beta 1 (TGFB1) and plasminogen activator inhibitor-1 (PAI-1) were upregulated specifically on Day 11, PGF2α-responsive CL. Functionally, THBS1 reversed FGF2 actions in luteal cells by inhibiting their proliferation, migration, and survival. Furthermore, the expression of THBS1 was suppressed by FGF2 on the contrary, TGFB1 elevated its gene expression. The mechanisms regulating THBS1 expression are not yet understood. microRNAs (miRNAs) represent a possible regulatory mechanism. We therefore aimed to identify relevant miRNA targeting THBS1 expression in luteal cells.
Methods: The TargetScan prediction tool was used to identify candidate miRNAs. Five miRNAs conserved in vertebrates were chosen for further investigation (miR-1, miR-18a, miR-144, miR-194 and miR-221). Luteal endothelial cells (LEC) were transfected with miRNA mimics, then mRNA and miRNA levels were determined by quantitative-PCR. Cell viability were estimated with XTT kit.
Results and discussion: Overexpression of miR-1, miR-194 and miR-221 significantly decreased THBS1 to levels 6070% lower than in the negative control. All these three miRNAs were endogenously expressed in CL, granulosa cells and LEC, with miR-221 being the most highly expressed. miR-221 was also the only one to be regulated by FGF2 and TGFB1, and in an opposite manner. FGF2 rapidly (after 2 h) upregulated miR-221, before inhibition of THBS1 was detected. Consistent with THBS1 inhibition, miR-221 significantly elevated viable LEC numbers by 160%. TGFB1, simultaneously increased THBS1 and reduced miR-221. Notably, PAI-1, a known TGFB1-induced protein was also reduced by miR-221. These finding suggest that miR-221 inhibits THBS1 in physiologically significant manner. The in vivo regulation of miR-221 in relationship to PGF2α remains to be determined.
11 Jul 2016 - 11 Jul 2016