Figure 2: miR-125b prevents GnRH activation of Gαq/11-, but not Gαs-dependent pathways.

(a) Effect of a GnRHa treatment on the recruitment into RISC of Gαq/11-medated pathway factors. Levels of miR-125b and selected potential target mRNAs were determined by qRT-PCR on pan-AGO-immunoprecipitated samples and normalized to input level. GnRHa treatment (10 nM for 4 h) of LβT2 cells reduced the recruitment of miR-125b and of all tested target mRNAs (n = 5). (b,c) Effect of alteration of miR-125b on the protein level of Gαq/11-related pathway factors in LβT2 cells. b; Cells were electroporated with a miR-125b expressing vector or an empty vector and protein levels were quantified on harvested cells by Western blot analysis. Overexpression of miR-125b led to a significant reduction of most basal or GnRH-induced selected Gαq/11-mediated pathway factor protein levels. p38 was not significantly affected (n = 7). (c) Cells were electroporated with anti-miR-125b or scrambled LNA. Blocking miR-125b increased most tested miR-125b potential targets with the exception of MAP2K7 which was significantly decreased (n = 7). (d,e) Effect of altering miR-125b on CREB phosphorylation status. LβT2 cells were electroporated either with anti-miR125b or scramble LNA, or with miR-125b or empty expression vector and then treated with 10 nM GnRHa for 30 min. Extracted proteins were analysed by western blotting using anti-CREB and anti-pCREB, successively and a representative blot is given below the analysis. d; Blocking miR-125b had no effect on the phosphorylation status of CREB. (e) Overexpression of miR-125b had no effect on the GnRH-induced phosphorylation of CREB (n ≥ 4). Representative western blot images are given on Fig. S2. *P < 0.05; **P < 0.01; ***P < 0.001.