Figure 5: miR-125b/miR-132 opposite regulation in gonadotrope cells.
From: A regulatory loop between miR-132 and miR-125b involved in gonadotrope cells desensitization to GnRH
(a) LβT2 cells were either treated by GnRHa (10 nM for 4 h) or the cAMP analogue 8Br-cAMP or were pre-treated with the PKA inhibitor Rp-cAMP before the same GnRHa treatment. Inhibition of PKA prevented the GnRH induction of miR-132 (n = 8) (b–d) Effect of alteration of miR-125b on miR-132 level. (b) LβT2 cells were electroporated with anti-miR-125b or scrambled LNA. Blocking miR-125b increased miR-132 level (n = 8). (c) Cells were electroporated with NSun2 or empty expressing vector. Overexpression of NSun2 led to a significant increase of miR-132 expression (n = 9). (d) Cells were electroporated with miR-125b or empty expression vector before a GnRHa treatment (10 nM for 4 h). Overexpression of miR-125b led to a significant reduction of the GnRHa-induced miR-132 expression (n = 4). (e) Effect of alteration of miR-125b on ERK1/2 phosphorylation status. LβT2 cells were electroporated with miR-125b or empty expression vector and then treated with 10 nM GnRHa for 2 h. Extracted proteins were analysed by western blotting using anti-ERK1/2 and anti-pERK1/2, successively. A representative blot is given on Fig. S2h). The antiserum recognizes both ERK1 (upper band) and ERK2 (lower band). The two signals were summed for quantification. Overexpression of miR-125b prevented the GnRH-induced phosphorylation of ERK1/2 (n = 3). (f) LβT2 cells were either treated by GnRHa (10 nM for 8 h) or were pre-treated with the MAPK kinase (MEK)1/2 (U0126) inhibitors before the same GnRHa treatment. Inhibition of ERK1/2 phosphorylation prevented the GnRH-induced expression of AK006051 mRNA encoding miR-132 and miR-212 (n = 3). (g,h) Effect of alteration of miR-132 on the miR-125b level in LβT2 cells. (g) Cells were electroporated with anti-miR-132 or scrambled LNA. Blocking miR-132 led to a significant decrease in miR-125b level (n = 6). (h) Cells were electroporated with a miR-132 expressing vector or an empty vector. Overexpression of miR-132 led to a significant increase of miR-125b expression (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001.
