Reciprocal regulation of autism-related genes MeCP2 and PTEN via microRNAs

Abstract

MeCP2 encodes a methyl-CpG-binding protein that plays a critical role in repressing gene expression, mutations of which lead to Rett syndrome and autism. PTEN is a critical tumor suppressor gene that is frequently mutated in human cancers and autism spectrum disorders. Various studies have shown that both MeCP2 and PTEN proteins play important roles in brain development. Here we find that MeCP2 and PTEN reciprocally regulate expression of each other via microRNAs. Knockdown of MeCP2 leads to upregulation of microRNA-137, which in turn represses expression of PTEN, thus PTEN would be down-regulated when MeCP2 is knockdown. Furthermore, we find that deletion of PTEN leads to phosphorylation of Serine 133 of CREB, then increases the expression of microRNA-132. miR-132 inhibits the expression of MeCP2 by targeting on the 3'UTR of MeCP2 mRNA. Our work shows that two critical disorders-related gene MeCP2 and PTEN reciprocally regulate expression of each other by distinct mechanisms, suggesting that rare mutations in various disorders may lead to dysregulation of other critical genes and yield unexpected consequences.

Figure 1. MeCP2 deficiency down-regulates PTEN.

(A) Primary cultured neurons were transduced with lentivirus expressing GFP (control) or MeCP2 RNAi (for Mecp2 knockdown). PTEN protein expression was down regulated in MeCP2-RNAi neurons. Glyceraldehyde phosphate dehydrogenase was used as a loading control. (B) MeCP2 and PTEN protein levels in (A) were quantified using Image J. (C) Mecp2 and Pten RNA levels were analyzed by qPCR. (D) AKT T308 phosphorylation status was increased by PTEN RNAi or MeCP2 RNAi compared with controls.

Figure 2. MeCP2 regulates PTEN expression via miR-137.

(A) Deep-sequencing data showed a > 2.5-fold increase in miR-132 expression in MeCP2 KO mice. (B) miR-137 expression was upregulated > 2-fold in MeCP2 RNAi neurons. (C) PTEN protein expression was down-regulated in neurons transduced with miR-137 mimic. (D) PTEN protein levels in (C) were quantified using Image J. (E) Signaling pathway for MeCP2 regulating PTEN expression in neurons.

Figure 3. MeCP2 is regulated by PTEN.

(A) Protein levels of PTEN and MeCP2 were downregulated in cultured neurons infected with lentivirus harboring PTEN RNAi (for PTEN knockdown), as demonstrated by Western blotting. (B) MeCP2 and PTEN protein levels in (A) were quantified using Image J. (C) Relative quantities of Mecp2, Pten, Slc2a3, Klhl24, and Prkcb showed that PTEN downregulated MeCP2 target genes.

Figure 4. PTEN regulates MeCP2 via CREB-mediated miR-132 expression.

(A) Western blotting analysis of CREB phosphorylation in PTEN RNAi neurons with 50 mM KCl stimulation for 30 min. (B) miR-132 levels were increased in Pten-knockdown neurons stimulated with 10 mM KCl for 2 h, as shown by quantitative real-time PCR analysis. (C) Mecp2 expression was significantly decreased in PTEN RNAi neurons with 10 mM KCl stimulation. (D) Signaling pathway for reciprocal regulation between MeCP2 and PTEN.