In the present study, we demonstrated that BMP2 stimulation directly increased the transcription of miR-17-92 and miR-106b-25 cluster via Smad activation, which leads to the up-regulation of mature miR-17/20a/93
In the present study, we demonstrated that BMP2 stimulation directly increased the transcription of miR-17-92 and miR-106b-25 cluster via Smad activation, which leads to the up-regulation of mature miR-17/20a/93. neural system [1,2]. The signaling pathway includes BMPs, BMPRs (Bone Morphogenetic Protein Receptors) and Smads, in which particular Smads Dxd are dedicated to different ligands, with R-Smad (Smad1, 5 and 8) and Co-Smad (Smad4) mediating signals from special members of the BMP subfamily [3,4]. There are substantial evidences that BMP signaling plays a crucial role in the neural development including proliferation, differentiation and maturation [57]. For example, BMP promotes the astroglial lineage commitment during the neural differentiation [8,9]. In addition, activation of Smad1 pathway through BMP2 or 4 facilitates the axonal growth in adult sensory neurons [10,11]. However, it is also reported that aberrant activation of BMP signaling can cause neuronal dysfunction which may lead to further disorders [12,13]. Therefore, it is of great significance to avoid the abnormality of BMP signaling in nervous system. MiRNAs are approximately 21-nucleotide small RNAs that are derived from hairpin precursors, which repress protein expression by Rabbit Polyclonal to Cox2 targeting 3-UTR (3-untranslated region) of mRNAs [14]. The miR-17 family consists Dxd of six members (miR-17, miR-20a, miR-20b, miR-93, miR-106a and miR-106b), which distribute in three genome clusters [15]. Unlike the miR-17-92 and miR-106b-25 Dxd cluster, which are both abundantly expressed in many sorts of tissues, the miR-106a-363 cluster is usually undetectable or unexpressed in most of the tissues [16,17]. There are several reports that miR-17 family functions in nervous system [1820]. Recently, it is reported that interleukin-6 modulates the expression of BMPRII in endothelial cells through miR-17-92 pathway [21]. Furthermore, there is also evidence that BMPs can increase the miR-17-92 expression in cardiac progenitor cells [22]. Based on these studies, we hypothesize that in neuron there may be a negative feedback in BMP-miR-17 family-BMPRII circle, which helps to maintain BMP signaling in a proper range under normal physiological conditions. To verify this notion, we first examined the expression of miR-17 family in primary neurons after BMP stimulation and found that BMP2 increased miR-17-92 and miR-106b-25 cluster transcription through the activation of Smads, which directly bound to the promoter region of those clusters. Further investigation showed that BMP2 mediated up-regulation of those miRNAs lead to the repression of BMPRII in neuronal cells. Moreover, we also exhibited that the unfavorable feedback in BMP-miR-17 family-BMPRII circle can alleviate neuronal apoptosis induced by over-stimulation of BMP2. Taken together, our data suggest Dxd that miR-17 family functions in a feedback loop of BMP signaling pathway in primary neuron, which helps to maintain cellular homeostasis. == Materials and Methods == == Animals == Animal studies were conducted in strict accordance with the principles and procedures approved by the Committee around the Ethics of Animal Experiments of Nanjing University. C57BL/6 Mice were housed under a 12-h light/12-h dark cycle and fed autoclaved water and laboratory rodent chow. The mother mice were sacrificed by cervical dislocation and brains were dissected out from embryos at E15.5. == Cell Culture == Primary mouse cortical neuron cultures from embryonic day 15.5 (E15.5) C57BL/6 pregnant mice were obtained and maintained as previously described [23]. In brief, neocortices from fetal mice were dissociated and plated onto six-well plates pre-coated with Poly-D-Lysine (Sigma) at a density of 1105per square centimeter for the neuron cultures, and maintained in Neurobasal Medium supplemented with 2%(v/v) B27 Supplement, 1 mM-glutamine and 1% (v/v) penicillin/streptomycin (all reagents provided by Gibco-Invitrogen). The cells were cultured for 3 days before transfection. Human neuroblastoma SH-SY5Y cells were purchased from ATCC. The cells were maintained in DMEM supplemented with 10 %10 % (v/v) FBS and 1% (v/v) penicillin/streptomycin (all reagents provided by Gibco-Invitrogen) at 37 C in an atmosphere made up of 5 % CO2. BMP2 (Prospec) was used in the cell culture.