RESUMEN
As multipotent progenitor cells, mesenchymal stem cells (MSCs) can renew themselves and give rise to multiple lineages including osteoblastic, chondrogenic and adipogenic lineages. It's previously shown that BMP9 is the most potent BMP and induces osteogenic and adipogenic differentiation of MSCs. However, the molecular mechanism through which BMP9 regulates MSC differentiation remains poorly understood. Emerging evidence indicates that noncoding RNAs, especially microRNAs, may play important roles in regulating MSC differentiation and bone formation. As highly conserved RNA binding proteins, Argonaute (AGO) proteins are essential components of the multi-protein RNA-induced silencing complexes (RISCs), which are critical for small RNA biogenesis. Here, we investigate possible roles of AGO proteins in BMP9-induced lineage-specific differentiation of MSCs. We first found that BMP9 up-regulated the expression of Ago1, Ago2 and Ago3 in MSCs. By engineering multiplex siRNA vectors that express multiple siRNAs targeting individual Ago genes or all four Ago genes, we found that silencing individual Ago expression led to a decrease in BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs. Furthermore, we demonstrated that simultaneously silencing all four Ago genes significantly diminished BMP9-induced osteogenic and adipogenic differentiation of MSCs and matrix mineralization, and ectopic bone formation. Collectively, our findings strongly indicate that AGO proteins and associated small RNA biogenesis pathway play an essential role in mediating BMP9-induced osteogenic differentiation of MSCs.
RESUMEN
The human insulin-like growth factor-II (IGF-II) gene transcribes four mRNAs (P1 mRNA-P4 mRNA), and P3 mRNA overexpression contributes to hepatocarcinogenesis. IGF-II-derived miR-483-5p is implicated in the development of cancers. Here, we investigated the involvement of miR-483-5p in P3 mRNA overexpression regulation and its role in hepatocellular carcinoma. Our results showed that miR-483-5p up-regulated P3 mRNA transcription by targeting the 5'-untranslated region (5'UTR) of P3 mRNA in hepatocellular carcinoma. The mechanism was involved in recruiting of an argonaute 1(Ago1)-argonaute 2 (Ago2) complex to the P3 mRNA 5'UTR and the P3 promoter of IGF-II gene by miR-483-5p, accompanied by increased enrichment of RNA polymerase II and activating histone marks histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 27 acetylation (H3K27ac), and histone 4 lysine 5/8/12/16 acetylation (H4Kac) at the P3 promoter. High miR-483-5p expression was an independent predictor for shorter survival of HCC patients. The findings suggest that miR-483-5p promotes P3 mRNA transcription by recruiting the Ago1-Ago2 complex to the P3 mRNA 5'UTR and is associated with poor prognosis of HCC. Our results display a potential new model for miRNAs to up-regulate gene expression.
RESUMEN
MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.