RESUMEN
Effect of micro ribonucleic acid (miR)-130a on neuronal apoptosis in rats with cerebral infarction (CI) was studied to explore whether phosphatase and tensin homolog deleted on chromosome ten (PTEN)/phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (Akt) is involved in the regulation of neuronal apoptosis. Thirty-six Sprague-Dawley (SD) rats were randomly divided into blank control group, model group and miR-130a low-expression group. miR-130a was determined by quantitative polymerase chain reaction (qPCR), the content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 was detected using the enzyme-linked immunosorbent assay (ELISA) kits, and the neuronal apoptosis level in each group was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The neurobehavioral score was significantly lower in model group than that in blank control group (P<0.01), while it was significantly higher in miR-130a low-expression group than that in model group (P<0.01). Compared with blank control group, the model group had obviously increased content of TNF-α and IL-6 (P<0.01), decreased content of IL-10 (P<0.01), more apoptotic neurons (P<0.01), higher expression of caspase-3 (P<0.01), and obviously lower Bcl-2/Bax (P<0.01). Moreover, expression of phosphorylated (p)-PTEN, PI3K and p-Akt in brain tissues was remarkably lower in the model group than those in the blank control group (P<0.01). The expression level of miR-130a in brain tissues of CI rats is significantly increased. miR-130a promotes the release of inflammatory factors and facilitates neuronal apoptosis through suppressing the PTEN/PI3K/Akt signaling pathway.
RESUMEN
Differentially expressed miRNAs in the GEO profile of ischemic stroke were analyzed to clarify the specific role of microRNA-324-5p (miRNA-324-5p) in ischemic stroke and the potential mechanism. After screening out miRNA-324-5p, its level in peripheral blood of stroke patients and in vitro oxygen-glucose deprivation (OGD)-induced primary rat neurons was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Regulatory effects of miRNA-324-5p on viability, and apoptosis of OGD-induced neurons were evaluated by CCK-8 and Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, respectively. Glucose uptake and caspase-3 activity in OGD-induced neurons transfected with miRNA-324-5p mimics or inhibitor were also examined. The binding of miRNA-324-5p to its target gene RAN was analyzed by dual-luciferase reporter gene assay and western blot analysis. By analyzing the data of GSE46266 profile, miRNA-324-5p expression was shown markedly lower in MCAO rats relative to controls. Identically, we also observed the downregulated miRNA-324-5p in peripheral blood of stroke patients and in vitro OGD-induced primary neurons. Overexpression of miRNA-324-5p accelerated viability, induced apoptosis and strengthened glucose uptake ability of OGD-induced neurons. Knockdown of miRNA-324-5p, conversely, obtained the opposite results. Furthermore, we confirmed the binding of miRNA-324-5p to RAN, the target gene that was negatively regulated by miRNA-324-5p. Importantly, RAN overexpression partially reversed the regulatory effect of miRNA-324-5p on viability and glucose uptake of OGD-induced neurons. miRNA-324-5p is downregulated after ischemic stroke, which aggravates the disease condition by inhibiting neuronal proliferation and glucose uptake via upregulating RAN.
RESUMEN
Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strategy for many incurable diseases including cancer. Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines. We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA: dual siRNA). VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT-PCR and western blot analysis. VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining. MG-63 and Saos-2 cell migration, proliferation, apoptosis, and angiogenesis were detected by scratch test, MTT assay, flow cytometry, and capillary tube assay respectively. The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels, compared with VEGF siRNA or Survivin siRNA alone. The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration, proliferation and angiogenesis, and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone, suggesting that the effects of the dual siRNA on inhibiting cell proliferation, migration, and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs. Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.