MicroRNA-15a inhibits inflammatory response and apoptosis after spinal cord injury via targeting STAT3.

OBJECTIVE: To clarify the function of microRNA-15a in the spinal cord injury (SCI) and its potential mechanism. PATIENTS AND METHODS: The plasma levels of microRNA-15a and signal transducer and activator of transcription 3 (STAT3) in SCI patients were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between the expressions of microRNA-15a and STAT3 was analyzed. The in vitro SCI model was established in H2O2-induced C8-D1A and C8B4 cells, and in vivo SCI model was established in mice by hitting T10. The mRNA and protein expressions of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) were detected in the SCI model. The apoptosis was examined by flow cytometry or TUNEL staining, respectively. The motor function of mouse hindlimb was evaluated using the Basso Beattie Bresnahan (BBB) standard scale. The target gene of microRNA-15a was predicted by bioinformatics and further verified by dual-luciferase reporter gene assay. The expression changes of target genes in C8-D1A and C8B4 cells with microRNA-15a overexpression or knockdown were examined by qRT-PCR and Western blot. Finally, rescue experiments were performed to evaluate the regulatory effects of microRNA-15a and STAT3 on cell apoptosis. RESULTS: MicroRNA-15a was lowly expressed in plasma of SCI patients, while STAT3 was highly expressed with a negative correlation to microRNA-15a. Identically, microRNA-15a was lowly expressed in H2O2-induced C8-D1A and C8B4 cells, and STAT3 was highly expressed. MicroRNA-15a overexpression downregulated mRNA and protein levels of TNF-α and IL-6 in C8-D1A and C8B4 cells. BBB score was markedly low in SCI mice relative to controls. SCI mice injected with microRNA-15a mimics had higher BBB score than those injected with negative control. Besides, SCI mice with microRNA-15a overexpression had downregulated expressions of STAT3, TNF-α, and IL-6 in the impaired spinal cord tissues, as well as lower apoptotic rate. Through bioinformatics, we found binding sites between STAT3 and microRNA-15a. Their binding conditions were further verified by dual-luciferase reporter gene assay. Moreover, STAT3 expression was negatively regulated by microRNA-15a. Finally, rescue experiments showed that STAT3 overexpression could reverse the regulatory effects of microRNA-15a on expressions of TNF-α and IL-6, as well as apoptosis. CONCLUSIONS: MicroRNA-15a expression decreases in the SCI model, which participates in the process of SCI by regulating inflammatory response and cell apoptosis via targeting STAT3.

Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells.

The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.