Conclusions
Up-regulation of miR-1b promoted rat neuron proliferation and regeneration yet inhibited apoptosis via targeting KLF7.
Material and methods
Neurons were successfully separated and identified using a microscope and immunofluorescence staining of microtubule-associated protein 2 (MAP-2). The expressions of miR-1b and Krüppel-like factor 7 (KLF7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Neuron viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Neuron regeneration states were observed using a microscope and analysed by the ImageJ software. Expressions of C-caspase-3 and cell regeneration-related proteins (nerve growth factor [NGF], ciliary neurotrophic factor [CNTF] and brain-derived neurotrophic factor [BDNF]) were measured by Western blot. Target genes and potential binding sites of KLF7 and miR-1b were predicted by TargetScan 7.2 and confirmed by dual luciferase reporter assay.
Methods
Neurons were successfully separated and identified using a microscope and immunofluorescence staining of microtubule-associated protein 2 (MAP-2). The expressions of miR-1b and Krüppel-like factor 7 (KLF7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Neuron viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Neuron regeneration states were observed using a microscope and analysed by the ImageJ software. Expressions of C-caspase-3 and cell regeneration-related proteins (nerve growth factor [NGF], ciliary neurotrophic factor [CNTF] and brain-derived neurotrophic factor [BDNF]) were measured by Western blot. Target genes and potential binding sites of KLF7 and miR-1b were predicted by TargetScan 7.2 and confirmed by dual luciferase reporter assay.
Results
Neurons were identified as MAP-2-positive. Up-regulation of miR-1b reduced neuron viability and regenerative ability, promoted neuron apoptosis and C-caspase-3 expression, and down-regulated the expressions of cell regeneration-related proteins. KLF7 was the target gene of miR-1b. Overexpressed KLF7 rescued the effects of up-regulation of miR-1b on neuron viability, regeneration and apoptosis. Expressions of NGF, CNTF and BDNF were suppressed yet C-caspase-3 expression was up-regulated by miR-1b mimic, which was partially rescued by overexpressed KLF7. Conclusions: Up-regulation of miR-1b promoted rat neuron proliferation and regeneration yet inhibited apoptosis via targeting KLF7.