脑损伤
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Figure 4｜MiR-502-3p modulates GABRα1 protein level at cellular junctions.
Figure 5｜Modulation of GABRα1 protein by miR-502-3p at cellular junctions.
Thus far it is evident that suppression of miR-502-3p increases GABRα1 levels and enhances cell viability. To determine the localization of miR-502-3p and GABRα1 in the cells, miRNA in situ hybridization and immunostaining analysis were performed. Hsa-miR-502-3p specific probe was used to detect the miR-502-3p overexpression in the cells. The miRNAScope analysis showed increased levels of miR-502-3p in agomiRs transfected cells relative to scramble control and antagomiRs treated cells, however, U6snRNA was detected in all groups of cells (Figure 4A). Further, immunofluorescence analysis showed an increased GABRα1 expression at the cell junctions in the miR-502-3p antagomiR transfected cells (Figure 4B), while GABRα1 protein was highly reduced at the cell junctions in miR-502-3p overexpressed cells. Fluorescence intensity analysis showed significant (P < 0.0001) upregulation of GABRα1 at the cell junction in the antagomiR-treated cells relative to control and agomiR-treated cells, while GABRα1 level was significantly reduced in miR-502-3p overexpressed cells relative to scramble control (Figure 4C). Further, we examined the cells with confocal microscopy to better understand the interaction of miR-502-3p with GABRα1 at the cellular junctions. Confocal images also showed increased levels of GABRα1 protein at the cell junctions in miR-502-3p antagomiR-treated cells, whereas the GABRα1 protein level was reduced in agomiR-treated cells (Figure 5A). Fluorescence intensity analysis showed significant levels of GABRα1 changes in response to miR-502-3p overexpression and under-expression (Figure 5B). These observations confirmed that overexpression of miR-502-3p negatively modulates the GABRα1 levels, particularly at cellular junctions, whereas suppression of miR-502-3p positively increases the GABRα1 levels in the cells and at the cellular junctions.