Upregulations of LINC00472 and proinflammatory cytokines in AD subjects.
RT-PCR was utilized to analyze the blood expression of LINC00472 in AD subjects and age-matched healthy individuals. As reported (5), LINC00472 was upregulated in AD subjects (Fig. 1A). Compared to healthy individuals, LINC00472 expression is 1.5-2 times higher in AD subjects (Fig. 1A). In addition, ELISA assays revealed significant upregulations of proinflammatory cytokines in serum such as TNF-α, IL-8, and IL-1β (12) (Fig. 1B; AD vs control, TNF-α is 150 vs 64 pg/ml, IL-8 is 120 vs 62 pg/ml, and IL-1β is 12 vs 2 pg/ml). Moreover, AD subjects have significantly reduced serum expression of superoxide dismutase (SOD) and catalase (CAT) and increase of malondialdehyde (MDA) (9) (Fig. 1C; AD vs control, SOD is 0.17 vs 0.22U/mol, MDA is 18 vs 9 nmol/L, and CAT is 1.64 vs 2.52 U/ml).
LINC00472 downregulation ameliorates Aβ induced oxidative stress.
In SH-SY5Y cells, we aimed to evaluate the therapeutic potential of LINC00472 downregulation in treating AD. The widely used Aβ was added into culture medium and the expression of LINC00472, proinflammatory cytokines, and ROS were compared with or without LINC00472 downregulation. Consistent with AD subjects, Aβ administration elevates LINC00472 in SH-SY5Y cells (Fig. 2A). Moreover, the Aβ treated cells revealed upregulations of TNF-α, IL-8, and IL-1β (Fig. 2B), reduced SOD and CAT (Fig. 2C), and increased MDA (Fig. 2C) and ROS (Fig. 2D). sh-LINC00472 transfection dramatically downregulated cellular LINC00472 (Fig. 2A), and accordingly, reversed the anomalies of TNF-α, IL-8, IL-1β (Fig. 2B), SOD, CAT, MDA (Fig. 2C), and ROS (Fig. 2D).
miR-141-3p is the target gene of LINC00472 and targets the FOXO3 gene.
Searching databases such as Starbase, we noticed the predicted bindings between LINC00472 and miR-141-3p (Fig. 3A), and miR-141-3p and FOXO3 (Fig. 4A). Luciferase assay revealed suppression of enzymatic activity by miR-141-3p overexpression, which was released by an intact but not mutated form of LINC00472 (Fig. 3B). Importantly, the miR-141-3p significantly pull down LINC00472 in RNA-pull down assay, and vice versa (Fig. 3C). In Aβ treated cells, miR-141-3p expression was significantly reduced, and could be attenuated by co-transfect sh-LINC00472 (Fig. 3D). Similarly, blood expression of miR-141-3p is reduced in AD subjects (Fig. 3E) and negatively correlated with LINC00472 expression (Fig. 3F). In addition, the suppression of enzymatic activity by miR-141-3p overexpression in luciferase assay was also released by an intact but not mutated form of FOXO3 (Fig. 4A, B). Cellular expression of FOXO3 was elevated after Aβ administration and was reversed by co-transfection with miR-141-3p mimic (Fig. 4C). Moreover, AD subjects have higher blood expression of FOXO3 by RT-PCR (Fig. 4D). Interestingly, blood expression of FOXO3 has a negative correlation with miR-141-3p (Fig. 4E) and, at the same time, a positive correlation with LINC00472 (Fig. 4F).
LINC00472 regulates oxidative stress through miR-141-3p/FOXO3
So far, we’ve shown increased oxidative stress in AD subjects and Aβ treated cells, as well as accumulated LINC00472. Then we wondered if specifically manipulating LINC00472/miR-141-3p/FOXO3 benefit controlling oxidative stress in AD. Indeed, in the Aβ treated cells, FOXO3 is accumulated (Fig. 5A). Consistent with the correlation assays, knocking down LINC00472 normalized the accumulation of FOXO3, and the normalization was attenuated by FOXO3 overexpression or miR-141-3p inhibitor (Fig. 5A). Similarly, Aβ administration induced upregulations of TNF-α, IL-8, and IL-1β were reversed by knocking down LINC00472 (Fig. 5B). Again, the knocking down effects were dismissed by FOXO3 overexpression or miR-141-3p inhibitor (Fig. 5B). Moreover, Aβ administration induced similar reductions of SOD and CAT and increase of malondialdehyde MDA as AD subjects (13), which have been partially rescued by knocking down LINC00472 (Fig. 5C). FOXO3 overexpression or miR-141-3p inhibitor treatment dismissed the rescue effects (Fig. 5C). Furthermore, the knocking down LINC00472 induced normalization of ROS was blocked by FOXO3 overexpression or miR-141-3p inhibitor treatment (Fig. 5C).