Effects of RNA interference targeting P2X7 receptor on microglial phagocytosis of amyloid-β protein

Abstract

To investigate the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial phagocytosis of amyloid-β (Aβ) protein and to explore its possible mechanism. The small interfering RNA (siRNA) targeting the P2X7R gene was identified. The microglial cells activated by Aβ1-42 were infected with the Lipofectamine-siP2X7R and it was designated as siP2X7R group. Microglia infected with Lipofectamine-siNC was designated as siNC group and non-infected cells as con group. The levels of P2X7R mRNA were detected by real-time PCR and the P2X7R protein was determined by Western blotting. The levels of IL-1β and TNF-α were measured by ELISA. The microglial phagocytosis of Aβ1-42 was observed by ELISA and immunocytochemistry staining. Detected by the Real-time PCR, the expression level of P2X7R mRNA of siP2X7R group decreased significantly versus siNC and con groups (P<0.05). The lowered expression of P2X7R protein detected by Western blotting was consistent with Real-time PCR. After RNA interference silencing P2X7R, the levels of IL-1β and TNF-α detected by ELISA in siP2X7R group less than those in con, siNC groups, significantly (P<0.05). In con, siNC and siP2X7R groups respectively, the levels of Aβ1-42 in supernatant were (423±20) pg/ml, (417±16) pg/ml, (296±30) pg/ml and the levels of Aβ1-42 in the microglia were (190±37) pg/ml, (187±39) pg/ml, (322±26) pg/ml. Compared to siNC and con groups, in siP2X7R group the levels of Aβ1-42 in supernatant decreased (P<0.05) and the levels of Aβ1-42 in the microglia increased (P<0.05). Aβ1-42 immunofluorescence staining showed that the red fluorescent products were seen in the cytoplasm of most microglias in siP2X7R group, but in con or siNC groups in only few microglias these products were depicted. The silence expression of P2X7R by RNA interference effectively decreases the levels of IL-1β and TNF-α released by microglia and promotes microglia to phagocytose Aβ. P2X7R could be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.