miR-34a Inhibitor May Effectively Protect against Sevoflurane-Induced Hippocampal Apoptosis through the Wnt/β-Catenin Pathway by Targeting Wnt1

Abstract

Purpose: Research has shown that sevoflurane-induced toxicity causes neurodegeneration in the developing brain. miR-34a has been found to negatively regulate ketamine-induced hippocampal apoptosis and memory impairment. However, the role of miR-34a in sevoflurane-induced hippocampal neurodegeneration remains largely unclear.

Materials and methods: C57/BL6 mice (7-day-old) inhaled 2.3% sevoflurane for 2 h/day over 3 consecutive days. miR-34a expression was reduced through intracerebroventricular injection with miR-34a interference lentivirus vector (LV-anti-miR-34a) into mouse hippocampus after anesthesia on the first day of exposure. Hippocampal apoptosis was detected by TUNEL assay and flow cytometry analysis. Spatial memory ability was evaluated by the Morris water maze test. The interaction between miR-34a and Wnt1 was confirmed by luciferase reporter assay, RNA immunoprecipitation, Western blot, and immunofluorescence staining. The effects of miR-34a on protein levels of B-cell lymphoma 2 (Bcl-2), bcl-2-like protein 4 (Bax), and Wnt/β-catenin pathway-related proteins were evaluated using Western blot analysis.

Results: Sevoflurane upregulated hippocampal miR-34a, and miR-34a inhibitor attenuated sevoflurane-induced hippocampal apoptosis and memory impairment. miR-34a negatively regulated Wnt1 expression by targeting miR-34a in hippocampal neurons. Moreover, forced expression of Wnt1 markedly undermined miR-34a-mediated enhancement of sevoflurane-induced apoptosis of hippocampal neurons, while Wnt1 silencing greatly restored anti-miR-34a-mediated repression of sevoflurane-induced apoptosis of hippocampal neurons. Increased expression of miR-34a inhibited the Wnt/β-catenin pathway in hippocampal neurons exposed to sevoflurane, while anti-miR-34a exerted the opposite effects.

Conclusion: miR-34a inhibitor may effectively protect against sevoflurane-induced hippocampal apoptosis via activation of the Wnt/β-catenin pathway by targeting Wnt1.

Keywords: Wnt/β-catenin pathway; Wnt1; hippocampal apoptosis; miR-34a; sevoflurane.

Fig. 1. miR-34a inhibitor greatly protected against sevoflurane-induced hippocampal apoptosis and memory impairment. After mice received sevoflurane exposure for 2 h/day on 3 consecutive days and subsequent hippocampal injection of lentiviruses, (A) the expression of miR-34a was checked by qRT-PCR in the isolated hippocampi; (B) the spatial memory ability of mice was evaluated by the MWM test; and (C) TUNEL assay was applied to evaluate the apoptosis of hippocampus. ^**p<0.01 or ^***p<0.001.

Fig. 2. Wnt1 was a target of miR-34a in hippocampal neurons. (A) Luciferase reporters carrying the fragments of Wnt1 3'UTR containing the WT or MUT miR-34a binding sites. (B) Luciferase activity was measured by luciferase reporter assay in hippocampal neurons after cotransfection with the constructed luciferase reporter plasmids and miR-34a or miR-NC. (C) RIP assay immunoprecipitated against Ago1. (D) The protein level of Wnt1 was detected by Western blot in hippocampal neurons transfected with miR-34a, anti-miR-34a, or matched controls. (E) The expression of Wnt1 was evaluated by immunofluorescence staining in hippocampal neurons transfected with miR-34a, anti-miR-34a, or matched controls. ^**p<0.01 or ^***p<0.001.

Fig. 3. Anti-miR-34a impedes sevoflurane-induced apoptosis in hippocampal neurons by upregulating Wnt1. (A) Flow cytometry analysis was conducted to analyze the apoptosis of hippocampal neurons exposed to sevoflurane. Hippocampal neurons were transfected with miR-34a, miR-NC, miR-34a+Wnt1, miR-34a+pcDNA, anti-miR-34a, anti-miR-NC, anti-miR-34a+si-Wnt1, or anti-miR-34a+si-NC, followed by treatment with sevoflurane. (B and C) Flow cytometry was then carried out to detect apoptosis of hippocampal neurons. (D–F) The expression of Bax and Bcl-2 protein was investigated in hippocampal neurons. ^**p<0.01 or ^***p<0.001.

Fig. 4. Anti-miR-34a activates the Wnt/β-catenin pathway by upregulating Wnt1 in hippocampal neurons exposed to sevoflurane. (A and B) Western blot analysis of protein levels of TCF-4, cyclin D1, β-catenin, and Wnt1 in hippocampal neurons after transfection with miR-34a, anti-miR-34a, or respective controls, followed by sevoflurane exposure. ^**p<0.01 or ^***p<0.001.