Overexpression of let-7a increases neurotoxicity in a PC12 cell model of Alzheimer's disease via regulating autophagy

Abstract

Increased deposition of β-amyloid (Aβ) protein is one of the typical characteristics of Alzheimer's disease (AD). Recent evidence has demonstrated that the microRNA let-7 family, which is highly expressed in the central nervous system, participates in the regulation of pathologic processes of AD. In the present study, the effect of let-7a overexpression on Aβ1-40-induced neurotoxicity was evaluated in PC12 and SK-N-SH cells. The results indicated that overexpression of let-7a enhanced the neurotoxicity induced by Aβ1-40 in PC12 and SK-N-SH cells. In addition, the apoptosis induced by Aβ1-40 in PC12 and SK-N-SH cells was increased by let-7a overexpression. Furthermore, Aβ1-40 treatment increased the protein levels of microtubule-associated protein 1A/1B-light chain 3 (LC3) and beclin-1 and increased the LC3 II/I ratio. The mRNA expression levels of beclin-1, autophagy protein 5 (Atg-5) and Atg-7 were also increased by Aβ1-40 treatment in PC12 cells. Let-7a overexpression further upregulated the above autophagy-related markers. Furthermore, the protein level of p62 was increased by Aβ1-40 treatment, and this was further enhanced by let-7a overexpression. Finally, the present results demonstrated that the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was involved in the autophagy regulation by let-7a. In conclusion, the present study demonstrates that the neurotoxicity induced by Aβ1-40 is augmented by let-7a overexpression via regulation of autophagy, and the PI3K/Akt/mTOR signaling pathway also serves a function in this process.

Keywords: Alzheimer's disease; autophagy; let-7a; phosphoinositide-3-kinase/Akt/mammalian target of rapamycin; β-amyloid 1-40.

Figure 1.

Effect of let-7a mimic transfection on let-7a expression in Aβ1-40-treated (A) PC12 and (B) SK-N-SH cells. The levels of let-7a were assessed by reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. Aβ1–40 group; ^&&&P<0.001 vs. Aβ1–40 + let-7a mimic group. Aβ, β-amyloid; miR-NC, negative control microRNA.

Figure 2.

Effect of let-7a overexpression on cell viability in Aβ1-40-treated (A) PC12 and (B) SK-N-SH cells. Cell viability was determined by MTT assay. Data are presented as the mean ± standard deviation (n=5). ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01 vs. Aβ1–40 group; ^&P<0.05 vs. Aβ1–40 + let-7a mimic group. Aβ, β-amyloid; miR-NC, negative control microRNA; OD, optical density.

Figure 3.

Overexpression of let-7a promoted the apoptosis of Aβ1-40-treated PC12 and SK-N-SH cells. Annexin-V/PI staining by flow cytometry was used to determine the apoptosis rate of (A) PC12 and (B) SK-N-SH cells. The percentages of apoptotic cells in the UR and LR quadrants are presented in the column charts. (C) Morphological changes of the nuclei in PC12 and SK-N-SH cells were assessed by Hoechst 33342 staining and representative images are shown (magnification, ×400). Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control group; ^###P<0.001 vs. Aβ1–40 group; ^&&P<0.01, ^&&&P<0.001 vs. Aβ1–40 + let-7a mimic group. UR, upper right; LR, lower right; Aβ, β-amyloid; miR-NC, negative control microRNA.

Figure 4.

Effect of let-7a overexpression on LC3, beclin-1 and p62 protein expression in Aβ1-40-treated PC12 and SK-H-SN cells. (A) Expression of LC3 in PC12 cells was evaluated by immunofluorescence staining. (B) Protein levels of LC3 I, LC3 II, beclin-1 and p62 in PC12 cells were assessed by western blot analysis. β-actin was used as a loading control. (C-E) Densitometric analysis of each protein band. (F) Protein levels of LC3 I and LC3 II in SK-H-SN cells were assessed by western blot analysis. β-actin was used as a loading control. (G) Densitometric analysis of each protein band. Data are presented as the mean ± standard deviation (n=3). *P<0.05, ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. Aβ1–40 group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. Aβ1–40 + let-7a mimic group. Aβ, β-amyloid; miR-NC, negative control microRNA; LC3, microtubule-associated protein 1A/1B-light chain 3.

Figure 5.

Effect of let-7a overexpression on beclin-1, Atg-5 and Atg-7 mRNA expression in Aβ1-40-treated PC12 cells. (A-C) mRNA expression of beclin-1, Atg-5 and Atg-7 in PC12 cells was assessed by reverse transcription-quantitative polymerase chain reaction. (D) Representative pictures of the morphological changes of nuclei in PC12 cells stained by Hoechst 33342 (magnification, ×400). (E) Annexin-V/PI staining by flow cytometry was used to determine the apoptosis rate of PC12 cells. The percentages of apoptotic cells in the UR & LR quadrants are presented in the column charts. Data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. control group; ^###P<0.001 vs. Aβ1–40 group; ^&&&P<0.001 vs. Aβ1–40 + let-7a mimic group; ^$P<0.05, ^$$$P<0.001 as indicated. UR, upper right; LR, lower right; Aβ, β-amyloid; miR-NC, negative control microRNA; Atg, autophagy protein; 3-MA, 3-methyladenine.

Figure 6.

Effect of let-7a overexpression on the PI3K/Akt/mTOR signaling pathway. (A) The protein levels of PI3K, p-Akt, Akt, p-mTOR and mTOR were detected by western blot analysis. β-actin was used as a loading control. (B-D) Densitometric analysis of each protein band. Data are presented as the mean ± standard deviation (n=3). **P<0.01, ***P<0.001 vs. control group; ^#P<0.05 vs. Aβ1–40 group; ^&P<0.05, ^&&P<0.01 vs. Aβ1–40 + let-7a mimic group. Aβ, β-amyloid; miR-NC, negative control microRNA; PI3K, phosphoinositide-3-kinase; p-, phosphorylated; mTOR, mammalian target of rapamycin.

Figure 7.

Effect of PI3K/Akt pathway activation on apoptosis and autophagy. (A) The protein levels of PI3K, p-Akt and Akt in PC12 cells were detected by western blot analysis. β-actin was used as a loading control. (B and C) Densitometric analysis of each protein band. (D) The apoptosis rate in PC12 cells was determined by flow cytometry. (E) The percentages of apoptosis cells in the UR & LR quadrants are presented in the column charts. (F) Representative images of the morphological changes of nuclei in PC12 cells stained by Hoechst 33342 (magnification, ×400). (G) Protein levels of LC3 I and LC3 II were detected by western blot analysis. β-actin was used as a loading control. (H) Densitometric analysis of each protein band. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 as indicated. UR, upper right; LR, lower right; Aβ, β-amyloid; miR-NC, negative control microRNA; LC3, microtubule-associated protein 1A/1B-light chain 3; PI3K, phosphoinositide-3-kinase; IGF-1, insulin-like growth factor 1.