Silencing of circFoxO3 Protects HT22 Cells from Glutamate-Induced Oxidative Injury via Regulating the Mitochondrial Apoptosis Pathway

Abstract

Recent studies demonstrated that FoxO3 circular RNA (circFoxO3) plays an important regulatory role in tumourigenesis and cardiomyopathy. However, the role of circFoxO3 in neurodegenerative diseases remains unknown. The aim of this study was to examine the possible role of circFoxO3 in neurodegenerative diseases and the underlying mechanisms. To model human neurodegenerative conditions, hippocampus-derived neurons were treated with glutamate. Using molecular and cellular biology approaches, we found that circFoxO3 expression was significantly higher in the glutamate treatment group than that in the control group. Furthermore, silencing of circFoxO3 protected HT22 cells from glutamate-induced oxidative injury through the inhibition of the mitochondrial apoptotic pathway. Collectively, our study demonstrates that endogenous circFoxO3 plays a key role in inducing apoptosis and neuronal cell death and may act as a novel therapeutic target for neurodegenerative diseases.

Keywords: Apoptosis; Circular RNA; FoxO3; Neurodegenerative; Neuroprotection; Reactive oxygen species.

Fig. 1

Involvement of circFoxO3 in glutamate-induced HT22 injury. a Cells were treated with glutamate for 24 h. The live and dead cells were observed using Calcein-AM and PI staining, respectively. Scale bar = 400 µm. b A Calcein-AM cell viability assay showed that glutamate treatment significantly decreased the cell survival rate in a dose-dependent manner. c qPCR showed that glutamate treatment increased the expression levels of circFoxO3 in a dose-dependent manner. d Schematic diagram illustrating the structure of circFoxO3. The backsplice junction sequences of circFoxO3 were validated by Sanger sequencing

Fig. 2

Efficiency of siRNA-mediated circFoxO3 knockdown in HT22 cells. a Sequences of two siRNAs targeting circFoxO3 and representative fluorescence microscopy images delineating transfection efficiency. Scale bar = 400 µm. b Both siRNA-1 and siRNA-2 significantly reduced the expression levels of circFoxO3. c siRNA-1 significantly reduced the expression levels of FoxO3 protein in HT22 cells

Fig. 3

Blocking circFoxO3 protects HT22 cells against glutamate-induced injury. a The Calcein-AM and PI cell viability assay demonstrated that blocking circFoxO3 protects HT22 cells from glutamate-induced injury. Scale bar = 400 µm. b Quantitative analysis of data from a. c Probe sequences used in the binding assay in RNA FISH. The probe crossed the backsplice junction and was specific for FoxO3 circular RNA. d FISH was performed to measure circFoxO3 expression, and it was found that the glutamate-treated HT22 cells had higher levels of circFoxO3 than the controls. However, circFoxO3 siRNA inhibited the expression of circFoxO3. Scale bar = 100 µm

Fig. 4

Blocking circFoxO3 affected the expression level and subcellular location of FoxO3. a Western blots demonstrated that glutamate treatment elevated the protein levels of FoxO3, and silencing of circFoxO3 partially reduced the protein levels of FoxO3. b Glutamate treatment induced the nuclear localization of FoxO3 in HT22 cells, while silencing of circFoxO3 reversed the nuclear translocation of FoxO3 induced by glutamate treatment. Scale bar = 40 µm

Fig. 5

Blocking circFoxO3 downregulates the expression of Bim. a Immunocytochemistry staining demonstrated that glutamate treatment increased the expression levels of Bim, and silencing of circFoxO3 downregulated the levels of Bim. Scale bar = 100 µm. b Quantitative analysis of the data from a. c Western blot demonstrated that glutamate treatment increased the expression levels of the Bim_EL protein, and silencing of circFoxO3 downregulated the levels of the Bim_EL protein. d Quantitative analysis of the data from c

Fig. 6

Silencing of circFoxO3 attenuates glutamate-induced MMP loss in HT22 cells. a JC-1 staining was used to measure the MMP. Red fluorescence indicated normal MMP. Green fluorescence indicated damaged mitochondria with loss of MMP. Scale bar = 100 µm. b Quantitative analysis of the ratio of green and red fluorescence intensity

Fig. 7

Silencing of circFoxO3 suppresses the glutamate-induced increase in intracellular ROS. a H2DFFDA staining showed that circFoxO3 siRNA attenuated the elevation of glutamate-induced ROS. Scale bar = 100 µm. b Quantitative analysis of the data from a

Fig. 8

Blocking circFoxO3 attenuates glutamate-induced mitochondrial apoptosis injury. a Western blots demonstrated that the inhibition of circFoxO3 abolished the glutamate-induced increase in Cytochrome c in HT22 cells. b Western blots demonstrated that the inhibition of circFoxO3 abolished the glutamate-induced increase in Cleaved caspase-3 in HT22 cells. c Immunocytochemistry staining demonstrated that glutamate treatment increased the expression levels of Cleaved caspase-3, and silencing of circFoxO3 downregulated the expression levels of Cleaved caspase-3. Scale bar = 100 µm. d Quantitative analysis of the data from c. e siRNA against circFoxO3 reversed the glutamate-induced increase in the activity of Caspase-3. f TUNEL assay revealed that circFoxO3 siRNA significantly attenuated glutamate-induced cell apoptosis. Scale bar = 400 µm

Fig. 9

Hypothetical model of this study. CircFoxO3 is upregulated during oxidative insult, which results in the nuclear translocation of FoxO3 and actually promoted mitochondrial apoptosis in HT22 cells. Blocking circFoxO3 attenuates ROS-induced apoptosis in HT22 cells