MicroRNA-668-3p regulates oxidative stress and cell damage induced by Aβ1-42 by targeting the OXR1/p53-p21 axis

Introduction

Alzheimer’s disease (AD) is a degenerative disease that is common in the elderly and is characterized by progressive memory loss and dementia (1,2). Due to increased longevity globally, the treatment of AD has become paramount for public healthcare systems (3,4). Although some drugs for AD have been approved for marketing (5,6), AD still has an unclear pathogenesis and no effective cure.

Increasing evidence shows that oxidative stress may be a key factor in the pathogenesis of AD (7,8). Excessive production of oxidative stress and free radicals may lead to neuronal death, thereby promoting the loss of cognitive ability (9,10). Additionally, amyloid β (Aβ) is closely related to the severity of cognitive decline, and Aβ1-42 is the main factor driving AD development (11,12). Studies have shown that oxidative stress is the earliest change in AD development, and it also participates in the subsequent stages of AD and is therefore the most important mechanism in AD pathogenesis (13-15). Chen et al. (16) identified that oxidative stress participates in AD progression by promoting Aβ deposition.

MicroRNAs (miRNAs) are important factors that affect the occurrence and development of various diseases such as breast cancer (17) and Parkinson’s disease (18). Studies have suggested that various physiological and pathological processes of the nervous system are also affected by miRNAs, which have been identified as potential biomarkers of AD (19-21). For example, miR-668-3p has an important regulatory effect on a variety of cells, such as liver cancer cells and cardiomyocytes, in terms of biological function (22,23). However, to the best of our knowledge, there has been no relevant research on the role of miR-668-3p in AD.

MiRNAs can affect the cell cycle, proliferation, differentiation, migration, invasion, and apoptosis by regulating downstream target genes and signaling pathways (24-28). Oxidation resistance 1 (OXR1) plays a role in antioxidative stress and has an effect on neurodegenerative diseases (29,30). Increased OXR1 expression can effectively protect against oxidative stress-induced cell damage (31,32).

Various factors that are upregulated in cancer cells to maintain growth and survival are downregulated in AD, leading to neurodegeneration. AD develops when the growth and antistress response of aging neurons are weakened, and the regulation of cell death and maintenance mechanisms are modified (33). Proteins p21 and p53 are important tumor-suppressor factors and have been the focus of various studies related to AD. The occurrence and development of AD can be promoted by p21 and p53 through excessive enhancement of their levels and continued maintenance of tau phosphorylation. Therefore, p21 and p53 have been identified as potential biomarkers for AD (34-36).

This study aimed to explore whether miR-668-3p acts downstream of OXR1 to affect p53-p21 signaling and mediate the oxidative stress induced by Aβ1-42, thereby alleviating AD progression. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-3598/rc).

Methods

Animals

Six 120-days-old female C57BL/6 and C57BL/6 (AD model) specific pathogen-free mice were obtained from the Guangdong Medical Experimental Animal Center (License No. SCXK 2018-0002; Guangzhou, China) and placed in a polystyrene cage in a room with constant temperature (23±2 ℃) and humidity (45%±15%) conditions, a 12-hour light/dark cycle, and ad libitum access to standard food and water. After a week of adaptive feeding, the mice were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg body weight). Thereafter, the brain tissue was aseptically removed, the cerebral cortex was carefully opened, the hippocampus was exposed, and the surrounding tissues of the hippocampus were separated using ophthalmic scissors and placed into Hank’s balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). A protocol was prepared before the study without registration. Animal experiments were approved by the Animal Care and Use Committee of Guangxi Medical University (approval No. 2017JJB10090) and were conducted in accordance with the National Institutes of Health guidelines for the care and use of animals.

Hematoxylin-eosin (HE) staining

The brain tissue was placed in 10% formalin for 48 hours, then rinsed with water for 24 hours to remove formalin from the tissue. After dehydration with a gradient concentration of ethanol, the brain tissue was soaked in xylene I for 40 minutes to render it transparent and then embedded in paraffin wax. The tissue was cut into 4-µm-thick slices, fixed on slides, dried, and then stained with HE staining solution (Solarbio) according to the manufacturer’s instructions. The slices were soaked in xylene, then in a gradient concentration of ethanol, and subsequently in hematoxylin, and then sealed with resin. After drying, the structure of the hippocampus was observed under a light microscope.

Determination of reactive oxygen species (ROS)

The brain tissue was cut to a size of 1 mm³ and washed twice with phosphate-buffered saline (PBS) to remove tissue debris and blood. Thereafter, the tissue was rapidly frozen with liquid nitrogen, homogenized using a homogenizer, centrifuged (1,500 ×g, 4 ℃, 20 minutes), and the supernatant was collected and resuspended in prechilled PBS. The obtained single-cell suspension was incubated at 37 ℃ for 1 hour with 10 µM 2',7'-dichlorodihydrofluorescein diacetate, centrifuged (1,000 ×g, 10 minutes), and washed twice with precooled PBS. The fluorescence intensity of dichlorofluorescein was measured at 488 nm using a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Enzyme linked immunosorbent assay (ELISA)

The cell supernatant or serum of each subgroup was collected after centrifugation (1,000 ×g, 10 minutes) to analyze the levels of nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH). The following kits were used according to the manufacturer’s instructions: NO content assay kit (BC1475, Solarbio), MDA assay kit (BC0025, Solarbio), GSH assay kit (BC1195, Solarbio), and SOD activity detection kit (BC5160, Solarbio).

Microarray raw dataset analysis using the Gene Expression Omnibus (GEO)

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Raw gene expression data (GSE157239_RAW.tar and GSE150696_RAW.tar) were downloaded from the GEO website (http://www.ncbi.nlm.nih.gov/geo) and divided into 4 groups: control case miRNAs (GSM4759791, GSM4759794, GSM4759796), AD case miRNAs (GSM4759793, GSM4759797, GSM4759804), control case mRNAs (GSM4556860, GSM4556861, GSM4556862), and AD case mRNAs (GSM4556851, GSM4556852, GSM4556853). The Affymetrix Transcriptome Analysis Console (version 4.0) was used to analyze the Affymetrix Human Genome U133 Plus 2.0 Array to generate a heatmap of differentially expressed miRNAs and mRNAs. Differentially expressed RNAs were considered significant at a P value of ≥0.01 and a log₂ (fold change, FC) >1.5. Sequences of the 3 small interfering RNAs (siRNAs) targeting OXR1, the miR-668-3p mimic or inhibitor, and the negative control (NC) are shown in Table S1.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay

Total RNA from neuronal stem cells (NSCs) was extracted using TRIzol^TM reagent (Thermo Fisher Scientific). After addition of 200 µL of chloroform and vigorous shaking and mixing for 30 seconds, the sample was left to stand for 10 minutes. Next, the upper aqueous phase was collected after centrifugation (14,000 ×g, 15 minutes) to obtain the RNA pellets. The cDNA was reverse transcribed using the PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s recommendations. RT-qPCR analysis was performed using the SYBR^® Premix Ex Taq™ II kit (Takara Bio, Shiga, Japan) and Applied Biosystems^® 7500 Real-Time PCR System (CXF96; Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 50 ℃ for 2 minutes, 95 ℃ for 2 minutes, followed by 40 cycles of 95 ℃ for 15 seconds and 60 ℃ for 32 seconds. The primers used for RT-qPCR analysis are listed in Table S2. MiR-668-3p and OXR1 RNA levels were normalized to those of U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated using the 2^−ΔΔCq method (37).

Immunohistochemistry (IHC) and immunofluorescence (IF) assay

Brain tissue sections were incubated in 3% H₂O₂ to quench the endogenous peroxidase activity. The sections were then incubated overnight at 4 ℃ with a rabbit polyclonal anti-OXR1 antibody (1:50 for IHC, 2 µg/mL for IF; ab251774; Abcam, Cambridge, UK), followed by incubation for 30 minutes at 37 ℃ with an appropriate horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (for IHC) or goat anti-rabbit Alexa Fluor^® 647 (1:50 for IF, ab190565, Abcam), followed by staining with 3,3'-diaminobenzidine or 4',6-diamidino-2-phenylindole (for IF).

Western blot assay

After the NSCs were lysed with lysis buffer (Solarbio), protein concentrations in the extracts were determined using a bicinchoninic acid (BCA) protein assay kit (Solarbio) according to the manufacturer’s instructions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10%) was used to separate denatured proteins (20 µg), which were then transferred to polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were rinsed with 10% tris-buffered saline (TBS)-Tween-20 (Solarbio), blocked with 5% bovine serum albumin (Solarbio), and incubated overnight at 4 ℃ with antibodies against OXR1 (0.4 µg/mL, ab251774, Abcam), p53 (1:10,000, ab32389, Abcam), and p21 (1:1,000, ab109520, Abcam). Membranes were then incubated at 25 ℃ for 2 hours with a secondary antibody (goat anti-rabbit, 1:10,000, ab205718, Abcam). GAPDH (1:10,000, ab181602, Abcam) was used as the loading control.

Isolation and culture of NSCs

The brain tissue was cut into pieces of approximately 1 mm³, digested with 0.125% trypsin, and incubated at 37 ℃ for 20 minutes, with gentle shaking 2–3 times during the incubation period. The stop solution and filter were added to prepare the cell suspension, which was then centrifuged (1,000 ×g, 10 minutes, 4 ℃). The pellet was resuspended in complete medium and inoculated into a culture flask (37 ℃, 5% CO₂). The NSCs were cultured for 6 hours, and the medium was then replaced with serum-free medium. After 3 days, Ara-c (Sigma-Aldrich, St. Louis, MO, USA) was added for 24 hours to inhibit the proliferation of nonneuronal cells. The medium was changed every 3 days for 21 days during the cell experiments.

Identification of NSCs

NSCs (5×10⁴) were inoculated into a 6-well plate (Corning, Corning, NY, USA) for 24 hours on round coverslips. After 3 rinses with PBS, Triton X-100 was added for 2 hours. NSCs were incubated with bovine serum albumin for 30 minutes, followed by overnight incubation with anti-MAP2 (1:500, ab254264, Abcam) and anti-β III tubulin (3 µg/mL, ab18207, Abcam) antibodies at 4 ℃ in the dark. After rinsing with PBS 3 times, the cells were incubated at 37 ℃ for 1 hour with goat anti-rabbit Alexa Fluor^® 488 (1:200, ab150077, Abcam) or 647 labeled secondary antibodies. Finally, the cells were mounted using a gold antifade mounting medium containing 4',6-diamidino-2-phenylindole (ProLong™, Thermo Fisher Scientific).

Dual luciferase assays

NSCs were transfected with 0.5 µg each of a miR-668-3p mimic/inhibitor, 1 µg each of the plasmids expressing wild-type (WT) or mutant (mut) OXR1, and 0.05 µg of pRL-SV40 reporter vector plasmid. After NSCs were incubated for 48 h, luciferase activity was measured at 490 nm (dual luciferase reporter assay system; Promega Corp., Fitchburg, WI, USA). The ratio of firefly to Renilla is normalized with firefly values.

Cell proliferation assay

NSCs were seeded in 96-well plates at 2.5×10³ cells/well and cultured. According to the manufacturer’s instructions, optical density values were measured at time nodes of 0, 24, 48, and 72 h with an enzyme-labeled instrument (Thermo Fisher Scientific) using the Cell Counting Kit-8 (Solarbio) assay.

Flow cytometry (FCM) analysis of apoptosis, cell cycle, and ROS of NSCs

For analysis of apoptosis, A dose of 5 µL of Annexin V-Fluorescein Isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ, USA) and 10 µL of propidium iodide (PI; BD Biosciences) were used in combination with the following conditions: dark, 15 min, 25 ℃. Then rinsed twice with PBS (Gibco, Thermo Fisher Scientific).

For cell cycle analysis, NSCs were washed twice with cold PBS and incubated at 37 ℃ for 30 minutes in the dark with PI (400 µL) and RNase (100 µL). The PI signal was detected using an FCM analyzer (BD Biosciences) and the ratio of G1, S and G2 phases in each group of cells was calculated.

For ROS analysis, NSCs were incubated with 2',7'-dichlorodihydrofluorescein diacetate was added to each group of cells at a dose of 1.0 µM and incubated under following conditions: 15 min and 37 ℃. Subsequently, Cells were then gently washed twice with PBS and via FCM to detect ROS level.

Statistical analysis

All experiments were performed in triplicate. Data are expressed as the mean ± standard deviation. All statistical analyses were performed using SPSS version 19.0 statistical analysis package (SPSS Corp., Armonk, NY, USA). Statistical analysis was performed using one-way analysis of variance with a Bonferroni post-hoc test. The means of the 2 groups were analyzed using the Student’s t-test. Differences were considered significant at P<0.05.

Results

Oxidative stress is elevated in AD

Analysis of HE staining showed the nerve cells in the brain tissue of the healthy control group to be neatly arranged and round, with a complete cell structure, clear cell membrane and cell nucleus, and no visible swelling or necrosis. Multiple nerve cells in the brain tissue of AD mice were extremely disordered and irregular in size and shape. The number of nerve cells decreased sharply, and the cell structure was blurry (Figure 1A). Additionally, the results suggested that the levels of ROS, MDA, and NO were increased, whereas the levels of SOD and GSH were decreased in the brain tissue of AD mice compared to those of the healthy control group (Figure 1B-1F). These results confirmed that oxidative stress levels were elevated in the AD mice.

Figure 1 Differences in hippocampus characteristics, oxidative stress levels, and miRNA levels between normal and AD mice. (A) HE staining analysis showing the hippocampal characteristics of normal mice and AD mice. Difference in ROS (B), MDA (C), NO (D), SOD (E), and GSH (F) content in the brain tissue of normal mice and AD mice. (G) Heatmap showing the differentially expressed miRNAs in the GSE157239 dataset. (H) RT-qPCR analysis of the differences in the expression of miR-668-3p in the brain tissues of normal mice and AD mice. *, P<0.05. AD, Alzheimer’s disease; ROS, reactive oxygen species; MDA, malondialdehyde; NO, nitric oxide; SOD, superoxide dismutase; GSH, glutathione peroxidase; HE, hematoxylin-eosin.

MiRNA screening

To explore the molecular mechanisms that affect AD progression, we analyzed the GSE157239 dataset using GEO. Compared to healthy individuals, in patients with AD, there were 67 dysregulated miRNAs (Figure 1G), of which 31 were upregulated and 36 were downregulated (Table S3). Subsequently, we selected miR-668-3p with the highest FC value as a potential therapeutic target for AD and explored its role in AD pathogenesis. In our subsequent RT-qPCR analysis, we found that miR-668-3p expression in the brain tissue of AD mice was significantly increased (Figure 1H). Therefore, miR-668-3p may be the key to AD development, which was consistent with the results of the GEO data analysis.

MiR-668-3p binds to OXR1

As miRNAs can regulate downstream signaling pathways by binding to mRNA, we conducted a joint analysis using the TargetScan and starBase databases and the mRNA mined from GEO (dataset GSE150696) (Figure 2A). We speculated that 40 mRNAs had the potential to bind to miR-668-3p. Genes with potential binding sites for miR-668-3p are listed in Table S4. Among these mRNAs, the role of the conserved gene family antioxidant gene 1 (OXR1) in protecting species from oxidative stress has been widely confirmed (29,32). Accordingly, OXR1 was downregulated in AD patients in GEO dataset GSE150696 (Figure 2B). However, there have been no studies discussing the role of OXR1 in AD. Subsequent analysis of RT-qPCR, IHC, and IF results revealed that the RNA and protein levels of OXR1 decreased in the brain tissue of AD mice compared to those in the healthy control group (Figure 2C-2E), indicating that reduced OXR1 levels correlated with AD progression. Additionally, multiple studies have shown that the p53 signaling pathway may be one of the main signaling pathways that affect AD development (38-40). We analyzed p53 signaling pathway-related proteins p53 and p21 by western blotting, and the results revealed that the protein levels of p53 and p21 increased significantly in AD mice (Figure 2F). Therefore, we believe that miR-668-3p may regulate p53 signal transduction by targeting the 3'UTR of OXR1 and promoting the oxidative stress response in brain tissue.

Figure 2 Screening of mRNA and protein levels in AD. (A) The GSE150696 dataset, TargetScan, and starBase databases jointly analyzed downstream mRNA expression. (B) Heatmap showing OXR1 gene expression in GSE150696 dataset. (C) RT-qPCR analysis of OXR1 expression in AD mice. (D) IHC analysis showing OXR1 protein levels in AD mice. (E) IF analysis indicating the location and levels of OXR1 in AD mice. (F) Western blot analysis indicating protein levels of p53 and p21 in AD mice. *, P<0.05. OXR1, oxidation resistance 1; AD, Alzheimer’s disease; IHC, immunohistochemistry; IF, immunofluorescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

MiR-668-3p inhibitor relieves the Aβ1-42-induced effects by regulating OXR1 expression

To explore the mechanism of the miR-668-3p/OXR1/p53-p21 axis, we extracted and isolated murine hippocampal NSCs. The IF results showed that the expression of MAP2 and β-tubulin in the isolated and cultured hippocampal neuronal cells were both positive (Figure 3A), indicating that the neuronal cells were successfully isolated. Subsequently, Aβ1-42 was used to induce AD in mouse neuronal cells. RT-qPCR and western blotting results suggested that compared to the healthy control group, after Aβ1-42 induction, the expression of miR-668-3p increased (Figure 3B), and the mRNA and OXR1 protein levels decreased (Figure 3C,3D). These results were consistent with those of previous in vivo experiments. We transfected the synthetic miR-668-3p mimic or inhibitor into mouse neuron cells. The results suggested that miR-668-3p RNA levels increased in the mimic group and decreased in the inhibitor group, confirming the effectiveness of the synthetic miR-668-3p mimic or inhibitor (Figure 3E). RT-qPCR results showed that after transfection of the 3 synthesized siRNAs targeting OXR1, the expression of OXR1 decreased to varying degrees. Among them, si-OXR1-1 showed the highest inhibitory efficiency (Figure 3F) and was therefore used to act as an OXR1 antagonist (si-OXR1) for further molecular mechanism research. The results of the dual luciferase assay performed to confirm the direct binding between miR-668-3p and OXR1 showed that in normal hippocampal neuronal cells, compared with the WT OXR1 + miR-668-3p mimic NC group, the fluorescence activity of the miR-668-3p mimic group was significantly decreased. Compared to the mut OXR1 + miR-668-3p inhibitor NC group, the mut OXR1 + miR-668-3p inhibitor group showed no significant difference in fluorescence activity (Figure 3G). RT-qPCR results suggested that suppression of miR-668-3p expression significantly increased the mRNA level of OXR1 (Figure 3H). These results demonstrated that miR-668-3p directly targeted OXR1.

Figure 3 The interaction between miR-668-3p and OXR1 in NSCs in vitro. (A) IF analysis results of NSCs isolated from murine brain tissue. RT-qPCR analysis examining the impact of Aβ1-42 on miR-668-3p (B) and OXR1 (C) expression. RT-qPCR analysis results of the impact of Aβ1-42 on the expression of OXR1. (D) Western blot analysis result investigating the impact of Aβ1-42 on OXR1 protein expression. (E) RT-qPCR analysis indicating the effectiveness of the synthesized miR-668-3p mimics and inhibitor. (F) RT-qPCR analysis showing the effectiveness of the 3 constructed siRNAs targeting OXR1. (G) Dual luciferase analysis showing the binding of miR-668-3p and OXR1. (H) RT-qPCR analysis examining the impact of si-OXR1 on miR-668-3p expression. *, P<0.05, Inhibitor group vs. Inhibitor NC group; ^#, P<0.05, Mimic group vs. Mimic NC group. Aβ1-42, amyloid β 1-42; OXR1, oxidation resistance 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IF, immunofluorescence; NC, negative control; si, small interfering; WT, wild-type; mut, mutant; NSCs, neuronal stem cells; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

MiR-668-3p negatively regulates OXR1 expression

The results of the rescue experiments showed that Aβ1-42 induced a significant reduction in hippocampal NSC proliferation (Figure 4A), increased apoptosis (Figure 4B) and cell cycle arrest (Figure 4C), and promoted oxidative stress (ROS, MDA, and NO levels increased, and SOD and GSH levels decreased; Figure 4D-4H). The addition of the miR-668-3p inhibitor partially reversed the effect induced by Aβ1-42, but suppression of OXR1 expression prevented the miR-668-3p inhibitor from exerting its effect. These results confirmed the negative regulatory relationship between miR-668-3p and OXR1.

Figure 4 The effect of miR-668-3p and OXR1 on NSCs. (A) CCK8 analysis examined the impact of Aβ1-42 induction, miR-668-3p inhibitor, and si-OXR1 on NSC proliferation. FCM analysis examining the effect of Aβ1-42 induction, miR-668-3p inhibitor, and si-OXR1 on NSC apoptosis (B), cell cycle (C), and ROS (D). ELISA analysis examining the impact of Aβ1-42 induction, miR-668-3p inhibitor, and si-OXR1 on the secretion of MDA (E), NO (F), SOD (G), and GSH (H) of NSCs. ^#, P<0.05, Inhibitor + si-OXR1 group vs. Inhibitor + si-NC group. *, P<0.05. Aβ1-42, amyloid β 1-42; NC, negative control; si, small interfering; OXR1, oxidation resistance 1; PI, propidium iodide; FITC, fluorescein isothiocyanate; G1, first gap of the cell cycle; S, synthesis of the cell cycle; G2, second gap of the cell cycle; ROS, reactive oxygen species; MDA, malondialdehyde; NO, nitric oxide; SOD, superoxide dismutase; GSH, glutathione peroxidase; NSCs, neuronal stem cells; CCK8, Cell Counting Kit-8; FCM, flow cytometry; ELISA, enzyme linked immunosorbent assay.

MiR-668-3p regulates the p53 signaling pathway via OXR1

The above in vivo experiments confirmed that the protein levels of p53 and p21 increased significantly in AD, which was verified in vitro using mouse hippocampal NSCs. Tenovin-1 (TEN; activator of the p53 signaling pathway) was cotransfected with the miR-668-3p inhibitor. Western blotting results showed that Aβ1-42 induced a decrease in OXR1 protein levels and an increase in p53/p21 protein levels, and TEN alone enhanced the effect of Aβ1-42 on p53/p21 but had no effect on OXR1 protein levels. The miR-668-3p inhibitor alone reversed the effect of Aβ1-42 on p53 and p21 protein levels by promoting the translation of OXR1, but the effect of the miR-668-3p inhibitor was partially weakened by the combined action of TEN (Figure 5A). Furthermore, the results showed that compared to the healthy control group, Aβ1-42 decreased cell proliferation (Figure 5B) and increased apoptosis (Figure 5C), cell cycle arrest (Figure 5D), and oxidative stress levels (Figure 5E-5I). TEN alone enhanced the effect induced by Aβ1-42, whereas the miR-668-3p inhibitor alone partially reversed the effect of Aβ1-42. Furthermore, the effect of the miR-668-3p inhibitor under the combined action of TEN and Aβ1-42 was significantly inhibited.

Figure 5 The effect of TEN and miR-668-3p on NSCs. (A) Western blot analysis showing the impact of Aβ1-42 induction, miR-668-3p inhibitor, and TEN on the protein levels of OXR1, p53, and p21. (B) CCK8 analysis showing the effect of Aβ1-42 induction, miR-668-3p inhibitor, and TEN on NSC proliferation. FCM analysis showing the effect of Aβ1-42 induction, miR-668-3p inhibitor, and TEN on NSC apoptosis (C), cell cycle (D) and ROS (E). ELISA analysis showing the impact of Aβ1-42 induction, miR-668-3p inhibitor, and TEN on the secretion of MDA (F), NO (G), SOD (H), and GSH (I) in NSCs. *, P<0.05, TEN group vs. Aβ1-42 group; ^#, P<0.05, TEN + inhibitor group vs. Inhibitor group. OXR1, oxidation resistance 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Aβ1-42, amyloid β 1-42; TEN, tenovin-1; PI, propidium iodide; FITC, fluorescein isothiocyanate; G1, first gap of the cell cycle; S, synthesis of the cell cycle; G2, second gap of the cell cycle; ROS, reactive oxygen species; MDA, malondialdehyde; NO, nitric oxide; SOD, superoxide dismutase; GSH, glutathione peroxidase; NSCs, neuronal stem cells; CCK8, Cell Counting Kit-8; FCM, flow cytometry; ELISA, enzyme linked immunosorbent assay.

In summary, miR-668-3p suppression was mediated by the p53-p21 pathway and negatively regulated OXR1 expression by targeting the 3'UTR of OXR1. Therefore, miR-668-3p may be a potential therapeutic target for AD.

Discussion

Dysregulated miRNAs, including miR-106b (36), miR-196a (37), and miR-124 (15), are important factors in the development of many degenerative diseases (41,42). For example, recent studies have shown that low levels of miRNAs miR-27a-3p (43) and high levels of miR-483-3p (44) can promote the development of AD. Moreover, miR-342-3p (45) and miR-103a-3p (46) affect the occurrence and subsequent stages of AD. The purpose of our study was to clarify the mechanisms underlying AD development by screening for miRNAs that affect AD progression. Among the murine homologous miRNAs, we found that the FC value of miR-668-3p was the highest. Currently, no follow-up study has investigated miR-668-3p as a therapeutic target for AD. Therefore, we screened miR-668-3p as a potential therapeutic target for AD and explored its role in AD pathogenesis. Subsequently we found that the suppression of miR-668-3p expression had a neuroprotective effect on cell damage induced by AD or Aβ1-42. Furthermore, this neuroprotective effect was related to the inhibition of oxidative stress and neuronal apoptosis as well as the regulation of the miR-668-3p/OXR1/p53-p21 axis.

Studies have shown that AD is characterized by the production of excessive amounts of ROS, MDA, and NO, and inhibition of the secretion of SOD, GSH, and other antioxidant enzymes (47,48), resulting in damage to the hippocampal neurons in the brain (49), which is consistent with our results. These large amounts of indelible ROS can induce damage to cell membranes and mitochondria, leading to NSC apoptosis (50). Additionally, we found that miR-668-3p was upregulated in the GEO data analysis, AD mouse models, and in vitro Aβ1-42-induced NSCs. These results were consistent with those of previous studies (51-53), and we found that in vitro Aβ1-42 induction decreased NSC proliferation and promoted apoptosis, cell cycle arrest, and oxidative stress. By suppressing miR-668-3p expression, the adverse effects of Aβ1-42 induction on cells could be reversed. This finding was similar to that of previous reports (54,55). Therefore, miR-668-3p expression may be the key to AD progression; however, the mechanism of miR-668-3p remains unclear.

It is well known that miRNAs can regulate the transcription and translation of mRNA by binding to the 3'UTR region of mRNA (28). OXR1 is an important protein known to protect cells from oxidative stress (56,57), and it plays a key role in preventing and alleviating neurodegeneration (29). Subsequent RT-qPCR, IHC, and IF experiments confirmed that OXR1 RNA and protein levels were downregulated in vivo and in vitro in the Aβ1-42 cell model of AD mice, a finding similar to that of Jiang et al. in degenerative diseases (Parkinson’s disease) (32). Subsequent luciferase experiments confirmed that miR-668-3p directly targeted the 3'UTR end of OXR1. Thus far, we can speculate that the positive effect of suppressing miR-668-3p expression in cells may have been achieved through OXR1.

A study has shown that various pathways, such as p53, TNF-α, and PI3K/AKT/MTOR, are negatively correlated with cancer in AD (33). We confirmed through rescue experiments that the suppression of OXR1 expression could reverse the effect of the miR-668-3p inhibitor on Aβ1-42 induction. As an activator of p53, the addition of TEN partially reversed the effect of the miR-668-3p inhibitor on Aβ1-42 induction. These results also confirmed the negative correlation between miR-668-3p and OXR1 or p53-p21, in which miR-668-3p regulates p53-p21 signaling by directly targeting OXR1, affecting the proliferation, apoptosis, cell cycle arrest, and oxidative stress levels of NSCs induced by Aβ1-42 in vitro. This study demonstrates that dysregulation of the miR-668-3p/OXR1/p53-p21 pathway is a key mediator of AD pathogenesis, highlights the importance of epigenetics and identifies novel therapeutic targets for AD.

There were some limitations to this study. First, there were no clinical data to support the role of miR-668-3p in patients with AD. Moreover, there are still many mRNAs derived from GEO data mining that have not been analyzed and verified. These are the directions and goals of our future research.

Conclusions

The suppression of miR-668-3p expression had a neuroprotective effect on cell damage induced by AD in vivo and Aβ1-42 in vitro through inhibition of cell cycle arrest, apoptosis, and oxidative stress. The neuroprotective mechanism involved the regulation of the miR-668-3p/OXR1/p53-p21 axis, and miR-668-3p may be a potential AD biomarker.

Acknowledgments

Funding: This work was supported by grants received from the Guangxi Natural Science Foundation for Young Scientists (No. 2017JJB140090z), Guangxi Zhuang Autonomous Region Health Care Commission Self-financing Research Projects (Nos. Z20200096, Z20201417), Guangxi Medicine and Healthy Self-Financing Research Projects (No. Z20180956), and Nanning Qingxiu District Key Research and Development Program (No. 2019041).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-3598/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-3598/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-3598/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. 2017JJB10090) granted by the Animal Care and Use Committee of Guangxi Medical University, in compliance with the National Institutes of Health guidelines for the care and use of animals. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: A. Muijlwijk)