Reduced serum miR‐202 may promote the progression of Alzheimer's disease patients via targeting amyloid precursor protein

Abstract

The current study investigated whether the expression of miR‐202 was abnormal in the serum of patients with Alzheimer's disease (AD) and evaluated the potential clinical significance, thereby shedding light on the diagnosis of AD. Here, our data showed that the level of miR‐202 decreased significantly in the serum of AD patients (n = 121) compared with that of healthy controls (n = 86). Further analysis showed that the level of serum miR‐202 was gradually decreased in the mild AD group (n = 31), moderate AD group (n = 52) and severe AD group (n = 38) compared with the healthy control group. Receiver operating characteristic (ROC) curve analysis demonstrated that serum miR‐202 could differentiate AD patients from healthy controls, with an AUC of 0.892. Spearman correlation analysis showed that serum miR‐202 was positively correlated with the Mini‐Mental State Examination (MMSE). Based on TargetScan, a conserved binding site was identified in the 3′UTR of amyloid precursor protein (APP). The dual luciferase assay showed that miR‐202 suppressed the relative luciferase activity of pmirGLO‐APP‐3′UTR. Western blot assays indicated that overexpression of miR‐202 suppressed the expression of APP, while the expression of APP was enhanced after inhibition of miR‐202 in PC12 cells, indicating that APP was a possible target gene of miR‐202. Moreover, the cell apoptosis induced by transfection of miR‐202 inhibitor was abolished by silencing APP. In summary, we showed novel data that downregulation of serum miR‐202 may be used as a potential biomarker for AD and may promote the development of AD by targeting APP.

INTRODUCTION

Currently, Alzheimer's disease (AD) is the fifth leading cause of death in people older than 65 years.¹ It not only seriously endangers the physical and mental health of the elderly but also places a heavy economic burden on the families and society of patients.² AD is a neurodegenerative disease characterized by progressive memory loss, cognitive impairment, and behavioral changes.³ The main pathological changes are abnormal deposition of β-amyloid (Aβ) and overphosphorylation of tau protein, which result in senile plaque and neurofibrillary tangles.⁴ At present, the clinical diagnosis of AD mainly depends on inquiring about the medical history, clinical examination, neuropsychological test, imaging examination, and laboratory examination.⁵ However, the sensitivity and specificity of these methods are not high enough to diagnose AD, especially in the early stage.⁶ Therefore, reliable biological indicators are urgently needed for the early diagnosis of AD to implement effective prevention and control measures as soon as possible.

MicroRNAs (miRNAs) are highly conserved small fragments of noncoding RNA that regulate gene expression at the posttranscriptional level.⁷ Previous studies have shown that miRs are abundantly expressed in the central nervous system with high temporal and spatial specificity, which is closely related to the formation and differentiation of neurons and the remodeling of synapses.^8,9 In addition, some studies have confirmed that microvesicles can penetrate the blood–brain barrier and release miRs into peripheral blood.¹⁰ Serum samples are convenient for sampling and induce minimal trauma, so they achieve better compliance in clinical application and have a higher detection frequency, which makes serum a very attractive source of biomarkers.^11,12 Therefore, serum miRs as biomarkers for the early diagnosis of AD are very promising.

It has been found that miR-202 is closely related to the occurrence and development of prostate cancer, breast cancer, oral cancer, and other tumors.^13–15 More importantly, in rat hippocampal tissues, upregulation of miR-202-3p was induced by chronic unpredictable mild stress.¹⁶ In the development of neuropathic pain, miR-202 plays a key role by suppressing the RAP1A gene.¹⁷ Overexpression of miR-202 was also found in the cerebellum of multiple-system atrophy.¹⁸ Loss of miR-202-3p in breast cancer cells increased the transmigration of breast cancer cells through the brain endothelium via elevating MMP-1 and disrupting the interendothelial junctions.¹⁹ Reduction of miR-202-5p is shown in N2a cells after oxygen–glucose deprivation/reoxygenation-induced injury and rats after middle cerebral artery occlusion.²⁰ Further study indicated that miR-202-5p could induce autophagy via suppressing AKT/GSK-3β signaling in N2a cells.²⁰ In the samples of cerebellar-type multiple-system atrophy, miR-202 is the most upregulated and is demonstrated to enhance oxidative stress-induced cell death.¹⁸ These results suggest that miR-202 may be associated with the occurrence of neurological diseases.

The aim of this study was to investigate whether the expression of miR-202 was abnormal in the blood of patients with AD and to evaluate its potential clinical significance, thereby shedding light on the diagnosis of AD.

MATERIALS AND METHODS Patient samples

From March 2016 to May 2018, serum samples from 121 AD patients and 86 healthy controls of the same age were collected in Rizhao People's Hospital. All subjects signed informed consent. The diagnostic criteria for AD patients were set by the National Institute of Neurology and the Institute of Alzheimer's Diseases.²¹ Briefly, the diagnosis of AD was made when patients demonstrated progressive memory decline for more than 6 months with a resulting impairment of self-care and social or occupational functioning. The presence of objective memory impairment should be recorded by the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) tests. Other essential diagnostic points included deficits in two or more areas of cognition, absence of disturbance in consciousness, disease onset between the ages of 40 and 90 years, absence of systemic disorders, or other brain diseases that could account for the progressive deficits in memory and cognition, evidence of cerebral atrophy on amyloid- and ¹⁸F-FDG positron emission tomography or magnetic resonance imaging without other significant organic lesions, and absence of any metabolic disorder.²¹

Patients with other nervous system diseases and common geriatric diseases were excluded by medical history investigation, clinical examination, imaging examination, and routine laboratory examination. The degree of cognitive impairment was assessed by the MMSE and CDR. The subjects were divided into four groups: healthy control group (MMSE 27–30, CDR-0), mild AD group (MMSE 20–26, CDR-1), moderate AD group (MMSE 10–19, CDR-2), and severe AD group (MMSE 0–9, CDR-3).

Fasting whole blood (3 ml) was collected from AD patients and healthy controls. After blood collection, severe shock was avoided, and serum was separated within 2 h. The supernatant was collected after centrifugation for 10 min at room temperature following 30 min at room temperature. The supernatant was centrifuged for 5 min at 4°C at 16,000 rpm, and serum samples were collected. Then, the serum was stored in a −80°C refrigerator to avoid repeated freezing and thawing.

Cell culture

PC12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured with 5 ng/ml NGF in Dulbecco's modified Eagle's medium (DMEM, HyClone; GE Healthcare Life Sciences). In total, 293 cells were purchased from the Peking Union Medical College Cell Culture Center (Beijing, China) and cultured in DMEM (HyClone; GE Healthcare Life Sciences). Cells were cultured in the appropriate medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 U/ml streptomycin at 37°C. The cells were seeded at a density of 1 × 10⁴ cells/ml and allowed to grow for 24 h prior to experimentation.

Transfection

miR-202 mimics, inhibitors, or negative control (NC) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China) and transfected into PC12 and 293 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were collected for subsequent experimentation following 48 h of transfection.

Reverse transcription polymerase chain reaction (RT-PCR)

To determine the levels of miR-202, qPCR was carried out using SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in an iCycleriQ real-time PCR detection system (Bio-Rad Laboratories, Inc.). The PCR amplifications were performed in a 10-μl reaction system, including 5 μl of SYBR Green Supermix, 0.4 μl of forward primer, 0.4 μl of reverse primer, 2.2 μl of double distilled H₂O, and 2 μl of template cDNA. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative mRNA expression was normalized to U6 using the 2^-∆∆Cq method.²² Primer sequences were as follows: miR-202-RT, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAAGA-3′; U6-RT, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATG-3′; miR-140-5p, forward 5′-GCGCTTCCTATGCATATACTTC-3′; U6, forward 5′-GCGCGTCGTGAAGCGTTC-3′; universal reverse primer, 5′-GTGCAGGGTCCGAGGT-3′.

Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL)

Nuclear fragmentation was examined using an In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the instructions. In brief, the cells were fixed using 4% paraformaldehyde for 30 min, followed by incubation with TUNEL buffer for 1 h at 37°C. After washing with phosphate buffered saline, the number of TUNEL-positive apoptotic cells and the total number of cells were counted using a microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 400×. The percentage of apoptotic cells was determined as the ratio of the number of TUNEL-positive cells to the total number of cells.

MTT assay

Aβ (Sigma-Aldrich; Merck KGaA) was used to establish the in vitro AD cell model. In brief, PC12 cells were seeded in 96-well tissue culture plates at a density of 5 × 10⁴ cells per well in DMEM. When the confluence reached 70%, Aβ was added to each well, and the cells were incubated at 37°C for 48 h. Cell viability was examined with MTT assay kits (Sigma-Aldrich; Merck KGaA). The blue formazan products in the cells were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich; Merck KGaA) and spectrophotometrically measured at a wavelength of 550 nm. All experiments were performed in triplicate.

Western blot

The cells were collected, and lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) was added. The protein was separated by 12% sodium alkyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. Five percent skimmed milk powder was added, membranes were sealed in this solution for 1 h, and then they were incubated with the corresponding primary antibody at 4°C overnight. On the second day, the secondary antibody labeled with horseradish peroxidase (HRP) was added and incubated at room temperature for 1 h. The protein concentration was detected by enhanced chemiluminescence.

Dual luciferase assay

Based on TargetScan, a conserved binding site was identified in the 3′UTR of amyloid precursor protein (APP). Then, the DNA fragment was cloned into the plasmid pmirGLO (Promega Corp., Madison, WI, USA), generating the pmirGLO-APP-3′UTR plasmid. Cells were inoculated into 24-well plates, and the density was adjusted to 2.0 × 10⁵. Then, PC12 cells were transfected with miR-202 mimic and pmiGLO-APP-3′UTR, NC and pmirGLO using Lipofectamine 2000 (Invitrogen). After 48 h of culture, the luciferase activity of cells in different groups was detected by a dual luciferase reporter gene kit (Promega Corp.).

Statistical analysis

Data are expressed as the mean ± standard deviation of the mean, as indicated. Each experiment was performed in triplicate. Multiple comparisons were performed using one-way analysis of variance followed by Tukey's multiple comparison test. The receiver operating characteristic (ROC) curves of miR-202 were analyzed to determine their diagnostic efficacy, sensitivity, and specificity. Spearman correlation analysis was used to evaluate the correlation between the expression level of miR-202 and the MMSE score. p < 0.05 was considered to indicate a statistically significant difference.

RESULTS Clinicopathological data

There were 207 participants, including 86 healthy controls and 121 AD patients. Among them, 31 were mild samples (25.6%), 52 were moderate samples (43.0%), and 38 were severe samples (31.4%). There was no significant difference in sex or age between the four groups. The average MMSE scores of the four groups were 28.9 ± 0.9, 24.1 ± 1.3, 15.5 ± 2.6, and 7.0 ± 3.7. The difference was significant and was calculated in post hoc analysis, as shown in Table 1. Moreover, there is no significant difference in terms of education background between healthy controls and AD patients (Table 2).

TABLE 1 Basic information of AD patients and healthy controls

	Healthy controls	Mild AD	Moderate AD	Severe AD	p
Cases	86	31	52	38	0.537
Age	69.0 ± 5.8	70.5 ± 6.2	69.6 ± 7.3	71.7 ± 6.4	0.528
Gender (male %)	52.3%	51.6%	53.8%	44.7	0.907
MMSE	28.9 ± 0.9	24.1 ± 1.3	15.5 ± 2.6	7.0 ± 3.7	0.004
CDR	0	1	2	3	—

Abbreviations: AD, Alzheimer's disease; CDR, Clinical Dementia Rating; MMSE: Mini-Mental State Examination.

TABLE 2 Comparison of education background between Alzheimer's disease (AD) patients and healthy controls

[Table omitted. See PDF]

Reduced level of serum miR-202 in AD patients

The RT-PCR results showed that the level of miR-202 decreased significantly in the serum of AD patients compared with that of healthy controls (Figure 1A). In addition, the level of serum miR-202 was significantly decreased based on the severity of AD (Figure 1B). Specifically, the level of serum miR-202 was decreased in the mild group compared with that of healthy controls. Meanwhile, lower serum miR-202 was shown in the moderate group and lowest serum miR-202 was found in the severe group.

FIGURE 1. Serum miR-202 was reduced in the serum of Alzheimer's disease (AD) patients compared with that of healthy controls. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that the level of miR-202 was decreased in the serum of AD patients. (B) The level of serum miR-202 was significantly decreased based on the severity of AD. ***p [less than] 0.001 versus control. ##p [less than] 0.01, ###p [less than] 0.001 versus as indicated

FIGURE 2. Spearman correlation analysis showed that serum miR-202 positively correlated with the Mini-Mental State Examination (MMSE) in Alzheimer's disease (AD) patients

FIGURE 3. Receiver operating characteristic (ROC) analysis demonstrated that serum miR-202 could differentiate Alzheimer's disease (AD) patients from healthy controls

Positive correlation between serum miR-202 and MMSE in AD patients

To determine whether AD-specific serum miR-202 can correctly reflect the clinical manifestations of the disease, we analyzed the correlation between miR-202 and MMSE scores. The results showed that serum miR-202 was positively correlated with the MMSE score (r = 0.7, p < 0.001) (Figure 2). As shown in Figure 3, serum miR-202 could differentiate ACI patients from healthy controls.

miR-202 reduced PC12 cell apoptosis and increased cell viability

In the Aβ-induced PC12 cellular AD model, we found that the level of miR-202 was decreased compared with the control (Figure 4A). Furthermore, we also analyzed cell viability after Aβ treatment. As shown in Figure 4(B), overexpression of miR-202 increased cell viability in PC12 cells induced by Aβ, while inhibition of miR-202 further inhibited the viability of PC12 cells. A previous study suggested that apoptosis plays a key role in the development of AD.²³ Hence, we evaluated the effect of miR-202 on the apoptosis of PC12 cells. Compared with the control, Aβ significantly promoted the apoptotic rate of PC12 cells (Figure 4C). Overexpression of miR-202 reduced the apoptotic rate induced by Aβ (Figure 4C). However, transfection of the miR-202 inhibitor further promoted the apoptotic rate induced by Aβ (Figure 4C).

FIGURE 4. miR-202 reduced PC12 cell apoptosis and increased cell viability. (A) In the Aβ-induced PC12 cellular Alzheimer's disease (AD) model, the level of miR-202 was decreased compared with the control. (B) The MTT assay showed that overexpression of miR-202 reduced the toxicity and side effects of Aβ. (C) TUNEL staining showed that overexpression of miR-202 can reduce the apoptotic rate induced by Aβ. *p [less than] 0.05, **p [less than] 0.01 versus control, #p [less than] 0.05 versus as indicated

APP was a target gene of miR-202

Based on these above observations, we explored the possible target gene of miR-202. A conserved binding site was identified in the 3′UTR of APP (Figure 5A). Dual luciferase assays showed that miR-202 suppressed the relative luciferase activity of pmirGLO-APP-3′UTR (Figure 5B). RT-PCR analysis showed that transfection with the miR-202 mimic significantly increased the relative level of miR-202 (Figure 5C). Western blot assays indicated that overexpression of miR-202 suppressed the expression of APP in PC12 cells (Figure 5D). In contrast, transfection with miR-202 inhibitor significantly suppressed the level of miR-202 (Figure 5E). Meanwhile, the expression of APP was enhanced after inhibition of miR-202 in PC12 cells (Figure 5F). These data suggested that APP was a possible target gene of miR-202.

FIGURE 5. Amyloid precursor protein (APP) was a target gene of miR-202. (A) Based on TargetScan, a conserved binding site was identified in the 3′UTR of APP. (B) Dual luciferase assays showed that miR-202 suppressed the relative luciferase activity of pmirGLO-APP-3′UTR. (C) Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that transfection with miR-202 mimic significantly increased the relative level of miR-202. (D) Western blot assays indicated that overexpression of miR-202 suppressed the expression of APP in PC12 cells. (E) Transfection with miR-202 inhibitor significantly suppressed the level of miR-202. (F) The expression of APP was enhanced after inhibition of miR-202 in PC12 cells. **p [less than] 0.01, ***p [less than] 0.001 versus control

Silencing APP abolished miR-202 inhibition-induced cell apoptosis

To further validate whether miR-202-mediated PC12 cell apoptosis was regulated by APP, a specific siRNA targeting APP was selected. As shown in Figure 6(A), the expression of APP was obviously reduced by si-APP, even in PC12 cells transfected with miR-202 inhibitor. More importantly, miR-202 inhibition-induced PC12 cell apoptosis could be largely reversed by knockdown of APP (Figure 6B). These observations indicated that reduced miR-202 promoted PC12 cell apoptosis by targeting APP.

FIGURE 6. Silencing amyloid precursor protein (APP) abolished miR-202 inhibition-induced cell apoptosis. (A) Western blot analysis showed that the expression of APP was obviously reduced by si-APP, even in PC12 cells transfected with miR-202 inhibitor. (B) TUNEL staining demonstrated that miR-202 inhibition-induced PC12 cell apoptosis could be largely reversed by knockdown of APP. **p [less than] 0.01, ***p [less than] 0.001 versus control

DISCUSSION

At present, there are no sensitive and specific biomarkers in the clinical diagnosis of AD.²⁴ Therefore, it is important to find potential biomarkers with high sensitivity and specificity in the diagnosis of AD, which can improve the early diagnosis ability of AD.²⁵ By RT-PCR, we found that the level of miR-202 in the serum of AD patients decreased significantly. ROC curve analysis showed that serum miR-202 could differentiate AD patients from healthy controls, which indicated that miR-202 has potential as a biomarker.

To further verify the relationship between miR-202 and AD, the correlation between miR-202 and the MMSE score was analyzed. The results showed that miR-202 was positively correlated with the MMSE score. This result again proved that miR-202 was closely related to AD. Furthermore, we also explored whether serum miR-202 can be used as a biomarker to classify diseases and monitor disease progression. The results showed that serum miR-202 was gradually decreased in accordance with the severity of the disease, which indicated that the abnormal change in miR-202 had taken place in the early stage of AD and that the abnormal changes remained stable during disease progression.

Apoptotic neurons are extensively identified in patients with AD.²⁶ During the progression of AD, a large quantity of neuronal loss is demonstrated, mainly due to apoptosis. Hence, the level of neuronal apoptosis may be an important indicator for AD to some extent. In the present study, we explored the effect of miR-202 on PC12 cell apoptosis. Interestingly, we found that suppression of miR-202 significantly promoted PC12 cell apoptosis. Then, we explored the possible target gene of miR-202. Here, we found that APP, the precursor substance of Aβ,²⁷ was the target gene of miR-202. Previous studies have shown that APP is closely related to the growth, development and pathological changes of the nervous system and may play an important regulatory role in the pathogenesis of AD.^28,29 Upregulation of APP is reported to induce abnormalities in hippocampal neurons and decrease cell survival, which results in neuronal dysfunction.³⁰

To further validate the inhibition of miR-202-induced PC12 cell apoptosis, we silenced APP using a specific siRNA targeting APP. Our data confirmed that si-APP reduced the expression of APP even in PC12 cells transfected with miR-202 inhibitor. The TUNEL assay showed that silencing APP reversed Aβ-induced cell apoptosis even in PC12 cells with miR-202 suppression. Based on these observations, we conclude that reduced expression of miR-202 contributes to cell apoptosis in the progression of AD by targeting APP.

There are limitations in the present study. First, the sample size is relatively small. Second, we did not explore the expression of miR-202 in the tissues of AD patients. Hence, the origin of circulating miR-202 could not been determined. Third, miR-202 may have other target genes and whether AD is totally or partially mediated through APP via miR-202 deserves further study. Fourth, increasing evidence has suggested that apolipoprotein E (APOE) increase AD risk.^31,32 However, we did not conduct APOE study in the present study. In the future, we will explore the correlation between miR-202 and APOE, which may further elucidate the pathological role of serum miR-202 in the progression of AD.

In summary, we show novel data that downregulation of serum miR-202 may be used as a potential biomarker for AD and that downregulation of miR-202 may promote the development of AD by suppressing APP.

CONFLICT OF INTEREST

All authors declare no conflict of interest.