Down regulation of miR‐218, miR‐124, and miR‐144 relates to Parkinson's disease via activating NF‐κB signaling

Abstract

Parkinson's disease (PD) is a neurological degenerative disorder that is partially induced by inflammation in the neural system. To explore the roles of disordered microRNAs in the development of PD, we screened 10 miRNAs in the brain samples of 15 postmortem PD patients and 10 postmortem healthy controls by qRT‐PCR. The direct targets of miRNAs were predicted by informatics tools and further confirmed by dual luciferase assay and immunoblotting. The function of miRNAs in regulating NF‐κB/p65 translocation was examined by immunoblotting, and the overactivation of NF‐κB signaling was examined by ELISA. The relationship between dysregulated miRNAs and cytokines was analyzed by correlation analysis. Three miRNAs were found to be reduced in the brains of patients with PD. KPNB1, KPNA3, and KPNA4 were identified as direct targets of miR‐218, miR‐124, and miR‐144. Additionally, KPNA3 was identified as a direct target of miR‐124, and KPNA4 was a direct target of both miR‐124 and miR‐218. The p65 translocation from the cytoplasm to the nucleus was repressed by miR‐124, miR‐218, and miR‐144 in the SH‐SY5Y cells. The NF‐κB signaling pathway was overactivated after miRNA inhibitor transfection. The upregulation of KPNB1, KPNA3, and KPNA4 in the brain samples of PD patients was confirmed by immunoblotting, and negative correlations were found between dysregulated miRNAs and cytokines. In conclusion, we identified that the downregulation of miR‐218, miR‐124, and miR‐144 in the brain was related to PD via activation of NF‐κB signaling, helping to unveil the role played by dysregulated miRNAs in the pathogenesis of PD and provide new potential targets for PD treatment.

1. INTRODUCTION

Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by the loss of dopaminergic neurons and usually leads to impairment of movement and speech. ¹ Until recently, the cause of PD has remained unclear, but abnormal inflammation and oxidative stress have been identified as two main factors related to the loss of dopaminergic neurons.2, 3

Nuclear factor κ light chain enhancer of activated B cells (NF‐κB) is a protein complex that regulates downstream gene transcription after stimulation. NF‐κB is a major transcription factor for immune responses and is broadly expressed in neural systems. Evidence shows that inflammation plays a key role in neurodegeneration in PD, and NF‐κB as the central inflammation mediator is an ideal target for anti‐inflammatory therapy.4, 5

MicroRNAs (miRNAs) are a group of short nonprotein coding RNAs that regulate mRNA translation through directly binding to the 3′UTR. It is estimated that more than 60% of human genes are modulated by miRNAs and almost all the pathophysiological processes related to miRNAs. ⁶ In the neural system, Kim et al. used Dicer conditional knockout (KO) mice and identified that Dicer is essential for dopaminergic neuron differentiation and maintenance. ⁷ It has also been reported that adults with a DGCR8 deletion have an increased occurrence of PD, ⁸ suggesting that miRNA biogenesis may play a role in the pathogenesis of PD. In humans, disordered miRNAs have been found in the brain, peripheral blood mononuclear cells, and serum from PD patients; but the underlying mechanisms are still not well understood. ⁹

In this study, we detected 10 candidate miRNAs in brain samples from 15 patients with PD and 10 healthy controls. We aimed to explore the role of disordered miRNAs in the development of PD.

2. MATERIALS AND METHODS

2.1. Subjects

All clinical samples were obtained from The First Affiliated Hospital of Jinzhou Medical University following the guidelines of the ethics committee of Jinzhou Medical University. PD symptoms were evaluated using the modified Heohn and Yahr scales. ¹⁰ Three patients had bilateral or midline involvement without the impairment of balance (HY‐2 stage). Six patients had postural reflex impairment (HY‐3 stage) and six patients had severe disability, but able to walk or stand unassisted (HY‐4 stage). Written informed consent was obtained from family members or authorized individuals. Brain samples of PD patients were obtained from the prefrontal cortex of the left cerebral hemisphere, cut into sections and stored at −80°C until use. Tissues from the prefrontal cortex region were selected for this study because the prefrontal cortex region plays an important role in PD ¹¹ and pathology does spread to this brain region. ¹²

Control brain samples were obtained from 10 healthy donors were identified as not suffering from neurological, metabolic, or mental disorders. The clinical characteristics of all participants are shown in Table 1.

TABLE 1.

Demographic and clinical characteristics of donors

	Control	PD	P value
Gender (female/male)	5/5	6/9	.65
Age (years)	70.60 ± 7.57	70.10 ± 7.05	.52
Age at onset (years)	—	62.57 ± 7.83	—
Disease duration (years)	—	5.93 ± 5.06	—
Heohn and Yahr scale	—	3.20 ± 0.77	—

Note: Data are expressed as mean ± SD. P values are calculated with Mann–Whitney test or un‐paired t test.

2.2. Cell culture

The human neuroblastoma cell line SH‐SY5Y was obtained from American Type Culture Collection (ATCC, Manassas, Virginia) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (HyClone Laboratories, Logan, Utah), 100 IU/mL of penicillin, and 100 IU/mL of streptomycin (HyClone). All cells were maintained at 37°C under an atmosphere of 5% CO₂.

2.3. MiRNA selection

We selected 10 candidate miRNAs that were found dysregulated in patients with PD.13, 14 Specifically, miR‐124 is a brain‐abundant miRNA that has been identified to inhibit NF‐κB signaling by targeting p65 in B‐cell lymphomas. ¹⁵ Moreover, miR‐218 was found to be related to hippocampal sclerosis. ¹⁶

2.4. RNA isolation and quantitative RT‐PCR

TRIzol reagent (Invitrogen, Carlsbad, California) was used to extract RNA from all the samples according to the manufacturer's instructions. Briefly, samples were resuspended in 100 μL phosphate‐buffered saline and then mixed with 1 mL TRIzol. After mixing with 200 μL chloroform and centrifuging, the supernatants were transferred into a new tube and the RNAs were precipitated by isopropanol. The RNA pellets were washed with 70% ethanol and then resolved. The RNA concentration and purity were determined using an ND‐2000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware). Only samples with absorbance ratios of ~2.0 at 260 nm/280 nm, and 1.9 to 2.2 at 260 nm/230 nm were considered for inclusion in the study.

Expression of miRNAs was determined by quantitative reverse transcription PCR (qRT‐PCR) using commercial TaqMan miRNA probes and primers. For normalization, U6 snRNA was used and each sample in each group was measured in triplicate. The relative expression of miRNAs was determined by the 2^−ΔΔct method ¹⁷ and the results were analyzed by Student's t‐test.

2.5. Dual luciferase assay

To construct the luciferase reporter vectors, the full length of KPNB1, KPNA3, or KPNA4 3′UTR was cloned into the pmirGLO plasmid (Promega, Madison, Wisconsin) downstream of the firefly luciferase gene. SH‐Y5Y cells were seeded in 48‐well plates attached overnight. One of the reporter vectors was co‐transfected into cells with miRNA mimic or inhibitor using Lipofectamine 2000 (Invitrogen) for 2 days followed by luciferase activity detection. The results were analyzed by Student's t‐test and shown as relative luciferase activity (Firefly Luciferase/Renilla Luciferase). miRNA mimic, miRNA inhibitor, and sequence scrambled single strand and double strand control oligo nucleotides were purchased from GenePharma Co., Ltd (Shanghai, China).

2.6. NF‐κB nuclear translocation

SH‐SY5Y cells were transfected with miRNA inhibitors for 48 hours and then treated with lipopolysaccharide (LPS). The cells were harvested for preparation of the cytoplasmic and nuclear extracts using NE‐PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, Rockford, Illinois) following the manufacturer's instructions. NF‐κB p65 levels in the extracts were examined by immunoblotting.

2.7. Immunoblotting

All protein samples were denatured by boiling in sodium dodecyl sulfate/β‐mercaptoethanol loading buffer and then separated using 10% PAGE gel. The proteins in the gels were blotted onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer and then incubated with one of the primary antibodies (anti‐KPNB1 rabbit polyclonal antibody (Abcam, Cambridge, UK), anti‐KPNA3 rabbit polyclonal antibody (Abcam), anti‐KPNA4 rabbit polyclonal antibody (Abcam) or anti‐β‐actin mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas)) overnight at 4°C after blocking with 5% nonfat milk. The membranes were incubated with horseradish peroxidase conjugated secondary antibody for another 2 hours at room temperature. The signals were then detected using an ECL kit (Pierce, Appleton, Wisconsin). The β‐actin signal was used as a loading control.

2.8. Statistical analysis

Statistical analysis was performed using the SPSS software version 19.0 (SPSS Inc., Chicago, Illinois). The data distributions were examined by D'Agostino‐Pearson normality test and. The data were considered to be normally distributed when P > .1, and the results were analyzed by unpaired t test. Otherwise, data were analyzed by Mann‐Whitney test. Correlations were evaluated by the χ ² test. A two‐tailed P value less than .05 was considered statistically significant.

3. RESULTS

To investigate the roles of miRNAs in the pathogenesis of PD, we detected the expression of 10 selected miRNAs in brain samples from 15 postmortem patients with PD and 10 postmortem healthy controls. Ten candidate miRNAs were all reported to be disordered in the brain samples from PD patients.13, 14 The levels of miR‐124, miR‐218, and miR‐144 were significantly reduced in patients with PD (Figure 1). Since miRNAs function as gene expression repressors targeting the 3′UTR of mRNAs, we predicted the direct target genes of miR‐124, miR‐218, and miR‐144 using the bioinformatics tool TargetScan (http://www.targetscan.org/vert_70/). Since the NF‐κB pathway plays a central role during immune responses in the neural system, we focused on the NF‐κB regulators as the predicted miRNA targets. We found three importins (KPNB1, KPNA3, and KPNA4), that regulate NF‐κB/p65 nucleo‐cytoplasmic transport,18, 19 and have the potential to be the direct targets of at least one of the three disordered miRNAs (Figure S1). To determine whether the expression of KPNB1, KPNA3, and KPNA4 was regulated by miR‐124, miR‐218, or miR‐144, we constructed the reporter vectors by cloning 3′UTRs of KPNB1, KPNA3, and KPNA4 into the pmirGLO plasmid. One of reporter vectors was co‐transfected with one of the candidate miRNAs into SH‐SY5Y cells for 48 hours, and the subsequent cell lysates were subjected to a dual luciferase assay. As shown in Figure 2A, the luciferase activity was repressed by miR‐218, miR‐124, and miR‐144 in cells transfected with the KPNB1 reporter vector. Additionally, the luciferase activity was repressed by miR‐124 in cells transfected with the KPNA3 reporter vector. Luciferase activity was also repressed by miR‐124 and miR‐218 in cells transfected with the KPNA4 reporter vector. These results indicated that these three selected miRNAs repressed luciferase expression by targeting the 3′UTR sequences of KPNB1, KPNA3, or KPNA4.

FIGURE 1.

miR‐124, miR‐144, and miR‐218 are reduced in patients with PD. The expression of 10 candidate miRNAs was detected by qRT‐PCR in brain samples from 15 postmortem patients with PD and 10 postmortem healthy controls. Results were analyzed by unpaired student's t‐test or Mann‐Whitney test. A two‐tailed P value less than .05 was considered statistically significant. *P < .05, **P < .01

FIGURE 2.

Confirmation of the direct targets of miR‐124, miR‐144 and miR‐218. One of the reporter vectors was transfected into SH‐Y5Y cells with one of the microRNA mimics for 48 hours. The lysates were subjected to luciferase assay. Results were analyzed by paired student's t‐test and P < .05 was considered as significant. *P < .05, **P < .01

To confirm whether endogenous KPNB1, KPNA3, and KPNA4 were repressed by selected miRNAs, the protein levels of KPNB1, KPNA3, and KPNA4 were detected with immunoblotting. The expression of KPNB1 was repressed by miR‐124, miR‐218, and miR‐144; the KPNA3 level was repressed by miR‐124, and the KPNA4 expression was repressed by miR‐124 and miR‐218 (Figure 2B).

To explore whether NF‐κB signaling was modulated by these three candidate miRNAs, inhibitors of miR‐124, miR‐144, and miR‐218 were transfected into SH‐SY5Y cells to mimic the conditions in the brain of PD patients. Then, the cells were treated with LPS to mimic LPS‐induced neuroinflammation. The repression of miR‐124, miR‐218, and miR‐144 was confirmed by qRT‐PCR(Figure 3A). The levels of KPNB1, KPNA3, KPNA4, and p65 were detected in the total cells lysate. The level of p65 was detected in both the nucleus and cytoplasm. The protein level of KPNB1 was elevated in the cells treated with the inhibitor of miR‐124, miR‐218, or miR‐144(Figure 3B). The KPNA3 level was increased by miR‐124 inhibitor, and the KPNA4 level was increased in cells transfected with miR‐124 or miR‐218 inhibitor(Figure 3B). In the miRNA inhibitor‐treated cells, the p65 level increased in the cell nucleus and decreased in the cytoplasm; whereas, the total p65 level was similar between these groups. This indicated that p65 translocation from the cytoplasm to the nucleus was promoted by the inhibitors of miR‐124, miR‐218, and miR‐144.

FIGURE 3.

Repression of miR‐124, miR‐144, and miR‐218 activates NF‐κB signaling by promoting p65 translocation. Inhibitors for miR‐124, miR‐144, and miR‐218 were transfected into SH‐SY5Y cells separately to mimic the conditions in the brain of PD patients. Forty‐eight hours after transfection, the cells were treated by LPS to mimic LPS‐induced neuroinflammation. The repression of miR‐124, miR‐218, and miR‐144 was confirmed by qRT‐PCR, A. The levels of KPNB1, KPNA3, KPNA4, and p65 in the total cells lysate, nucleus and cytoplasm were detected by immunoblotting, B. The levels of IL‐1β, TNF‐α, and IFN‐γ in the cell culture medium were examined by ELISA, C

To confirm whether NF‐κB signaling was activated, we measured the levels of IL‐1β, TNF‐α, and IFN‐γ in the cell culture medium by enzyme‐linked immunosorbent assay (ELISA). Significantly increased IL‐1β, TNF‐α, and IFN‐γ levels were observed in the cells transfected with the inhibitors of miR‐124, miR‐218, or miR‐144 (Figure 3C).

To understand whether KPNB1, KPNA3, and KPNA4 were upregulated in vivo, the protein levels of these three proteins were determined by immunoblotting and the results were quantified by Quantity One software. The levels of IL‐1β, TNF‐α, and IFN‐γ were also detected by immunoblotting. Significantly increased levels of KPNB1 and KPNA4 in brain samples from patients with PD (Figure 4A and B). Elevated IL‐1β, TNF‐α, and IFN‐γ levels were found in the brains of patients with PD (Figure 4C). Subsequently, the relationship between miRNAs and cytokines were analyzed. A strong negative correlation between TNF‐α and miR‐218(r = −.63, P = .0012), a weak negative correlation between TNF‐α and miR‐124(r = −0.47, P = 0.0017), and a weak negative correlation between TNF‐α and miR‐144(r = −.26, P = .039) were all found (Figure 5A). A strong negative correlation was found between IL‐1β and miR‐218 (r = −.52, P = .046) (Figure 5B). A weak negative correlation was found between IFN‐γ and miR‐218 (r = −0.43, P = .042) (Figure 5C).

FIGURE 4.

KPNB1, KPNA3, and KPNA4 are up‐regulated and the NF‐κB signaling is activated in patients with PD. The protein levels of KPNB1, KPNA3, and KPNA4 were determined by immunoblotting, the results were quantified and analyzed by unpaired student's t‐test or Mann‐Whitney test. Results were analyzed by unpaired student's t‐test. A two‐tailed P value less than .05 was considered statistically significant. *P < .05, **P < .01

FIGURE 5.

Correlation analysis of disordered miRNA and cytokines in patients with PD. The correlations between disordered miRNAs and cytokines were evaluated by the χ ² test. A two‐tailed P value less than .05 was considered statistically significant

4. DISCUSSION

Abnormal inflammation is one of the main factors that contribute to the loss of dopaminergic neurons in patients with PD. ²⁰ The NF‐κB pathway plays a central role during immune responses in the neural system. Overactivated NF‐κB signaling has been found to be related to the pathogenesis of PD. ²¹ While, researchers have found disturbed miRNAs levels in PD patients, the roles of most of these miRNAs during the PD pathogenesis are still controversial or unknown.13, 14 In the present study, we detected the levels of 10 candidate miRNAs in the brains of 15 PD patients and 10 controls. The levels of miR‐124, miR‐218, and miR‐144 were reduced significantly in the brains of PD patients. Regarding miR‐124, it is a brain abundant miRNA that was found to be reduced in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treated mouse model of PD. ²² Regarding miR‐218 and miR‐144, miR‐218 was found to be related to hippocampal sclerosis and miR‐144 was found to be downregulated in Patients with PD. ¹³ In B‐cell lymphomas, miR‐124 has been identified to inhibit NF‐κB signaling by targeting p65. ¹⁵ In the present study, it was identified for the first time that miR‐124, miR‐218, and miR‐144 inhibited NF‐κB signaling by targeting KPNB1, KPNA3, and KPNA4. Inhibition of miR‐124, miR‐218, or miR‐144 promoted the translocation of p65 from the cytoplasm to the nucleus and activated NF‐κB signaling. This finding partially unveiled the mechanism underlying the disordered miRNAs during the pathogenesis of PD.

The present study reported the overexpression of KPNB1, KPNA3, and KPNA4 in the brains of patients with PD for the first time. Through correlation analysis, it was found that TNF‐α level negatively correlated with miR‐218, miR‐124, and miR‐144. However, only miR‐218 level negatively correlated with IL‐1β and IFN‐γ levels in the brain. This suggested that other key regulators may be involved in the NF‐κB signaling regulation system that are partially antagonizing the functioning of miR‐124 and miR‐144.

Finally, miR‐124 has been found to be downregulated in the plasma sample of patients with PD ²³ and thus has the potential to be used as a biomarker for PD diagnosis. However, the relationship between the level of miR‐124 in the plasma and the brain needs to be further investigated.

Limitations were present in this study. First, brain samples used were obtained from the prefrontal cortex of the left hemisphere which plays an important role in PD because the pathology spreads to that region. However, it is not confirm whether this region was affected in every patient. Second, the mechanism studied only depends on SH‐SY5Y cells which is a human neuroblastoma cell line. These findings need to be confirmed for human primary neurons in the future.

In conclusion, this study identified that the downregulation of miR‐218, miR‐124, and miR‐144 in the brain was related to PD via activation of NF‐κB signaling, helping to which unveil the role played by dysregulated miRNAs during the pathogenesis of PD and provide new potential targets for PD treatment.

Supporting information

Figure S1 The schematic diagram of predicted interaction between miRNAs and target genes.

The interactions between dysregulated miRNAs and direct targets were predicted using TargetScan.

Xing R‐X, Li L‐G, Liu X‐W, Tian B‐X, Cheng Y. Down regulation of miR‐218, miR‐124, and miR‐144 relates to Parkinson's disease via activating NF‐κB signaling. Kaohsiung J Med Sci. 2020;36:786–792. 10.1002/kjm2.12241