Regulatory mechanism of miR-146a on MPTP-induced neuroinflammation in mice with Parkinson's disease

Purpose: To study the regulatory influence of microRNA-146a (miR-146a) on 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced neuroinflammation in mice with Parkinson's disease (PD). Methods: Forty specific pathogen-free (SPF) male C57BL/6 mice were divided into 2 groups: normal control and PD groups. The 2 groups were each divided into 2 subgroups: miR-146a inhibitor and inhibitor control groups. The mRNA and protein expressions of miR-146a, interleukin-1 receptorassociated kinase-1 (IRAK-1) and P65-NF-κB were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblot assay, respectively. Levels of interleukin (IL)-1, IL-6 and TNF-α were assayed by enzyme-linked immunosorbent assay (ELISA). Results: The level of expression of miR-146a was significantly and time-dependently increased in PD mice, relative to control (p < 0.05). In PD group, mRNA and protein expressions of IRAK-1 were markedly higher in miR-146a inhibitor group than in inhibitor control (p < 0.05). Protein expression of P65-NF-κB was significantly upregulated in brains of PD mice, relative to normal mice (p < 0.05). Moreover, IL-1, IL-6 and TNF-α levels were significantly higher in brains of PD mice than in control Conclusion: These results show the involvement of miR-146a in the etiology of PD, and that its regulation of neuroinflammation occurs via inhibition of IRAK1 gene expression. This finding may be useful in the development of new anti-PD drugs based on miR-146a.


INTRODUCTION
Parkinson's disease (PD), a degenerative disease of the CNS, is prevalent among older adults. It is characterized by tremor, myotonia and bradykinesia (reduced movement). The main pathological changes in PD are degeneration and death of dopaminergic neurons in substantia nigra of the brain. With a total incidence of 0.3 %, the disease ranks second only to Alzheimer's disease (AD) [1]. Parkinson's disease (PD) has a strong positive correlation with age [1]. The rise in its incidence places serious burden on the family of sufferers and society. Till date, the pathogenesis of PD is largely unknown, although it may entail environmental and genetic factors [2]. Studies have shown that damage to dopaminergic neurons in the substantia nigra results in mitochondrial dysfunction, neuronal immune response, inflammatory response, oxidative stress, apoptosis and autophagy. Moreover, defective mitochondrial function leads to apoptosis of dopaminergic neurons in dense area of substantia nigra. At present, there is a dearth of clinical biomarkers for early diagnosis of PD.
Only recently, the role of miRNA in the immune system received attention. A non-coding RNA with regulatory functions, miRNA participates in physiological processes of the cell via regulation of genes after transcription [4]. A role exists for miRNAs in the regulation of the progression of neurodegenerative diseases, including PD [5]. MicroRNA-146a (miR-146a) is highly expressed in the CNS. Its expression has been reported to be significantly downregulated in serum of patients with AD. It has also been demonstrated to promote inflammatory response of cells via increased expression of TLR2 gene [6]. The aim of this research was to study the regulatory mechanism of miR-146a on MPTP-induced neuroinflammation in PD mice.

Mice
Specific-pathogen-free (SPF) male C57BL/6 mice (n = 40) aged 8 -10 weeks and weighing 20 -26 g (mean age = 9 ± 1 weeks; mean weight = 23 ± 3 g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co. Ltd. The mice were accommodated in plastic cages under equal light/dark photoperiod, and were permitted unlimited access to water and feed. The study protocol received approval from the Institutional Animal Ethics Committee of People's Hospital of Ningxia Hui Autonomous Region (Approval No.200903041), according to "Principles of Laboratory Animal Care" (NIH publication no. 85-23, revised 1985) [7].
Two groups of mice were used, each with 20 animals: normal control and PD groups. The 2 groups were each divided into 2 subgroups (5 mice/group): miR-146a inhibitor and inhibitor control groups. Parkinson's disease was induced with single i.p. administration of MPTP (30 mg/kg). Normal control mice received equivalent volume of normal saline. The mice were euthanized at different time points: day 0, 1, 5, 10 and 20 post-induction. Complete brain tissue of each mouse was excised and the midbrain removed and used for biochemical analysis. Moreover, lateral ventricles of mice in the subgroups were injected with miR-146a inhibitor or inhibitor control. Two days after induction of PD, the mice were anesthetized and dissected to obtain brain tissues which were stored at -80 ℃ prior to analysis.

qRT-PCR assay
After thawing, frozen mouse brain was homogenized with TRizol reagent. Total RNA was also obtained with TRIzol extraction, and its quality and concentration were measured colorimetrically. The total RNA was then converted to cDNA according to the instructions of reverse transcription kit. Reverse transcription was carried out on PCR machine at 42 o C for 50 min, with incubation for 5 min at 95 o C. The synthesized cDNA was amplified via PCR for determination of the expression levels of miR-146a, IRAK1 and P65-NF-κB under standard assay conditions. The relative mRNA levels were estimated using 2 -ΔΔCt procedure, with β-actin as standard gene.

ELISA
The levels of IL-1, IL-6 and TNF-α were determined using ELISA. Mid-brain tissues of mice were homogenized with ice-cold normal saline and centrifuged at 3500 rpm for 10 min to obtain clear supernatant which was stored overnight at 4 o C. After washing with PBS for 1 min, 200 µL of blocking solution was added, and after 1 h, washing was done thrice with PBS within 3 min. Blank control, standard and sample were added to their respective wells and incubated for 1 h at 37 o C. The mixture was rinsed with PBS for 3 min. This was followed by the addition of enzyme-labeled secondary antibody, and after 30 min, rinsing was done with PBS for 3 min. Exactly 50 µL of substrate A/B was put in well and incubated for 20 min at 37 o C. After a 10-min agitation, the optical density of each well was determined at 450 nm.

Statistics
Measurement results are presented as mean ± SEM, and were statistically analyzed with SPSS (21.0). Group comparisons were done with t-test, with p < 0.05 indicating statistically significant differences.

Expression level of miR-146a in brain tissues of mice with PD
The level of expression of miR-146a was significantly and time-dependently increased in PD mice, relative to normal control group ( Figure  1).

Expression levels of IRAK-1 mRNA and protein in mice brain tissues
These were markedly lower in brain tissues of PD mice than in normal control mice (p < 0.05).
In PD group, IRAK-I mRNA and protein were markedly higher in miR-146a inhibitor subgroup than in inhibitor control subgroup ( Table 2).

Expression levels of NF-κB protein in brains of mice with PD
As shown in Figure 2 and Figure 3, protein expression of P65-NF-κB was markedly increased in brains of mice with PD, relative to normal control group. In contrast, in PD group, there was no marked difference in P65-NF-κB protein expression between miR-146a inhibitor and inhibitor control subgroups.   Table 3 and Figure 4 show that inflammatory cytokines were markedly higher in brains of PD mice than in normal control group, but the levels of these inflammatory cytokines were comparable between miR-146a inhibitor and inhibitor control subgroups in mice with PD.

DISCUSSION
Parkinsonism is a neurodegenerative ailment marked by a sustained loss of dopaminergic neurons from brain stem. The pathology of the disease is based on degeneration of dopaminergic neurons and attendant decrease in dopamine levels in dense region of substantia nigra [8]. Statistics show that PD is prevalent among older adults (age ≥ 60 years), with total incidence of 1 -2 %. The pathogenesis of PD is complex, and its diagnosis is based mainly on clinical manifestations presented.  In recent times, pathogenic genes have been associated with PD [9]. The involvement of miRNAs in the pathogenesis of PD has been reported [9]. MicroRNAs (miRNAs) are noncoding RNAs which possess regulatory function. These miRNAs which contain approximately 20 -24 nucleotides, exist freely in cells. MicroRNAs (miRNAs) participate in the regulation of gene transcription via complementary binding to 3' untranslated region (3' UTR) of the gene. Their interactions with genes inhibit or induce mRNA degradation, thereby affecting the stability of mRNA.
Studies have shown that miRNA genes make up one-third of the human genome [10,11]. While a single miRNA can regulate multiple target genes, multiple miRNAs may combine to regulate a single target gene. MicroRNA-mediated gene expression are vital in the maintenance of normal cell division cycle, apoptosis and intermediary metabolism [12]. It has been reported that multiple miRNAs participate in the pathogenesis of PD [13]. The mechanism involved in posttranscriptional regulation by miRNA has been demonstrated to be involved in the pathogenesis of PD, and it is thought to serve as sensitive index for diagnosis of PD as well as evaluation of disease progression and prognosis [14].
MicroRNA-146a (miR-146a) is an NF-κBdependent molecule located on chromosome 5 in humans. Indeed, miR-146a is an immunemodulatory molecule implicated in tumorigenesis and inflammatory response, and it is abnormally expressed in a variety of nervous system tumors. A study has reported that miR-146a was markedly lower in cerebrospinal fluid (CSF) of PD subjects than in normal healthy patients [15].
Interleukin-1 receptor-associated kinase-1 (IRAK-1) gene which encodes 712 amino acids, is located on chromosome Xq28. Its 3' UTR and exon region with two polymorphism loci (rs3027898 and rs1059703), promote NF-κB activation. Polymorphism in IRAK1 is associated with increased susceptibility to certain diseases such as whooping cough and systemic lupus erythematosus (SLE) [16]. Studies have shown that miR-146a exerts its role in disease via regulation of IRAK-1 gene. In addition, miR-146a suppresses inflammation via mechanisms involving inhibition of NF-κB pathway and downregulation of IRAK-1 protein expression [17].
The present study has demonstrated that miR-146a expression level was significantly and timedependently increased in PD mice, relative to normal control group, an indication that miR-146a may be implicated in the etiology of PD. In PD mice, IRAK-1 mRNA and protein expression levels were markedly higher in miR-146a inhibitor group than in inhibitor control group. Similarly, P65-NF-κB protein, IL-1, TNF-α and IL-6 in brains of mice with PD were markedly higher than those in normal control mice, an indication that miR-146a may negatively regulate IRAK-1 gene and activate NF-κB pathway downstream via regulation of expression of related inflammatory cytokines. Results of previous studies show that the promoter region of miR-146a contains a binding site for NF-κB, which enhances the transcription of miR-146a, while downregulating IRAK1 protein expression [17][18][19].

CONCLUSION
The results obtained in this study show that miR-146a is implicated in the etiology of PD, and its regulation of neuroinflammation occurs via inhibition of IRAK-1 gene expression. This finding may be useful in the quest for new anti-PD drugs based on miR-146a.