MiR-9-5p Inhibits the MMP+-Induced Neuron Apoptosis through Regulating SCRIB/β-Catenin Signaling in Parkinson's Disease

The pathogenesis of Parkinson's disease remains unclear that there is no cure for Parkinson's disease yet. The abnormal expressions of certain miRNA are closely related to the occurrence and progression of Parkinson's disease. Here, we demonstrate that miR-9-5p inhibits the dopaminergic neuron apoptosis via the regulation of β-catenin signaling which directly targets SCRIB, a tumor suppressor gene. Besides, miR-9-5p improved the motor function of mice with Parkinson's disease. The results of this study suggest that miR-9-5p might be a potential therapeutic target against Parkinson's disease.

2.6. Flow Cytometry. Cell apoptosis was evaluated by flow cytometry, using FITC Annexin-V (Becton Dickinson). The washed cells were resuspended in binding buffer at a final concentration of 1 × 10 6 /ml. According to the manufactures' instruction, AV-FITC and/or PI were added into the tube and incubated at room temperature for 15 min in dark. Then, samples were detected in an hour.  The apoptosis rate of MN9D cells in the mmp + group was higher than that in the control group (P < 0:05). (c, d) The expression of cleaved-caspase 3 was higher in the mmp + group than in the control group, and the expression of bcl-2 was lower (P < 0:05). (e, f) The expression of cleaved-caspase 3 was higher in PD mice (P < 0:001). The expression of TH was lower ðP > 0:05Þ. (g) The expression of mmu-miR-9-5p was decreased in the mmp + group than in the control group (P < 0:05). Scale bar: 100 μm.   Oxidative Medicine and Cellular Longevity 2.7. Luciferase Reporter Assay. Putative binding site between mmu-miR-9-5p and SCRIB was predicted by the miRNA database (http://www.targetscan.org). The mmu-miR-9-5p sequence binding to the 3 ′ UTR of SCRIB, either wildtype or mutant, was cloned into the pMIRREPORT vector (Ambion, USA). MN9D cells were cultured in 24-well plates and transfected with 0.1 μg of luciferase reporter vectors contained mmu-miR-9-5p mimics or control miRNA. Renilla luciferaseexpressing vector (pRL-TK, Promega, USA) was cotransfected for normalization. Cells were harvested after 48 h transfection.

Oxidative Medicine and Cellular Longevity
According to the manufacturer's instruction, Firefly and Renilla luciferase activities were detected using the Dual-Luciferase Reporter Assay System (Promega).

Statistical
Analysis. Data were expressed as mean ± standard error. Statistical analysis was performed with SPSS 20.0 software. Differences between means were assessed by Student's t-test for normal distribution data or Mann-Whitney U test for nonnormal distribution data. In multiple comparisons, one-way analysis of variance (ANOVA) was adopted. A value of P < 0:05 was considered statistically significant.

MPTP Induces Apoptosis in Dopaminergic Neurons.
Abnormal apoptotic signaling is involved in the progression of neurodegenerative diseases, including PD [21]. In this study, apoptosis was increased in MPTP-treated MN9D cells. Flow cytometry showed that the apoptosis rate in MN9D cells treated with MMP + was significantly higher than that of the control group (Figures 1(a) and 1(b)). Meanwhile, the protein expression of cleaved-caspase 3 was higher in the later than the former, and the protein expression of bcl-2 was the opposite (Figures 1(c) and 1(d)). In vivo, cleaved-caspase 3 was upregulated in nigrostriatal system in MPTP-treated mice (Figures 1(e) and 1(f)). To investigate whether miR-9-5p is involved in PD, we examined the level of mmu-miR-9-5p. Results showed that the expression of mmu-miR-9-5p was reduced in MMP + -treated MN9D (Figure 1(g)).
3.2. mmu-miR-9-5p Alleviates the MPTP-Induced Apoptosis in Dopaminergic Neurons. We tried to restore the expression of mmu-miR-9-5p in the PD cell model. With gene transfection, we successfully enhanced the expression of mmu-miR-9-5p in MN9D cells (Figure 2(a). Then, we evaluated the trend of apoptosis. The apoptosis rate of mmu-miR-9-5p treated cells was decreased (Figures 2(b) and 2(c)). Western blot showed that the expression of cleaved-caspase 3 was reduced while the expression of bcl-2 was upregulated in mmu-miR-9-5p treated cells (Figures 2(d) and 2(e)).

Oxidative Medicine and Cellular Longevity
3.6. mmu-miR-9-5p Improves the Behavior of PD Mice. To verify the effect of mmu-miR-9-5p in vivo, mmu-miR-9-5p was administered in PD mice model. It seemed that athletic ability of PD mice was improved (Figures 5(a) and 5(b)). Furthermore, brain tissue staining showed more TH-positive cells in the mmu-miR-9-5p group than in the control group (Figures 5(c) and 5(d)). Immunofluorescence results showed that the expression of TH in the mir-9 treated group was higher than that in the untreated group, and there was no statistical significance between the two groups in the cleaved-caspase 3 expression (Figures 5(e) and 5(f)).

Discussion
The decrease or dysfunction of dopaminergic neurons is the main cause for Parkinson's disease [23]. Apoptosis, a programmed cell death, is an effective way to eliminate the aged or aberrant cells, thus maintain the self-renewal of organs [24]. The abnormality of the apoptosis is one of the pathogenesis of many neurodegenerative diseases, such as Parkinson's disease, and subsequently leads to the loss of dopaminergic neurons in the substantia nigra pars compact [25,26]. The measurements that promote apoptosis could be used against the progression of Parkinson's disease and to improve the patients' prognosis. MiR-9-5p is crucial in the development of the nervous system, targeting different mRNAs. It regulates several physiological processes in neural precursor cells, such as proliferation, migration, and differentiation [27]. The role of miR-9-5p in neurodegenerative diseases is complex. Studies have shown that the expression of miR-9-5p is changed with time and lesion site depended in Alzheimer's disease [15,16,[28][29][30], which means the roles of miR-9-5p relying on neuron types. The expression of serum miR-9-5p was significantly higher in treated Parkinson's patients than untreated Parkinson's patients and healthy people [31]. MiR-9-5p is upregulated in PD patients' dopaminergic neurons via somatic cell reprogramming and induced pluripotent stem cells' differentiation [18]. Our evidence demonstrated that miR-9-5p protects dopaminergic neuron from apoptosis which induced by MMP + . Taken together, the neuroprotective effect of miR-9-  Figure 4: SCRIB inhibits the protective effect of mmu-miR-9-5p on apoptotic cell. Western blot showed that after the SCRIB expression was restored, mmu-miR-9-5p lost its regulation of β-catenin (a, b, e), while the expression of p-p38,p-JNK, and p-AKT was not affected (a, c, d, f).
(g, h) The apoptosis rate of MN9D cells was also out of control of mmu-miR-9-5p after the SCRIB expression was restored (P > 0:05).

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Oxidative Medicine and Cellular Longevity 5p is undisputed. The upregulation of miR-9-5p in Parkinson's disease might be a self-protective feedback. Drug-induced β-catenin signaling is effectively counteracted the toxicity of dopaminergic neurons, leading to neuroprotection and neurorestoration [32][33][34]. Our data showed that miR-9-5p activates β-catenin signaling which was inhibited by MPTP in dopaminergic neurons. MiR-9-5p has potential to be a therapeutic agent for PD. Besides, studies suggest that βcatenin is a vital pathway for dopaminergic neurogenesis. The unusual β-catenin pathway may precede and/or accompany PD onset and progression [35]. In MPTP treated mice, cell proliferation in subventricular zone (SVZ), riched in neural stem cells, was significantly inhibited with decrease of β-catenin signal [34]. Therefore, miR-9-5p administration may promote the proliferation and differentiation of neural stem cells through activating β-catenin signaling and regain the dopaminergic neurons as well.
Studies have shown that SCRIB bind to β-catenin form stable complexes, promoting β-catenin degradation [22]. Our data suggested that SCRIB is the target of miR-9-5p, and the regulation of miR-9-5p on β-catenin is not direct but realized by SCRIB.

Conclusion
MiR-9-5p inhibits the apoptosis of dopaminergic neurons in PD and improves the symptoms of Parkinson's disease, involving in a variety of signaling pathways. MiR-9-5p upregulates β-catenin signaling pathway by directly targeting SCRIB. In conclusion, miR-9-5p has great potential to be a therapeutic target for Parkinson's disease.

Data Availability
The article data used to support the findings of this study are included within the article. The mir-9 database is on the http://mirdb.org.

Additional Points
Contribution to the field. Parkinson's disease is a complex challenge of neuroregulatory disorder. There is still no cure for Parkinson's disease, and even the pathogenesis of Parkinson's disease remains unclear. The abnormal expression of miRNA is closely related to the occurrence and progression of Parkinson's disease. Here, we demonstrated that miR-9-5p inhibited the apoptosis of dopaminergic neurons by regulating β-catenin signaling, directly targeting SCRIB, which was regard as a tumor suppressor gene. Besides, miR-9-5p improved the motor function of mice with Parkinson's disease. The results of this study suggest a potential therapeutic target for Parkinson's disease.

Conflicts of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors' Contributions
Zhenyong Xiao, Xiang Sun, and Zhenxing Yan planned and designed the experimental scheme and the initial draft of the paper, animal model sample collection, and performed experiment. Baoyan Wang, Mengqi Gao, Fengfei Lu, and Jian Liu participated in the cell culture and microRNA extraction experiments and participated in the collation of experimental data, statistics, and results analysis. Zhitao Zong, Hongbo Zhang, and Yanwu Guo were fully responsible for the implementation and supervision of the subject. Zhenyong Xiao, Zhenxing Yan, and Xiang Sun contributed equally to the research.  Figure 5: mmu-miR-9-5p improved the behavior of PD mice. Pole test (a) and hang test (b) were used to evaluate the motor function of mice. (c, d) Immunofluorescence showed that TH positive cells were significantly higher than the control group (P < 0:05).