LncRNA SOX2-OT Participates in Parkinson’s Disease Through Regulating miRNA-942-5p/NAIF1 Axis

Parkinson’s disease (PD) is a neurodegenerative disease. Studies have shown that lncRNA SOX2-OT was highly expressed in PD patients, but its specic functions and mechanisms still need further research. This study aimed to explore whether lncRNA SOX2-OT could regulate oxidative stress, inammation and neuronal apoptosis in PD in vitro model and explored the underlying mechanism. An in vitro PD cell model was induced by 1-methyl-4-phenylpyridinium (MPP + ). The results of the biological software analysis and luciferase reporter assay indicated that miR-942-5p was a direct target of lncRNA SOX2-OT, and NAIF1 was a direct target of miR-942-5p. Experiments showed that the expression levels of lncRNA SOX2-OT and NAIF1 were increased, and miR-942-5p expression was decreased in SH-SY5Y cells following MPP + treatment. In addition, MPP + treatment reduced SH-SY5Y cell viability, induced apoptosis, increased cleaved-Caspase3 protein expression, and increased cleaved-Caspase3/Caspase3 ratio, increased LDH viability, and increased the levels of TNF-α, IL-1β and ROS in SH-SY5Y cells, reduced SOD activity, however, all these effects were inhibited by SOX2-OT-siRNA, and these inhibitions were reversed by miR-942-5p inhibitor. Moreover, the protective role of miR-942-5p mimic in MPP + induced SH-SY5Y cells was signicantly eliminated by NAIF1-plasmid. In summary, this study conrmed that lncRNA SOX2OT regulated oxidative stress, inammation and neuronal apoptosis via directly regulating the miR-942-5p/NAIF1 signal axis, and then participated in the occurrence and development of PD. These data provide a new potential targets for PD diagnosis and treatment.


Introduction
Parkinson's disease (PD) is a universal neurodegenerative disease, this disease results primarily from the death of dopaminergic neurons in the substantia nigra [1,2]. There were many theories about the pathogenesis of PD. Patel et al pointed out that this disease was caused by a combination of age, genetic and environmental factors [3], These results showed that the pathogenesis was complicated. At present, more and more evidence indicated that de cits in mitochondrial function, oxidative stress, in ammation and apoptosis were the most important factors [4,5], but the speci c mechanism is not yet clear. Therefore, it is very necessary to study the mechanism and develop treatment methods of PD.
Long non-coding RNA (LncRNA) has a signi cant function in the development and disease of the central nervous system (CNS), a number of lncRNAs were highly expressed in the adult and developing brain [6].
Recent research demonstrated that lncRNAs interfere in transcriptional and translational processes, and there were 5 lncRNAs that were clearly differentially expressed in PD [7]. There were increasing evidences that lncRNA plays a crucial role in the development of PD [8,9]. Long et al showed that miRNA and lncRNA co-regulate the pathogenesis of PD [9]. Lu et al found that lncRNA MALAT1 targeting miR-4 contributes to cell apoptosis in PD [10], and the research results of Xie et al showed that up-regulated lncRNA small nucleolar RNA host gene 1 promotes MPP + -induced reactive oxygen generation and cytotoxicity in human dopaminergic SH-SY5Y cells through the miR-15b-5p /GSK3β axis [11]. Moreover, SOX2-OT as a lncRNA, and some microRNAs (miRNAs) have a targeting relationship in disease [12]. The role and speci c mechanism of SOX2-OT in PD remain to be investigated.
As we all know, miRNAs regard as one kind of small non-coding RNAs that modulate gene expression at the post-transcriptional level [13]. In addition, miRNAs can participate in a variety of pathological processes in a variety of pathways [14]. miR-942-5p as a miRNA was related to the pathogenesis of many neurodegenerative diseases, such as attention de cit hyperactivity disorder [15], however, there were few reports on the role of miR-942-5p in PD. Past studies had shown that cell apoptosis is involved in the pathological mechanisms of PD [16,17], however, the nuclear apoptosis-inducing factor 1 (NAIF1) was a very important substance in the process of cell apoptosis [18,19]. Moreover, study has shown that the expression levels of miRNA and NAIF1 were related [20].
Therefore, this study aimed to explore whether lncRNA SOX2-OT participated in PD through regulating oxidative stress, in ammation and neuronal apoptosis via directly regulating miR-942-5p. Our research will provide new strategies for PD diagnosis and treatment.

Materials And Methods
Acquisition and culture of human neuroblastoma SH-SY5Y cells Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were stored in Dulbecco's modi ed Eagle medium (DMEM) medium (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin/streptomycinin, 10% fetal bovine serum (FBS, Gibco) and incubated in a humidi ed atmosphere containing 5% CO 2 at 37℃.

Dual-luciferase reporter veri cation
The association between lncRNA SOX2-OT and miR-942-5p was identi ed by bioinformatics software (Starbase). In addition, TargetScan determined the relationship between miR-942-5p and NAIF1. We then used a dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China) and the QuikChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA) to generate NAIF1-WT, NAIF1-MUT, SOX2-OT-WT and SOX2-OT-MUT according to the manufacturer's instructions. Finally, the luciferase activity was analyzed by the dual luciferase reporter gene analysis system (Promega, USA).
RNA extraction and quantitative real-time PCR (qRT-PCR) Total cellular RNA was isolated from SHSY5Y cells using TRIzol reagent (Invitrogen, USA), and reverse transcribed into rst-strand cDNA using a cDNA Synthesis Kit (Invitrogen) according to the instructions provided by the manufacturer. The expression levels of miR-942-5p, lncRNA SOX2-OT and NAIF1 mRNA were quanti ed by the Prism 7000 real-time PCR system using Power SYBR Green Master mix (Vazyme, Piscataway, NJ, USA) according to the instructions provided by the manufacturer. The ampli cation conditions were as follows: denaturation at 94℃ for 35 cycles of 60 seconds, anneal at 60℃ for 60 seconds, then extend at 72℃ for 1 minute, and then at 72℃ for 10 minutes. U6 and GAPDH were used as inner control genes. Calculate the relative expression levels of miR-942-5p, lncRNA SOX2-OT and NAIF1 mRNA by the 2 -ΔΔCt method [22].
3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay SH-SY5Y cells were inoculated into 96-well plates in triplicate, incubating overnight. Subsequently, the medium was removed, and after transfection of the cells at 37℃, the cells were treated with 1 mM MPP + . The cells were incubated with 10 μl MTT solution (Beyotime, Shanghai, China) for 4 hours. Subsequently, after removing the solution, adding 100 μl dimethyl sulfoxide (DMSO) to each well to dissolve the formazan product. Detection was achieved by monitoring the absorbance at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). The optical density value was used to normalize the relative cell viability relative to the control group.
Flow cytometry (FCM) to detect apoptosis SH-SY5Y cells were seeded into 6-well plates overnight, and collected by trypsinization following treatment. Washing the cells once with PBS buffer and subsequently re-suspended in 1 × binding buffer. A total of 100 µl of cell suspension was transferred into a 5 ml test tube and mixed with 5 μl uorescein isothiocyanate (FITC)-Annexin V and 5 μl propidium iodide (PI) (BD Biosciences, San Diego, CA), respectively, according to the manufacturer's speci cations. We analyzed the induction of apoptosis with a FACSCalibur ow cytometer (BD Biosciences, USA) within one hour, and analyzed the data with FlowJo software (version 7.6.1; FlowJo LLC).

Western blot analysis
After 48 hours of transfection and cell culture, SH-SY5Y cells were treated with 1 mM MPP + for 24 hours, washed three times with cold PBS, and immediately lysed with RIPA lysis buffer (Beyotime, Shanghai, China). The lysate was centrifuged at 12,000 rpm at 4℃ for 10 minutes, and the total protein level was measured by the BCA protein kit (Pierce, USA). Equal amounts of protein samples were separated using a sodium dodecyl sulfate (SDS) gel-polyacrylamide gel electrophoresis (PAGE), and then transfer it to the PVDF membranes. After sealing with 5% skim milk for 1 hour, the PVDF membranes were incubated with NAIF1 (1: 1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved-Caspase3 (1: 1,000; Cell Signaling Technology, Inc.), Caspase3 (1: 1,000; Cell Signaling Technology, Inc.), GAPDH (1:1,000; Cell Signaling Technology, Inc.) antibodies, immediately overnight at 4℃. The next day, the membranes were washed 3 times with PBST buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; Cell Signaling Technology, Inc.) at 37℃ for 1 hour. We nally visualized the protein bands using ECL luminescent substrate (Pierce) according to the manufacturer's instructions. The experiments were repeated for 3 times at least.
Lactate dehydrogenase (LDH) activity assay SH-SY5Y cells were cultured with 1 mM MPP + for 24 hours, then the activity of LDH released into the culture medium was measured using a lactate dehydrogenase assay kit (Jiancheng Institute of Bioengineering, China) according to the manufacturer's instructions. A micro-plate reader (Bio-Rad, Hercules, CA, USA) was used to record the absorbance at 490 nm.
ELISA assay SH-SY5Y cells were treated with MPP + for 24 hours, gathered and centrifuged to detect the expression levels of TNF-α and IL-1β using an ELISA kit (BioLegend, Inc., CA, USA) according to the instructions provided by the manufacturer. A microplate reader (Bio-Rad, Hercules, CA, USA) was employed to measure the absorbance at 450 nm.

Reactive oxygen species (ROS) release and superoxide dismutase (SOD) activity test
The treated cells were incubated with 10 μM DCFH-DA (Sigma) at 37°C for 45 minutes in the dark. A uorescence microplate reader (Labsystems Oy, Helsinki, Finland) was used to quantify the uorescence intensity, using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
SH-SY5Y cells were gathered and lysed using cell lysis buffer (Beyotime, Shanghai, China). According to the manufacturer's instructions, SOD activity assay kit (China Jiancheng Institute of Biological Engineering) was used to determine the SOD activity.

Statistical analysis
The experimental data were indicated as the mean ± standard deviation (SD) of at least three independent experiments. SPSS 13.0 software was performed for statistical analysis. The difference between the two groups were determined by Student's t-test, and the one-way analysis of variance followed by Bonferroni post-hoc test was applied to analyze the difference between multiple groups. The p-value below 0.05 (P <0.05) was identi ed as a signi cant difference.
LncRNA SOX2-OT in uences neuronal apoptosis, in ammatory response and the induction of oxidative stress in MPP + -induced SHSY5Y cells To explore the mechanism of lncRNA SOX2-OT affecting PD, the SH-SY5Y cells were transfected with control-siRNA, SOX2-OT-siRNA, SOX2-OT-siRNA + inhibitor control or SOX2-OT-siRNA + miR-942-5p inhibitor for 48 h, and then treated with 1mM MPP + for 24 h. These cells were divided into the following groups: control group; MPP + group; MPP + + control-siRNA group; MPP + + SOX2-OT-siRNA group; MPP + + SOX2-OT-siRNA + inhibitor control group; MPP + + SOX2-OT-siRNA + miR-942-5p inhibitor group. Subsequently, MTT detected the cell viability; LDH assay detected LDH activity; ow cytometry detected apoptosis; western blotting detected the protein expression of cleaved-Caspase3 and calculated the ratio of cleaved-Caspase3/Caspase3; ELISA was used to examine the section of TNF-α and IL-1β; ROS release and SOD vitality were also tested. The results revealed that compared to the control group, MPP + treatment observably decreased SH-SY5Y cell viability ( Figure 4A), increased the activity of LDH ( Figure  4B), induced apoptosis ( Figure 4C and D), increased cleaved-Caspase3 protein expression ( Figure 4E), and enhanced the ratio of cleaved-Caspase3/Caspase3 ( Figure 4F); compared with the MPP + + control-siRNA treatment group, SOX2-OT-siRNA increased the viability of SH-SY5Y cells, decreased the activity of LDH, reduced cell apoptosis, and decreased cleaved-Caspase3 protein expression, and reduced the ratio of cleaved-Caspase3/Caspase3, and these effects were signi cantly eliminated by miR-942-5p inhibitor.

NAIF1 is a target of miR-942-5p
In order to study the molecular mechanism of miR-942-5p and NAIF1 in PD, the relationship between miR-942-5p and NAIF1 was examined. TargetScan results indicated the binding sites between miR-942-5p and NAIF1 ( Figure 6A). Moreover, a luciferase reporter assay was performed in 293T cells in order to ascertain the predicted binding sites of NAIF1 and miR-942-5p. The experimental results con rmed the direct targeting relationship between miR-942-5p and NAIF1( Figure 6B).
Besides, the expression level of NAIF1 were detected by qRT-PCR in SH-SY5Y cells treated with MPP + .
And SH-SY5Y cells were treated with 0, 0.25, 0.5, 1 or 2 mM MPP + (Sigma, St. Louis, MO, USA) for 24 h, or exposed to 1 mM MPP + for 0, 6, 12, 24 or 48 h. The experimental results revealed that compared to the control group, MPP + increased the mRNA expression level of NAIF1 ( Figure 6C, 6D) in a dose-dependent and time-dependent manner in SH-SY5Y cells.
MiR-942-5p negatively regulate NAIF1 expression in SH-SY5Y cells To explore the contribution of miR-942-5p and NAIF1 to the development of PD, the SH-SY5Y cells were transfected with mimic control, miR-942-5p mimic, control-plasmid, NAIF1-plasmid, miR-942-5p mimic + control-plasmid or miR-942-5p mimic + NAIF1-plasmid for 48 h. The transfection e ciency was determined by qRT-PCR and western blotting. The experimental results revealed that compared to the mimic control group, miR-942-5p mimic observably increased the expression of miR-942-5p in SH-SY5Y cells ( Figure 7A); Compared to the control-plasmid group, NAIF1-plasmid observably increased the expression of NAIF1 mRNA in SH-SY5Y cells ( Figure 7B). Compared with the mimic control group, miR-942-5p mimic signi cantly reduced the expression of NAIF1 mRNA and protein in SH-SY5Y cells, and these reductions were signi cantly eliminated by NAIF1-plasmid ( Figure 7C and D).

Discussion
LncRNAs had appeared as a novel regulator of neurogenesis [23]. SOX2OT as a lncRNAs which harbors one of the primary regulator of pluripotency [24], at present, studies had proved that lncRNA was signi cantly highly expressed in PD [25][26][27]. In addition, a mass of studies had proved that miRNAs can participate in the development of various diseases by regulating the expression levels of key factors for cell growth and apoptosis [28][29][30], there were reports in the literature that there was a targeting relationship between NAIF1 and certain miRNAs [31]. However, in ammation, oxidative stress and neuronal apoptosis were also related to the pathogenesis of PD. These evidences indicated that lncRNA may have a certain relationship with miRNAs and NAIF1 to participate in the development of PD, but the speci c mechanism is still unclear.
In this study, the prediction analysis con rmed that lncRNA SOX2-OT directly targeted miR-942-5p and NAIF1. This research revealed that the expression level of lncRNA SOX2-OT and NAIF1 were increased signi cantly, while the expression level of miR-942-5p was decreased following an increase in the concentration levels of MPP + and in the treatment period, which was similar to the results of previous studies [1,21]. In addition, the effects of lncRNA SOX2-OT, miR-942-5p and NAIF1 were studied on the in vitro PD cell model, MPP + was a commonly used substance for establishing PD in vitro models [32]. The results demonstrated that the downregulation of the expression level of lncRNA SOX2-OT signi cantly increased SH-SY5Y cell viability, reduced cell apoptosis, and decreased LDH viability in MPP + -treated SH-SY5Y cells, and these effects were signi cantly eliminated by miR-942-5p inhibitor. Apoptosis or necrosis can cause cell membrane rupture to release LDH, the released LDH activity can represent the amount of cell necrosis and was widely used in cytotoxicity testing [33]. Furthermore, preceding studies had shown that proin ammatory factors were related to the pathogenesis of PD [34]. In our current research, ELISA was employed to examine IL-1β and TNF-α and other pro-in ammatory factors, and western blotting was employed to examine the expression level of cleaved-Caspase3 protein. The results manifested that the down-regulated expression of lncRNA SOX2-OT leads to decrease of the expression levels of TNF-α, IL-1β and cleaved-Caspase3 protein in SH-SY5Y cells treated with MPP + , moreover, reduces the cleaved-Caspase3/Caspase3 ratio. In addition, we further noticed that the down-regulated expression of lncRNA SOX2-OT signi cantly reduced the level of ROS and enhanced the SOD activity in MPP + -treated SH-SY5Y cells in the in vitro PD cell model. These results showed that the lncRNA SOX2-OT inhibition suppresses MPP + -induced oxidative stress, however, miR-942-5p inhibitor signi cantly reversed all the above effects in SH SY5Y cells treated with MPP + . Further studies had shown that miR-942-5p mimic signi cantly improves SH-SY5Y cell viability, reduces cell apoptosis, the expression of cleaved-Caspase3 protein, the ratio of cleaved-Caspase3/Caspase3, the LDH activity, the levels of TNF-α, IL-1β and ROS in SH-SY5Y cells, and increase the activity of SOD, but these effects were signi cantly eliminated by NAIF1-plasmid; these results indicated that lncRNA SOX2-OT modulated the in ammatory response and the induction of oxidative stress and neuronal apoptosis by targeting miR-942-5p and NAIF1 in an in vitro PD cell model.
In summary, our experiments have proved that lncRNA SOX2-OT regulates in ammation, oxidative stress and neuronal apoptosis by directly regulating the miR-942-5p/NAIF1 signal axis, and then participates in the occurrence and development of Parkinson's. The present study provides a novel potential target for PD diagnosis and therapy.

Declarations Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Figure 1 LncRNA SOX2-OT directly targeted miR-942-5p. A. The Starbase discovered the binding site between lncRNA SOX2-OT and miR-942-5p. B. Dual-luciferase reporter assay was used to reveal the relationship between lncRNA SOX2-OT and miR-942-5p. C. The level of miR-942-5p in 293T cells transfected with mimic control or miR-942-5p mimic was determined using qRT-PCR.   Effects of lncRNA SOX2-OT inhibition on MPP+-induced SH-SY5Y cells viability and apoptosis. A. The cell viability in SH-SY5Y cells following treatments was measured by MTT assay. B. LDH release assay was applied to detect LDH activity of treated SH-SY5Y cells. C and D. Flow cytometry analysis was employed to detect apoptotic rate of treated SH-SY5Y cells. E and F. Western blotting was employed to examine the expression of cleaved-Caspase3 protein and calculated the ratio of cleaved-Caspase3/Caspase3 of treated SH-SY5Y cells.   Effects of miR-942-5p up-regulation on expression of NAIF1 in SH-SY5Y cells. A. The level of miR-942-5p in SH-SY5Y cells was examined by qRT-PCR. B. The level of NAIF1 mRNA in treated SH-SY5Y cells was detected by qRT-PCR assay. C. The levels of NAIF1 mRNA and protein expression in SH-SY5Y cells transfected with mimic control, miR-942-5p mimic, miR-942-5p mimic+control-plasmid, or miR-942-5p mimic+NAIF1-plasmid were detected by qRT-PCR and western blotting assay.

Figure 8
Effects of miR-942-5p on MPP+-induced SH-SY5Y cells viability and apoptosis. A. The cell viability in SH-SY5Y cells following treatments was measured by MTT assay. B. LDH release assay was applied to detect LDH activity of treated SH-SY5Y cells. C and D. Flow cytometry analysis was employed to detect apoptotic rate of treated SH-SY5Y cells. E and F. Western blotting was employed to examine the expression of cleaved-Caspase3 protein and calculated the ratio of cleaved-Caspase3/Caspase3 of treated SH-SY5Y cells.