LincRNA-p21 Inhibits Cell Viability and Promotes Cell Apoptosis in Parkinson's Disease through Activating α-Synuclein Expression

Long intergenic noncoding RNA-p21 (lincRNA-p21) has been reported to be increased in Parkinson's disease (PD). However, the function and underlying mechanisms of lincRNA-p21 remain not clear. In order to explore the role of lincRNA-p21 in PD, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce in vivo PD model (C57BL/6 mice) and utilized N-methyl-4-phenylpyridinium (MPP+) to create in vitro PD model (SH-SY5Y cells). Results showed that the expression level of lincRNA-p21 was increased significantly in PD models. High abundance of lincRNA-p21 inhibited viability and promoted apoptosis markedly in SH-SY5Y cells treated with MPP+. Mechanistically, further experiments demonstrated that upregulation of lincRNA-p21 could sponge miR-1277-5p and indirectly increase the expression of α-synuclein to suppress viability and activate apoptosis in SH-SY5Y cells. In short, our study illustrated that lincRNA-p21/miR-1277-5p axis regulated viability and apoptosis in SH-SY5Y cells treated with MPP+ via targeting α-synuclein. LincRNA-p21 might be a novel target for PD.


Introduction
Parkinson's disease (PD) is a neurodegenerative disease owing to a reduction in dopaminergic neurons in the substantia nigra [1,2]. The progressive loss of dopamineproducing is the critical pathological hallmark of PD [3]. Myotonia, bradykinesia, depression, anxiety, static tremors, and cognitive dysfunction are typical symptoms of PD [4]. Various factors such as heredity, environment, and life style are closely related to the occurrence of PD, and the prevalence of PD all over the world is 1-2% of populations among populations over 65 years of age [5,6]. Despite huge attempts to investigate the molecular mechanisms of occurrence of PD, novel targeted drugs are missing and now there is still a lack of known cure for PD [7].
Long noncoding RNAs (lncRNAs) with length of more than 200 nucleotides are involved in various biological processes, such as tumor development, transcriptional regulation, and cell apoptosis [8]. Long intergenic noncoding RNA-p21 (lincRNA-p21), 3100 nt, is located on chromosome 6, involved in cell proliferation, metabolism, and reprogramming, and regarded as a potential diagnostic marker in various diseases [9]. LincRNA-p21, a p53-dependent transcriptional target gene, functions as a transcriptional repressor and triggers cell apoptosis [10]. A study has shown that lincRNA-p21 promotes cell apoptosis in hepatocellular carcinoma through inducing ER stress [11]. LincRNA-p21 plays a suppressive role in prostate cancer through modulating p53 [12]. LincRNA-p21 can activate hepatic stellate cells through lincRNA-p21-miR-181b-PTEN pathway in liver 2 BioMed Research International fibrosis [13]. However, the function of this lncRNA in PD is still unknown.
The expression levels of different lncRNAs were illustrated in 30 brain specimens derived from patients diagnosed with PD, and lincRNA-p21, SNHG1, MALAT1, and TncRNA expression levels were increased significantly [14].
-Synuclein is natively unfolded protein associated with Parkinson's disease (PD) [15]. After overexpressingsynuclein in the substantia nigra pars compacta of presymptomatic mice, 756 lncRNAs were expressed differently via microarray analysis [16]. Ye et al. reported that lincRNA-p21 sponged miR-181 promoted microglial activation through enhancing the expression of PKC-in PD models [17]. However, more indepth researches are needed to investigate the role of lincRNA-p21 and its pathogenic mechanism of PD.
In this study, we investigate the effect of lincRNA-p21 on cell viability and apoptosis in PD model cells (SH-SY5Y) treated with N-methyl-4-phenylpyridinium (MPP+). Besides, further mechanism of lincRNA-p21 on cell viability and apoptosis was manifested. Our work may provide another theoretical basis that lincRNA-p21 might be a novel target for PD.

Materials and Methods
. . Animals. Male C57BL/6 mice (5-10 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. 12 mice were randomly divided into two groups equally: the negative control group and the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) group. Sterile saline solution was injected intraperitoneally with a dose of 20 mg/kg body weight (four times/per day, two hour intervals) in negative control group. MPTP (Sigma, St. Louis, MO, USA) was injected intraperitoneally with an equal volume of sterile saline solution in MPTP group. The midbrains were isolated and harvested for the subsequent analysis after sacrifice with the last MPTP injection in the 21st day. The experiments procedures were approved by the Institutional Ethics Review Board of Peking University Shenzhen Hospital and were carried out according to the Guide for Care and Use of Laboratory Animals.
. . Cell Culture. SH-SY5Y cell line (human neuroblastoma cells) was purchased from American Type Culture Collection (ATCC) (Manassas, Va., USA). The SH-SY5Y cell line was cultured according to the suggestions of ATCC. SH-SY5Y was pretreated with 100 M 1-Methyl-4-phenylpyridine (MPP + ) (Sigma) for 24 hours to get the in vitro PD model.

. . Plasmid Construction and Cell Transfection.
The sequence of lincRNA-p21 was synthesized by GenePharma Co. Ltd. (Shanghai, China) and then cloned into pcDNA3.1 vector between the XhoI and BamHI sites to create the pcDNA3.1-lincRNA-p21 plasmid, and then the CMV promoter derived the expression of lincRNA-p21. The siRNAs targeting lincRNA-p21 or -synuclein were synthesized by GenePharma Co. Ltd. (Shanghai, China). The miR-1277-5p mimics and inhibitors were purchased from GeneCopoeia Co. Ltd. (Guangzhou, China). The sequence of 3 UTR of -synuclein or mutant 3 UTR of -synuclein was inserted into 3 UTR of renilla luciferase gene digested with Xhol and Notl in the psiCHECKTM-2 luciferase reporter plasmid purchased from Promega, Madison, WI, USA. Plasmids, siRNAs, or miR-1277-5p mimics/inhibitors were transfected into SH-SY5Y cells using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. All experiments were repeated at least three times.
. . Cell Nucleus and Cytoplasm Fraction Isolation. NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA) were used to prepare cytoplasmic and nuclear extracts from SH-SY5Y cells pretreated with MPP + . RNAs separated from each of the fractions were used for qRT-PCR analysis to illustrate the levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH), and lincRNA-p21.
. . Western Blot Analysis. The protein concentration was measured by the bicinchoninic acid (BCA) quantification assay (Pierce Biotechnology, Rockford, IL, USA). Equal amounts (20 g) of whole protein extract were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes using a semidry transfer cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated overnight at 4 ∘ C with specific primary antibodies against -synuclein (1:1000; Abcam, USA) and -actin (1:1000; Abcam, USA) after being blocked with 5% nonfat dry milk to prevent the nonspecific binding of the primary antibody. Horseradish peroxidase (HRP) conjugated secondary antibody (1:10000; Amersham, Arlington Heights, IL) was used to incubate the blot for one hour at room temperature on a rocking platform. Besides, the blot was washed with 1x TBST three times. Finally, super signal chemiluminescence reagents (Thermo Fisher Scientific, Inc., Massachusetts, USA) were used to detect signal intensities.
. . Dual-Luciferase Activity Assay. SH-SY5Y cells were seeded in 24-well plates (1 × 10 5 per well). SH-SY5Y cells were transfected with psi--synuclein or mutantsynuclein psi-mut--synuclein, along with miR-1277-5p mimics or inhibitors utilizing transfection reagent (Invitrogen). After 48-hour transfection, luciferase activity was measured through the dual-luciferase assay system (Promega. Madison, WI, USA) in accordance with the manufacturer's instructions. To calculate the activity of luciferase, the activity of renilla luciferase was normalized to the activity of firefly luciferase in psiCHECKTM-2 vector.
. . Cell Viability Assay. The cell viability was measured using Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China). 5 × 10 3 SH-SY5Y cells per well were seeded into 96-well plates. 10 l of CCK-8 reagent was added to each well and then incubated at 37 ∘ C for 2 hours. Absorbance at a wavelength of 450 nm was detected by a microplate reader (Bio-Rad, Hercules, CA, USA) following the manufacturer's protocols. Each test was performed at least three times.
. . Cell Apoptosis Assay. Cells were seeded in 12-well plates and transfected with plasmid, siRNAs or miRNAs. The activity of caspase-3 represented the levels of apoptosis and was detected employing the caspase-3 enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, China) according to the manufacturer's protocol. And this assay was widely used to detect cell apoptosis [18]. All experiments were carried out at least three times.
. . Statistical Analysis. All experimental data from three independent experiments were presented as mean ± standard deviation (SD) and processed by SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Student's t-test was used to analyze the difference of two groups. For more than two groups, the difference was analyzed by one-way ANOVA followed by a post hoc Tukey's test. Difference was considered statistically significant when a two-tailed value of P < 0.05 was exhibited.

Results
. . Overexpression of LincRNA-p Inhibited Cell Viability and Induced Apoptosis. Compared with the corresponding negative control, the expression levels of lincRNA-p21 were increased significantly in PD mice and in vitro PD model SH-SY5Y (Figures 1(a) and 1(b)). Then, knockdown and overexpression of lincRNA-P21 were performed through siRNAs and overexpression plasmid pcDNA3.1. As shown in Figure 1(c), cell viability was decreased significantly in only MPP + group (P < 0.01) compared with the untreated with MPP + group. After knockdown of lincRNA-p21 in SH-SY5Y cells treated with MPP + , cell viability was increased remarkably compared with the si-NC+ MPP + group (P < 0.01). After overexpression of lincRNA-p21 in SH-SY5Y cells treated with MPP + , cell viability was reduced obviously compared with the pcDNA3.1+ MPP + (P < 0.01). Next, the effects of lincRNA-p21 on cell apoptosis were also measured. As presented in Figure 1(d), compared with the untreated SH-SY5Y cells, the caspase-3 activity was improved significantly in SH-SY5Y cells treated with MPP + (P < 0.01). Compared with si-NC+ MPP + group, the activity of caspase-3 was inhibited dramatically in si-lincRNA-p21 group+ MPP + group (P < 0.01). However, after overexpression of lincRNA-p21, cell apoptosis was increased significantly in cells treated with MPP + (P < 0.001). Thus, high abundance of lincRNA-p21 suppressed cell viability and promoted cell apoptosis in PD models.
. . LincRNA-p Sponged miR--p. In order to investigate the mechanisms of lincRNA-p21 in PD, we firstly tested the subcellular localization of lincRNA-p21. As shown in Figure 2(a), lincRNA-p21 was mainly located in cytoplasm. Thus, lincRNA-p21 may inhibit cell viability and apoptosis at posttranscriptional level. Next, we speculated that lincRNA-p21 may sponge miRNAs. According to the miRDB, TargetScan, and miRWalk database, miR-1277-5p can bind to lincRNA-p21. Then, qRT-PCR data showed that the expression of miR-1277-5p was decreased significantly in PD mice (Figure 2(b), P < 0.01) and SH-SY5Y cells treated with MPP + (Figure 2(c), P < 0.001). To verify whether lincRNA-p21 sponged miR-1277-5p, we performed RIP and dual-luciferase assays. It has been known that miRNAs degrade mRNA and inhibit translation in an Argonaute 2 (AGO2)-dependent manner by binding to their targets [19]. We conducted anti-AGO2 RIP in SH-SY5Y cells transiently overexpressing miR-1277-5p to pull down lincRNA-p21 using control IgG or AGO2 antibodies, followed by PCR analysis for lincRNA-p21 levels. As presented in Figure 2 mimics. However, GAPDH was not pulled down with anti-AGO2 antibodies. This result suggested that miR-1277-5p could directly target lincRNA-p21 in AGO2 manner. Besides, the wide-type lincRNA-p21 sequence (WT) or the sequence with mutated binding sites of miR-1277-5p (Mut) was inserted into the 3 UTR of the renilla luciferase in ps-CHECK2 vector (Figure 2(e)). As shown in Figure 2(f), after overexpressing of miR-1277-5p, the luciferase activities of WT reporter were decreased significantly compared with the Mut reporter. On the contrary, the luciferase activities of WT reporter were increased remarkably compared with the Mut control after inhibition of expression of miR-1277-5p. These results demonstrated that lincRNA-p21 suppressed the activity of miR-1277-5p via direct binding between them and lincRNA-p21 might be regarded as a competing endogenous RNA (ceRNA) for miR-1277-5p. The expression of miR-1277-5p was downregulated significantly in PD mice and PD model cells, SH-SY5Y. (d) RIP was performed and coprecipitated RNA was utilized for PCR assay. IgG, negative control group; input, total RNA group; AGO2, AGO2 antibody group. (e) The sequence of wide-type lincRNA-p21 or mutated lincRNA-p21 was inserted into the 3 UTR of renilla luciferase (hRluc). (f) The luciferase activity was decreased significantly after transfection of miR-1277-5p mimics in lincRNA-p21 WT group. The luciferase activity was increased obviously after transfection of miR-1277-5p inhibitor in lincRNA-p21 WT group. However, there was no difference in lincRNA-p21 Mut group. All data are presented as the mean ± SD ( * * P < 0.01, * * * P < 0.001); bar, SD.

. . -Synuclein Was a Target Gene of miR--p.
According to the online database TargetScan (http://www.targetscan .org), -synuclein is a target gene of miR-1277-5p. In order to verify whether miR-1277-5p bound to -synuclein, we performed the luciferase assay. The wide-type -synuclein sequence (WT) or the sequence with mutated binding sites of miR-1277-5p (Mut) was inserted into the 3 UTR of the renilla luciferase in ps-CHECK2 vector (Figure 3(a)). As shown in Figure 3(b), after cotransfecting with -synuclein WT luciferase reporter and miR-1277-5p mimics, the activity of luciferase was decreased significantly compared with the NC mimics group (P < 0.01). However, the luciferase activity was increased remarkably after cotransfecting with -synuclein WT luciferase reporter and miR-1277-5p inhibitor (P < 0.001). Besides, there was no difference in -synuclein Mut group.

Discussion
Long noncoding RNAs (lncRNAs) participate in the regulation of gene expression, affecting neural plasticity, development, and aging [20,21]. LncRNAs have been illustrated to function as mediators in miRNAs, mRNA degradation, and protein function [22]. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been utilized to create PD animal models and 1-methyl-4-phenylpyridine (MPP + ) was used to establish in vitro PD cell model [23,24]. LincRNA-p21 was one kind of lncRNAs and, in this study, results indicated that the expression of lincRNA-p21 was increased significantly in MPTP-induced PD mice and SH-SY5Y cells treated with MPP + , which was consistent with the previous study [14]. This data suggested that lincRNA-p21 may play vital roles in the regulation of neurons cells in PD.
Results of cell viability and apoptosis assays showed that high abundance of lincRNA-p21 inhibited viability and induced apoptosis notably in SH-SY5Y cells treated with MPP + . Thus, the underlying mechanism of lincRNA-p21 was further investigated in this study. The miRNA degraded mRNA through binding to the 3 UTR of mRNA completely and inhibited translation processes via incomplete complementarity between miRNA and downstream mRNA [25]. Various studies demonstrated that miRNAs played vital roles in the pathogenesis of PD [26]. High abundance of lncRNA SNHG1 promoted cytotoxicity and ROS production via miR-15p/ GSK3 axis in SH-SY5Y cells treated with MPP + [27]. We suspected that lincRNA-p21 may take effect in PD through miRNA. Our results suggested that lincRNA-p21 was mainly located in cytoplasm, and through bioinformatic analysis, we found that lincRNA-p21 may bind to miR-1277-5p. RIP assay and dual-luciferase assay results verified that lincRNA-p21 bound to miR-1277-5p and regulated the expression of miR-1277-5p. Further experiments indicated that overexpression of miR-1277-5p could abrogate the effects of lincRNA-p21 on cell viability and apoptosis.
PD is one kind of "synucleinopathies" and neurodegenerative diseases [28]. The -synuclein protein of 140 amino acids with an incompletely defined function was involved in synaptic vesicle trafficking [29]. The -synuclein has been verified as a hallmark and critical pathogenic protein in PD [30]. High abundance of -synuclein has developed the PDlike behavioral abnormalities [31]. In this study, we found that -synuclein was the target gene of miR-1277-5p through bioinformatic analysis. Results of dual-luciferase assay, qRT-PCR, and western blot assay proved that -synuclein was really the downstream gene of miR-1277-5p. Effects of miR-1277-5p and -synuclein on cell viability and apoptosis were also measured, and data showed that effects of high expression of miR-1277-5p on cell viability and apoptosis were  (a, b) The luciferase activity was detected after cotransfection of lincRNA-p21 and miR-1277-5p mimics/inhibitor in -synuclein group. (c, d) The expression of -synuclein was measured after cotransfection of lincRNA-p21 and miR-1277-5p mimics/inhibitor. (e) Cell viability was measured after cotransfection of lincRNA-p21 and miR-1277-5p mimics in SH-SY5Y cells. (f) Cell apoptosis was observed after cotransfection of lincRNA-p21 and miR-1277-5p mimics in SH-SY5Y cells. All data are shown as the mean ± SD ( * P < 0.05, * * P < 0.01, * * * P < 0.001); bar, SD. counteracted by overexpression of -synuclein. Besides, in order to illustrate the relationship among lincRNA-p21, miR-1277-5p, and -synuclein, we also carried out dual-luciferase assay and qRT-PCR to measure the effects on the expression of -synuclein between lincRNA-p21 and miR-1277-5p. Data indicated that high expression of lincRNA-p21 promoted the expression of -synuclein. The expression level of -synuclein was inhibited and abrogated after cotransfection of lincRNA-p21 and miR-1277-5p mimics. Cell viability was inhibited by overexpression of lincRNA-p21. However, cell viability of SH-SY5Y cells treated with MPP + was increased after cotransfection of lincRNA-p21 and miR-1277-5p mimics. Similarly, cell apoptosis was promoted by high expression of lincRNA in SH-SY5Y cells. But after transfection of lincRNA-p21 and miR-1277-5p simultaneously, apoptosis was suppressed significantly. These results demonstrated effects of lincRNA-p21 on cell viability and apoptosis through regulatingsynuclein.

Conclusion
Our study illustrated that lincRNA-p21 inhibited viability and promoted apoptosis of SH-SY5Y cells induced by MPP + via downregulation of miR-1277-5p and upregulation ofsynuclein protein. LincRNA-p21 might be regarded as a promising target for PD.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors report no conflicts of interest.