Knockdown of SNHG14 Alleviates MPP+-Induced Injury in the Cell Model of Parkinson’s Disease by Targeting the miR-214-3p/KLF4 Axis

Background Parkinson’s disease (PD) is the second most common neurodegenerative disease. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) has been demonstrated as an important regulator in PD pathology. However, the functional mechanisms played by SNHG14 in PD remain largely unclear. Methods We used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) to establish PD mouse and cell models. The levels of SNHG14, miR-214-3p, and Krüppel-like factor 4 (KLF4) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot analysis. Cell viability and apoptosis were determined using the Cell Counting-8 Kit (CCK-8) assay and flow cytometry, respectively. The levels of inflammatory cytokines were evaluated by ELISA. The relationships among SNHG14, miR-214-3p, and KLF4 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Results Our data indicated that SNHG14 was upregulated and miR-214-3p was downregulated in PD models. SNHG14 knockdown ameliorated MPP+-stimulated damage in SK-N-SH cells, as evidenced by the enhancement in cell viability and the suppression in cell apoptosis and pro-inflammatory cytokine production. Mechanistically, SNHG14 directly targeted miR-214-3p via binding to miR-214-3p, and SNHG14 knockdown protected SK-N-SH cell from MPP+-stimulated cytotoxicity by upregulating miR-214-3p. KLF4 was a direct target of miR-214-3p, and SNHG14 regulated KLF4 expression by acting as a miR-214-3p sponge. Furthermore, miR-214-3p overexpression alleviated MPP+-stimulated damage in SK-N-SH cells by downregulating KLF4. Conclusion Our current study first demonstrated the protective effect of SNHG14 knockdown on MPP+-stimulated cytotoxicity in SK-N-SH cells at least partially by targeting the miR-214-3p/KLF4 axis, illuminating a promising target for PD intervention and treatment.


INTRODUCTION
Parkinson's disease (PD) is a degenerative disorder and the second common neurodegenerative disease (Barnett, 2016). PD has a high incidence with an estimated 0.1-0.2% in unselected populations, 1% in people above 60 years of age, and 4% in people more than 80 years worldwide (Tysnes and Storstein, 2017). Despite decades of research, effective treatment for PD is still limited (Waldthaler and Timmermann, 2019). A better understanding of what drives PD pathology is of particular importance for developing innovative therapeutic interventions.
Long non-coding RNAs (lncRNAs) are longer than 200 nucleotides transcripts that are emerging as diverse regulators in development and disease programs (Kopp and Mendell, 2018). Thousands of lncRNAs have been shed light on their critical roles on gene expression modulation through sequestering available microRNAs (miRNAs), illuminating the importance of such interactions in human diseases, including PD (Wu et al., 2013;Kraus et al., 2017;Lekka and Hall, 2018). For instance, Peng et al. (2019) identified that HAGLR opposite strand lncRNA (HAGLROS) expression was elevated in PD mouse and cell models, and negatively regulated 1-methyl-4-phenylpyridinium (MPP + )-evoked cell autophagy and apoptosis via mediating autophagy related 10 (ATG10) expression by sponging miR-100. Cao et al. (2018) ascertained that small nucleolar RNA host (SNHG) gene 1 (SNHG1) level was overexpressed in PD, and the silencing of SNHG1 protected microglia against lipopolysaccharide-evoked inflammatory injury by the regulation of miR-7/nod-like receptor protein 3 (NLRP3) axis. Lin et al. (2019) reported that HOX transcript antisense RNA (HOTAIR) functioned as a sponge of miR-126-5p to contribute to PD development via protecting against RAB3A interacting protein (RAB3IP) expression inhibition. Interestingly, lncRNA SNHG gene 14 (SNHG14) was reported to be highly expressed in PD, and its deficiency performed a protective effect on PD neurotoxicity, highlighting a promising target for PD treatment (Zhang et al., 2019). Nevertheless, the functional mechanisms of SNHG14 on PD pathology remain largely unknown.
Numerous researches have underscored that the deregulation of miRNAs is implicated in PD etiology (Mushtaq et al., 2016). A low serum level of miR-214 was reported as a potential biomarker for PD early diagnosis (Dong et al., 2016;Ma et al., 2016), and miR-214-3p was prominently differentially expressed in PD blood compared with normal controls using meta-analysis (Schulz et al., 2019). Moreover, the downregulation of miR-214 was associated with PD pathogenesis by targeting α-synuclein (Wang et al., 2015). However, it is not unclear whether miR-214-3p was involved in SNHG14-mediated regulation in PD pathology.
For these reasons, we undertook to identify the biological role and mechanism played by SNHG14 in PD pathology, with the hope that SNHG14 might serve as a potential target for PD intervention and treatment.

Animal and Treatment
Animal experimental protocols in this study were approved by the Ethics Committee of Huaihe Hospital, Henan University, and all procedures were in line with the National Guidance of the Care and Use of Laboratory Animals. Twelve male C57BL/6 mice (10 weeks of age; Guangdong Medical Laboratory Animal Center, Guangdong, China) were maintained on a specific-pathogenfree environment and used for the establishment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD mouse models as reported (Cao et al., 2018). All mice were divided into two groups (n = 6 per group): (1) MPTP group, intraperitoneal injection of MPTP-HCl (30 mg/kg; Meilunbio, Dalian, China) every day for 7 consecutive days; (2) control group, injection of the same volume of PBS in the same way. Three days after the last injection, these mice were sacrificed, and the ventral midbrain tissues were collected and stored in liquid nitrogen until use.

Primary Neuron Preparation, Cell Culture, and Treatment
The cortices from C57BL/6 embryos (Guangdong Medical Laboratory Animal Center) were stripped out and were digested in 0.025% trypsin (Invitrogen, Paisley, Scotland, United Kingdom). The supernatant was plated in culture dishes; maintained in neurobasal medium; enriched with B27 supplement, 1% penicillin/streptomycin, and 1 × glutamax (all from Invitrogen); and cultured for 7 days for further experiments. Primary cells at ∼70% confluence were stimulated with MPP + (Meilunbio) at different final concentrations of 0.5, 1, and 1.5 mM for 24 h in an atmosphere of 5% CO 2 at 37 • C.
SK-N-SH cells (ATCC HTB-11; ATCC, Manassas, VA, United States) were maintained using standard protocols (composed of Eagle's Minimum Essential Medium and 10% fetal bovine serum) provided by the ATCC. The cells at ∼70% confluence were stimulated with 1 mM of MPP + for 24 h at 37 • C.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
TRIzol reagent (Invitrogen) was used for RNA preparation from midbrain tissues and cells following the manufacturer's recommendations. The expression levels of SNHG14 and Krüppel-like factor 4 (KLF4) were gauged using PrimeScript One Step TB Green RT-PCR Kit (TaKaRa, Beijing, China) in 25 µl of reaction containing 200 ng of total RNA with the specific primers sets (provided in Table 1; BGI, Shenzhen, China). The quantification of miR-214-3p was performed using TaKaRa miRNA qRT-PCR TB Green Kit and primers specific for miR-214-3p (provided in Table 1; BGI). The Cycle Dice System (TaKaRa) was used for all reactions as recommended by the manufacturers. The relative levels of SNHG14, KLF4, and miR-214-3p were calculated by the 2 − Ct method (Cao et al., 2018) and normalized to β-actin or U6.

Cell Viability and Apoptosis Assays
SK-N-SH cells of ∼70% confluence were transfected with the indicated oligonucleotides or plasmids and then treated with 1 mM of MPP + for 24 h. The sensitive colorimetric assay for cell viability was carried out using Cell Counting-8 Kit (CCK-8; Abcam, Cambridge, United Kingdom) as recommended by the manufacturers. The flow cytometry for cell apoptosis was carried out using the double staining of fluorescein isothiocyanatetagged Annexin V and propidium iodide (PI; Elabscience, Wuhan, China) based on the manufacturer's guidance.

RNA Immunoprecipitation (RIP) Assay
Cell lysates were prepared using the Pierce IP Lysis buffer (Thermo Fisher Scientific, Hemel Hempstead, United Kingdom) and subsequently incubated with antibody against Argonaute 2 (Ago2, MA5-23515; Invitrogen) or isotype IgG (ab200699; Abcam) for 3 h at 4 • C, followed by the addition of magnetic A/G beads for 3 h. Beads were harvested, and total RNA was extracted. The levels of SNHG14, miR-214-3p, and KLF4 were gauged by qRT-PCR.

Western Blot for KLF4 Expression
Total protein of midbrain tissues and cells was obtained using Invitrogen Lysis buffer and quantified by the BCA method (Thermo Fisher Scientific). An equal amount of protein (50 µg) was subjected to electrophoresis on 10% SDS-polyacrylamide gels and then blotted on nitrocellulose membranes (Thermo Fisher Scientific). After being blocked in 5% non-fat milk, the membranes were probed with anti-KLF4 (ab129473; Abcam) or anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, ab9485; Abcam) antibody, followed by the addition of horseradish peroxidase-conjugated secondary antibody (ab205718; Abcam). Immunoblots were developed by the enhanced chemiluminescence method (Thermo Fisher Scientific) as recommended by the manufacturers.

Statistical Analysis
Data were presented as the mean ± standard deviation. Student's t-test (two-tailed) or ANOVA was used for the determination

SNHG14 Was Upregulated and MiR-214-3p Was Downregulated in PD Models
To preliminarily observe the involvement of SNHG14 and miR-214-3p in PD pathology, we first established the in vivo and in vitro models of PD by MPTP-mediated mouse and MPP +stimulated primary neuronal cells. As shown by qRT-PCR, SNHG14 expression was significantly increased and miR-214-3p level was remarkably decreased in MPTP-mediated PD mouse (Figures 1A,B). Moreover, MPP + stimulation elevated SNHG14 level and reduced miR-214-3p expression in a dose-dependent manner in primary neuronal cells (Figures 1C,D).

SNHG14 Knockdown Ameliorated MPP + -Stimulated SK-N-SH Cell Damage
To determine the role of SNHG14 in PD pathology, we performed "phenocopy" silencing by si-SNHG14. Transient introduction of si-SNHG14, but not the si-NC control, prominently reduced SNHG14 expression in MPP + -stimulated SK-N-SH cells (Figure 2A). The results of CCK-8 and flow cytometry assays showed that in contrast to the normal control, the stimulation of MPP + led to a significant reduction in cell viability ( Figure 2B) and a striking promotion in cell apoptosis ( Figure 2C). MPP + stimulation also resulted in increased levels of IL-1β, IL-6, and TNF-α in SK-N-SH cells (Figure 2D), indicating the enhancement of MPP + on cell inflammation. Subsequently, in comparison with the control group, the knockdown of SNHG14 caused a strong enhancement in cell viability (Figure 2B), a remarkable repression in cell apoptosis (Figure 2C), as well as a distinct reduction in cell inflammation ( Figure 2D) in MPP + -stimulated SK-N-SH cells. In addition, our FIGURE 4 | SNHG14 knockdown alleviated MPP + -stimulated SK-N-SH cell injury by upregulating miR-214-3p. MiR-214-3p expression was gauged by qRT-PCR (A); cell viability was assessed by CCK-8 assay (B); cell apoptosis was determined by flow cytometry (C); the levels of IL-1β, IL-6, and TNF-α were evaluated by ELISA (D) in MPP + -stimulated SK-N-SH cells transfected with si-NC, si-SNHG14, si-SNHG14 + anti-miR-NC, or si-SNHG14 + anti-miR-214-3p. *P < 0.05. data showed that SNHG14 overexpression significantly repressed cell viability when comparing with the negative control in MPP +treated SK-N-SH cells (Supplementary Figure 1A).

SNHG14 Sequestered MiR-214-3p via Binding to MiR-214-3p in SK-N-SH Cells
We then determined the mechanism by which SNHG14 regulated MPP + -stimulated cytotoxicity. Computer algorithms predicted that SNHG14 harbors eight nucleotides that match the seed sequence of miR-214-3p ( Figure 3A). To verify this finding, we carried out luciferase activity assays using SNHG14 wildtype reporter (WT-SNHG14). With WT-SNHG14 and miR-214-3p, overexpression triggered a remarkable reduction in luciferase activity (Figure 3B). To validate whether the miR-214-3p-binding sites were required for this effect, a mutant SNHG14 reporter (MUT-SNHG14), in which all eight predicted complementary sites were mutated, was tested. Notably, this mutant no longer elicited such an effect ( Figure 3B). Ago2 is the core component of the RNA-induced silencing complex (RISC), where miRNAs silence gene expression (Golden et al., 2017). Hence, we performed RIP experiments using anti-Ago2 antibody. These results revealed that in contrast to the negative control, the levels of SNHG14 and miR-214-3p were synchronously elevated by anti-Ago2 antibody (Figure 3C), demonstrating their potential endogenous interplay in SK-N-SH cells. The data of qRT-PCR showed that the introduction of SNHG14 overexpression vector caused a significant augment in SNHG14 expression in SK-N-SH cells ( Figure 3D). Furthermore, in comparison with their counterparts, miR-214-3p expression was remarkably increased by SNHG14 knockdown, and it was markedly decreased by the upregulation of SNHG14 (Figure 3E).

MiR-214-3p Directly Interacted With the 3 UTR of KLF4
The potential targets of miR-214-3p were predicted by the starBase software, and the data revealed a putative target sequence (CUGCUG) for miR-214-3p within KLF4 3 UTR ( Figure 5A). To confirm this, KLF4 3 UTR wild-type reporter (KLF4 3 UTR-WT) or KLF4 3 UTR mutant-type construct (KLF4 3 UTR-MUT) was transfected into SK-N-SH cells. In contrast to the control group, the transfection of miR-214-3p mimic dramatically reduced the luciferase activity of KLF4 3 UTR-WT but barely affected the luciferase of the mutant ( Figure 5B), demonstrating the validity of the target sequence for interaction. Moreover, miR-214-3p and KLF4 were concurrently enriched by anti-Ago2 antibody in SK-N-SH cells (Figure 5C). In addition, in comparison with the corresponding negative control, KLF4 protein level was prominently increased in MPTPmediated PD mouse and MPP + -stimulated primary neuronal cells (Figures 5D,E).

MiR-214-3p Overexpression Alleviated MPP + -Stimulated SK-N-SH Cell Damage by Downregulating KLF4
As demonstrated by western blot, in comparison with the miR-NC control, miR-214-3p upregulation resulted in decreased expression of KLF4 protein (Figure 6A), indicating that KLF4 was a direct target of miR-214-3p in MPP + -stimulated SK-N-SH cells. Functional analyses data showed that the overexpression of miR-214-3p led to a striking enhancement in cell viability (Figure 6B), a prominent suppression in cell apoptosis ( Figure 6C), as well as a strong reduction in IL-1β, IL-6, and TNFα production ( Figure 6D) in MPP + -stimulated SK-N-SH cells. These data together pointed that the upregulation of miR-214-3p alleviated MPP + -stimulated cytotoxicity. Besides, by contrast, the data of CCK-8 revealed that miR-214-3p knockdown remarkably suppressed cell viability (Supplementary Figure 1B).

SNHG14 Regulated KLF4 Expression by Acting as a MiR-214-3p Sponge
A key question was whether SNHG14 could regulate KLF4 expression. The data of qRT-PCR and western blot revealed that MPP + treatment triggered a significant upregulation in KLF4 expression at both mRNA and protein levels (Figures 7A,B). Moreover, as expected, KLF4 mRNA and protein levels were remarkably reduced by SNHG14 silencing in MPP + -treated SK-N-SH cells, and these effects were prominently abolished by the cotransfection of anti-miR-214-3p in MPP + -stimulated SK-N-SH cells (Figures 7A,B).

DISCUSSION
PD is a common age-related neurodegenerative disease, leading to impaired quality of life (Beitz, 2014). Owing to the inadequate knowledge in PD pathology, current treatments focus on symptom mitigation instead of PD prevention and fundamental treatment (Ng, 2018). LncRNAs are emerging as key regulators in PD pathogenesis, and the identification of their function remains a major challenge (Majidinia et al., 2016;Riva et al., 2016). In the present work, our data supported that SNHG14 knockdown protected against MPP + -stimulated cytotoxicity, as has been reported (Zhang et al., 2019). Mechanistically, SNHG14 knockdown alleviated MPP + -induced PD progression in vitro via the regulation of the miR-214-3p/KLF4 axis.
Recent studies indicated that SNHG14 functioned as a tumor promoter by sponging different miRNAs in a series of cancers, such as retinoblastoma, pancreatic ductal adenocarcinoma, and osteosarcoma (Hou and Mao, 2020;Sun et al., 2020;Xie et al., 2020). SNHG14 was uncovered to be highly expressed in ischemic stroke, and the inhibition of SNHG14 mitigated neuron inflammatory damage (Qi et al., 2017;Zhong et al., 2019). In addition, the silencing of SNHG14 was reported to relieve LPS-triggered inflammation in the model of acute lung injury by targeting the miR-34c-3p/WISP1 axis (Zhu et al., 2019). Here, we demonstrated a significant overexpression of SNHG14 in PD mice and cells, in line with the findings by Zhang et al. (2019). We also substantiated that the knockdown of SNHG14 ameliorated MPP + -stimulated damage in SK-N-SH cells, as evidenced by the enhancement in cell viability and the suppression in cell apoptosis and pro-inflammatory cytokine production.
LncRNAs can fine-tune gene expression through acting as miRNA sponges to sequester available miRNAs. Here, we were first to demonstrate that SNHG14 targeted miR-214-3p via directly binding to miR-214-3p in SK-N-SH cells. Among thousands of candidates predicted by the starBase software, miR-214-3p was fascinating because of its key role in multitudinous human diseases. For example, miR-214-3p regulated the tumorigenesis of ovarian cancer, endometrial cancer, and papillary thyroid carcinoma (Fang et al., 2019;Yang C. et al., 2019;Sui et al., 2020). MiR-214-3p was identified to repress cardiac fibrosis through the inhibition of the NOD-like receptor family CARD domain containing 5 (Yang K. et al., 2019). In addition, miR-214-3p expression was downregulated in the rat model of spinal nerve ligation, and the elevated miR-214-3p expression mitigated neuroinflammation and neuropathic pain by targeting colonystimulating factor-1 (Yang K. et al., 2019). MiR-214-3p also associated with the neuroprotection of Sigma 1 receptor in retinitis pigmentosa (Wang and Smith, 2019). We subsequently uncovered a protective effect of miR-214-3p overexpression on MPP + -stimulated cytotoxicity. More interestingly, for the first time, we identified that SNHG14 knockdown mitigated MPP + -stimulated cytotoxicity in SK-N-SH cells by upregulating miR-214-3p.
Here, we first confirmed that KLF4 in SK-N-SH cells was directly targeted and suppressed by miR-214-3p. KLF4, a transcription factor and cell-cycle suppressor, has been shown to be implicated in various human diseases Xu et al., 2020). In addition, KLF4 was uncovered to be relevant to the pathogenesis of Alzheimer's disease (Cheng et al., 2018). Previous evidence also reported that KLF4 silencing protected against MPP +evoked cytotoxicity in PD (Chen et al., 2013;Kong et al., 2016). In this research, we were first to demonstrate that miR-214-3p overexpression alleviated MPP + -evoked cytotoxicity via targeting KLF4. Similarly, Kong et al. (2016) confirmed that miR-7 protected SH-SY5Y cell from MPP + -triggered apoptosis by targeting KLF4. Song et al. (2018) uncovered that miR-212 directly targeted KLF4 to relieve MPP +stimulated cell injury. More interestingly, we first uncovered that SNHG14 modulated KLF4 expression via acting as a sponge of miR-214-3p.
In conclusion, the current study provided evidence for the protective role of SNHG14 knockdown on MPP +stimulated cytotoxicity in SK-N-SH cells through targeting the miR-214-3p/KLF4 axis. It is the first research for the SNHG14/miR-214-3p/KLF4 regulatory network in PD pathogenesis (Figure 7C), illuminating a promising target for intervention and treatment.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal experimental protocols in this study were approved by the Ethics Committee of Huaihe Hospital, Henan University and all procedures were in line with the National Guidance of the Care and Use of Laboratory Animals.

AUTHOR CONTRIBUTIONS
SZ and DZ conceived and designed the experiments. JG performed the experiments and funding acquisition. JZ contributed reagents, materials, and analysis tools. YC wrote the article. All authors contributed to the article and approved the submitted version.