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Long noncoding RNA NORAD regulates MPP+-induced Parkinson’s disease model cells
Journal of Chemical Neuroanatomy 101 (2019) 101668
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Long noncoding RNA NORAD regulates MPP+-induced Parkinson’s disease model cells Qingxin Songa, Yunming Gengb, Yue Lic, Liuli Wangd, Jinyan Qina,c,e,
T
⁎
a
Department of Neurosurgery, Linyi People's Hospital, Linyi, Shandong Province, 276000, China Department of Neurosurgery, Linyi Fourth People's Hospital, Linyi, Shandong Province, 276000, China c Department of Neurology, Linyi People's Hospital, Linyi, Shandong Province, 276000, China d Department of Health Care, Linyi Lanshan Maternal and Child Health Care Hospital, Linyi, Shandong Province, 276000, China e Department of Hyperbaric Oxygen, Linyi People's Hospital, Linyi, Shandong Province, 276000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parkinson’s disease MMP+ lncRNA NORAD
Background: Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in human diseases. Yet, the functions of lncRNAs in neurodegenerative disorders, such as Parkinson's disease (PD) are poorly understood. In this study, we used human neuroblastoma SH-SY5Y cell line as a cell-basedin vitro PD model, and investigated the role of lncRNA, Non-Coding RNA Activated By DNA Damage (NORAD) in 1-methyl-4-phenylpyridinium (MPP+)-induced PD-like cytotoxicity. Methods: SH-SY5Y cells were culturedin vitro, and treated with MPP + at various concentrations, or of various durations of times to induce PD-like cytotoxic events. qRT-PCR was used to measure MPP+-induced NORAD expression changes. Lentiviral transduction was applied to stably upregulate or downregulate NORAD in SHSY5Y cells. The effects of NORAD upregulation or downregulation on MPP+-induced cytotoxic events, including dose-dependent and time-dependent cell death, apoptosis, caspase 3/7, reactive Oxygen Species (ROS) and lactate dehydrogenase (LDH) activities, were quantitatively investigated. Results: MPP + induced cytotoxicity, and downregulated NORAD in both dose- and time- dependent manners in SH-SY5Y cells. Lentiviral-induced NORAD upregulation was found to protect against MPP+-induced cytotoxicity in SH-SY5Y cells, as it rescued MPP+-induced cellular destruction and apoptosis, as well as decreased MPP +-induced caspase 3/7, ROS and LDH activities. Alternatively, NORAD downregulation was found to significantly deteriorate MPP+-induced cytotoxicity in SH-SY5Y cells. Conclusion: We presented a novel functional role of lncRNA NORAD in regulating human Parkinson’s disease.
1. Introduction Parkinson's disease (PD) is one of the most progressive neurodegenerative disorders for both men and women patients, affecting at least 2% of the population above 65 years at any given time (Tysnes and Storstein, 2017; Ascherio and Schwarzschild, 2016; Poewe et al., 2017). While the cellular features of PD pathophysiology are characteristic and clear, mainly caused by neuronal loss in the substantia nigra due to striatal dopamine deficiency and intracellular aggregation of α-synuclein, the underlying genetic mechanisms contributing to those features are largely unknown (Tysnes and Storstein, 2017; Bastide et al., 2015; Lill, 2016). Long noncoding RNAs (lncRNAs) are families of non-protein-coding, long-length (> 200 nucleotides) RNAs, that had been recently shown to
play critical roles in many pathological processes in human diseases (Wapinski and Chang, 2011; Esteller, 2011; Taft et al., 2010). In neurodegenerative disorders, several lncRNAs were demonstrated to be dysregulated and having functional roles in regulating the progression of neurodegenerations in human brains (see review (Wan et al., 2017)). Specifically, in Parkinson's disease, the expression of lncRNA antisense to Uchl1 (AS Uchl1) was found to be close associated with early-onset of PD (Carrieri et al., 2015). In addition, de-regulation of lncRNA phosphatase and tensin homologue–induced kinase1 (PINK1) was found to be linked to mitochondrial dysfunction leading to dopamine release deficit (Scheele et al., 2007). Of many of the human diseases-associated lncRNAs, Noncoding RNA activated by DNA damage (NORAD) was initially identified as an exon encoding transcript at Chr20q11.23 that functionally contributes
⁎ Corresponding author at: 27 Jiefang Road East Section, Department of Hyperbaric Oxygen, Linyi Fourth People's Hospital, Linyi, Shandong Province, 276000, China. E-mail address: [email protected] (J. Qin).
https://doi.org/10.1016/j.jchemneu.2019.101668 Received 23 May 2019; Received in revised form 13 July 2019; Accepted 13 August 2019 Available online 14 August 2019 0891-0618/ © 2019 Published by Elsevier B.V.
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specific absorbance measured using equation, [Abs450 nm(Test) – Abs450 nm(Blank)] – Abs660 nm(Test), and then normalized against the specific absorbance for control wells.
to genomic stability by sequestering PUMILIO proteins (Lee et al., 2016). Recently, NORAD was found to be de-regulated in several types of human cancers, and may have significant impact on cancer cell oncogenesis, development, metastasis and apoptosis (see review (Yang et al., 2019)). However, there have been no reports showing any functional involvement of NORAD during the development or progression in human neurodegenerative diseases. In this study, we took advantage of a cell-based Parkinson's disease model, in which human neuroblastoma SH-SY5Y cell line was treated with 1-methyl-4-phenylpyridinium (MPP+) in vitro, to induce both dose- and time- dependent PD-like cytotoxic cellular events (Xie et al., 2010; Fall and Bennett, 1999; Xicoy et al., 2017; Shishido et al., 2019; Zhu et al., 2018). During the meantime, we used qRT-PCR to explore the dynamic expression change of NORAD in response to MPP + treatment in SH-SY5Y cells. Most importantly, we used lentiviral transduction to successfully created SH-SY5Y cell lines with stable NORAD upregulation or NORAD downregulation. Then, the effects of NORAD upregulation and downregulation on MPP+-induced cytotoxic events were carefully examined.
2.5. RNA extraction and quantitative real time-PCR
2. Materials and methods
From SH-SY5Y cells, RNA extraction and purification was completed using a PureLink Pro 96 total RNA Purification Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. Reverse Transcription to obtain complementary DNA (cDNA) was completed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer’s instruction. To assess lncRNA NORAD gene expression, quantitative real time-PCR (qRT-PCR) was conducted on an ABI PRISM 7700 Sequence Detector System (Applied Biosystems, USA), and using a TaqMan Universal PCR Master Mix Kit (Applied Biosystems, USA) and a TaqMan non-coding RNA assay with a customized human NORAD qRT-PCR probe (Invitrogen, USA), according to the manufacturers’ instructions. In addition, a 18S rRNA TaqMan assay (Invitrogen, USA) was conducted as control. Finally, relative expression level of NORAD was calculated using the 2(−ΔΔCt) method.
2.1. Ethics statement
2.6. NORAD upregulation and downregulation in SH-SY5Y cells
In this study, all experimental protocols were thoroughly reviewed and approved by the Clinical Research & Ethics Committee at the Linyi People's Hospital, Linyi Fourth People's Hospital and Linyi Lanshan maternal and child health care hospital in Linyi, Shandong Province, China. Also, all procedures related to animals were conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in the United States of America (https://grants.nih.gov/grants/olaw/Guide-for-thecare-and-use-of-laboratory-animals.pdf).
Lentiviral vectors containing the full sequence of human NORAD (L/ NORADm), an empty lentiviral vector (L/Cm), a short hairpin RNA (shRNA) targeting NORAD (L/NORADi) and a non-specific shRNA (L/ Ci) were all synthesized and purchased from RiboBio (RiboBio Biotechnology Ltd., Guangzhou, China). In addition, transfection of lentiviral vectors, along with lentivirus packaging vectors in human HEK293 T cells to yield high-titer lentiviruses were also conducted by RiboBio (RiboBio Biotechnology Ltd., Guangzhou, China). In the in vitro culture of SH-SY5Y cells, cells were transduced with lentivirus of L/ NORADm, L/Cm, L/NORADi or L/Ci and 8 μg/ml polybrene (Thermo Fisher Scientific, USA) at multiplicity of infection (MOI) of 3 for 48 h, followed by a blasticidin selection procedure (15 μg/ml, Thermo Fisher Scientific, USA) for additional 5 days. After discarding the floating colonies, the attached, healthy, round-shape multi-cell colonies were carefully selected and re-plated in 6-well plates to proliferate. After 5˜7 passages, qRT-PCR was conducted to confirm the upregulation or downregulation of NORAD in SH-SY5Y cells.
2.2. Cell culture In this study, human neuroblastoma cell line, SH-SY5Y was commercially purchased from the American Type Culture Collection in the United States of America (ATCC, USA). Cells were maintained in 6-well plate (VWR, USA) in the Dulbecco's Modified Eagle Medium with Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific, USA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, USA) and 100 U/ml Penicillin-Streptomycin (Gibco, USA) in a tissue-culture chamber with circulating gas of 95% O2 / 5% CO2 at 37 °C.
2.7. Measurement of apoptosis SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, cellular apoptosis was assessed using a Click-iT™ Plus TUNEL Assay for in situ Apoptosis Detection Kit (Alexa Fluor™ 594 dye, Invitrogen, USA) according to the manufacturer’s instruction. Also, a DAPI (blue) staining was performed for 15 min. Then, 96-well plates were moved onto an Evos microscope system (Thermo Fisher Scientific, USA). Apoptotic cells were characterized as those positive to both DAPI and TUNEL stainings. Then, the percentage of non-apoptotic cells in each well was measured.
2.3. Cell-based Parkinson's disease model In this study, we adapted a cell-based Parkinson's disease (PD) model, which was well established in previous publications (Xie et al., 2010; Fall and Bennett, 1999; Xicoy et al., 2017; Shishido et al., 2019; Zhu et al., 2018). To induced PD-like cytotoxicity in the culture of SHSY5Y cells, cells were treated with 1-methyl-4-phenylpyridinium (MPP +) (Thermo Fisher Scientific, USA) in vitro. The treatment of MMP + may vary at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM, or in times of 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h.
2.8. Measurement of caspase-3/7 activity SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, caspase-3 and caspase-7 activities were assessed in SH-SY5Y culture using a CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the relative caspase 3/7 activity was calculated as fluorescence measurement at 530 nm (excited at 500 nm) and normalized to
2.4. Cell viability assay SH-SY5Y cells were collected and re-seeded in 96-well plates (VWR, USA). Post MPP + treatment, the death and viability of SH-SY5Y cells was assessed using a CyQUANT™ XTT Cell Viability Assay (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the absorbance (Abs) was read at 450 nm and 660 nm. Relative cell viability was characterized as the 2
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the fluorescence measurement for control wells.
SY5Y cells. After transduction was stabilized, qRT-PCR showed that, SH-SY5Y cells transduced with L/NORADm had significantly higher NORAD expression level than cells transduced with L/Cm (Fig. 2A, * P < 0.05). Conversely, SH-SY5Y cells transduced with L/NORADi had significantly lower NORAD expression level than cells transduced with L/Ci (Fig. 2B, * P < 0.05). Therefore, these results showed that we successfully created SH-SY5Y cells with stably upregulated or downregulated NORAD. Then, we tested the effects of NORAD upregulation / downregulation on MPP+-induced dose-dependent and time-dependent SHSY5Y cytotoxicity. First, L/Cm-, L/NORADm-, L/Ci- and L/NORADitransduced SH-SY5Y cells were treated with MPP + for 24 h, at concentrations ranging from 0.05 to 10 mM, respectively. The result of viability assay showed that, for cells treated with MPP + at 0.25 mM or higher concentrations for 24 h, MPP+-induced cytotoxicity was significantly reduced by NORAD upregulation (Fig. 2C, * P < 0.05). On the other hand, in SH-SY5Y cells treated with MPP+ at 0.1 mM or higher concentrations for 24 h, MPP+-induced cytotoxicity was significantly enhanced by NORAD downregulation (Fig. 2D, * P < 0.05). Second, L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SHSY5Y cells were treated with 5 mM MPP + for different periods of time, ranging from 0.5 to 24 h, respectively. The result of viability assay showed that, for cells treated with 5 mM MPP + between 16 and 24 h, MPP+-induced cytotoxicity was significantly reduced by NORAD upregulation (Fig. 2E, * P < 0.05). On the other hand, in SH-SY5Y cells treated with 5 mM MPP+ between 2 and 24 h, MPP+-induced cytotoxicity was significantly enhanced by NORAD downregulation (Fig. 2F, * P < 0.05). Thus, these results clearly demonstrated that NORAD upregulation could protect against, whereas NORAD downregulation could further deteriorate MPP+-induced cytotoxicity in SH-SY5Y cells.
2.9. Measurement of reactive oxygen species activity SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, reactive Oxygen Species (ROS) activity was assessed using a Total Reactive Oxygen Species Assay Kit (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the relative ROS activity was calculated as fluorescence measurement at 520 nm (excited at 490 nm) and normalized to the fluorescence measurement for control wells. 2.10. Measurement of lactate dehydrogenase (LDH) level SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, supernatants were transferred to other 96-well plates. The lactate dehydrogenase (LDH) activity was measured using a Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific, USA) according to the manufacturer’s instruction. Then, 96well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). Absorbance was measured at 490 and 680 nm. For each well, LDH activity was calculated as an absorbance ratio at 490/680 nm and normalized to the absorbance ratio in control wells. 2.11. Statistical analysis In this study, all statistical analyses were done using a one-way ANOVA followed by post-hoc test on a Windows-Based SPSS software (SPSS, Version 20.0, USA). All experiments were independently repeated for at least 3 times. The averaged data were presented as means +/- standard errors of the mean (SEM). P < 0.05 was considered as statistically different.
3.3. Genetic modification of NORAD regulated MPP+-induced apoptosis in SH-SY5Y cells L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SH-SY5Y cells, as well un-transduced (N/A) SH-SY5Y cells were treated with 5 mM MPP + for 6 h. A TUNEL assay demonstrated that, in un-transduced (N/A) SH-SY5Y cells, and those transduced with L/Cm and L/Ci, moderate number of cells were apoptotic due to MPP + treatment (Fig. 3A, B, D). However, significantly less apoptotic cells were observed in L/NORADm-transduced SH-SY5Y cells (Fig. 3C), whereas significantly more apoptotic cells were observed in L/NORADi-transduced SH-SY5Y cells (Fig. 3E). Then, quantitative measurement on the percentages of apoptotic SH-SY5Y cells showed that, while more apoptotic cells were induced by MPP + treatment in un-transduced (N/ A) and all lentiviral-transduced SH-SY5Y cells (Fig. 3F, * P < 0.05), NORAD upregulation rescued whereas NORAD downregulation increased the apoptotic effects induced by MPP+ (Fig. 3F, ** P < 0.05). Thus, our results clearly demonstrated that NORAD upregulation could reduce whereas NORAD downregulation would further increase MPP+-induced apoptosis in SH-SY5Y cells.
3. Results 3.1. LncRNA NORAD was downregulated during the process of MPP +-induced cytotoxicity in SH-SY5Y cells SH-SY5Y cells were treated with MPP + for 24 h, at concentrations ranging from 0.05 to 10 mM. MPP+-induced cytotoxicity was assessed using a viability assay. It demonstrated that, significant cellular destruction was observed in SH-SY5Y cells treated with 0.1 mM or higher concentrations of MPP+ (Fig. 1A, * P < 0.05, vs. 0 mM MPP+). During the meantime, qRT-PCR showed that, NORAD expression was significantly downregulated in SHSY5Y cells treated with 0.25 mM or higher concentrations of MPP+ (Fig. 1B, * P < 0.05, vs. 0 mM MPP+). In addition, SH-SY5Y cells were treated with 5 mM MPP + for different periods of time, ranging from 0.5 to 24 h. Cytotoxic measurement showed that, significant cellular destruction was observed in SH-SY5Y cells treated with 5 mM MPP + for 2 h or longer (Fig. 1C, * P < 0.05, vs. 0 h). Subsequently, qRT-PCR showed that NORAD expression was significantly downregulated in SHSY5Y cells treated with 5 mM MPP+ for 6 h or longer (Fig. 1D, * P < 0.05, vs. 0 h). Therefore, these results showed that MPP+-induced cytotoxicity also induced dose-dependent and time-dependent NORAD downregulation in SH-SY5Y cells.
3.4. Genetic modification of NORAD regulated MPP+-induced caspase 3/ 7, ROS activities and LDH level in SH-SY5Y cells Finally, for L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SH-SY5Y cells, as well as un-transduced (N/A) SH-SY5Y cells who were treated with (or without) 5 mM MPP + for 6 h, Parkinson’s Disease-like cytotoxic indicators, including caspase 3/7 activity, ROS activity and LDH level, were quantitatively measured. The results showed that, in both lentiviral-transduced and un-transduced SH-SY5Y cells, caspase 3/ 7 activity, ROS activity and LDH level were enhanced by MPP + treatment (Fig. 4A, B, C, * P < 0.05). In addition, as compared to L/Cm-transduced SH-SY5Y cells, MPP+-induced caspase 3/7 activity, ROS activity and LDH level were all decreased in L/NORADm-
3.2. Genetic modification of NORAD regulated MPP+-induced cytotoxicity in SH-SY5Y cells Genetic modification, including both upregulation and downregulation of NORAD, was performed by lentiviral transduction in SH3
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Fig. 1. MPP + downregulated NORAD in SHSY5Y cells. (A) SH-SY5Y cells were treated with MPP + at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM for 24 h. MPP +-induced dose-dependent cytotoxicity was measured by a viability assay (* P < 0.05, vs. 0 mM MPP+). (B) In addition, MPP+-induced dose-dependent NORAD downregulation was measured by qRT-PCR (* P < 0.05, vs. 0 mM MPP+). (C) SHSY5Y cells were treated with 5 mM MPP + for 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h. MPP+-induced time-dependent cytotoxicity was measured by a viability assay (* P < 0.05, vs. 0 h). (D) In addition, MPP+-induced time-dependent NORAD downregulation was measured by qRT-PCR (* P < 0.05, vs. 0 h).
4. Discussions
transduced SH-SY5Y cells (Fig. 4A, B, C, ** P < 0.05). Conversely, as compared to L/Ci-transduced SH-SY5Y cells, MPP+-induced caspase 3/ 7 activity, ROS activity and LDH level were all enhanced in L/NORADitransduced SH-SY5Y cells (Fig. 4A, B, C, ** P < 0.05). Thus, our results demonstrated that NORAD upregulation could reduce whereas NORAD downregulation would further increase MPP +-induced cytotoxic events in SH-SY5Y cells.
Mounting evidence has demonstrated that lncRNAs may play important roles in human neurodegenerative diseases (Wan et al., 2017). In Alzheimer’s disease, lncRNA β-secretase-1 antisense RNA (BACE1AS) was found to be associated with hippocampal neurogenesis in animal models (Modarresi et al., 2011), and knockdown of BACE1-AS dramatically reduced cortical β-amyloid synthesis and aggregation in vivo (Faghihi et al., 2008). In Huntington’s disease, the results from an in vivo mouse model demonstrated that overexpression of lncRNA 4
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Fig. 2. The effects of NORAD upregulation and downregulation on MPP+-induced toxicity in SH-SY5Y cells. (A) SH-SY5Y cells were transduced with L/Cm or L/NORADm lentiviruses, followed by qRT-PCR to assess their endogenous NORD expressions (* P < 0.05). (B) SH-SY5Y cells were transduced with L/Ci or L/ NORADi lentiviruses, followed by qRT-PCR to assess their endogenous NORD expressions (* P < 0.05). (C–D) SH-SY5Y cells transduced with L/Cm, L/NORADm, L/Ci and L/NORADi were treated with MPP + at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM for 24 h. MPP+-induced dose-dependent cytotoxicity was measured by a viability assay, and compared between L/Cm- and L/NORADmtransduced SH-SY5Y cells (C), and L/Ci- and L/ NORADi- transduced SH-SY5Y cells (D) (* P < 0.05). (E–F) SH-SY5Y cells transduced with L/Cm, L/NORADm, L/Ci and L/NORADi were treated with 5 mM MPP + for 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h. MPP+-induced timedependent cytotoxicity was measured by a viability assay, and compared between L/Cmand L/NORADm- transduced SH-SY5Y cells (E), and L/Ci- and L/NORADi- transduced SHSY5Y cells (F) (* P < 0.05).
models or human subjects are certainly needed. Another intriguing feature of our findings is that, while NORAD was found to be downregulated in response to MPP + treatment, it was predominantly found to be upregulated in several human cancer tumors (see review (Yang et al., 2019)). It may suggest that, the exact NORAD dys-regulation patterns in human diseases may vary due to different pathological conditions. The most important findings in this study was that we identified a protective mechanism of NORAD upregulation during the process of MPP+-induced cytotoxicity in SH-SY5Y cells. Using lentiviral transduction, we were able to create SH-SY5Y cell lines with stable NORAD upregulation or NORAD downregulation. Then, we were able to demonstrated that NORAD upregulation could protect against, whereas NORAD downregulation could enhance MPP+-induced cytotoxicity in SH-SY5Y cells, in both dose- and time- dependent manners.
Abhd11os had neuro-protective effect against N-terminal fragment of mutant huntingtin protein (Francelle et al., 2015). Then, in Parkinson's disease, aberrant expression of lncRNA PINK1 was demonstrated to be directly associated with dopamine release dysfunction (Scheele et al., 2007). In this study, we focused on a novel lncRNAs NORAD, whose biological function was just discovered recently (Lee et al., 2016), and explored its expression and functions during the process of MPP+-induced cytotoxicity in SH-SY5Y cells. First, we discovered that NORAD was downregulated in MPP+-injured SH-SY5Y cells, in both dose- and time- dependent manners. This novel observation is very important, suggesting that NORAD expression may be closely associated with Parkinson’s Diseases’ pathological conditions, at least under in vitro environment. It would be interesting to find out if it’s still the case under in vivo conditions, as further in vivo studies involving animal 5
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Fig. 3. The effects of NORAD upregulation and downregulation on MPP+-induced apoptosis in SH-SY5Y cells. (A–E) Un-transduced (NA) SH-SY5Y cells (A), L/Cm-transduced SH-SY5Y cells (B), L/NORADm-transduced SH-SY5Y cells (C), L/Ci-transduced SH-SY5Y cells (D), L/NORADi-transduced SH-SY5Y cells (E) were either treated with 5 mM MPP+, or not treated with MPP + for 6 h, followed by a TUNEL assay (Red) to identify apoptotic cells. A DAPI antibody (Blue) was also applied to identify the nuclei of SH-SY5Y cells. (F) The relative percentages of apoptotic SH-SY5Y cells were quantitatively measured for MPP+-untreated and MPP+-treated SH-SY5Y cells, which were un-transduced with lentivirus (NA), or transduced with L/Cm, L/NORADm, L/Ci and L/ NORADi (* P < 0.05, ** P < 0.05).
While it’s truly remarkable to reveal a possible functional role of NORAD in human PD, we also acknowledge that the limitation of our study. As mentioned, it is critical to test NORAD functions in in vivo PD animal models, or explore its expression in human PD patients. In addition, it is important to explore the associated signaling pathways, which could be either pre- or post-transcriptional, to be responsible NORAD regulations in PD. Thus, future experiments are much needed.
In addition, we explored the functions of NORAD on MPP+-induced SH-SY5Y cell apoptosis, and its associated caspase 3/7 signaling pathway. Specifically, we looked into MPP+-induced mitochondrial dysfunctions in SH-SY5Y cells, including ROS activity and LDH level. In all examined biochemical assays, it was demonstrated that NORAD upregulation rescued, whereas NORAD downregulation deteriorated MPP+-induced apoptosis and mitochondrial dysfunction, suggesting that NORAD might be able to regulate intrinsic onset, development or inhibition of PD-associated apoptotic or mitochondrial signaling pathways (Tatton et al., 2003; Anglade et al., 1997; Schapira et al., 1990; Abou-Sleiman et al., 2006). It is worth noting that in all experiments, the results obtained from SH-SY5Y cells transduced with control lentiviruses (L/Cm or L/Ci) were similar to those obtained from cells without lentiviral transduction (N/A). These data further confirmed our findings that genetic modification of NORAD could functional affect MPP+-induced cytotoxic events in SH-SY5Y cells.
5. Conclusion To conclude, in this study we presented a novel functional role of lncRNA NORAD in regulating MPP+-induced cytotoxicity in an in vitro model of Parkinson’s disease. Further investigations would help to identify the role of NORAD in human patients with Parkinson’s disease.
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Fig. 4. The effects of NORAD upregulation and downregulation on MPP+-induced caspase 3/7, ROS and LDH activities in SH-SY5Y cells. Un-transduced (NA), L/ Cm-, L/NORADm-, L/Ci- and L/NORADi- transduced SH-SY5Y cells were either treated with 5 mM MPP+, or not treated with MPP + for 6 h. Then, their caspase 3/7 activities (A), ROS activities (B) and LDH levels (C) were quantitatively measured and compared (* P < 0.05, ** P < 0.05).
Declaration of Competing Interest
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Francelle, L., Galvan, L., Gaillard, M.C., Petit, F., Bernay, B., Guillermier, M., Bonvento, G., Dufour, N., Elalouf, J.M., Hantraye, P., et al., 2015. Striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo. Neurobiol. Aging 36 (3), e1607–1616 1601. Lee, S., Kopp, F., Chang, T.C., Sataluri, A., Chen, B., Sivakumar, S., Yu, H., Xie, Y., Mendell, J.T., 2016. Noncoding RNA NORAD regulates genomic stability by sequestering PUMILIO proteins. Cell 164 (1-2), 69–80. Lill, C.M., 2016. Genetics of Parkinson's disease. Mol. Cell. Probes 30 (6), 386–396. Modarresi, F., Faghihi, M.A., Patel, N.S., Sahagan, B.G., Wahlestedt, C., Lopez-Toledano, M.A., 2011. Knockdown of BACE1-AS nonprotein-coding transcript modulates betaamyloid-related hippocampal neurogenesis. Int. J. Alzheimers Dis. 2011, 929042. Poewe, W., Seppi, K., Tanner, C.M., Halliday, G.M., Brundin, P., Volkmann, J., Schrag, A.E., Lang, A.E., 2017. Parkinson disease. Nat. Rev. Dis. Primers 3, 17013. Schapira, A.H., Cooper, J.M., Dexter, D., Clark, J.B., Jenner, P., Marsden, C.D., 1990. Mitochondrial complex I deficiency in Parkinson’s disease. J. Neurochem. 54 (3), 823–827. Scheele, C., Petrovic, N., Faghihi, M.A., Lassmann, T., Fredriksson, K., Rooyackers, O., Wahlestedt, C., Good, L., Timmons, J.A., 2007. The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function. BMC Genomics 8, 74. Shishido, T., Nagano, Y., Araki, M., Kurashige, T., Obayashi, H., Nakamura, T., Takahashi, T., Matsumoto, M., Maruyama, H., 2019. Synphilin-1 has neuroprotective effects on MPP(+)-induced Parkinson’s disease model cells by inhibiting ROS production and
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Long noncoding RNA NORAD regulates MPP+-induced Parkinson’s disease model cells Qingxin Songa, Yunming Gengb, Yue Lic, Liuli Wangd, Jinyan Qina,c,e,
T
⁎
a
Department of Neurosurgery, Linyi People's Hospital, Linyi, Shandong Province, 276000, China Department of Neurosurgery, Linyi Fourth People's Hospital, Linyi, Shandong Province, 276000, China c Department of Neurology, Linyi People's Hospital, Linyi, Shandong Province, 276000, China d Department of Health Care, Linyi Lanshan Maternal and Child Health Care Hospital, Linyi, Shandong Province, 276000, China e Department of Hyperbaric Oxygen, Linyi People's Hospital, Linyi, Shandong Province, 276000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parkinson’s disease MMP+ lncRNA NORAD
Background: Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in human diseases. Yet, the functions of lncRNAs in neurodegenerative disorders, such as Parkinson's disease (PD) are poorly understood. In this study, we used human neuroblastoma SH-SY5Y cell line as a cell-basedin vitro PD model, and investigated the role of lncRNA, Non-Coding RNA Activated By DNA Damage (NORAD) in 1-methyl-4-phenylpyridinium (MPP+)-induced PD-like cytotoxicity. Methods: SH-SY5Y cells were culturedin vitro, and treated with MPP + at various concentrations, or of various durations of times to induce PD-like cytotoxic events. qRT-PCR was used to measure MPP+-induced NORAD expression changes. Lentiviral transduction was applied to stably upregulate or downregulate NORAD in SHSY5Y cells. The effects of NORAD upregulation or downregulation on MPP+-induced cytotoxic events, including dose-dependent and time-dependent cell death, apoptosis, caspase 3/7, reactive Oxygen Species (ROS) and lactate dehydrogenase (LDH) activities, were quantitatively investigated. Results: MPP + induced cytotoxicity, and downregulated NORAD in both dose- and time- dependent manners in SH-SY5Y cells. Lentiviral-induced NORAD upregulation was found to protect against MPP+-induced cytotoxicity in SH-SY5Y cells, as it rescued MPP+-induced cellular destruction and apoptosis, as well as decreased MPP +-induced caspase 3/7, ROS and LDH activities. Alternatively, NORAD downregulation was found to significantly deteriorate MPP+-induced cytotoxicity in SH-SY5Y cells. Conclusion: We presented a novel functional role of lncRNA NORAD in regulating human Parkinson’s disease.
1. Introduction Parkinson's disease (PD) is one of the most progressive neurodegenerative disorders for both men and women patients, affecting at least 2% of the population above 65 years at any given time (Tysnes and Storstein, 2017; Ascherio and Schwarzschild, 2016; Poewe et al., 2017). While the cellular features of PD pathophysiology are characteristic and clear, mainly caused by neuronal loss in the substantia nigra due to striatal dopamine deficiency and intracellular aggregation of α-synuclein, the underlying genetic mechanisms contributing to those features are largely unknown (Tysnes and Storstein, 2017; Bastide et al., 2015; Lill, 2016). Long noncoding RNAs (lncRNAs) are families of non-protein-coding, long-length (> 200 nucleotides) RNAs, that had been recently shown to
play critical roles in many pathological processes in human diseases (Wapinski and Chang, 2011; Esteller, 2011; Taft et al., 2010). In neurodegenerative disorders, several lncRNAs were demonstrated to be dysregulated and having functional roles in regulating the progression of neurodegenerations in human brains (see review (Wan et al., 2017)). Specifically, in Parkinson's disease, the expression of lncRNA antisense to Uchl1 (AS Uchl1) was found to be close associated with early-onset of PD (Carrieri et al., 2015). In addition, de-regulation of lncRNA phosphatase and tensin homologue–induced kinase1 (PINK1) was found to be linked to mitochondrial dysfunction leading to dopamine release deficit (Scheele et al., 2007). Of many of the human diseases-associated lncRNAs, Noncoding RNA activated by DNA damage (NORAD) was initially identified as an exon encoding transcript at Chr20q11.23 that functionally contributes
⁎ Corresponding author at: 27 Jiefang Road East Section, Department of Hyperbaric Oxygen, Linyi Fourth People's Hospital, Linyi, Shandong Province, 276000, China. E-mail address: [email protected] (J. Qin).
https://doi.org/10.1016/j.jchemneu.2019.101668 Received 23 May 2019; Received in revised form 13 July 2019; Accepted 13 August 2019 Available online 14 August 2019 0891-0618/ © 2019 Published by Elsevier B.V.
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specific absorbance measured using equation, [Abs450 nm(Test) – Abs450 nm(Blank)] – Abs660 nm(Test), and then normalized against the specific absorbance for control wells.
to genomic stability by sequestering PUMILIO proteins (Lee et al., 2016). Recently, NORAD was found to be de-regulated in several types of human cancers, and may have significant impact on cancer cell oncogenesis, development, metastasis and apoptosis (see review (Yang et al., 2019)). However, there have been no reports showing any functional involvement of NORAD during the development or progression in human neurodegenerative diseases. In this study, we took advantage of a cell-based Parkinson's disease model, in which human neuroblastoma SH-SY5Y cell line was treated with 1-methyl-4-phenylpyridinium (MPP+) in vitro, to induce both dose- and time- dependent PD-like cytotoxic cellular events (Xie et al., 2010; Fall and Bennett, 1999; Xicoy et al., 2017; Shishido et al., 2019; Zhu et al., 2018). During the meantime, we used qRT-PCR to explore the dynamic expression change of NORAD in response to MPP + treatment in SH-SY5Y cells. Most importantly, we used lentiviral transduction to successfully created SH-SY5Y cell lines with stable NORAD upregulation or NORAD downregulation. Then, the effects of NORAD upregulation and downregulation on MPP+-induced cytotoxic events were carefully examined.
2.5. RNA extraction and quantitative real time-PCR
2. Materials and methods
From SH-SY5Y cells, RNA extraction and purification was completed using a PureLink Pro 96 total RNA Purification Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. Reverse Transcription to obtain complementary DNA (cDNA) was completed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer’s instruction. To assess lncRNA NORAD gene expression, quantitative real time-PCR (qRT-PCR) was conducted on an ABI PRISM 7700 Sequence Detector System (Applied Biosystems, USA), and using a TaqMan Universal PCR Master Mix Kit (Applied Biosystems, USA) and a TaqMan non-coding RNA assay with a customized human NORAD qRT-PCR probe (Invitrogen, USA), according to the manufacturers’ instructions. In addition, a 18S rRNA TaqMan assay (Invitrogen, USA) was conducted as control. Finally, relative expression level of NORAD was calculated using the 2(−ΔΔCt) method.
2.1. Ethics statement
2.6. NORAD upregulation and downregulation in SH-SY5Y cells
In this study, all experimental protocols were thoroughly reviewed and approved by the Clinical Research & Ethics Committee at the Linyi People's Hospital, Linyi Fourth People's Hospital and Linyi Lanshan maternal and child health care hospital in Linyi, Shandong Province, China. Also, all procedures related to animals were conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in the United States of America (https://grants.nih.gov/grants/olaw/Guide-for-thecare-and-use-of-laboratory-animals.pdf).
Lentiviral vectors containing the full sequence of human NORAD (L/ NORADm), an empty lentiviral vector (L/Cm), a short hairpin RNA (shRNA) targeting NORAD (L/NORADi) and a non-specific shRNA (L/ Ci) were all synthesized and purchased from RiboBio (RiboBio Biotechnology Ltd., Guangzhou, China). In addition, transfection of lentiviral vectors, along with lentivirus packaging vectors in human HEK293 T cells to yield high-titer lentiviruses were also conducted by RiboBio (RiboBio Biotechnology Ltd., Guangzhou, China). In the in vitro culture of SH-SY5Y cells, cells were transduced with lentivirus of L/ NORADm, L/Cm, L/NORADi or L/Ci and 8 μg/ml polybrene (Thermo Fisher Scientific, USA) at multiplicity of infection (MOI) of 3 for 48 h, followed by a blasticidin selection procedure (15 μg/ml, Thermo Fisher Scientific, USA) for additional 5 days. After discarding the floating colonies, the attached, healthy, round-shape multi-cell colonies were carefully selected and re-plated in 6-well plates to proliferate. After 5˜7 passages, qRT-PCR was conducted to confirm the upregulation or downregulation of NORAD in SH-SY5Y cells.
2.2. Cell culture In this study, human neuroblastoma cell line, SH-SY5Y was commercially purchased from the American Type Culture Collection in the United States of America (ATCC, USA). Cells were maintained in 6-well plate (VWR, USA) in the Dulbecco's Modified Eagle Medium with Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific, USA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, USA) and 100 U/ml Penicillin-Streptomycin (Gibco, USA) in a tissue-culture chamber with circulating gas of 95% O2 / 5% CO2 at 37 °C.
2.7. Measurement of apoptosis SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, cellular apoptosis was assessed using a Click-iT™ Plus TUNEL Assay for in situ Apoptosis Detection Kit (Alexa Fluor™ 594 dye, Invitrogen, USA) according to the manufacturer’s instruction. Also, a DAPI (blue) staining was performed for 15 min. Then, 96-well plates were moved onto an Evos microscope system (Thermo Fisher Scientific, USA). Apoptotic cells were characterized as those positive to both DAPI and TUNEL stainings. Then, the percentage of non-apoptotic cells in each well was measured.
2.3. Cell-based Parkinson's disease model In this study, we adapted a cell-based Parkinson's disease (PD) model, which was well established in previous publications (Xie et al., 2010; Fall and Bennett, 1999; Xicoy et al., 2017; Shishido et al., 2019; Zhu et al., 2018). To induced PD-like cytotoxicity in the culture of SHSY5Y cells, cells were treated with 1-methyl-4-phenylpyridinium (MPP +) (Thermo Fisher Scientific, USA) in vitro. The treatment of MMP + may vary at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM, or in times of 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h.
2.8. Measurement of caspase-3/7 activity SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, caspase-3 and caspase-7 activities were assessed in SH-SY5Y culture using a CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the relative caspase 3/7 activity was calculated as fluorescence measurement at 530 nm (excited at 500 nm) and normalized to
2.4. Cell viability assay SH-SY5Y cells were collected and re-seeded in 96-well plates (VWR, USA). Post MPP + treatment, the death and viability of SH-SY5Y cells was assessed using a CyQUANT™ XTT Cell Viability Assay (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the absorbance (Abs) was read at 450 nm and 660 nm. Relative cell viability was characterized as the 2
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the fluorescence measurement for control wells.
SY5Y cells. After transduction was stabilized, qRT-PCR showed that, SH-SY5Y cells transduced with L/NORADm had significantly higher NORAD expression level than cells transduced with L/Cm (Fig. 2A, * P < 0.05). Conversely, SH-SY5Y cells transduced with L/NORADi had significantly lower NORAD expression level than cells transduced with L/Ci (Fig. 2B, * P < 0.05). Therefore, these results showed that we successfully created SH-SY5Y cells with stably upregulated or downregulated NORAD. Then, we tested the effects of NORAD upregulation / downregulation on MPP+-induced dose-dependent and time-dependent SHSY5Y cytotoxicity. First, L/Cm-, L/NORADm-, L/Ci- and L/NORADitransduced SH-SY5Y cells were treated with MPP + for 24 h, at concentrations ranging from 0.05 to 10 mM, respectively. The result of viability assay showed that, for cells treated with MPP + at 0.25 mM or higher concentrations for 24 h, MPP+-induced cytotoxicity was significantly reduced by NORAD upregulation (Fig. 2C, * P < 0.05). On the other hand, in SH-SY5Y cells treated with MPP+ at 0.1 mM or higher concentrations for 24 h, MPP+-induced cytotoxicity was significantly enhanced by NORAD downregulation (Fig. 2D, * P < 0.05). Second, L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SHSY5Y cells were treated with 5 mM MPP + for different periods of time, ranging from 0.5 to 24 h, respectively. The result of viability assay showed that, for cells treated with 5 mM MPP + between 16 and 24 h, MPP+-induced cytotoxicity was significantly reduced by NORAD upregulation (Fig. 2E, * P < 0.05). On the other hand, in SH-SY5Y cells treated with 5 mM MPP+ between 2 and 24 h, MPP+-induced cytotoxicity was significantly enhanced by NORAD downregulation (Fig. 2F, * P < 0.05). Thus, these results clearly demonstrated that NORAD upregulation could protect against, whereas NORAD downregulation could further deteriorate MPP+-induced cytotoxicity in SH-SY5Y cells.
2.9. Measurement of reactive oxygen species activity SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, reactive Oxygen Species (ROS) activity was assessed using a Total Reactive Oxygen Species Assay Kit (Invitrogen, USA) according to the manufacturer’s instruction. Then, 96-well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). For each well, the relative ROS activity was calculated as fluorescence measurement at 520 nm (excited at 490 nm) and normalized to the fluorescence measurement for control wells. 2.10. Measurement of lactate dehydrogenase (LDH) level SH-SY5Y cells were lifted off from 6-well plates and equally replated in 96-well plates (5000 cells / well), and then treated with 5 mM MPP + for 6 h. Post MPP + treatment, supernatants were transferred to other 96-well plates. The lactate dehydrogenase (LDH) activity was measured using a Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific, USA) according to the manufacturer’s instruction. Then, 96well plates were moved onto a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). Absorbance was measured at 490 and 680 nm. For each well, LDH activity was calculated as an absorbance ratio at 490/680 nm and normalized to the absorbance ratio in control wells. 2.11. Statistical analysis In this study, all statistical analyses were done using a one-way ANOVA followed by post-hoc test on a Windows-Based SPSS software (SPSS, Version 20.0, USA). All experiments were independently repeated for at least 3 times. The averaged data were presented as means +/- standard errors of the mean (SEM). P < 0.05 was considered as statistically different.
3.3. Genetic modification of NORAD regulated MPP+-induced apoptosis in SH-SY5Y cells L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SH-SY5Y cells, as well un-transduced (N/A) SH-SY5Y cells were treated with 5 mM MPP + for 6 h. A TUNEL assay demonstrated that, in un-transduced (N/A) SH-SY5Y cells, and those transduced with L/Cm and L/Ci, moderate number of cells were apoptotic due to MPP + treatment (Fig. 3A, B, D). However, significantly less apoptotic cells were observed in L/NORADm-transduced SH-SY5Y cells (Fig. 3C), whereas significantly more apoptotic cells were observed in L/NORADi-transduced SH-SY5Y cells (Fig. 3E). Then, quantitative measurement on the percentages of apoptotic SH-SY5Y cells showed that, while more apoptotic cells were induced by MPP + treatment in un-transduced (N/ A) and all lentiviral-transduced SH-SY5Y cells (Fig. 3F, * P < 0.05), NORAD upregulation rescued whereas NORAD downregulation increased the apoptotic effects induced by MPP+ (Fig. 3F, ** P < 0.05). Thus, our results clearly demonstrated that NORAD upregulation could reduce whereas NORAD downregulation would further increase MPP+-induced apoptosis in SH-SY5Y cells.
3. Results 3.1. LncRNA NORAD was downregulated during the process of MPP +-induced cytotoxicity in SH-SY5Y cells SH-SY5Y cells were treated with MPP + for 24 h, at concentrations ranging from 0.05 to 10 mM. MPP+-induced cytotoxicity was assessed using a viability assay. It demonstrated that, significant cellular destruction was observed in SH-SY5Y cells treated with 0.1 mM or higher concentrations of MPP+ (Fig. 1A, * P < 0.05, vs. 0 mM MPP+). During the meantime, qRT-PCR showed that, NORAD expression was significantly downregulated in SHSY5Y cells treated with 0.25 mM or higher concentrations of MPP+ (Fig. 1B, * P < 0.05, vs. 0 mM MPP+). In addition, SH-SY5Y cells were treated with 5 mM MPP + for different periods of time, ranging from 0.5 to 24 h. Cytotoxic measurement showed that, significant cellular destruction was observed in SH-SY5Y cells treated with 5 mM MPP + for 2 h or longer (Fig. 1C, * P < 0.05, vs. 0 h). Subsequently, qRT-PCR showed that NORAD expression was significantly downregulated in SHSY5Y cells treated with 5 mM MPP+ for 6 h or longer (Fig. 1D, * P < 0.05, vs. 0 h). Therefore, these results showed that MPP+-induced cytotoxicity also induced dose-dependent and time-dependent NORAD downregulation in SH-SY5Y cells.
3.4. Genetic modification of NORAD regulated MPP+-induced caspase 3/ 7, ROS activities and LDH level in SH-SY5Y cells Finally, for L/Cm-, L/NORADm-, L/Ci- and L/NORADi-transduced SH-SY5Y cells, as well as un-transduced (N/A) SH-SY5Y cells who were treated with (or without) 5 mM MPP + for 6 h, Parkinson’s Disease-like cytotoxic indicators, including caspase 3/7 activity, ROS activity and LDH level, were quantitatively measured. The results showed that, in both lentiviral-transduced and un-transduced SH-SY5Y cells, caspase 3/ 7 activity, ROS activity and LDH level were enhanced by MPP + treatment (Fig. 4A, B, C, * P < 0.05). In addition, as compared to L/Cm-transduced SH-SY5Y cells, MPP+-induced caspase 3/7 activity, ROS activity and LDH level were all decreased in L/NORADm-
3.2. Genetic modification of NORAD regulated MPP+-induced cytotoxicity in SH-SY5Y cells Genetic modification, including both upregulation and downregulation of NORAD, was performed by lentiviral transduction in SH3
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Fig. 1. MPP + downregulated NORAD in SHSY5Y cells. (A) SH-SY5Y cells were treated with MPP + at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM for 24 h. MPP +-induced dose-dependent cytotoxicity was measured by a viability assay (* P < 0.05, vs. 0 mM MPP+). (B) In addition, MPP+-induced dose-dependent NORAD downregulation was measured by qRT-PCR (* P < 0.05, vs. 0 mM MPP+). (C) SHSY5Y cells were treated with 5 mM MPP + for 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h. MPP+-induced time-dependent cytotoxicity was measured by a viability assay (* P < 0.05, vs. 0 h). (D) In addition, MPP+-induced time-dependent NORAD downregulation was measured by qRT-PCR (* P < 0.05, vs. 0 h).
4. Discussions
transduced SH-SY5Y cells (Fig. 4A, B, C, ** P < 0.05). Conversely, as compared to L/Ci-transduced SH-SY5Y cells, MPP+-induced caspase 3/ 7 activity, ROS activity and LDH level were all enhanced in L/NORADitransduced SH-SY5Y cells (Fig. 4A, B, C, ** P < 0.05). Thus, our results demonstrated that NORAD upregulation could reduce whereas NORAD downregulation would further increase MPP +-induced cytotoxic events in SH-SY5Y cells.
Mounting evidence has demonstrated that lncRNAs may play important roles in human neurodegenerative diseases (Wan et al., 2017). In Alzheimer’s disease, lncRNA β-secretase-1 antisense RNA (BACE1AS) was found to be associated with hippocampal neurogenesis in animal models (Modarresi et al., 2011), and knockdown of BACE1-AS dramatically reduced cortical β-amyloid synthesis and aggregation in vivo (Faghihi et al., 2008). In Huntington’s disease, the results from an in vivo mouse model demonstrated that overexpression of lncRNA 4
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Fig. 2. The effects of NORAD upregulation and downregulation on MPP+-induced toxicity in SH-SY5Y cells. (A) SH-SY5Y cells were transduced with L/Cm or L/NORADm lentiviruses, followed by qRT-PCR to assess their endogenous NORD expressions (* P < 0.05). (B) SH-SY5Y cells were transduced with L/Ci or L/ NORADi lentiviruses, followed by qRT-PCR to assess their endogenous NORD expressions (* P < 0.05). (C–D) SH-SY5Y cells transduced with L/Cm, L/NORADm, L/Ci and L/NORADi were treated with MPP + at concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mM for 24 h. MPP+-induced dose-dependent cytotoxicity was measured by a viability assay, and compared between L/Cm- and L/NORADmtransduced SH-SY5Y cells (C), and L/Ci- and L/ NORADi- transduced SH-SY5Y cells (D) (* P < 0.05). (E–F) SH-SY5Y cells transduced with L/Cm, L/NORADm, L/Ci and L/NORADi were treated with 5 mM MPP + for 0, 0.5, 1, 2, 6, 8, 16, 20 and 24 h. MPP+-induced timedependent cytotoxicity was measured by a viability assay, and compared between L/Cmand L/NORADm- transduced SH-SY5Y cells (E), and L/Ci- and L/NORADi- transduced SHSY5Y cells (F) (* P < 0.05).
models or human subjects are certainly needed. Another intriguing feature of our findings is that, while NORAD was found to be downregulated in response to MPP + treatment, it was predominantly found to be upregulated in several human cancer tumors (see review (Yang et al., 2019)). It may suggest that, the exact NORAD dys-regulation patterns in human diseases may vary due to different pathological conditions. The most important findings in this study was that we identified a protective mechanism of NORAD upregulation during the process of MPP+-induced cytotoxicity in SH-SY5Y cells. Using lentiviral transduction, we were able to create SH-SY5Y cell lines with stable NORAD upregulation or NORAD downregulation. Then, we were able to demonstrated that NORAD upregulation could protect against, whereas NORAD downregulation could enhance MPP+-induced cytotoxicity in SH-SY5Y cells, in both dose- and time- dependent manners.
Abhd11os had neuro-protective effect against N-terminal fragment of mutant huntingtin protein (Francelle et al., 2015). Then, in Parkinson's disease, aberrant expression of lncRNA PINK1 was demonstrated to be directly associated with dopamine release dysfunction (Scheele et al., 2007). In this study, we focused on a novel lncRNAs NORAD, whose biological function was just discovered recently (Lee et al., 2016), and explored its expression and functions during the process of MPP+-induced cytotoxicity in SH-SY5Y cells. First, we discovered that NORAD was downregulated in MPP+-injured SH-SY5Y cells, in both dose- and time- dependent manners. This novel observation is very important, suggesting that NORAD expression may be closely associated with Parkinson’s Diseases’ pathological conditions, at least under in vitro environment. It would be interesting to find out if it’s still the case under in vivo conditions, as further in vivo studies involving animal 5
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Fig. 3. The effects of NORAD upregulation and downregulation on MPP+-induced apoptosis in SH-SY5Y cells. (A–E) Un-transduced (NA) SH-SY5Y cells (A), L/Cm-transduced SH-SY5Y cells (B), L/NORADm-transduced SH-SY5Y cells (C), L/Ci-transduced SH-SY5Y cells (D), L/NORADi-transduced SH-SY5Y cells (E) were either treated with 5 mM MPP+, or not treated with MPP + for 6 h, followed by a TUNEL assay (Red) to identify apoptotic cells. A DAPI antibody (Blue) was also applied to identify the nuclei of SH-SY5Y cells. (F) The relative percentages of apoptotic SH-SY5Y cells were quantitatively measured for MPP+-untreated and MPP+-treated SH-SY5Y cells, which were un-transduced with lentivirus (NA), or transduced with L/Cm, L/NORADm, L/Ci and L/ NORADi (* P < 0.05, ** P < 0.05).
While it’s truly remarkable to reveal a possible functional role of NORAD in human PD, we also acknowledge that the limitation of our study. As mentioned, it is critical to test NORAD functions in in vivo PD animal models, or explore its expression in human PD patients. In addition, it is important to explore the associated signaling pathways, which could be either pre- or post-transcriptional, to be responsible NORAD regulations in PD. Thus, future experiments are much needed.
In addition, we explored the functions of NORAD on MPP+-induced SH-SY5Y cell apoptosis, and its associated caspase 3/7 signaling pathway. Specifically, we looked into MPP+-induced mitochondrial dysfunctions in SH-SY5Y cells, including ROS activity and LDH level. In all examined biochemical assays, it was demonstrated that NORAD upregulation rescued, whereas NORAD downregulation deteriorated MPP+-induced apoptosis and mitochondrial dysfunction, suggesting that NORAD might be able to regulate intrinsic onset, development or inhibition of PD-associated apoptotic or mitochondrial signaling pathways (Tatton et al., 2003; Anglade et al., 1997; Schapira et al., 1990; Abou-Sleiman et al., 2006). It is worth noting that in all experiments, the results obtained from SH-SY5Y cells transduced with control lentiviruses (L/Cm or L/Ci) were similar to those obtained from cells without lentiviral transduction (N/A). These data further confirmed our findings that genetic modification of NORAD could functional affect MPP+-induced cytotoxic events in SH-SY5Y cells.
5. Conclusion To conclude, in this study we presented a novel functional role of lncRNA NORAD in regulating MPP+-induced cytotoxicity in an in vitro model of Parkinson’s disease. Further investigations would help to identify the role of NORAD in human patients with Parkinson’s disease.
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Fig. 4. The effects of NORAD upregulation and downregulation on MPP+-induced caspase 3/7, ROS and LDH activities in SH-SY5Y cells. Un-transduced (NA), L/ Cm-, L/NORADm-, L/Ci- and L/NORADi- transduced SH-SY5Y cells were either treated with 5 mM MPP+, or not treated with MPP + for 6 h. Then, their caspase 3/7 activities (A), ROS activities (B) and LDH levels (C) were quantitatively measured and compared (* P < 0.05, ** P < 0.05).
Declaration of Competing Interest
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