LncRNA EPIC1 downregulation mediates hydrogen peroxide-induced neuronal cell injury

Excessive oxidative stress causes neuronal cell injury. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a MYC-interacting LncRNA. Its expression and potential functions in hydrogen peroxide (H2O2)-stimulated neuronal cells are studied. In SH-SY5Y neuronal cells and primary human neuron cultures, H2O2 downregulated Lnc-EPIC1 and key MYC targets (Cyclin A1, CDC20 and CDC45). Ectopic overexpression of Lnc-EPIC1 increased expression of MYC targets and significantly attenuated H2O2-induced neuronal cell death and apoptosis. Contrarily, Lnc-EPIC1 siRNA potentiated neuronal cell death by H2O2. MYC knockout by CRISPR/Cas9 method also facilitated H2O2-induced SH-SY5Y cell death. Significantly, MYC knockout abolished Lnc-EPIC1-induced actions in H2O2-stimulated neuronal cells. Together, these results suggest that Lnc-EPIC1 downregulation mediates H2O2-induced neuronal cell death.


INTRODUCTION
Neurons in the central nerve system (CNS) are vulnerable to reactive oxygen species (ROS) overproduction and excessive oxidative injury. It is possibly due to the high rate of oxygen consumption, enrichment of polyunsaturated fatty acids and defective Nrf2 cascade in neurons [1][2][3][4][5]. Excessive oxidative stress will induce profound neuronal cell injury, serving as a key pathogenesis mechanism of neurodegenerative diseases [1][2][3][4]. Hydrogen peroxide (H2O2), produced during the redox process, has been widely utilized to treat cultured neurons and neuronal cells in vitro, mimicking oxidative injury [6][7][8]. H2O2 overproduction can induce profound lipid peroxidation, DNA breaks, protein damage and eventually neuronal cell death and apoptosis [7,9,10].
Understanding the molecular mechanisms of H2O2induced neuronal cell death is vital for developing possible intervention strategies.
Long non-coding RNAs (LncRNAs) are traditionally considered as transcriptional noise. Recently, there are growing literatures showing that LncRNAs are key regulators of almost all cellular and physiological processes [11,12]. LncRNAs' functions in CNS and neuronal cells are still largely elusive [13,14]. The transcription factor MYC is vital for the transcription and expression of key genes of cell survival in neuronal cells [15,16]. The activity of MYC is mainly controlled by transcriptional and posttranscriptional mechanisms [17,18]. Wang et al., recently demonstrated that LncRNA EPIC1 AGING (epigenetically-induced LncRNA1, "Lnc-EPIC1") directly associates to MYC [19].
In the primary human neuronal cultures, siRNA-mediated knockdown of Lnc-EPIC1 (by "EPIC1-siRNA1", see Figure 3I) augmented H2O2-induced viability reduction ( Figure 3J) and cell death ( Figure 3K). Collectively, H2O2-induced neuronal cytotoxicity is exacerbated by Lnc-EPIC1 silencing, further supporting that Lnc-EPIC1 downregulation mediates H2O2-induced cytotoxicity in the neuronal cells. (I-K) were transfected with applied Lnc-EPIC1 siRNA ("EPIC1-siRNA1/2", 100-500 nM) or scramble control siRNA ("scr-siRNA", 500 nM) for 48h, then treated with/without hydrogen peroxide (H2O2, 300 μM), cells were further cultured for indicated time, expression of Lnc-EPIC1 (A and I) and listed mRNAs (B and C) were tested by qPCR assay; Expression of listed proteins was tested by Western blotting assay (D); Cell viability (by the CCK-8 assay, E and J), cell death (by the LDH assay, F and K) and apoptosis (by the caspase-3 activity and ssDNA ELISA, G and H) were tested. Listed proteins were quantified, with the values normalized to Tubulin (D). Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. "Ctrl" treatment in "scr-siRNA" cells. # P < 0.05 vs. H2O2 treatment of "scr-siRNA" cells. Experiments in this figure were repeated three times, and similar results were obtained. AGING
MYC-KO cells presented with decreased cell viability and increased cell death, when compared to the control cells with lenti-CRISPR control construct ("Cas9-C") ( Figure 4B and 4C). Importantly, MYC-KO cells were more vulnerable to H2O2, by showing significantly enhanced viability reduction and death versus control cells ( Figure 4B and 4C). Therefore, MYC-KO cells presented with similar phenotypes to the Lnc-EPIC1silenced cells (Figure 3). Notably, MYC-KO did not significantly change Lnc-EPIC1 expression in SH-SY5Y cells with/without H2O2 stimulation ( Figure 4D).

DISCUSSION
LncRNAs are involved in regulating almost all key cellular behaviors, by functioning as molecular signals, guides, scaffolds, enhancers or microRNA spongers, and altering gene transcription and/or functions [11,12,23]. Moreover, LncRNAs are important for genomic imprinting, cell cycle progression, cell survival, differentiation and development [11,12,23]. A growing number of studies have reported that aberrant LncRNA expression is detected in neuronal cells and human brain, participating in the pathogenesis of brain and even in neurodegenerative diseases [14]. LncRNA can function as a endogenous RNA (ceRNA) for various miRNAs to inhibit or promote neuronal cell apoptosis. For example, Li et al., have shown that LncRNA KCNQ1OT1 promoted SH-SY5Y neuronal cell apoptosis by sponging miR-296 and upregulating Bax [24]. In ischemic stroke LncRNA SNHG6 (small nucleolar RNA host gene 6) promoted neuronal cell apoptosis by inhibiting miR-181c but upregulating its target Bim [25]. Here we tested the involvement of Lnc-EPIC1 in H2O2-induced neuronal cell death.
LncRNA-mediated regulation on MYC transcriptional activity is largely unknown until recently [19]. MYC by itself is unable to form a homodimer nor binding to DNA. Certain LncRNAs interact directly with proteins and can influence the structural state and activity of these proteins [14]. Wang et al., have demonstrated that Lnc-EPIC1 regulates MYC's occupancy on several key MYC target genes, including Cyclin A1, CDC20 and CDC45 [19]. By directly binding to double-strand DNA, Lnc-EPIC1 functions as the "guide" RNA to promote expression of MYC targets [19]. Furthermore, MYC-MAX association can also be facilitated by Lnc-EPIC1 [19]. Here our evidence suggest that Lnc-EPIC1 is also vital for the survival of neuronal cells.
We here reported a new mechanism of Lnc-EPIC1 in promoting neuronal cell survival via directly interacting with MYC, and Lnc-EPIC1 is important for MYC function in neuronal cells. In both SH-SY5Y cells and primary neuron cultures, forced overexpression of Lnc-EPIC1 significantly increased mRNA and protein expression of key MYC targets, including Cyclin A1, CDC20 and CDC45. Contrarily, their levels were significantly reduced by siRNA-mediated silencing of Lnc-EPIC1. Importantly, Lnc-EPIC1 was decreased in H2O2-treated SH-SY5Y cells and primary neuronal cultures. Therefore, H2O2 downregulates Lnc-EPIC1 to inhibit MYC functions, which could be an important mechanism responsible for H2O2-induced neuronal cell death.
A number of recent data have implied that Lnc-EPIC1, by directly interacting with MYC, promotes survival of human cells [20][21][22]. However, Zhao et al., have reported that it inhibited osteosarcoma cell survival by inducing MEF2D ubiquitination [26]. Results from this study support that Lnc-EPIC1 exerts neuronal protective functions. We show that Lnc-EPIC1 downregulation mediated H2O2-induced neuronal cytotoxicity. Importantly, ectopic overexpression of Lnc-EPIC1 using a lentiviral construct significantly attenuated H2O2induced neuronal cytotoxicity. On the contrary, siRNAmediated silencing of Lnc-EPIC1 potentiated neuronal cell death and apoptosis by H2O2. Lnc-EPIC1 siRNA alone was cytotoxic to neuronal cells as well. Thus, targeting Lnc-EPIC1 could be a novel strategy for neuronal protection against oxidative injury.
MYC is a transcription factor regulating expression of a wide variety of genes of cell survival. In the present study, we show that MYC KO by CRISPR/Cas9 method induced neuronal cell death and apoptosis. MYC KO also intensified H2O2-induced neuronal cytotoxicity.

Reagents
H2O2, puromycin, polybrene, fetal bovine serum (FBS) and other cell culture reagents were provided by Sigma-Aldrich Co. (St. Louis, MO). Antibodies were purchased from Cell Signaling Technology Co. (Shanghai, China). mRNA primers and sequences were synthesized by Genechem (Shanghai, China). All the transfection reagents were purchased from Invitrogen-Thermo Fisher Co. (Shanghai, China).

Primary human neuron cultures
The primary human neuron cultures were provided by Dr. Zhang [28]. Briefly, the normal fetal brains were digested and filtered. The dissociated neurons were centrifuged and resuspended in MEM medium with the described supplements [29]. Neurons were plated in poly-L-lysine-coated six-well plates. Astrocytes were abandoned [29]. Neuron cultures were grown for eight days (day in vitro 8, DIV8) before any further experiments. The protocols of using human cells were approved by the Ethics Committee of authors' institutions.

Quantitative real-time PCR assay (qPCR)
Following the applied treatment, the TRIzol reagents (Promega) was applied to extract total cellular RNA. The Fast-Start Universal SYBR Green Master mix (Roche) was used for the qPCR. Melting curve analysis was performed to calculate the product melting temperature. qPCR quantification was through 2 -ΔCt method using the following formula: 2 -(Ct of target gene-Ct of reference gene) . The data presented were normalized to GAPDH transcripts. mRNA primers for MYC, Cyclin A1, CDC20, and CDC45 [19] and Lnc-EPIC1 [22] were synthesized by Genechem (Shanghai, China). Lnc-EPIC1 expression was normalized to U6 RNA.

Western blotting assay
For each treatment, 40 μg protein lysates were separated by 10% SDS-PAGE gels, transferred to PVDF blots.
After blocking in 10% non-fat milk, the blots were probed with the appropriate primary antibodies, followed by incubation in the HRP-conjugated secondary antibodies. The blots were visualized by the ECL system (Sigma). Quantification of the band intensity was performed via using the Image J software.

LDH assay
Following the applied treatment, lactate dehydrogenase (LDH) release to the conditional medium of dying cells was tested by a two-step LDH detection kit (Takara, Tokyo, Japan). Medium LDH was normalized to total LDH.

ssDNA ELISA assay
Single strand DNA (ssDNA) contents were measured as a characteristic marker of cell apoptosis. In brief, thirty μg cell lysates per treatment were analyzed by the ssDNA ELISA (Roche, Basel, Switzerland) to quantify DNA fragmentations. The ssDNA ELISA absorbance values were recorded at 405 nm.

JC-1 assay of mitochondrial depolarization
JC-1, a fluorescence dye, can form green monomers and aggregate in mitochondria in stressed cells with mitochondrial depolarization [31]. Following the applied H2O2 treatment, neuronal cells were first stained with JC-1 (10 μg/mL), washed and tested immediately via a fluorescence spectrofluorometer at 550 nm. The representative JC-1 images, integrating the green (at 550 nm) and red (at 625 nm) fluorescence wavelength were presented.

Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analyses among different groups were performed by the one-way analysis of variance (ANOVA) with Scheffe's test using SPSS18.0 software (SPSS Inc., Chicago, IL). The two-tailed paired T tests (Excel 2007) were calculated to compare significance between two groups. Values of P < 0.05 were considered statistically significant. Experiments were repeated at least three times.

AUTHORS CONTRIBUTIONS
All listed authors designed the study, performed the experiments and the statistical analysis, and wrote the manuscript. All authors have read the manuscript and approved the final version.

CONFLICTS OF INTEREST
The authors declare no conflict of interests.