LncRNA EPIC1 downregulation mediates hydrogen peroxide-induced neuronal cell injury

H₂O₂ downregulates Lnc-EPIC1 in neuronal cells

In order to study the potential effect of LncRNA EPIC1 (Lnc-EPIC1) in neuronal cells, differentiated SH-SY5Y neuronal cells were treated with H₂O₂. Expression of Lnc-EPIC1 was tested by qPCR assay using the described primers [21, 22]. Its level was normalized to U6 RNA. As demonstrated, H₂O₂ treatment (300 μM) time-dependently decreased Lnc-EPIC1 level in SH-SY5Y cells (Figure 1A). Its levels reduced to 83.51 ± 6.56%, 61.79 ± 3.31%, 43.34 ± 7.72%, 37.84 ± 2.10% of control after 2h, 4h, 8h and 16h of H₂O₂ treatment, respectively (Figure 1A). Further, in a dose-dependent manner H₂O₂ downregulated Lnc-EPIC1 in SH-SY5Y cells (Figure 1B). Lnc-EPIC1 downregulation was significant following 100-300 μM of H₂O₂ treatment (Figure 1B). At the lowest concentration (50 μM) H₂O₂ failed to significantly alter Lnc-EPIC1 expression (P > 0.05 vs. “Ctrl”, Figure 1B).

Figure 1. H₂O₂ downregulates Lnc-EPIC1 in neuronal cells. SH-SY5Y cells (A–D) or the primary human neuron cultures (E–G) were treated with hydrogen peroxide (H₂O₂, 50-300 μM), cells were further cultured for indicated time periods, expression of Lnc-EPIC1 (A, B and E, vs. U6 RNA) and listed mRNAs (C and F, vs. GAPDH) were tested by qPCR assay. Listed proteins in total cell lysates were tested by Western blotting assay (D and G). Listed proteins were quantified, with the values normalized to Tubulin (D and G). “Ctrl” stands for untreated control cells (same for all Figures). “LDH%” stands for medium LDH ratio, indicating cell death (same for all Figures). Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Ctrl” cells. Experiments in this figure were repeated three times, and similar results were obtained.

Lnc-EPIC1 directly interacts with MYC, essential for MYC function and expression of key MYC targets [19, 21, 22]. qPCR assay results, in Figure 1C, demonstrated that mRNA levels of the MYC targets, Cyclin A1, CDC20 and CDC45 [19, 21, 22], were significantly downregulated after H₂O₂ (100/300 μM) treatment in SH-SY5Y cells. Cyclin A1, CDC20 and CDC45 protein levels were decreased as well (Figure 1D), where MYC mRNA and protein expression were unchanged (Figure 1C and 1D).

In the primary human neuronal cultures, H₂O₂ treatment (300 μM, 16h) significantly downregulated Lnc-EPIC1 expression (28.24 ± 1.21% of control, Figure 1E). mRNA and protein expression levels of MYC targets, Cyclin A1, CDC20 and CDC45, were also downregulated (Figure 1F and 1G). MYC expression was again unchanged by H₂O₂ (Figure 1F and 1G). These results show that H₂O₂ downregulates Lnc-EPIC1 and MYC targets in neuronal cells, indicating a functional activity of Lnc-EPIC1 in H₂O₂-induced neuronal cytotoxicity.

Ectopic overexpression of Lnc-EPIC1 inhibits H₂O₂-induced neuronal cytotoxicity

In order to test the potential function of Lnc-EPIC1 in H₂O₂-induced neuronal cytotoxicity, a lentiviral Lnc-EPIC1-expression construct was transfected to SH-SY5Y cells. Three stable SH-SY5Y cell lines (“Lnc-EPIC1-OE-1/-2/-3”) were established following puromycin selection (see Methods). qPCR assay was performed to show that Lnc-EPIC1 levels were significantly elevated in the Lnc-EPIC1-OE cells, with/without H₂O₂ treatment (Figure 2A). Expression of key MYC targets, including CDC20 and CDC45, were dramatically increased in Lnc-EPIC1-OE cells (Figure 2B and 2C). Further, H₂O₂-induced downregulation of CDC20 and CDC45 was reversed by Lnc-EPIC1 overexpression (Figure 2B and 2C).

Figure 2. Ectopic overexpression of Lnc-EPIC1 inhibits H₂O₂-induced neuronal cytotoxicity. Stable SH-SY5Y cells with the lentiviral Lnc-EPIC1-expression construct (three lines, “Lnc-EPIC1-OE-1/-2/-3”) or the vector control cells (“Vector”) were treated with hydrogen peroxide (H₂O₂, 300 μM), cells were further cultured for indicated time, expression of Lnc-EPIC1 (A) and listed mRNAs (B and C) were tested by qPCR assay; Cell survival (by the CCK-8 assay, D), death (by the LDH assay, E) and apoptosis (by the caspase-3 activity, ssDNA ELISA and JC-1 staining assays, F–H) were tested. The primary human neuron cultures were infected with the lentivirus with Lnc-EPIC1 construct (“LV-Lnc-EPIC1”) or empty vector (“Vector”) for 48h, treated with hydrogen peroxide (H₂O₂, 300 μM) for applied time, Lnc-EPIC1 expression (I), neuronal survival (by the CCK-8 assay, J) and death (by the LDH assay, K) were tested. Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Ctrl” treatment of “Vector” cells. ^#P < 0.05 vs. H₂O₂ treatment of “Vector” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar= 100 μm (H).

Significantly, H₂O₂-induced cell viability (CCK-8 OD) reduction (Figure 2D) and cell death (medium LDH release, Figure 2E) were largely attenuated by Lnc-EPIC1 overexpression (Figure 2D and 2E). H₂O₂ induced significant apoptosis activation in control SH-SY5Y cells, evidenced by caspase-3 activation (Figure 2F) and single-strand DNA (“ssDNA”) accumulation (Figure 2G), which were significantly attenuated in Lnc-EPIC1-OE cells (Figure 2F and 2G). Furthermore, H₂O₂-induced mitochondrial depolarization, evidenced by JC-1 green fluorescence accumulation, was largely inhibited with Lnc-EPIC1 overexpression (Figure 2H).

In primary human neuron cultures, transfection of the lentiviral Lnc-EPIC1-expression construct (“LV-Lnc-EPIC1”) significantly increased Lnc-EPIC1 expression, even after H₂O₂ stimulation (Figure 2I). H₂O₂-induced neuronal death, reflected by CCK-8 OD reduction (Figure 2J) and medium LDH release (Figure 2K), was significantly attenuated by LV-Lnc-EPIC1 (Figure 2I–2K). Collectively, these results show that ectopic overexpression of Lnc-EPIC1 attenuates H₂O₂-induced neuronal cytotoxicity.

Lnc-EPIC1 siRNA potentiates H₂O₂-induced neuronal cell death

Since Lnc-EPIC1 overexpression protected neuronal cells from H₂O₂ (see Figure 2), Lnc-EPIC1 silence should then potentiate H₂O₂-induced neuron injury. To test this hypothesis, two different Lnc-EPIC1 siRNAs (“EPIC1-siRNA1/2”), with non-overlapping sequences [19, 21, 22], were transfected to SH-SY5Y cells. As demonstrated, each of the applied EPIC1-siRNA (at 500 nM) further decreased Lnc-EPIC1 level in H₂O₂-treated SH-SY5Y cells (Figure 3A). MYC targets, including CDC20 and CDC45, were downregulated by EPIC1-siRNA (Figure 3B and 3C). There levels went further decreased following H₂O₂ treatment (Figure 3B and 3C). CDC20 and CDC45 protein levels were also decreased in Lnc-EPIC1-silneced SH-SY5Y cells after H₂O₂ treatment (Figure 3D).

Figure 3. Lnc-EPIC1 siRNA potentiates H₂O₂-induced neuronal cell death. SH-SY5Y cells (A–H) or the primary human neuron cultures (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.

Importantly, in SH-SY5Y cells, H₂O₂-induced viability (CCK-8 OD) reduction (Figure 3E), cell death (medium LDH release, Figure 3F) and apoptosis (increases in caspase-3 activity and ssDNA accumulation, Figure 3G and 3H) were significantly potentiated by Lnc-EPIC1 siRNAs (Figure 3E–3H). Lnc-EPIC1 siRNA alone also induced significant SH-SY5Y cell death and apoptosis (Figure 3E–3H).

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.

MYC knockout abolishes Lnc-EPIC1-induced actions in H₂O₂-stimulated neuronal cells

If MYC is the primary target of Lnc-EPIC1 in neuronal cells, Lnc-EPIC1 should be ineffective in the MYC-knockout cells. To test this hypothesis, CRISPR/Cas9 strategy was applied. Two different lenti-CRISPR/Cas9-MYC-KO-GFP constructs, targeting non-overlapping DNA sequence of MYC, were transfected to SH-SY5Y neuronal cells. Via FACS-mediated GFP sorting and puromycin selection, stable monoclonal cells were established (“MYC-KO-L1/L2”), showing completely depleted MYC mRNA (Figure 4A). Consequently, mRNA expression of MYC targets, including Cyclin A1, CDC20 and CDC45, were significantly downregulated (Figure 4A, left panel). Additionally, cyclin A1, CDC20 and CDC45 protein levels were largely reduced in MYC-KO SH-SY5Y cells (Figure 4A, right panel).

Figure 4. MYC knockout abolishes Lnc-EPIC1-induced actions in H₂O₂-stimulated neuronal cells. Expression of listed mRNAs and proteins in the stable SH-SY5Y cells with the lenti-CRISPR/Cas9-MYC-KO-GFP constructs (“MYC-KO-L1/L2”) or the control construct (“Cas9-C”) as well as in the parental control cells (“Pare”) were shown (A). Above cells were treated with/without hydrogen peroxide (H₂O₂, 300 μM) for the applied time, cell viability (by CCK-8 assay, B) and death (by LDH assay, C) were examined; Lnc-EPIC1 levels were tested by qPCR assay (D). “MYC-KO-L1” cells were transfected with lentiviral Lnc-EPIC1 construct (“LV-Lnc-EPIC1”) or Lnc-EPIC1 siRNA-1 (“EPIC1-siRNA1”, 500 nM) for 48h, followed by hydrogen peroxide (H₂O₂, 300 μM) stimulation for the applied time, cell viability (E), cell death (F) and Lnc-EPIC1 expression (G) were tested. Listed proteins were quantified, with the values normalized to Tubulin (A). Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Ctrl” treatment of “Pare” cells (A–D). * P < 0.05 vs. “Ctrl” treatment (E–G). ^#P < 0.05 vs. H₂O₂ treatment of “Pare” cells (A–C). Experiments in this figure were repeated three times, and similar results were obtained.

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-EPIC1-silenced cells (Figure 3). Notably, MYC-KO did not significantly change Lnc-EPIC1 expression in SH-SY5Y cells with/without H₂O₂ stimulation (Figure 4D).

Further studies demonstrated that in MYC KO cells (“MYC-KO-L1”), Lnc-EPIC1 overexpression (by “LV-Lnc-EPIC1”) or silencing (by “EPIC1-siRNA1”) failed to change H₂O₂-induced cytotoxicity (Figure 4E and 4F). Although both LV-Lnc-EPIC1 or the siRNA altered Lnc-EPIC1 expression in H₂O₂-stimulated neuronal cells (Figure 4G). Similar results were also obtained in the “MYC-KO-L2” cells (Data not shown). Therefore, MYC knockout abolished Lnc-EPIC1-induced actions in H₂O₂-stimulated SH-SY5Y cells, suggesting that MYC is the primary target of Lnc-EPIC1.

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).

SH-SY5Y cells

Human neuronal SH-SY5Y cells (from Dr. Gao [27]) were cultured in DMEM plus 10% FBS. Before H₂O₂ treatment, SH-SY5Y cells were cultured for 10 days in retinoic acid (RA, 10 μM)-containing complete DMEM medium, followed by another five days culture in serum-free DMEM with BDNF (50 ng/mL) and glutamine (2 mM).

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.

Ectopic Lnc-EPIC1 overexpression

The pLenti6-GFP-puro expression vector with Lnc-EPIC1 sequence (“LV-EPIC1”) was provided by Dr. Zhang [22], transfected to SH-SY5Y cells (in polybrene-containing medium) through Lipofectamine 2000. Stable cells were selected via puromycin (2.0 μg/mL). Three stable SH-SY5Y cell lines with LV-EPIC1 were established, named as “Lnc-EPIC1-OE1/2/3”. qPCR assay was performed to verify Lnc-EPIC1 overexpression. Control cells were transfected with empty vector (“Vector”). For the primary human neuron cultures, LV-EPIC1 was directly transfected to neurons, and no puromycin-mediated selection was performed.

Cell viability assay

Neuronal cells were seeded in the 96-well plates (2 × 10⁴ cells/cm²). Following treatment, cell viability was examined by the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). CCK-8 optical density (OD) values at 550 nm were recorded.

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.

Lnc-EPIC1 siRNA

Two Lnc-EPIC1 siRNAs, 5′-CCUUCAGACUGUCUUUGAA-3′ (“EPIC1-siRNA1”) and 5′-GCUUUCUCUCGGAAACGUG-3′ (““EPIC1-siRNA2”), were utilized [21, 22]. SH-SY5Y cells or the primary neurons were seeded in the six well tissue culture plates (2 × 10⁴ cells/cm²), transfected with Lnc-EPIC1 siRNA or the scramble control siRNA (“scr-siRNA”) using the Lipofectamine RNAiMAX Reagent for 48h. Lnc-EPIC1 knockdown was verified by qPCR assay.

Caspase-3 activity assay

The caspase-3 activity assay was described in detail elsewhere [30].

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.

MYC knockout

MYC small guide RNA (sgRNA) (sgRNA-1: 5′-GCCGTATTTCTACTGCGACG-3′ [22] or sgRNA-2: 5′-CTATGACCTCGACTACGACT-3′) was packed into the lenti-CRISPR-GFP vector (From Dr. Zhang [22]). By using Lipofectamine 2000 each of the two constructs was individually transfected to SH-SY5Y cells. GFP-positive cells were FACS sorted. After culturing for another 3 weeks, cells were distributed to six well plates, followed by genotyping of depleted MYC. The monoclonal cells were further selected by puromycin for seven days. Control cells were transfected with lenti-CRISPR/Cas9-GFP construct with scramble sgRNA (“Cas9-c”).

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.