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Green tea polyphenols protect PC12 cells against H2O2-induced damages by upregulating lncRNA MALAT1

It is of significance to alleviate oxidative damages for the treatment of spinal cord injury (SCI). Studies have ascertained that green tea polyphenols (GTPs) exert protective activities against oxidative damages. In this study, we aimed to investigate the protective effects of GTP against H2O2-caused injuries in PC12 cells as well as the molecular underpinnings associated with long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). PC12 cells were preincubated with GTP prior to H2O2 stimulation. Furthermore, MALAT1-deficient PC12 cells were constructed by transfection and identified by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Next, viability and apoptosis were detected by cell counting kit-8 and flow cytometry, respectively. Meanwhile, Western blot assay was carried out to monitor the expression alteration of proteins associated with apoptosis (Bcl-2, Bax, pro-Caspase-3/9, and cleaved Caspase-3/9) and autophagy (microtubule-associated protein 1 light chain 3 (LC3)-II, LC3-I, Beclin-1, and p62). Moreover, we examined the expression of β-catenin and dissected the phosphorylation of phosphatidylinositol 3′-kinase (PI3K) and protein kinase B (AKT). We found that H2O2 decreased the viability of PC12 cells while initiated apoptosis and autophagy processes. GTP-preincubated PC12 cells maintained the viability and resisted the apoptosis and autophagy induced by H2O2. Pointedly, GTP-pretreated PC12 cells showed an increase in MALAT1 after H2O2 stimulation. Of note, the protective effects of GTP were buffered in MALAT1-deficient cells in response to H2O2. The expression of β-catenin and phosphorylation of PI3K and AKT were upregulated by GTP, while MALAT1 knockdown led to opposite results. To sum up, GTP protected PC12 cells from H2O2-induced damages by the upregulation of MALAT1. This process might be through activating Wnt/β-catenin and PI3K/AKT signal pathways.


Introduction
Spinal cord injury (SCI) is referred to as the damage in spinal cord which is characterized by paralysis and loss of sensation. 1 Even worse, it is intractable to repair the central nervous system (CNS) or restore its function. 2 Mostly, the damages in the SCI are ascribed to three causative factors, including the secondary effects of glutamate excitotoxicity, 3 calcium overload, 4 and oxidative stress. 5 Although it still remains elusive about the etiology and pathogenesis of SCI, studies have proposed a notion that reactive oxygen species (ROS) and oxidative pressure were responsible for the SCI. 1,6,7 As a consequence, it becomes extremely important to alleviate oxidative stress for therapeutic intervention of SCI.
Green tea polyphenol (GTP) is an active pharmaceutical ingredient which shows a strong potential in the treatment of various diseases, 8 such as cardiovascular disease, 9 diabetes, 10 neurodegenerative disease, 11 and cancer. 12 Specially, it has been documented that GTP effectively reduces oxidative damages in different diseases. 10,13 Meanwhile, a handful of studies focused on the effects of GTP on SCI. For example, a recent report illustrated that green tea extract apparently attenuates the adverse inflammation in an experimental model of SCI. 14 Furthermore, epigallocatechin gallate (EGCG), a bioactive component from GTP, exhibits a protective effect on rats after contusive SCI. 15 Even though significant advances have achieved to relieve SCI, the underlying mechanisms are still uncharacterized. In this study, we established cell model in vitro to further investigate the possible mechanisms.
Long non-coding RNAs (lncRNAs) are characterized as thousands of RNA transcripts (⩾200 nt) with no protein-coding potential, and lncRNAs have seized great attention throughout the world in recent years. 16 Studies revealed that kinds of lncR-NAs are involved in the onset and process of SCI. 17 For example, lncRNA spinal cord injury related 1 (SCIR1) downregulation is closely associated with the expression alteration of several mRNAs in SCI. 18 Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is first identified as a monitoring factor for lung adenocarcinoma or squamous cell cancer patients. 19 In recent years, MALAT1 has been confirmed to function in various diseases, such as in cancers, 20 vascular diseases, and neurological disorders. 21 However, there are little data or studies on the roles of MALAT1 in SCI according to our current knowledge.
In this study, we used H 2 O 2 to induce the damages of PC12 cells. Studies were performed to explore the functions of GTP on H 2 O 2 -induced cell injury and the underlying mechanisms associated with MALAT1. This study might provide a theoretical basis for the treatment of SCI.

Cell culture and treatment
Rat pheochromocytoma adrenal gland PC12 cells (Item No. CRL-1721) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Considering the preponderance of easiness for cultivation and passage and characters of nerve cells, PC12 cells are widely used to study nervous physiology and pharmacology. 22 As a consequence, PC12 cells were exploited in this study to confirm the neuroprotective role of GTP. The growth medium for PC12 cells was ATCC-formulated Roswell Park Memorial Institute (RPMI)-1640 medium (Catalog No.  with fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) at the concentration of 5% (v/v), heat-inactivated horse serum (Thermo Scientific) at the concentration of 10%, 100 U/mL penicillin (Sigma-Aldrich, St Louis, MO, USA), and 100 μg/mL streptomycin (Sigma-Aldrich). The cells were cultured in a humidified incubator containing 5% CO 2 and 95% air, at 37°C. The culture medium was refreshed once in 2 days. H 2 O 2 (Sigma-Aldrich) was prepared in different concentrations (12.5, 50, 100, and 200 μM) according to the experiment requirement. GTPs (Thea-flan; Itoen, Tokyo, Japan) were resolved in distilled water and diluted into different concentrations (50, 100, 150, and 200 μM) according to the experiment requirement. The cells were treated by GTP for 24 h prior to H 2 O 2 stimulation.

Cell counting kit-8
Cell counting kit-8 (CCK-8; Yeasen, Shanghai, China) was used for detecting cell viability. In brief, the cells were seeded in a 96-well plate at a density of 5000 cells/well. Second, 10 μL of the CCK-8 solution was added to each well of the plate, and the cells were cultured for 1 h at 37°C in a humidified incubator. Third, the absorbance at 450 nm was read using a Microplate Reader (Bio-Rad, Hercules, CA, USA).

Apoptosis assay
Flow cytometry analysis was carried out to measure the apoptotic cells which were stained using propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V staining (Yeasen). In brief, the cells were seeded in a six-well plate at a density of 100,000 cells per well. After stimulation by GTP and H 2 O 2 , the cells were washed twice in precooling phosphate buffer saline (PBS; Sigma-Aldrich), and then centrifuged and resuspended in binding buffer. Then, 5 μL of Annexin V-FITC were added. Next, the culture was mixed gently and incubated in the dark for 15 min. In addition, the plates were added with 5 μL of PI. The apoptotic cells were measured with a flow cytometer (Beckman Coulter, IN, USA) according to the manufacture's instruction.

Cell transfection
To silence MALAT1, si-MALAT1 (Genepharma, Shanghai, China) was transfected into PC12 cells in the presence of Lipofectamine 3000 reagent (Thermo Scientific) by following the manufacturer's protocol. Meanwhile, transfection with negative control (NC) was simultaneously conducted. The efficiency of transfection was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR).

qRT-PCR
Following the manufacturer's instructions, total RNA was isolated from all experimental cells by using TRIzol reagent (Thermo Scientific) and DNaseI (Promega, Madison, WI, USA). The MultiscribeRT kit (Applied Biosystems, Foster City, CA, USA) and random hexamers or oligo (dT) were applied to quantify RNA expression. MALAT1 level was calculated by the 2 -△△Ct method and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which served as an internal control.

Statistical analysis
All results were imported as mean ± standard deviation (SD) from three independent experiments. GraphPad Prism 5 software (GraphPad, San Diego, CA, USA) was used for statistical analyses. A oneway analysis of variance (ANOVA) was used for calculating P-values. If a P-value was less than 0.05, it was accepted to indicate the significance.

GTP alleviated H 2 O 2 -induced cell injury
PC12 cells were exposed to H 2 O 2 at different concentrations (0, 12.5, 50, 100, and 200 μM) in order to obtain an applicable concentration of H 2 O 2 for inducing cellular damages. Results showed that the cell viability was not impacted by H 2 O 2 at a lower concentration (12.5 μM). However, the significant difference was found in the viability of PC12 cells after being treated by H 2 O 2 at higher concentrations (50 μM, P < 0.05, 100 μM, P < 0.01, and 200 μM, P < 0.01; Figure 1(a)). Consequently, 200 μM of H 2 O 2 was chosen for the following experiments in this study. In addition, cell apoptosis was statistically increased by H 2 O 2 (P < 0.001; Figure 1(b)). The expression of anti-apoptotic protein Bcl-2 was downregulated while pro-apoptotic proteins Bax and cleaved Caspase-3/9 were all upregulated by H 2 O 2 compared with control ( Figure 1(c)). To assess the effects of GTP against H 2 O 2 -evoked damages, PC12 cells were exposed to GTP at different concentrations (50, 100, 150, and 200 μM). Results showed that there was no significant change by GTP at the concentration of 50 and 100 μM compared with control (P > 0.5), while the significant changes were observed when GTP was at the concentration of 150 and 200 μM compared with control (both P < 0.05; Figure  1(d)). Furthermore, the viability was increased by GTP at a concentration of 50 μM (P < 0.05) and 100 μM (P < 0.01) compared with control in H 2 O 2treated cells (Figure 1(e)). Hence, GTP at a concentration of 100 μM was used in the subsequent experiment. Obviously, GTP reduced apoptosis in H 2 O 2 -treated cells (P < 0.05; Figure 1(f)). The expression of anti-apoptotic protein Bcl-2 was upregulated while pro-apoptotic protein Bax and cleaved Caspase-3/9 were downregulated by GTP in H 2 O 2 -treated PC12 cells (Figure 1(g)). These results showed that GTP could increase cell viability and decrease apoptosis in H 2 O 2 -treated PC12 cells.

GTP alleviated cell autophagy induced by H 2 O 2
Then, we measured the ratio of LC3-II to LC3-I, and expression of Beclin-1 and p62 in H 2 O 2 -treated PC12 cells. The ratio of LC3-II to LC3-I is an important factor for autophagosome formation. 23 In this study, we investigated whether GTP could influence cell autophagy induced by H 2 O 2 treatment. Results showed that the ratio of LC3-II to LC3-I (P < 0.001) was increased, and Beclin-1 expression (P < 0.01) was upregulated while p62 expression was downregulated (P < 0.05) by H 2 O 2 treatment compared with control (Figure 2(a) and  (b)). However, administration with GTP changed the trend by decreasing the ratio of LC3-II to LC3-I, downregulating Beclin-1 (both P < 0.01), and upregulating p62 (P < 0.05) compared with H 2 O 2 treatment (Figure 2(a) and (b)). Taken together, GTP alleviated cell autophagy induced by H 2 O 2 .

GTP upregulated the expression of MALAT1
As shown in Figure 3, the expression of MALAT1 was upregulated by H 2 O 2 in PC12 cells (P < 0.05). Interestingly, GTP further upregulated MALAT1 expression in H 2 O 2 -treated cells (P < 0.05; Figure 3), indicating that MALAT1 might be involved in the protective effects of GTP against H 2 O 2 -induced injury.

GTP alleviated H 2 O 2 -induced injury via upregulating MALAT1
In order to clarify the functions of MALAT1 in the protective effects of GTP, si-MALAT1 (MALAT1 knockdown) was transfected into PC12 cells. qRT-PCR assay was carried out to confirm the downregulation of MALAT1 in PC12 cells (P < 0.01; Figure 4(a)). Obviously, transfection with si-MALAT1 impaired the protective effects of GTP by decreasing cell viability (P < 0.05; Figure 4

GTP activated Wnt/β-catenin and PI3K/AKT signal pathways by upregulating MALAT1
The protein expression of β-catenin, PI3K, and AKT was detected by Western blot. As shown in Figure 5(a) and (b), β-catenin was significantly downregulated (P < 0.05), while the phosphorylation of PI3K and AKT was upregulated (both P < 0.01) by H 2 O 2 treatment in PC12 cells. In addition, we found that GTP increased the expression of β-catenin (P < 0.01) and also increased the phosphorylation of PI3K and AKT in H 2 O 2 -treated PC12 cells. However, transfection with si-MALAT1 led to the opposite results, indicating that GTP activated Wnt/β-catenin and PI3K/AKT signal pathways by upregulating MALAT1.

Discussion
In this study, we investigated the functions of GTP on H 2 O 2 -induced cell injury in PC12 cells. Results demonstrated that GTP could maintain the viability of PC12 and dampen the apoptosis and autophagy processes in response to H 2 O 2 . Furthermore, our results revealed that GTP could upregulate MALAT1 and then prevent PC12 cells from H 2 O 2triggered lesions. This process was accompanied by the activation of Wnt/β-catenin and PI3K/AKT signal pathways. H 2 O 2 treatment was often used to establish cellular model of SCI. 24 In this study, cell viability was significantly decreased, while cell apoptosis and autophagy were increased by H 2 O 2 treatment in PC12 cells, which indicated that H 2 O 2 treatment successfully induced cell injury. Further studies were enacted to evaluate viability, apoptosis, and autophagy for confirming the functions of GTP in H 2 O 2 -treated PC12 cells. Increased viability and decreased apoptosis were observed in GTP-treated PC12 cells compared with the H 2 O 2 group. Interestingly, previous studies demonstrated that GTP shows an inhibitory effect on the growth of cancer cells while promotes the growth of non-cancer cells. 25 Our results were consistent with the previous studies that GTP has protective functions against cell injuries. 26,27 As a highly conserved cellular process, autophagy is involved in lipid, protein, and organelle degradation through the lysosomal pathway. 28 LC3 is a commonly used way for monitoring autophagy. Next, we detected the conversion of LC3 (LC3-I to LC3-II) because the amount of LC3-II is closely correlated with the number of autophagosomes. 29 In addition, the alteration of the ratio of LC3-II to I is a key factor reflecting the autophagy process. 23 Beclin-1 plays a central role in autophagy since it mediates the movement of autophagy proteins to the preautophagosomal membrane. 30,31 p62 protein can directly bind to LC3 and then enhance the degradation of aggregating ubiquitinated proteins. 32 In this study, the ratio of LC3-II to LC3-I and Beclin-1 were upregulated, while p62 expression was downregulated, indicating that H 2 O 2 treatment induced cell autophagy. This result was consistent with the previous studies that H 2 O 2 induces p62 degradation, 33 causes a significant increase in the ratio of LC3-II to LC3-I, 34 and induces Beclin-1-independent autophagic death. 35 Further study demonstrated that GTP alleviated cell autophagy, which was consistent with a previous report that GTP administration led to a significant reduction in the formation of LC3-II and autophagosomes. 36 MALAT1 was reported to contribute to inflammatory response of microglia after SCI. 21 In this study, the expression of MALAT1 was upregulated by H 2 O 2 treatment and was further upregulated by the administration of GTP. In addition, further studies were carried out to investigate the  functions of MALAT1 in the protective effects of GTP. Our results demonstrated that the protective effects of GTP were through the upregulation of MALAT1, which was consistent with the previous study that MALAT1 attenuated H 2 O 2 -induced death and apoptosis. 37 In this study, we found that the protective mechanism might be dependent on MALAT1 upregulation. However, the further studies are required to address the possible mechanisms. Of note, MALAT1 has recently been reported to modulate autophagy process via regulating microRNA, 38,39 which reveals that MALAT1 might serve as a mediator of GTP to regulate microRNA. Wnt/β-catenin and PI3K/AKT signal pathways were closely correlated with SCI. 40,41 Several research demonstrated that the Wnt/β-catenin signaling pathway is activated after SCI and is helpful for functional recovery. 42 The PI3K/AKT signaling pathway is of importance in mediation of cellular growth and survival. 43 GTP triggered the activation of Wnt/β-catenin and PI3K/AKT pathways by the upregulation of MALAT1. Previous studies also revealed similar results, such as MAPK1 regulates the expression of MALAT1 via activating PI3K/AKT signaling; 44 MALAT1 is involved in glucose-induced injury via activating Wnt/β-catenin. 45 In conclusion, our result showed that GTP possessed protective effects against H 2 O 2 -caused damages by increasing cell viability and decreasing cell apoptosis and autophagy. Further results revealed that GTP could upregulate MALAT1 expression and then fulfill its protective functions against H 2 O 2 . In addition, GTP also activated Wnt/β-catenin and PI3K/AKT signal pathways by upregulating MALAT1.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.