MiR-29c Inhibits TNF-α-Induced ROS Production and Apoptosis in Mouse Hippocampal HT22 Cell Line

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.


Introduction
It is well known that microRNAs (miRNAs) are a novel and abundant class of ~ 22-nucleotide (nt) RNAs that regulate gene expression at the post-transcriptional level by binding to the 3′-UTR of target mRNAs. Abnormal miR-NAs expression profile in hippocampus has emerged as a crucial factor in chronic neurological diseases characterized by oxidative stress and apoptosis [1,2]. The miR-29 family consists of three members, including miR-29a, miR-29b and miR-29c. Among them, the content of miR-29c in cerebrospinal fluid and peripheral blood samples was found significantly downregulated in Alzheimer's disease (AD) patients [3,4]. More importantly, the upregulation of miR-29c in hippocampus improved the learning and memory deficits in AD mice [4]. One recent report from Hori et al. found that the expression of miR-29c in hippocampus has declined by 70% in peripheral nerve injury-induced neuropathic pain rats [5]. Decreased levels of miR-29c were also observed in both in vivo (mouse hippocampus) and in vitro (HT22 cells) ischemia/reperfusion injury models [6]. It seems that miR-29c may be a promising potential therapeutic target against some neurological diseases. However, there are some contradictory reports. For example, intracerebroventricular infusion of miR-29c agomir resulted in increased sensitivity to ischemia/ reperfusion injury, whereas down regulation of miR-29c with antagomir in vivo was neuroprotective [7]. In addition, miR-29c may be involved in the inhibitory action of chondroitin sulfate proteoglycans on axonal growth [8]. These puzzling experimental results promote us to further explore the regulate role of miR-29c in neuronal function under pathological condition. Mouse hippocampal neuronal HT22 cell is widely used as an in vitro model for neuroinflammation and cellular signal pathway investigations. For this reason, we investigate the effect of miR-29c on the neuronal function of HT22 cells. Please check and confirm the affiliation It has been checked The hippocampus is an important region that plays essential roles in learning and memory formation, emotional regulation and pain perception. The association between reactive oxygen species (ROS) production and hippocampal neuron injury has been the focus of a number of recent studies [1,2]. Ischemic insult induced ROS production and excessive mitochondrial fragmentation in mice hippocampus [9]. The increased ROS and malondialdehyde (MDA) production, and decreased the activity of superoxide dismutase (SOD) were observed in brains of middle cerebral artery occlusion and reperfusion (MCAO/R) rats and primary culture of rat hippocampal neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R) [10]. In HT22 cells, OGD/R initiates a series of devastating cascades that lead to the overproduction of ROS and damage to mitochondrial functions [11]. It is clear that tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine regulating neuroinflammatory response. The elevated levels of TNF-α in hippocampus have been associated with various neurological diseases such as AD, depression and chronic pain [12][13][14]. The contents of TNF-α and the expression levels of apoptotic factors such as cleaved caspase-3 and Bcl-2 Associated X Protein (Bax), and the TUNEL-positive cell rate were increased in hippocampus of MCAO rats [15]. It is reported that tumor necrosis factor receptor 1(TNFR1)mediated TNF signalling contributes to ROS accumulation and cell apotosis in HT-22 cells [16]. More importantly, bioinformatics data analysis identified a regulatory relationship between miR-29c and TNFR1 [17]. In addition, in miR-29c overexpression inhibited OGD/R-induced HT22 cells apotosis by inhibiting the expression of mitogenactivated protein kinase 6(MAP2K6) at the transcriptional level [6]. Then, in the present study, we detected the effect and possible mechanism of miR-29c on TNF-α-induced ROS production and cell apoptosis in HT22 cells.

Construction of the EGFP-miR29c Expression Vector and Lentivirus Packaging
The Enhanced Green Fluorescent Protein (EGFP)-miR-29c/ sponge expression vector was constructed with a lentiviral expression system. The precursor sequence of miR-29c was used for overexpression as follows: sense primer: 5′-ATG  GAC GAG CTG TAC AAG TAA AGG CAG TGA TAGT GAG  AAA GTT TTT G-3′; anti sense primer: 5′-TTC CAG ACG  CGG TCT AGA AAAA ATA GTA GAT AAA ACA GAC ATGA-3′. The precursor sequence of miR-29c sponge was used as follows: sense primer: 5′-TCG GCA TGG ACG AGC TGT AC-3′; anti sense primer: 5′-TTG ATT GTT CCA GAC GCG GTC-3′. Mouse precursor miR-29c and miR-29c sponge were synthesized and further constructed into the pCMV-EGFP-miRNA(sponge)-WPRE-Vector, and packaged into lentivirus by Sagon Biotech (Shanghai) co., ltd. An EGFP control vector was also constructed using the same expression system without miR-29c gene. Every step of the vector construction was verified by DNA sequencing. The final titers ranged between 3 × 10 9 and 4 × 10 9 transducing units (TU)/ml.

Cell Transfection
The lentiviral vectors carrying the miR-29c/sponge gene were added into the HT22 cells at a multiplicity of infection (MOI) of 120. HT22 cells were seeded in a 24-well plate, cultured overnight, and then infected with 100 µL of the lentivirus for 4 h followed by the addition of 500 µL of DMEM medium with 10% FBS in each well. After transfection for 48 h, the expression of the green fluorescent protein was observed under the fluorescence microscope. Then, puromycin was added to the medium at concentration of 3 µg/mL for 14 days. At last, HT22 cells stably expressing EGFP-miR-29c, EGFP-miR-29c sponge or EGFP alone were generated and these infected cells (puromycin-resistant HT22 cells) could be easily viewed under a fluorescence microscope. In the following experiments, the cell line over expressing miR-29c stable was named LV-miR-29c. The cell line infected with miR-29c sponge virus was named LV-miR-29c sponge. When vectors encoding these sponges are transfected into cultured cells, sponges depress microRNA targets at least as strongly as chemically modified antisense oligonucleotides [18]. The control vector cell line was named LV-Vector. Real-time PCR assay was used to detect the expression of miR-29c. The sequence of miR-29c is: 5′-ATC TCT TAC ACA GGC TGA CCG ATT TCT CCT GGT  GTT CAG AGT CTG TTT TTG TCT AGC ACC ATT TGA AAT  CGG TTA TGA TGT AGG GGG A-3′. The sequence of miR-29c sponge is: 5′-TAA CCG ATT TTT TTG GTG CTA CGC G  TAA CCG ATT TTT TTG GTG CTA CGC G TAA CCG ATT  TTT TTG GTG CTA CGC GTA ACC GAT TTT TTT GGT GCT  ACG CGT AAC CGA TTT TTT TGG TGC TAC GCG TAA CCG  ATT TTT TTG GTG CTA CGC GTA ACC GAT TTT TTT GGT  GCT ACG CGT AAC CGA TTT TTT TGG TGC TA-3′. The binding sites between miR-29c and miR-29c sponge were shown in metallic gray.

Cell Viability Assay
The Cell Counting Kit-8 method (CCK-8; Solarbio, Beijing, China) was used for cell viability assay. HT22 cells grown in 96-well plates (7000 cells/well) were incubated in the presence of TNF-α. TNF-α (ab259411, Abcam, Cambridge, MA, USA) was dissolved in 0.01 M PBS. After 24 h incubation at 37 °C, the 10 µL CCK-8 reagent was then added and the incubation continued for 4 h. The absorbance of each well was measured at 450 nm with a microplate reader (Thermo Fisher Scientific). Assays were performed in triplicate. Cell survival rate (%) = (OD the experimental group − OD the blank group )/ (OD the control group − OD the blank group )×100%.

LDH Release Assay
At the end of cell death, cell membrane integrity is damaged and the membrane becomes leaky. LDH is a soluble enzyme located in the cytoplasm. The enzyme is released into the surrounding environment upon cell damage. Then, LDH assay kit (Beyotime Technology, Nantong, China) was used to evaluate both the TNF-α neurotoxicity and the protective effects of miR-29c. In brief, the HT22 cells grown in 96-well plates (7000 cells/well) were treated with the TNF-α, then the supernatant were collected by centrifugation (400×g, 5 min), and the LDH release was evaluated according to the protocol instruction. The absorbance was detected at 490 nm with a microplate reader (Thermo Fisher Scientific), and all values of % LDH released were normalized to the untreated control group.

Intracellular ROS Detection
HT22 cells were seeded into confocal dishes at a density of 2 × 10 5 cells/well. After TNF-α application, ROS assay kit (KeyGen, Nanjing, China) was used to evaluate the intracellular ROS level. NAC (N-acetyl-l-cysteine) is commonly used to inhibit ROS production. In the present study, NAC (616-91-1, Abmole Bioscience Inc.) was dissolved in 0.01 M PBS. At the end of treatment period, the cells were incubated with 5 µM DHE (Dihydroethidium, Beyotime Institute of Biotechnology, Shanghai, China) for 20 min. The dish was washed three times with PBS, and then the photos were taken by using a confocal microscope (excitation = 510 nm; emission = 560 nm, LSM900; Carl Zeiss MicroImaging). Finally, the fluorescence intensity per cell was analyzed with ZEN lite software (ZEISS, Jena, Germany). At least 120 cells from randomly selected fields per group were analyzed to determine the content of ROS.

Mitochondrial Membrane Potential Assay
Mitochondrial function was assessed by monitoring mitochondrial membrane potential (MMP) using a TMRE MMP assay kit (a cationic potentiometric fluorescent dye, Beyotime Technology, Shanghai, China). TMRE (100 nM) was added to each well (1 × 10 4 cells/well) and incubated for 30 min. The HT22 cells were then washed three times in PBS and the fluorescent intensity of TMRE was measured by using a confocal microscope (excitation = 514 nm; 1 3 emission = 610 nm). Fluorescent images were taken using a confocal microscope. Finally, the fluorescence intensity per cell was analyzed with ZEN lite software (ZEISS, Jena, Germany). At least 120 cells from randomly selected fields per group were analyzed to determine the changes of MMP.

Immunofluorescence
HT22 cells were seeded on the coverslip in 6-well plates at 6 × 10 5 cells per well the day before the experiment. After 24 h incubation of TNF-α or vehicle (0.01 M PBS), HT22 cells were fixed in 4% paraformaldehyde for 20 min, then washed with PBS. Cultures were stained with mouse anti-TNFR1 antibody (300 µg/mL, 1:300, Proteintech) overnight. Subsequently, CY3-conjugated goat anti-mouse IgG (600 µg/mL, 1:300 in PBS, Proteintech) was applied for 1 h at room temperature. All incubations were held at 25 °C and separated by three 5-min washes in 0.01 M PBS in a dark place. Fluorescent images of TNFR1-immunolabeled specimens were taken by a confocal microscope. Finally, the fluorescence intensity per cell was analyzed with ZEN lit software (ZEISS, Jena, Germany). At least 120 cells from randomly selected fields per group were analyzed to determine the percentage of apoptotic cells. In control experiments, the cells on coverslips were treated as described above, except that incubation with the primary antibody was omitted.

Hoechst 33258 Staining
Cell apoptosis was determined by Hoechst 33258 staining. Briefly, cells were seeded into 24-well plates at a density of 1 × 10 5 cells/well for 24 h. The cells were challenged with TNF-α or Vehicle (0.01 M PBS) for 24 h and then washed with PBS. All cells were fixed using paraformaldehyde for 20 min at room temperature followed by washing twice with PBS. Afterwards, cells were stained with Hoechst 33258 solution (10 µmol/L) for 5 min at room temperature. After being washed with PBS, the images of the cells were visualized by a fluorescence-capable microscope (Leica, Axio Vert, Germany). The percentage of apoptotic cells was calculated by counting the total number of cells and the number of Hoechst-stained cell.

TUNEL Assay
Cellular apoptosis was also assessed using a TdT-mediated dUTP-Cy3 nick end-labeling (TUNEL) Apoptosis Assay kit (Beyotime Technology, Shanghai, China) according to the manufacturer's directions. Briefly, the HT22 cells were treated with TNF-α, and the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then incubated with 0.3% Triton X-100 in PBS for 15 min. Fixed cells were stained with TUNEL(Red) and DAPI (blue). Thereafter, TUNEL-positive cells were observed using a confocal microscope (excitation/emission: 550/750 nm). The percentage of apoptotic cells was calculated by counting the total number of nuclei and the number of TUNEL-stained cells. At least 120 cells from randomly selected fields were analyzed to determine the percentage of apoptotic cells.

Realtime Fluorescence Quantitative PCR (RTFQ-PCR)
The total RNA of cultured HT22 cells was immediately isolated by using Trizol reagent (Takara Bio Inc.) after TNF-α application. The concentration and purity of RNA samples were measured using Spectrophotometer (Thermo Fisher Scientific). The ratios of OD260/OD280 were between 1.9 and 2.1. RTFQ-PCR is carried out in iCycler IQ Real-Time PCR Detection System (Thermo Fisher Scientific) with SYBR Green PCR Master Mix (Takara). Subsequently, cDNA was synthesized from RNA by reverse transcription reaction using the PrimeScript™ RT Reagent Kit (Takara). RTFQ-PCR was performed in a final volume of 20 µL (8 µL H 2 O, 10 µL mastermix, 2 µL cDNA) on the QuantStudio™ 6 Flex Real-Time PCR Systems (Thermo Fisher Scientific). PCR conditions were as follows: 95 °C 30 s 1 Cycle; 95 °C 5 s, 60 °C 30 s, 40 Cycles. The cycle threshold (Ct) value represents the cycle number at which a fluorescent signal rises statistically above background. The relative quantification of gene expression was analyzed by the 2-ΔΔ Ct method. The expression of the mRNA and miRNA were normalized using β-actin or U6 as the internal control respectively.
In the present study, the nucleotide sequences of the primers were synthesized by TaKaRa Biological Engineering Company. Primer preparations were devised according to the sequence searched on GenBank. The nucleotide sequences of the primers used in this experiment were as follows:

Western Blot
Total proteins were extracted from cultured HT22 cells lysed by radioimmunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitor cocktails (Solarbio Science & Technology Co., Ltd, China), and the protein concentration was determined by using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd, China). Then 20 µg equal amounts of denatured proteins were subjected to SDS-PAGE gel electrophoresis and transferred onto polyvinylidenefluoride (PVDF) membranes (Merck Millipore Ltd, Ireland). Next, the transferred membranes were blocked with 5% Albumin Bovine V (Solarbio) for 2 h and incubated with the primary antibodies (Table 1) overnight at room temperature. The following day, the membranes were washed three times with 0.1% Tween-Tris-buffered saline (TBST), and then incubated with secondary antibodies (Table 1) at room temperature for 1 h. Finally, the membranes were imaged using an Electro-Chemi-Luminescence-Kit. Images of the blots were captured using the G: BOX Chemi XX9 system (Synoptics Group). The image was scanned, and band intensity was semi-quantified using Image J (version 1.8.0, National Institutes of Health). β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference, and the gray value of the target protein was divided by the gray value of the internal reference to correct the error. The result represents the relative content of the target protein in the sample.

Statistical Analysis
All data were presented as mean ± standard deviation (SD). Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test by using SPSS17.0. Differences at the P < 0.05 level were considered statistically significant.

Establishment of Stable HT-22 Cell Lines Overexpressing MiR-29c
To elucidate the effect of miR-29c on HT22 cells, we established stable HT-22 cell lines that overexpressed miR-29c or miR-29c sponge. The lentiviral vectors carrying the gene of miR-29c or miR-29c sponge were added into the HT22 cells. Then, positive monoclonal cells expressing EGFP were selected by puromycin resistance screening and expanded to obtain stable transfected cell lines (Fig. 1A). We extracted total RNA from the three established stable cell lines in which miR-29c were detected. Figure 1B shows that miR-29c expression was significantly higher in the LV-miR-29c group than that in the LV-Vector group (P < 0.05). Simultaneously, PCR results corroborated that the level of miR-29c was lower in LV-miR-29c sponge group than that in LV-Vector group and LV-miR-29c group (P < 0.05). These results suggested that we successfully established stable HT-22 cell lines that overexpressed miR-29c or miR-29c sponge.

MiR-29c Attenuated TNF-α-Induced ROS Production, MMP Decreases and iNOS Expression in HT22 Cells
It is well known that ROS production and MMP decrease play a key role in neuronal mitochondria dysfunction [25]. Then, we examined whether miR-29c is able to repress ROS production by TNF-α. DHE staining was used to detect the accumulation of intracellular ROS, and intracellular red fluorescence intensity was an indicator showing the level of ROS. More studies have demonstrated that ROS can cause apoptosis and decrease the MMP. In turn, decreased MMP further exacerbated mitochondrial dysfunction, further increasing ROS production and perpetuating a vicious cycle by which ROS production continually increases [26][27][28]. Then, to further confirm the inhibitory effects of miR-29c on ROS production and MMP decreases induced by TNFα, the ROS scavenger NAC (5 mM, 2 h) was used. First, as shown in Fig. 3A and C, TNF-α stimulation induced ROS accumulation in LV-Vector + TNF-α group than that in LV-Vector + Vehicle group (P < 0.05). miR-29c decreased and miR-29c sponge increased TNF-α-induced ROS accumulation significantly (P < 0.05). TNF-α-induced ROS production was completely reversed by NAC (P < 0.05). After TNF-α stimulation, miR-29c sponge led to a greater increase in ROS content, while the effect was potentially alleviated by NAC (P < 0.05).
Subsequently, the change of MMP in HT22 cells was monitored with TMRE (Red) staining and intracellular red fluorescence intensity was an indicator showing the level of MMP. The result showed that ( Fig. 3B and D), TNF-α stimulation significantly reduced the TMRE fluorescence intensity of HT22 cells in LV-Vector + TNF-α group than that in LV-Vector + Vehicle group (P < 0.05). miR-29c attenuated and miR-29c sponge substantially deteriorated TNF-α-induced decreased TMRE fluorescence intensity (P < 0.05), which means that miR-29c attenuate MMP decrease while miR-29c sponge led to greater reduction of MMP level. In addition, TNF-α-induced decreased TMRE fluorescence intensity was completely reversed by NAC (P < 0.05). After TNF-α stimulation, miR-29c sponge led to a greater decrease in TMRE fluorescence intensity, while the effect was potentially alleviated by NAC (P < 0.05). It seems that miR-29c may protect HT22 cells against TNF-α-induced, at least partly through inhibiting oxidative stress.

MiR-29c Overexpression Attenuated TNF-α-Induced Apoptosis in HT22 Cells
In previously studies, TNFR1 has been found to be present in HT22 cells and is involved in TNF-α-induced neuronal apoptosis [19]. However, it is not clear whether miR-29c overexpression can protect HT22 cells from TNF-α-induced apoptosis. Then, TNFR1 expression and apoptosis rate were observed. Our results showed that TNF-α stimulation significantly enhanced TNFR1 fluorescence intensity, which was roughly decreased by miR-29c ( Fig. 4A and B, P < 0.05). However, after TNF-α stimulation, miR-29c sponge led to more elevated TNFR1 fluorescence intensity (P < 0.05). In addition, after TNF-α stimulation, TNFR1 expression at the mRNA and protein level was significantly upregulated in LV-Vector-modified HT22 cells ( Fig. 4C and D, P < 0.05). At the same time, miR-29c attenuated and miR-29c sponge promopted TNFR1 expression induced by TNF-α (P < 0.05). It is reasonable to speculate that miR-29c may exert a neuroprotective effect by suppressing TNFR1 expression.
Hoechst and Tunel assay were used to evaluate TNF-αinduced apoptosis. Hoechst staining showed that (Fig. 4E and F), after TNF-α stimulation, the percentage of apoptotic cells were remarkably increased in LV-Vector-modified HT22 cells (P < 0.05), while the effect was impaired by miR-29c (P < 0.05). After TNF-α stimulation, the percentage of apoptotic cells were remarkably increased, and the cells appeared more nuclear condensation (brightening) in cells transfected with miR-29c sponge than that in LV-miR-29c-modified HT22 cells (P < 0.05). Furthermore, as shown in Fig. 4G and H, after TNF-α stimulation, TUNELpositive expression of HT22 cells increased significantly in LV-Vector-modified HT22 cells (P < 0.05), while the effect was impaired by miR-29c (P < 0.05). In contrast, after TNF-α stimulation, the number of TUNEL-positive cells was substantially elevated in LV-miR-29c sponge-modified HT22 cells than that in LV-miR-29c-modified HT22 cells (P < 0.05). Taken together, these results demonstrated that miR-29c effectively ameliorated the TNFR1 expression and apoptosis in TNF-α-stimulated HT22 cells.

MiR-29c Affected TNF-α-Induced the Decreased Expression of MAP-2, GAP-43 and SYN-1
As indicated by the above experimental results, TNF-αinduced HT22 cell injury was attenuated by miR-29c and was further deteriorated by miR-29c sponge. On the other hand, hippocampal synaptic pathology, including abnormal density and morphology of dendritic spines, synapse loss is involved in the pathophysiology of some neurodegenerative and neuropsychiatric diseases [35][36][37]. Then, we explored the expression of synapse-related proteins, including growth-associated protein-43 (GAP-43, a marker of neurite outgrowth), microtubule-associated protein-2 (MAP-2, a marker of neuronal morphology) and synapsin I (SYN-1, a marker of presynaptic protein) in HT22 cells. As can be seen in Fig. 7, TNF-α stimulation induced the decreased expression of MAP-2, GAP-43 and SYN-1 in LV-Vectormodified HT22 cells (Fig. 7, P < 0.05), while miR-29c significantly reversed the effect (P < 0.05). However, TNFα-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was significantly enhanced in LV-miR-29c sponge-modified cells than that in LV-Vector-and LV-miR-29c-modified cells (P < 0.05). It appears that the miR-29c may attenuate TNF-α-induced cell injury by upregulating the expression of MAP-2, GAP-43 and SYN-1.

Discussion
Numerous studies have shown that inflammatory response and cell apoptosis are central features in many neurological diseases, including brain trauma, stroke and AD. miR-29c is an important member of the miR-29 family, and recent studies reported that miR-29c is expressed in hippocampus of humans and rodents, where it is implicated in cognitive impairment, pain perception and emotion dysregulation [4,38,39]. Thus, it is required to explore how miR-29c  regulates hippocampal neuronal functions. In the present study, mouse HT22 cells, an immortalized hippocampal neuronal cell line, was utilized as an in vitro model. We found that TNF-α stimulation inhibited cell viability, promoted oxidative stress and triggered cell apoptosis, as evidenced by the elevated level of ROS, iNOS and Bax, the reduced level of Bcl-2 and MMP, and the increased cleavage of caspase-9, caspase-8 and caspase-3. In LV-miR-29c-modified HT22 cells, TNF-α-induced cell injury was attenuated, as evidenced by reduced level of ROS, iNOS and Bax, the increased level of Bcl-2 and MMP, and the decreased cleavage of caspase-9, caspase-8, caspase-3 and PARP-1. In contrast, in LV-miR-29c sponge-modified HT22 cells, TNF-α-induced injury appeared to be aggravated. Although in vitro experiments have some limitations, our observations may support a potential therapeutic target of miR-29c against neurological diseases related to oxidative stress and cell apoptosis.
Excessive production of cytokine TNF-α could induce neuroinflammation and such damage is related with many neurological diseases. TNF-α signaling occurs through the TNF-α receptor (TNFR), where ligand binding recruits adaptor proteins into a large complex to regulate the activity of key cellular proteins during the signal transduction process [40]. TNF-α affects mitochondrial function through TNFR1 because soluble TNF-α signaling is primarily through TNFR1 [41]. It is clear that mitochondrial dysfunction can augment ROS production in many cell types [42]. On the other hand, ROS accumulation aggravates mitochondrial dysfunction, disrupts intracellular calcium balance, opens mitochondrial permeability transition pores, and leads to a decrease in mitochondrial membrane potential, thereby contributing to a vicious cycle [43]. Doll et al. also indicated that TNF-α caused a rapid and profound reduction in mitochondrial function and the neurotoxic effect is mediated through TNFR1 in HT22 cells [23]. In this study, we found that TNF-α stimulation increased ROS production and reduced mitochondrial membrane potential, whereas these effects were attenuated in LV-miR-29c-modified HT22 cells and further deteriorated in LV-miR-29c sponge-modified cells. At the same time, NAC pretreatment alleviated TNFα-induced ROS production and MMP collapse in LV-miR-29c sponge-modified HT22 cells. More importantly, TNF-α stimulation increased TNFR1 expression at the mRNA and protein expression, and the effect was impaired by miR-29c and was enhanced by miR-29c. It appears that miR-29c may exert the antioxidant effect via downregulating TNR1 expression.
Nitric oxide, a gaseous free radical and ubiquitous neurotransmitter, is one of the contributor to ROS generation. In HT22 cells, TNF-α administration increased iNOS expression but had a minor effect on nNOS expression [24]. It is reported that TNF-α induced iNOS expression and nitric oxide release, caused the accumulation of in tracellular Ca 2+ and endoplasmic reticulum stress, and therefore leading to mitochondrial dysfunction [24]. OGD/R-induced HT22 cells damage is related to the iNOS-derived NO production [44]. We found that NAC pretreatment suppressed TNF-α increased iNOS expression in LV-Vector-and LV-miR-29c sponge-modified cells, which imply that TNF-α increased iNOS expression is related to ROS production. Simultaneously, miR-29c could attenuate TNF-α-induced iNOS expression, but miR-29c sponge had the opposite effect, as indicated by more elevated iNOS expression. These data revealed that one possible mechanism for miR-29c-mediated neuropretective effect may via inhibiting iNOS expression.
Recent studies have shown that TNF-α induced the decline of MMP, the release of cytochrome c from mitochondrial and the cleavage of caspase 9 and caspase 3 in HT22 cells [19,20,24]. The released cytochrome c induces the formation of an apoptosome consisting of cytochrome c, apoptotic protease activating factor 1(Apsf-1), dATP and pro-caspase 9 [23,45]. Caspase 9 has long been considered to be the initiator caspase in the mitochondrial apoptotic pathway triggering the cleavage of caspase 3. We found miR-29c can inhibit TNF-α-induced cell apoptosis, as evidenced by the decreased cleavage of caspase-9, caspase-3, the decreased Bax expression, the decreased number of TUNEL-positive cells and the increased Bcl-2 expression. In addition, excessive cleavage of PARP-1 by activated caspase 3 hampers DNA repair and compels cells towards death. Our results showed that TNF-α-induced cleavage of PARP-1 was attenuated by miR-29c and was enhanced by miR-29c sponge. It appears that the protective role of miR-29c against TNF-α-induced neuronal apoptosis may be mediated through inhibiting mitochondrial apoptotic pathway. On the other hand, in LV-miR-29c sponge-modified cells, the decreased miR-29c level and the increased TNFR1 level may leave cells more vulnerable to TNF-α stimulation.
The binding of TNF-α to its receptor TNFR1 is a critical initiation factor for TNF-α-induced neuronal apoptosis [16]. One recent study reported that the binding site for miR-29c was located in the 3′UTR of TNFRSF1A mRNA using the online bioinformatics analysis, and the regulatory relationship between miR-29c and TNFRSF1A was further verified using the luciferase assay [17]. We stained the HT22 cells with Hoechst to observe chromatin condensation, which is a morphological hallmark of apoptotic cell death. Our result showed that miR-29c obviously inhibited the chromatin condensation induced by TNF-α. However, TNF-α-induced nuclear condensation appeared to be aggravated after transfection of HT22 cells with miR-29c sponge. Similar result was also obtained when the nuclei were stained with TUNEL. miR-29c significantly reduced the number of apoptotic cells generated following the incubation with TNF-α. However, the apoptosis rate induced by TNF-α and the number of apoptotic cells was enhanced in LV-miR-29c sponge-modified HT22 cells. It seems that the protective effect of miR-29c on TNF-α-induced apoptosis in HT22 cells was at least partly mediated through inhibiting TNFR1 expression.
There is a growing body of evidence indicating that an overactived TNF-α/TNFR1 signaling plays a vital role in nervous system during inflammation, cell proliferation, differentiation and neuronal apoptosis [17,19,23,45]. How to reduce the overactived TNF-α/TNFR1 signaling has become the key issue for treatment of neurological disorders. It is reported that TRADD was critical for the accumulation of ROS in TNF-α-treated HT22 cells [16]. TNFR1 possesses a cytoplasmic death domain, which can bind the adaptor proteins TRADD and FADD. Caspase 8 can be recruited by FADD to the TNFR1 complex, where it becomes activated and initiates a protease cascade that leads to apoptosis [34,45]. We found that, in TNF-α-treated HT22 cells, the cell apoptosis rate increased and the expression of TNFR1, TRADD and FADD increased. MiR-29c suppressed the apoptosis rate and decreased the expression of TNFR1, TRADD, FADD and cleaved caspase-8, which was further deteriorated in LV-miR-29c sponge-modified cells. A recent study performed in rat hippocampus suggested that the upregulated expression of TNFR1, TRADD and FADD is accompanied by the TNFR1 complex formation with TRADD and FADD [46]. Liu et al. also described that the increased expression of TRADD, FADD and caspase-8 plays a role in initiating caspase cell death cascades in rat adolescent binge drinking model [47]. Furthermore, the increased expression of FADD and caspase-8 was observed in Aconitine-induced HT22 cell apoptosis [48]. Although our experiment do not directly address the role of miR-29c in the TNFR1 complex formation, the present finding also alerts us to the possibility that miR-29c reduces TNF-αinduced HT22 cell apoptosis due to the inhibition of TNFR1 signaling pathway. The present study points to a potential role of miR-29c on the suppression of TNFR1 signaling. In deed, the effect of miR-29c on TRADD-FADD binding remains to be further studied.
MiRNAs play an important role in the regulation of a wide assortment of cellular processes by sequestering target mRNAs and inhibiting their translation, and thereby downregulating the expression of the targeted proteins [49]. Multiple miRNAs can regulate a single mRNA target and, conversely, a single miRNA can regulate many different mRNA targets. For example, luciferase reporter assay and WB analysis indicated that overexpression of the miR-29 family inhibited the OGD/R-induced ROS production and MMP collapse by targeting a pro-apoptotic BCL2 family member PUMA in HT22 cells [49]. OGD/R induced the decreased levels of miR-29c and the increased MAP2K6 expression in HT22 cells. MAP2K6 was also identified as a direct target of miR-29c [6]. Additionally, in U251 human glioblastoma cells and SH-SY5Y human neuroblastoma cells, the regulatory relationship between miR-29c and TNFR1 was verified using the luciferase assay [17]. Zong et al. indicated that the NAV3 (Neuron Navigator 3) mRNA has a functional miR-29c binding site in the 3′UTR [38]. Thus, there is a possibility that the protective effect of miR-29c on TNF-α-induced HT22 cell apoptosis may be due to regulate more than one target mRNA. Then, RNA sequencing and luciferase assay should be performed in the future study.
Neuronal MAP-2 is considered to be essential for microtubule stability. The decreased MAP-2 expression has been observed in ischemia brain injury and AD, and been suggested to trigger neurons apoptosis [50]. In OGD/R-or amyloid beta-treated HT22 cells, the decreased MAP-2 expression has been observed during mitochondrial dysfunction and the cell death process [51,52]. We found that TNF-αinduced the decreased expression of MAP-2, and the effect was reversed by miR-29c and was further deteriorated by miR-29c sponge in HT22 cells. It appears that miR-29c may play a role in maintain microtubule stability by upregulating MAP-2 expression, which is important for cell survival in response to high concentration of TNF-α. In addition, SYN1 and GAP-43 are two specific proteins located presynaptic membrane. SYN1 is reported to be involved in the neurotransmitters release from presynaptic membrane [53]. GAP-43 is required for neuronal axonal elongation and synaptic development [54,55]. We found that TNF-α-induced the decreased expression of SYN1 and GAP-43, and the effect was attenuated by miR-29c and was further deteriorated by miR-29c sponge in HT22 cells. These results indicated that miR-29c overexpression may exert neuroprotective effect against TNF-α by upregulating the expression level of SYN1 and GAP-43, which is important for neuronal morphology and function.
In summary, the present study demonstrated that, in HT22 neuronal cells, TNF-α-induced cell apoptosis is inhibited in transfection of HT22 cells with miR-29c. The neuroprotective effects of miR-29c on TNF-α-treated HT22 cells may be associated with reduced TNFR1 and iNOS expression, restore mitochondrial function and reduce oxidative stress, thereby mitigating cell apoptosis. In addition, it seems that TNFR1/TRADD/FADD pathway-mediated apoptosis was also impaired by miR-29c in TNF-α-treated HT22 cells. Despite some limitation of the in vitro study, our finding also provides a line of evidence supporting the protective effect of miR-29c on HT22 cells injury induced by TNF-α.