The mouse BV-2 microglial cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Beijing, China) and the mouse HT22 hippocampal neuron cell line obtained from the BeNa Culture Collection (Beijing, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) mixed 1:1 with Ham's F-12 (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ atmosphere at 37°C.

BV-2 microglial cells were maintained in serum/glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) in an anoxic environment for 1 h at 37°C. The cells were subsequently transferred into an anoxic incubator and reserved in the serum-free medium (Gibco; Thermo Fisher Scientific, Inc.; added with 1% B27, 2 mmol/l glutamine and 10 µl/ml penicillin-streptomycin). After 48 h treatment, the MCM was harvested. Centrifugation (1,000 × g; 1 min; 4°C) was used to purify the obtained conditioned medium. The MCM was diluted with serum-free medium to 1:1. Subsequently, the MCM was used as the culture medium for hippocampal neuron cells in the subsequent experiments.

Four treatment groups were prepared in the present study, including the control group (BV-2 microglial cells or hippocampal neuron cells), mimic control group [BV-2 microglial cells or hippocampal neuron cells transfected with control scrambled sequences (5′-GAGUAUGUGAGAUUAACUGGUGGC-3′; Shanghai GenePharma Co., Ltd., Shanghai, China)], miR-155 mimics group [BV-2 microglial cells or hippocampal neuronal cells transfected with miR-155 mimics (5′-UUAAUGCUAAUCGUGAUAGGGGUU-3′; Shanghai GenePharma Co., Ltd.)], and lipopolysaccharide (LPS) group. BV-2 microglial cells or hippocampal neuron cells were treated with LPS (10 µg/ml; dissolved in PBS; Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) for 24 h at 37°C. Cells were seeded into 6-well plates at a density of 1×10⁴ cells/well. The cells were starved overnight and then transfected with 75 pmol mimic control or miR-155 mimic using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were harvested 24 h after transfection and then used for subsequent experiments.

Overexpression of suppressor of cytokine signaling 1 (SOCS1) was induced by transfecting cells (1×10⁵ cells/ml) for 24 h at 37°C with 2.5 µg pcDNA3.1-SOCS1 plasmid (Shanghai GenePharma Co., Ltd, Shanghai, China) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc), according to the manufacturer's protocol. pcDNA3.1 served as the negative control.

Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China) was used to determine the cell viability of BV-2 microglial cells and hippocampal neuron cells from the four treatment groups described above. A density of ~6×10⁴ cells/ml in the logarithmic phase were seeded into the wells of 96-well plates, and subsequently incubated in a 5% CO₂ atmosphere at 37°C for 12 h. The cells were maintained for 12, 24 and 48 h, respectively. Subsequently, 10 µl CCK reagent was supplemented into the wells. Cells were maintained for 3 h. A microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to record the absorbance at 450 nm. Cell viability was evaluated by the percentage of cell survival compared with control.

Commercially available ELISA kits were used to detect interleukin-6 (IL-6; cat. no. M6000B; R&D systems), IL-10 (cat. no. M1000B; R&D systems), TNF-α (cat. no. MTA00B; R&D systems), TNF-β (cat. no. E-EL-M1210; Elabscience, Wuhan, China) and indoleamine 2,3-dioxygenase 1 (IDO1; cat. no. CSB-EL010996MO; CUSABIO TECHNOLOGY LLC, Wuhan, China). Cell culture supernatants were added into the corresponding wells, and the wells were sealed using adhesive tape and maintained at 37°C for 90 min. A total of 100 µl biotinylated antibody fluids were supplemented into wells. Subsequently, the wells were sealed by adhesive tape and incubated for 60 min at room temperature. Chromogenic substrate was added into the wells, excluding for the blank wells. Plates were incubated for 10–15 min in the dark at 37°C. Stop solution was added into each well and immediately mixed for 10 min at room temperature. The optical density (OD) 450 value was detected using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell apoptosis was evaluated by flow cytometry (FCM). Following washing with PBS, cells were trypsinized using 0.25% trypsin (Beyotime Institute of Biotechnology). Following centrifugation (1,000 × g; 1 min; 4°C), the supernatant was removed and the cells for assessment were suspended in the incubation buffer at a density of 1×10⁶ cells/ml. Cells were incubated with Annexin V-fluorescein isothiocyanate and propidium iodide (Xilong Scientific, Co., Ltd., Guangdong, China) at room temperature for 15 min in the dark. Cell apoptosis was subsequently assessed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes NJ, USA) and ModFit LT software version 2.0 (Verity Software House, Topsham, ME, USA).

Total protein was isolated from cells using NP40 lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was measured using Bradford Protein Assay kit (Bio-Rad Laboratories, Inc.). Proteins (30 µg/lane) were separated by 12% SDS-PAGE. The separated products were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk at room temperature for 2 h. Blotting was performed with specific primary antibodies at 4°C overnight: Anti-TNF-α (1:1,000; Abcam, Cambridge, UK; cat. no. ab6671; rabbit anti-mouse); anti-TNF-β (1:500; Abcam; cat. no. ab106353; rabbit anti-mouse); anti-IDO1 (1:50; Abcam; cat. no. ab106134; rabbit anti-mouse); anti-apoptosis regulator Bax (Bax; 1:1,000; Abcam; cat. no. ab32503; rabbit anti-mouse); anti-apoptosis regulator Bcl2 (Bcl-2; 1:500; Abcam; cat. no. ab59348; rabbit anti-mouse); anti-pro-caspase-3 (1:10,000; Abcam; cat. no. ab32499; rabbit anti-mouse); anti-cleaved-caspase-3 (1:1,000; Abcam; cat. no. ab2302; rabbit anti-mouse); anti-SOCS1 (1:200; Abcam; cat. no. ab3691; rabbit anti-mouse) and anti-actin (1:5,000; Abcam; cat. no. ab179467; rabbit anti-mouse). Horseradish peroxidase-conjugated secondary antibodies (1:5,000; Abcam; cat. no. ab205718; goat anti-rabbit) were added and incubated at room temperature for 1 h. Enhanced chemiluminescent (ECL) reagents (EMD Millipore) with an ECL system (GE Healthcare, Chicago, IL, USA) were used for the evaluation of results. Quantity One Analysis software version 4.6.2 (Bio-Rad Laboratories, Inc.) was used for densitometric analysis of the blots.

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed to cDNA using the BeyoRT™ First Strand cDNA Synthesis kit (cat. no. D7166; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The protocol for RT was as follows: 37°C for 60 min, 85°C for 5 min and then hold at 4°C. RT-qPCR analysis was performed using a SYBR^® Green PCR Master Mix kit (Takara Bio, Inc., Otsu, Japan) with an ABI 7500 Thermocycler (Applied Biosystems, Foster City, CA, USA). PCR cycles were as follows: 10 min pretreatment at 95°C, 93°C for 15 sec, 67°C for 45 sec (45 cycles), 93°C for 15 sec, 67°C for 1 min, 95°C for 15 sec, a final extension at 75°C for 10 min and held at 4°C. The primers were designed by Invitrogen (Thermo Fisher Scientific, Inc.): miR-155, forward: 5′-CTGTTAATGCTAATCGTGAT-3′ and reverse: 5′-AACTGACTCCTACATATTAG-3′ (product 215 bp); IL-6, forward: 5′-TTCTTGGGACTGATGCTGGT-3′ and reverse: 5′-CAAGTGCATCATCGTTGTTCA-3′ (product 211 bp); IL-10, forward: 5′-AGTACAGCCGGGAAGACAAT-3′ and reverse: 5′-TTTCTGGGCCATGCTTCTCT-3′ (product 249 bp); TNF-α, forward: 5′-CAGAAAGCATGATCCGCGAC-3′ and reverse: 5′-GGTCTGGGCCATAGAACTGA-3′ (product 224 bp); TNF-β, forward: 5′-CATCCTGAAACCTGCTGCTC-3′ and reverse: 5′-GGAGGAAAAGAGCTGGACCT-3′ (product: 244 bp); IDO1, forward: 5′-GACTGTGTCCTGGCAAACTG-3′ and reverse: 5′-GTAGCTATGTCGTGCAGTGC-3′ (product 233 bp); and actin, forward: 5′-TCTGAACTCCAACGATGCCT-3′ and reverse: 5′-TCTTGTCCTTAAGCCTGGGG-3′ (product 221 bp). Actin was used as the control of the input RNA level. Relative gene expression was determined using the 2^−ΔΔCq method (29).

All experiments were repeated at least three times. The results in the present study are presented as the mean ± standard error. All of the experimental data was analyzed by one-way analysis of variance following Dunnett's t-test method. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-155 in BV-2 cells was investigated. The results demonstrated that the expression of miR-155 was upregulated by LPS stimulation (Fig. 1A; P<0.05). LPS is able to induce inflammation injury (30). It was suggested that miR-155 was closely associated with inflammation response of microglial cells. In the present study, the miR-155 mimics were transfected into BV-2 microglial cells. According to the RT-qPCR data, the expression level of miR-155 in BV-2 microglial cells was significantly increased by transfecting with miR-155 mimics compared with the mimic control (Fig. 1A; P<0.01). Furthermore, a CCK-8 assay was performed to measure the cell viability of BV-2 microglial cells in the treatment groups. As demonstrated in the results, compared with other groups, miR-155 mimics significantly decreased the cell viability of BV-2 microglial cells, particularly at 48 h (Fig. 1B; P<0.05). The results indicated that the miR-155 reduces the cell viability of BV-2 microglial cells.

In order to investigate the associations between miR-155 and inflammatory injury, the expression levels of interleukin-6 (IL-6), IL-10, TNF-α, TNF-β and IDO1 were assessed in BV-2 microglial cells from each treatment group. On the basis of the ELISA data, significant increases were observed in the protein levels of IL-6, IL-10, TNF-α, TNF-β and IDO1 in BV-2 microglial cells transfected with miR-155 mimics (Fig. 2A; P<0.01). Additionally, the RT-qPCR results indicated that the IL-6, IL-10, TNF-α, TNF-β and IDO1 mRNA expression in BV-2 microglial cells was significantly upregulated by transfection with miR-155 mimics (Fig. 2B; P<0.01). Additionally, the protein expression levels of pro-inflammatory cytokines, TNF-α, TNF-β and IDO1, were measured by western blotting (Fig. 3). It was revealed that miR-155 significantly increased the expression levels of TNF-α, TNF-β and IDO1 in BV-2 microglial cells compared with mimic controls (P<0.01). These results confirmed that miR-155 increased the expression levels of pro-inflammatory cytokines in BV-2 microglial cells.

Hippocampal neuron cells were cultured with MCM in vitro. A CCK-8 assay was conducted to assess the cell viability of hippocampal neuron cells following culture in the MCM of microglial cells that received the treatment/transfection described above. Based on the results, LPS-stimulation of microglia significantly decreased the cell viability of hippocampal neuron cells. According to the CCK-8 results, it was additionally identified that the cell viability of hippocampal neuron cells was significantly decreased by miR-155-stimulation of microglia (Fig. 4; P<0.05).

FCM data revealed that the percentage of apoptotic cells in the miR-155 mimic-transfected hippocampal neuron group was 29.24%, which was significantly increased compared with the control and mimic control groups (6.29 and 6.41%, respectively; P<0.01). Furthermore, following treatment with MCM from the LPS-treated microglia, the apoptosis rate of hippocampal neuron cells was increased to 28.63%. These data indicated that miR-155-stimulation of microglia significantly increased the apoptosis of hippocampal neuron cells, which was even more marked compared with the positive control (LPS; Fig. 5). In order to further investigate the associated apoptosis mechanisms in hippocampal neuron cells, the expression levels of several apoptosis-associated proteins were assessed, including Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3 in the hippocampal neuronal cells. It was observed that the expression level of Bcl-2 in hippocampal neuronal cells was significantly decreased in the miR-155 mimics group (Fig. 6; P<0.01). However, there was no significant difference in the expression level of pro-caspase-3 in hippocampal neuronal cells among the treatment groups. The western blot analysis additionally revealed similar trends in Bax, Bcl-2, pro-cleaved-3 and cleaved-caspase-3 protein expression in hippocampal neuron cells from each treatment group (Fig. 6). Based on these results, it was suggested that miR-155-stimulation of microglia modulates the expression levels of Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3 in hippocampal neuron cells. Therefore, it was concluded that miR-155 contributed to increasing the apoptosis of hippocampal neuronal cells by modulating the levels of Bax, Bcl-2, and cleaved-caspase-3.

The effect of miR-155 on cell cycle progression of hippocampal neuronal cells was examined. It was demonstrated that MCM from LPS-treated microglial cells decreased the cell numbers in the G1 phase and increased the cell numbers in the S phase compared with the control group. The cell cycle was also arrested by miR-155 group; identified by the decreasing cell numbers in the G1 phase and increasing cell numbers in the S phase compared with the mimics control group (Fig. 7; P<0.05).

It has been reported that SOCS1 is a direct target of miR-155 during microglia activation (31,32). Therefore, the function of SOCS1 was investigated in the present study. The results demonstrated that the expression of SOCS1 was reduced in the presence of miR-155 mimics (Fig. 7; P<0.05). Furthermore, the expression of Bcl-2 and Bax was upregulated and downregulated by overexpression of SOCS1 in hippocampal cells, respectively, compared with the miR-155 mimics group (Fig. 8; P<0.05). It was suggested that miR-155 mediates hippocampal neuron cell injury via SOCS1.