The present study recruited 74 suspected cases of HEV71 encephalitis, including mild cases (n=40) and severe cases (n=34), at the Department of Pediatrics, Qilu Hospital of Shandong University (Shandong, China). All patients were admitted between August 2013 and December 2014 (54 males and 20 females; age, 15.2–41.2 months old; weight, 10.39–15.67 kg). All 74 cases were diagnosed as HEV71 encephalitis. The patients were divided into two groups, comprising a HEV71-infected encephalitis group and an HFMD without complications group. HFMDs were diagnosed according to the following clinical features: Typical ulcers on lips, and rashes on the hands and feet. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the HEV71 virus infection. On the day of admission, throat swabs or cerebrospinal fluid (CSF) samples were collected from each subject for RNA extraction. A diagnosis of encephalitis was made when subjects met the following criteria: White blood cell count >5/mm³ in the CSF; parenchymal damage detected by MRI or CT scan. Each subject went through a regular physical examination and other laboratory tests in the following 3 days, from which baseline values of clinical and laboratory characteristics were collected. The study protocol was approved by the Clinical Research Ethics Committee of Shandong University. Written Informed consent was obtained from each subjects' guardian prior to commencement of the study.

CSF (2 ml) was collected and centrifuged at 1,600 × g for 10 min to remove particles. The levels of CCL2 were determined using a Cusabio ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol. In brief, the kit contained a microplate precoated with monoclonal antibody against CCL2 and a peroxidase-bonded anti-CCL2 polyclonal antibody. The detecting sensitivity was 5 pg/ml.

Total RNA were extracted using an RNA extraction kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The RNA extract was used to generate the first strand cDNA using the SuperRT cDNA Synthesis kit (Kangwei Biotech Co., Ltd., Beijing, China). The qPCR reaction (total volume, 20 µl) was performed using UltraSYBR Green qPCR Master mix (with ROX I) (Kangwei Biotech Co., Ltd.) and the LightCycler 480 Real-Time PCR system (Roche Diagnostics GmbH, Mannheim, Germany). The primer sequences were as follows: CCL2 (forward 5′-ATGCAGTTAATGCCCCACTC-3′, reverse 5′-TTCCTTATTGGGGTCAGCAC-3′), miR-206 (3′-UGGAAUGUAAGGAAGUGUGUGG-5′), GAPDH (forward 5′-GAAGATGGTGATGGGATTTC-3′, reverse 5′-GAAGGTGAAGGTCGGAGTC-3′), U6 (forward 5′-CTCGCTTCGGCAGCACATATACT-3′, reverse 5′-ACGCTTCACGAATTTGCGTGTC-3′). All samples were run in duplicate and the reaction conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 40 sec and extension at 72°C for 40 sec. The 2^−ΔΔCq relative quantification method was used to calculate the expression levels of miRNA (17). The expression of U6 was used as an internal control. Values were expressed as fold differences of the expression of miR-206 and CCL2 relative to that of U6.

U-251 cells (Ybiotech, Beijing, China) were maintained in DMEM (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂ humidified atmosphere. miR-206 mimics or inhibitor and CCL2 small interfering (si)RNA (50 nM) (Shanghai GenePharma Co., Ltd., Shanghai, China) were transfected into the U-251 cells (1×10⁵ cells) using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. In addition, scrambled controls were transfected into cells as negative controls. The transfection efficiency of the U-251 cells was confirmed using RT-qPCR analysis.

The 3′untranslated region (UTR) of CCL2 containing the putative binding site of miR-206 was identified by searching the online miRNA database (18,19). The CCL2 3′UTR was amplified using PCR, and the PCR products were isolated using agarose gel (2%). The fragments were then purified and inserted into a pGL3-control vector (Promega Coproration, Madison, WI, USA). Mutagenesis was performed for the same site and inserted into the control vector (Promega Corporation) using PCR. The miR-206 primers were added into the reverse transcription system. The miRNA reverse transcription program is consistent with the common mRNA procedures (20).

A dual-luciferase reporter assay (Promega Corporation) was used to investigate the effects of the miR-206 (targeting the putative 3′UTR binding site) on luciferase activity. In brief, the cells were seeded into a 96-well plate (3×10⁴ cells/ml; 200 µl/well) overnight prior to transfection. On the day of transfection, the U-251 cells were cotransfected with the wild-type (WT) or mutant type (MT) vector and miR-206 or the negative control vector. At 2 days post-transfection, the cells were subjected to dual-luciferase reporter assays. The luciferase activity of a Renilla control plasmid was also analyzed as in internal control. The results were recorded as ratios of miR-206 luciferase activity relative to that of Renilla.

Western blot analysis was performed to assess the effect of miR-206 on protein expression in the U-251 cells. The U-251 cells were lysed and purified, and the protein level was determined using an Enhanced BCA Protein Assay kit (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The extracts, containing 25 µg of total proteins, were loaded on 10% SDS-polyacrylamide gel. Following electrophoresis, the protein blots were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was washed with PBS and then blocked in 10% skimmed milk at 4°C overnight. The PVDF membranes were incubated with the following antibodies at 4°C overnight: Rabbit anti-CCL2 (IC479A; 1:700; R&D Systems, Inc., Minneapolis, MN, USA) or rabbit anti β-actin (a8227; 1:300; Abcam, Cambridge, UK), and subsequently incubated with goat anti-rabbit IgG (CW0111S; 1:2,200; Kangwei Biotech Co., Ltd.) for 2.5 h at room temperature. The conjugated proteins were visualized using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.). Fluor Chen 2.0 software (Olympus, Yokohama, Japan) was used to analyzed the density values. β-actin was used as an internal control, with values presented as the expression of CCL2 relative to that of β-actin.

For each experiment, replicate samples were subjected to analyses at least three times and the results were compared using Student's t test. All data analyses were performed using SPSS 19.0 (IBM SPSS, Armonk, NY, USA). Comparisons among more than three groups were performed using one-way, two-way or three-way analysis of variance (ANOVA) without repeated measures, as appropriate. Comparisons among less than three were performed using one-way ANOVA followed by Dunnett's post hoc test. Comparisons among three or groups were performed using two- or three-way ANOVA followed by a stratified independent t-test with Bonferroni corrections for multiple comparisons. P≤0.05 was considered to indicate a statistically significant difference. The values from all experiments are expressed as the mean ± standard deviation.

To identify miRNAs involved in regulating the severity of HEV71 encephalitis, the present study compared miRNA expression levels between severe HEV71 encephalitis and mild HEV71 encephalitis samples. It was found that miR-206 was consistently downregulated in severe HEV71 encephalitis (Fig. 1). Therefore, the present study focused on miR-206, and it was hypothesized that miR-206 may regulate the transformation of mild HEV71 encephalitis into severe HEV71 encephalitis.

One of the primary challenges in examining miRNAs is to identify target genes and correlate their downregulation with cellular properties. Computational algorithms have been used to predict putative miR-206 targets based on complementarity to the 3′UTR of the target mRNA. Using bioinformatics analysis, miR-206 was predicted to target the human CCL2 3′-UTR (Fig. 2). To investigate whether miR-206 is a regulator of CCL2, further experiments were performed. Firstly, luciferase reporter vectors were constructed containing a WT or MT CCL2 3′-UTR downstream of Renilla luciferase (Fig. 2). The miR-206 mimic and corresponding reporter vectors were co-transfected into U-251 cells, and a dual-luciferase assay was performed 48 h following transfection. The results of the dual-luciferase assay showed that miR-206 significantly suppressed the expression of luciferase if the reporter vector contained the WT CCL2 3′-UTR. By contrast, target region mutation completely eradicated this interaction (Fig. 3). To determine whether miR-206 disrupted the expression of CCL2 in a human cell line, miR-206 mimics were transfected into U-251 cells, with blank and scramble serving as a negative control, and CCL2 siRNA serving as a positive control. The data showed that the mRNA and protein expression levels of CCL2 were suppressed by the miR-206 mimic (Fig. 4A and B). Similar experiments were performed using an miR-206 inhibitor, and the decrease in miR-206 led to an increase in the mRNA and protein levels of CCL2 (Fig. 4A and B). To determine whether miR-206 negatively regulates endogenous CCL2 in human HEV71 encephalitis, the expression levels of miR-206 and CCL2 were measured in the CSF samples using RT-qPCR analysis. The association between the expression of miR-206 and CCL2 were also evaluated using Pearson rank correlation analysis (Fig. 5). The results indicated a statistically significant inverse correlation between miR-206 and CCL2, indicating that miR-206 suppressed the expression of CCL2 in the human brain. These results showed that miR-206 directly targeted the CCL2 3′UTR and suppressed the expression of CCL2.

The present study hypothesized that the reduction in miR-206 leads to a further increase in the expression of CCL2 in severe HEV71 encephalitis. To support this, RT-qPCR analysis was used to determine the mRNA expression level of CCL2 in the two groups (Fig. 6). The results showed that the expression of CCL2 was upregulated in the transformation of mild to severe HEV71 encephalitis.

CCL2 is important in regulating inflammation, and high expression levels of CCL2 indicate severe inflammation. Consequently, the present study hypothesized that miR-206 also regulates cell proliferation and apoptosis. To confirm this, the miR-206 mimics and inhibitor were introduced into the U-251 cells, with blank and scramble siRNA as a negative control, and CCL2 siRNA as a positive control. The results showed that miR-206 induced U-251 cell proliferation and reduced apoptosis (Fig. 7).