Peripheral blood samples were collected from a total of 96 patients with COPD hospitalized at the ICU of Qinghai Red Cross Hospital from September, 2015 to August, 2017 for clinical analysis. Although all patients treated with mechanical ventilation were eligible for the screening of the study, all enrolled patients must have experienced at least one episode of VAP. Peripheral blood samples were collected from all subjects to isolate their monocytes and to determine their genotypes of rs1056629 SNP. The isolation process was accomplished using the Human Peripheral Blood Mononuclear Cell Isolation and Viability kit (ab234628; Abcam) following the instructions of the manufacturer. Subsequently, based on the results of rs1056629 SNP genotyping, all the subjects were divided into 3 groups as follows: The CC group (n=18), the CA group (n=33) and the AA group (n=45). The protocol of the study, as well as the template of informed consent form (ICF) was reviewed and approved by the Clinical Ethics Committee of our Qinghai Red Cross Hospital for the retrospective use of these blood samples, and written informed consent was obtained from all subjects or their family members prior to the initialization of the study.

The CPIS of each subject was assessed using an established method as described in a previous study (17).

First, peripheral blood samples were collected under fasting conditions from each subject using EDTA blood collection tubes to isolate mononuclear cells. The genomic DNA in each sample of mononuclear cells was isolated from archived pellets utilizing a QIAamp genomic DNA extraction assay kit (Qiagen, Inc.) following the instructions provided with the assay kit. The genotypes of rs1056629 SNP in the genomic DNA isolated from the mononuclear cells of each subject were determined using quantitative PCR (qPCR), which was carried out using a TaqMan genotyping assay kit (cat. no. 4381657; Applied Biosystems; Thermo Fisher Scientific, Inc.) on a 7900HT fast real-time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.) following a standard protocol provided by the manufacturer. For the purpose of quality control, both negative control wells, which contained blank samples free of DNA, and positive control wells, which contained genomic DNA samples of a known genotype of rs1056629 SNP, were set up in each microtiter plate of the qPCR reaction.

Peripheral blood samples collected from each subject and the A549 and H1299 cells (described below) were subjected to treatment with a mirVana assay kit (Ambion; Thermo Fisher Scientific, Inc.) following the instructions provided by the kit manufacturer to collect and purify the total RNA content in each sample. Subsequently, the integrity and concentration of each purified RNA sample were evaluated utilizing an Agilent Bioanalyzer (Model 2100, Agilent Technologies, Inc.). Reverse transcription was then performed utilizing a MiScript Reverse Transcription kit (cat. no. 218160; Qiagen GmbH) to produce cDNA templates, which were then subjected to qPCR utilizing specific TaqMan probes and Universal TaqMan Master Mix (cat. no. 4304437; Applied Biosystems; Thermo Fisher Scientific, Inc.) in accordance with the instructions provided by the manufacturer. All qPCR reactions were carried out in triplicate wells of a 384-well qPCR plate, which was then loaded into a 7900HT fast real-time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.) for operation. The thermocycling conditions were 95°C for 15 min (initial activation), 94°C for 15 sec (denaturation), 55°C for 30 sec (annealing), and 72°C for 60 sec (extension). Finally, the relative expression of miR-491 and MMP-9 mRNA in each sample was calculated by normalization vs. the expression of the U6 (for miR-491) and GAPDH (for MMP-9 mRNA) housekeeping genes using the 2^−ΔΔCq method, respectively (18). The primers used for PCR were as follows: miR-491 forward, 5′-AGTGGGGAACCCTTCC-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′; MMP-9 forward, 5′-GCCACTACTGTGCCTTTGAGTC-3′ and reverse, 5′-CCCTCAGAGAATCGCCAGTACT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

The human lung adenocarcinoma cell line, A549 (cat. no. CRM-CCL-185™), and the human lung carcinoma cell line, H1299 (cat. no. CRL-5803), were obtained from the American Type Culture Collection and incubated in a Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2 mM L-glutamine, 10% heat inactivated FBS and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The culture conditions were 95% air, 5% CO₂, saturated humidity and a temperature of 37°C. These cell lines were selected due to the fact that they exhibited good growth conditions and were easier to obtain during the study. When the cells reached the logarithmic growth, they were divided into 3 groups as follows: The NC group, the 50 nM miR-491 mimics (Thermo Fisher Scientific, Inc.) group and the 100 nM miR-491 mimics (Thermo Fisher Scientific, Inc.) group, and transfected with either a scramble miRNA control sequence (5′-UGGGCGUAUAGACGUGUUACAC-3′) or the corresponding concentrations of miR-491 mimics (Thermo Fisher Scientific, Inc.) using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 48 h following a standard transfection protocol provided by the manufacturer. Subsequent observations were performed at 48 h post-transfection.

To determine the regulatory association between miR-491 and MMP-9, as well as the effect of different genotypes of rs1056629 SNP on the binding affinity between miR-491 and MMP-9 3′UTR, the 3′UTR of MMP-9 containing the rs1056629-A or rs1056629-C allele in its miR-491 binding site were respectively sub-cloned into pcDNA luciferase vectors (Promega Corporation). The A549 and H1299 cells were then co-transfected with different vectors of MMP-9 3′UTR in conjunction with 20 pmol miR-491 mimics or a scramble control for 24 h at 4°C using Lipofectamine 3000^®, followed by the detection of luciferase activity of the transfected cells at 48 h following the start of transfection using a Bright-Glo luciferase assay kit (Promega Corporation) following the protocol provided with the kit. The relative luciferase activity was normalized to the Renilla luciferase activity.

The collected clinical samples, as well as the cultured cell samples were first lysed in a RIPA lysis buffer (pH 7; Cell Signaling Technology, Inc.) containing 0.5% sodium deoxycolate, 10 mM EDTA, 0.5% NP-40, 100 mM NaCl, 100 mM Tris, and a cocktail of phosphatase and protease inhibitors (Cell Signaling Technology, Inc.). The supernatant of each sample was then collected via 30 min of centrifugation at 4°C and 14,000 × g, followed by the quantification of the protein concentration using a BCA assay kit (Pierce; Thermo Fisher Scientific, Inc.). Subsequently, the protein was resolved using 10% SDS-PAGE and transferred onto a PVDF membrane, which was then blocked with 5% skim milk, incubated at 4°C for 12 h with primary anti-MMP-9 antibody (1:1,000; ab38898; Abcam) and subsequently incubated at room temperature for 1 h with corresponding HRP-labeled secondary antibody (1:2,000; ab6721; Abcam) consecutively, developed using an enhanced chemiluminescence reagent (Amersham; Cytiva), visualized using a Bio-Rad imager (Bio-Rad Laboratories, Inc.) and processed using ImageJ software (V1.4.1; National Institutes of Health) to determine the relative expression of MMP-9 proteins utilizing β-actin as the internal reference.

The levels of TNF-α and IL-6 in monocytes isolated from the peripheral blood samples of all subjects were determined using commercial ELISA kits (E-EL-H0109 for TNF-α, E-EL-H0102 for IL-6, Elabscience) following the kit manuals, and the absorbance values were measured utilizing a Multiskan GO microplate reader (Thermo Fisher Scientific, Inc.).

All statistical analyses were carried out utilizing SPSS 16.0 statistical software (SPSS, Inc.). Continuous parameters are presented as the mean ± SD, and inter-group comparisons were carried out using one-way ANOVA with the Student-Newman-Keuls post hoc test. All statistical tests were bilateral and P-values <0.05 were considered to indicate statistically significant differences.

All 96 patients with COPD in the present study were genotyped for their rs1056629 SNP, which was located within the 3′UTR of MMP-9 mRNA. As shown in Fig. 1, carriers of either one or two C alleles had a significantly shorter time to develop VAP when compared to the carriers of the wild-type AA (Fig. 1A). Consistently, the CPIS was notably increased in both the CC and CA groups (Fig. 1B).

ELISA was carried out to evaluate the expression of TNF-α and IL-6 in the monocytes of the 3 groups of patients. The expression of TNF-α and IL-6 was evidently elevated in patients with the CC and AC genotypes than in those with the AA genotype (Fig. 2). Furthermore, the expression of miR-491 and MMP-9 mRNA was also analyzed using RT-qPCR, and no marked differences in the expression of miR-491 were found between all 3 groups (Fig. 3A). However, the expression of MMP-9 mRNA was significantly increased in the CC and AC groups (Fig. 3B).

The screening of potential binding sites of miR-491 identified a miR-491 binding site at the 3′UTR of MMP-9 mRNA. Subsequently, luciferase reporter plasmids of MMP-9 3′UTR containing different genotypes of rs1056629 SNP were constructed, and were then co-transfected with miR-491 into A549 cells (Fig. 4A). The luciferase activity of wild-type rs1056629-A, but not that of mutant rs1056629-C SNP was markedly inhibited by miR-491 (Fig. 4B). Furthermore, the inhibitory effect of miR-491 on MMP-9 expression was also confirmed in H1299 cells (Fig. 4C). Thus, these results indicated that miR-491 suppressed the expression of MMP-9 by binding to its 3′UTR.

To further confirm the inhibitory role of miR-491 in MMP-9 expression, 50 and 100 nM miR-491 mimics were transfected into the A549 and H1299 cells, and this led to the overexpression of miR-491 (Figs. 5A and 6A) in a concentration-dependent manner. MMP-9 mRNA expression was also significantly reduced in the A549 (Fig. 5B) and H1299 cells (Fig. 6B) transfected with miR-491 in a concentration-dependent manner. Similarly, the expression of MMP-9 protein in the A549 (Fig. 5C) and H1299 (Fig. 6C) cells transfected with miR-491 was also decreased in a concentration-dependent manner. Collectively, these results suggested that miR-491 inhibited MMP-9 expression in a concentration-dependent manner.