Effect of rs4719839 polymorphism on risk of ventilator‐associated pneumonia, expression of microRNA‐148 and autophagy‐related 16‐like 1 (ATG16L1)

Abstract MiR‐148 is a negative regulator of autophagy 16‐like 1 (ATG16L1), a gene implicated in the pathogenesis of ventilator‐associated pneumonia (VAP). Therefore, the role of miR‐148 polymorphism in the pathogenesis of VAP was studied here. The expression of miR‐148, ATG16L1, Beclin‐I, LC3‐II, TNF‐α and IL‐6 in serum and peripheral blood mononuclear cells (PBMCs) of VAP patients was detected to study their relationship in the pathogenesis of VAP. Chronic obstructive pulmonary disease patients carrying the AA/AG genotypes of miR‐148 rs4719839 single nucleotide polymorphism (SNP) were more prone to VAP due to the higher expression of miR‐148, TNF‐α and IL‐6 along with suppressed expression of ATG16L1, Beclin‐I and LC3‐II in their serum and PBMCs. Transfection of miR‐148 mimics to primary PBMCs genotyped as GG and AA decreased the expression of ATG16L1, Beclin‐I and LC3‐II. Finally, cells carrying the AA genotype of rs4719839 SNP were more sensitive to the role of LPS stimulation in suppressing ATG16L1, Beclin‐I and LC3‐II expression while activating TNF‐α and IL‐6 expression. Our work presented detailed evidence, suggesting that the rs4719839 polymorphism can affect the risk of VAP.

Autophagy-related 16-like 1 (ATG16L1) plays critical roles in the regulation of autophagy and formation of autophagosomes by forming a complicated complex with ATG12 and ATG5. 11 A single nucleotide polymorphism (SNP), that is rs2241880 Thr300 Ala c.898A>G, located in ATG16L1 impairs autophagy functions to increase the risk of tuberculosis. 12 Furthermore, NOD2 and NOD1 receptors can recruit ATG16L1 proteins upon bacterial infection to activate autophagy, while the A allele of rs2241880 impairs the activation of autophagy to increase the risk of septic shock in VAP patients. 13 As a type of short RNAs containing about 22 nucleotides, mi-croRNA (miRNA) possesses dual tumour-suppressing and oncogenic properties based on actual cellular context. 14 Deregulated expression of miRNAs can lead to multiple malignancies such as lung and breast cancer. [15][16][17] The G allele of rs4719839 SNP located in miR-148a affects the size and TNM score of certain tumours. 18 The G allele in the rs2241880 SNP of ATG16L1 affects the activation of autophagy by modifying the polarity of ATG16L1 protein, thus increasing the risks of Crohn's disease (CD). [19][20][21] Furthermore, rs2241880 SNP was identified as a risk factor in certain European population. [22][23][24] It has been reported that rs2241880 polymorphism may affect the expression level of ATG16L1, while the rs4719839 polymorphism may affect the expression of miR-148, a negative regulator of ATG16L1. 18,25 ATG16L1 has been implicated in the pathogenesis of VAP. 26 In this study, we enrolled chronic obstructive pulmonary disease (COPD) patients with or without VAP to study the role of rs2241880 and rs4719839 polymorphisms in the pathogenesis of VAP.

| Human sample collection
A total of 280 patients provided consent to take part in our research, which included 142 COPD patients suffering from VAP and 132 COPD patients free of VAP who were enrolled during May 2013 to August 2016 at China-Japan Friendship Hospital. Peripheral blood samples were collected from above patients. Information of all participants including their age, gender, APACHE II score 27 and underlying diseases (type 2 diabetes, non-Hodgkin's lymphoma, Parkinson's disease, coronary heart disease, heart failure and chronic kidney diseases) was collected and summarized. Institutional ethical committee has approved the protocol of this study.

| Genotyping
The peripheral blood samples collected from the COPD patients with or without VAP were subjected to isolation of genomic DNA utilizing a Genomic DNA Isolation Assay Kit (Qiagen). Then, 25 ng of isolated genomic DNA from each patient was mixed with an appropriate amount of Maxima Hot Start Green Master Mix (Thermo Fisher Scientific) and corresponding primers were designed for miR-148 rs4719839 SNP and ATG16L1 rs2241880 SNP. In the next step, real-time PCRs were carried out on a Bio-Rad real-time PCR machine (Bio-Rad) using a TaqMan Assay Kit (ABI) in accordance with the instructions provided on kit manual to determine the genotypes of miR-148 rs4719839 SNP and ATG16L1 rs2241880 SNP in various peripheral blood samples.

| RNA isolation and real-time PCR
Peripheral blood samples collected from the COPD patients with or without VAP were treated to separate monocytes. Then, total RNA content in the monocytes was isolated utilizing a TRIzol reagent (Invitrogen) following the instructions of the manufacturer. In the next step, the extracted RNA was reversely transcribed to cDNA using a reverse transcription assay kit (Thermo Fisher Scientific) following kit instruction and then loaded onto the Bio-Rad real-time

| Cell culture and treatment
Primary PBMCs genotyped as GG and AA were obtained and cultured in DMEM containing appropriate antibiotics and 10% serum.
The routine culture operation was carried out in a 5% CO 2 incubator at 37°C and saturated humidity. Upon 80% confluency, primary PBMCs genotyped as GG and AA were divided into the following

| Luciferase assay
The TargetScan database was used to obtain the wild-type sequence for the 3′-untranslated region (UTR) of ATG16L1. Then, the mutant type or wild-type fragments of ATG16L1 3′-UTR containing the miR-148 binding site were subcloned into pcDNA3.1 luciferase vectors (Promega), which were transfected into primary PBMCs genotyped as GG and AA in conjunction with miR-148 using Lipofectamine 2000. After 48 hours of incubation, the luciferase activity of cell lysates in each well of the tissue culture plate was measured using a luciferase reporter assay (Promega) following a routine procedure recommended by the producer. The luciferase activity of the cells transfected with the mutant type or wild-type fragments of ATG16L1 3′-UTR was compared to determine the regulatory relationship between ATG16L1 and miR-148 expression.

| Western blotting analysis
The peripheral blood samples collected from the COPD patients with or without VAP, as well as the transfected cell samples, were lysed to isolate their protein content. Then, 50 µg of protein from

| ELISA
Peripheral blood samples collected from the COPD patients with or without VAP were treated to separate monocytes. Then, the expression levels of TNF-α and IL-6 in both the peripheral blood samples and the separated monocytes were determined using commercially available ELISA kits (Thermo Fisher Scientific) following the routine assay methods recommended in the kit instructions.

| Statistical analysis
The regulatory relationship between ATG16L1 and miR-148 expression was evaluated utilizing the Mann-Whitney U tests. The comparisons among multiple groups were carried out using oneway ANOVA or Student's t tests. The correlation between miR-148 rs4719839 SNP and the expression level of miR-148 as well as the correlation between ATG16L1 rs2241880 SNP and the expression level of ATG16L1 was analysed utilizing Spearman's rank tests.
Multivariate logistic regression analysis was used to evaluate the association between the risk of VAP in COPD patients and the miR-148 rs4719839 or ATG16L1 rs2241880 polymorphisms. All statistical analyses were carried out using GraphPad Prism 7.0 (GraphPad) and SPSS 21.0 (SPSS, IBM). The level of statistical significance was set to 0.05.

| Patient characteristics in experimental and control groups
As shown in Table 1 TA B L E 1 Basic information of the participants of this study disease, heart failure and chronic kidney diseases) was collected from the COPD participants with or without VAP. Student's t test was carried out to compare the above characteristics between COPD + VAP and COPD-VAP groups, and no difference was observed.

| Rs4719839 and rs2241880 polymorphisms were not associated with the risk of VAP
Multivariate logistic regression analysis was used to evaluate the association between the risk of VAP in COPD patients and the miR-148 rs4719839 or ATG16L1 rs2241880 polymorphisms, as shown in Table 2. Both polymorphisms showed no obvious association with the risk of VAP in COPD patients.

| AA and AG genotypes of miR-148 rs4719839 SNP reduced the incidence of VAP in COPD patients
We collected 142 COPD patients suffering from VAP to evaluate the relationship between the genotypes of rs4719839/rs2241880 SNPs and the incidence of VAP. Rs4719839 AA and AG SNPs showed a higher incidence of VAP than the GG genotype ( Figure 1A), while no obvious difference was found among rs2241880 AA, AG and GG genotypes ( Figure 1B).

| Differential expression of ATG16L1 in different groups
Next, we evaluated the expression of ATG16L1, which has been implicated in the pathogenesis of VAP, in the PBMC of VAP patients carrying different genotypes of rs4719839 SNP. As shown in Figure 4, the mRNA ( Figure 4A) and protein ( Figure 4B) expression of ATG16L1 was significantly enhanced in patients with the GG genotype. Moreover, the expression of Beclin-I ( Figure 4C) and

| MiR-148 down-regulated ATG16L1 and other genes related to VAP
To understand the regulatory role of miR-148 in VAP, we trans-

| Divergent response of cells carrying different genotypes of rs4719839 SNP upon LPS stimulation
Primary PBMCs genotyped as GG and AA carrying GG and AA genotypes of rs4719839 SNP were subjected to LPS stimulation to compare their responses. MiR-148 expression was significantly higher in primary PBMCs genotyped as AA carrying AA genotype, but no obvious change was detected when the cells were treated with LPS ( Figure 6A). ATG16L1 expression was elevated in primary PBMCs genotyped as GG carrying GG genotype and was decreased after LPS was added to the cells. In addition, the decrease in ATG16L1 expression was more pronounced in the primary PBMCs genotyped as AA carrying AA genotype ( Figure 6B).
Besides, the expression of ATG16L1, Beclin-I and LC3-II proteins showed the same trend as that of ATG16L1 mRNA in different groups ( Figure 6C).

| D ISCUSS I ON
In this study, we enrolled a group of COPD patients carrying dif- MyD88 in many fish species to reduce the level of inflammation. 33 MiR-148 overexpression also substantially attenuates the synthesis of inflammatory cytokine via targeting MyD88 functions directly to inhibit NF-κB signalling. 33 As a miR-148a target, Gas1 promotes Hh signalling activation to reduce the activity of autophagy in HSC. Upon starvation of cells, miR-148a can also induce autophagy by suppressing the level of Hh signalling, while the overexpression of miR-148a inhibits the proliferation of HSC and promotes their apoptosis. 34 35 Therefore, the formation of autophagosomes depends on ATG16L1 displacement and the activation of its upstream pathways.
Moreover, the elongation of autophagosomes and their curvature can promote their release. 35 For example, the activation of the ATG16L1 signalling can protect cells against ER-induced stress and reduce IKK/NF-κB-induced pro-inflammatory events. 36 The characterization studies on IRGM and ATG16L1 mutants also pointed to the essential role of autophagosomes in controlling gastrointestinal inflammation and the progression of CD. Moreover, the inhibited ATG16L1 expression can increase the in vivo pro-inflammatory responses upon NOD2 activation. 37 Other studies also showed that ATG16L1 deletions in mice impaired the autophagy machinery and increased the severity of inflammation in CD. 30,38

| CON CLUS ION
Taken together, our study demonstrates that the rs4719839 SNP in ATG16L1 can influence the expression of miR-148 and negatively regulate ATG16L1 expression to increase the risk of VAP.

ACK N OWLED G EM ENTS
This work was supported by the Discipline Construction Project of Peking Union Medical College (201920102304).

CO N FLI C T O F I NTE R E S T
None. F I G U R E 6 Divergent response of cells carrying different genotypes of rs4719839 upon LPS stimulation. A, No obvious difference in expression was detected after both primary PBMCs genotyped as GG and AA were stimulated by LPS. B, Decrease in ATG16L1 mRNA expression was more pronounced after LPS stimulation in primary PBMCs genotyped as AA carrying AA genotype of rs4719839 (*P value < 0.05 compared with unstimulated PBMCs). C, Down-regulation of ATG16L1, Beclin-I and LC3-II protein was more pronounced after LPS stimulation in primary PBMCs genotyped as AA carrying AA genotype of rs4719839. PBMCs, peripheral blood mononuclear cells

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.