miR‐126‐5p expression in the plasma of patients with sepsis‐induced acute lung injury and its correlation with inflammation and immune function

Abstract Objective This work was implemented to elucidate the miR‐126‐5p expression in the plasma of patients with sepsis‐induced acute lung injury (ALI) and its correlation with inflammation and immune function. Methods The peripheral blood of patients with sepsis‐induced ALI was obtained, and the levels of inflammatory factors (interleukin‐6 [IL‐6], C‐reactive protein [CRP], and procalcitonin [PCT]) were determined. Meanwhile, T lymphocyte subsets (CD3+, CD4+, and CD8+), and immunoglobulins (IgA, IgM, and IgG) were tested. miR‐126‐5p and TRAF6 mRNA expression in plasma was assessed. Receiver operating characteristic (ROC) curve was performed to assess the diagnostic accuracy of miR‐126‐5p in sepsis without ALI and sepsis with ALI. Correlation between miR‐126‐5p expression and clinical indicators was analyzed. The targets of miR‐126‐5p were predicted using the bioinformatics method, and the direct targets were verified through investigations. Results miR‐126‐5p expression in plasma of patients with sepsis‐induced ALI was reduced than that of patients with sepsis without ALI. miR‐126‐5p expression was negatively correlated with IL‐6, CRP, and PCT but positively correlated with IgA, IgM, and IgG as well as CD3+, CD4+, and CD8+ in patients with sepsis‐induced ALI. ROC curve suggested that miR‐126‐5p (AUC: 0.777; 95%CI: 0.689–0.866) could distinguish patients with sepsis with ALI from patients with sepsis without ALI. TRAF6 expression in patients with sepsis‐induced ALI was higher than that in patients with sepsis without ALI. TRAF6 was a target gene of miR‐126‐5p, Conclusion This research highlights that miR‐126‐5p is reduced in the plasma of patients with sepsis‐induced ALI, and miR‐126‐5p relates to systemic inflammation and immune function indicators.


| INTRODUCTION
Sepsis is a complicated and life-threatening disease that develops from dysregulation of the host's response to infection and leads to organ dysfunction. 1 The prevention, diagnosis, and treatment of sepsis have become a global health priority, with the developed treatments substantially diminishing organ dysfunction and alleviating the prognosis of patients with sepsis. 2 Respiratory failure is one of the most significant complications of sepsis, and about half of the patients with severe sepsis may develop into acute lung injury (ALI) or even acute respiratory distress syndrome (ARDS). 3 Patients with sepsis-induced ALI have a poor prognosis with a mortality rate of more than 40%. 4 Therefore, it is urgent to seek for useful therapeutic methods for sepsisinduced ALI.
Recently, the modulation of microRNA (miRNA) has raised increasing attention in sepsis. 5,6 miRNAs are noncoding endogenous RNAs that are implicated in the diagnosis of sepsis via the control of various genes at the posttranscriptional level through translational repression or mRNA degradation. 7 Some articles have mentioned that miRNAs could be considered to be a novel biomarker in sepsis. 8,9 Moreover, there are also studies highlighting that miRNAs could induce or suppress ALI during sepsis. 10,11 An immune storm leads to organ damage. In addition, sepsis is also a severe infection with organ dysfunction; thus, it is essential to modulate the immune and inflammatory effects in sepsis. 12 Nowadays, miRNAs have been reported to function in immune modulation. 13 As one of the miRNAs, miR-126 could be considered to be a biomarker for the therapy of infection, or autoimmune diseases. 14 It is also demonstrated that miR-126-5p is involved in the regulation of sepsis-induced liver injury. 15 miRNA has been indicated to lead to regulatory and protective roles in barrier functions. 16 Interestingly, miR-126-5p enhances the tight junction protein expression, indicating a potent mechanism by which miR-126 may alleviate lipopolysaccharide (LPS)-induced lung injury. 11 It has been reported that nuclear transcription factor-κB (NF-κB) specifically binds to the B lymphocyte immunoglobulin kappa light chain gene enhancer B sequence GGGACTTTCC, which was first identified in 1986. To date, NF-κB has been found to be present in almost all mammalian cells and is involved in a variety of biological processes such as inflammatory response 17 and immune response 18 through the regulation of genes encoding acute phase response proteins, cytokines, and immune regulatory molecules. NF-κB-mediated signaling pathways are an important pathogenesis of ALI. 19 It has been shown that miR-126 is involved in regulating the NF-κB signaling pathway. [20][21][22] Evidence has demonstrated that tumor necrosis factor (TNF) receptorassociated factor 6 (TRAF6) can activate the NF-κB signaling pathway. 23,24 Meanwhile, the bioinformatics website StarBase (https://starbase.sysu.edu.cn/) and a previous study have shown that TRAF6 is a target gene of miR-126. 25 Therefore, in this study, we speculated that miR-126 targeted TRAF6 gene to regulate the NF-κB signaling pathway, which may be associated with inflammatory response and immune response in sepsis-induced ALI. Nevertheless, miR-126-5p expression in the plasma of patients with sepsis-induced ALI and its relationship with inflammation and immune function remains to be uncovered. Therefore, we investigated the relationship between miR-126-5p expression and the levels of inflammatory factors, T lymphocyte subsets, and immunoglobulins in patients with sepsis-induced ALI and determined the diagnostic value of miR-126-5p.

| Ethics
This research was ratified by the ethics committee of The Second People's Hospital of Zhuji, and all participants provided informed consent.

| Participants
One hundred and twenty patients with sepsis admitted to the intensive care unit (ICU) center of The Second People's Hospital of Zhuji between January 2017 and January 2020 were enrolled as the research subjects, including 68 males and 52 females (age: 60.92 ± 9.16 years old). Inclusion criteria are the following: (1) patients aged ≥18 years; (2) patients conformed to The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) 26 and diagnosed as sepsis before enrollment; (3) patients admitted to ICU within 24 h after onset of symptoms. Exclusion criteria are as follows: (1) patients died within 24 h of admission; (2) patients with other serious illnesses or complications such as cardiothoracic surgery, severe liver or kidney dysfunction, acute coronary syndrome, malignant tumor, or autoimmune disease; (3) patients who are pregnant or lactating women. Among these 120 patients, there were 60 patients with sepsis-induced ALI, including 36 males and 24 females (age: 61.43 ± 9.46 years old). All of them were in line with the diagnosis of sepsis-induced ALI 27 : corresponding primary disease; acute onset, dyspnea, or distress symptoms; chest X-ray showed bilateral lung infiltration, pulmonary artery wedge pressure less than 18 mmHg, and without signs of left atrial hypertension. Additionally, 120 healthy subjects were selected as a control group, including 63 males and 57 females (age: 59.98 ± 9.09 years).

| RT-qPCR
Peripheral blood was harvested (for patients with sepsis, fasting peripheral blood was collected within 24 h of admission; for healthy subjects, fasting peripheral blood was harvested on the enrollment). Total RNA was extracted using TRIzol (Takara Bio, Dalian, China). A Nanodrop2000 device was then utilized for the testing of the RNA concentration. RNA was reverse transcribed into cDNA using the Mir-X miR First-Strand Synthesis Kit (Takara Bio) or the PrimeScript RT reagent kit with gDNA Eraser Kit (Takara Bio). Subsequently, RT-qPCR was performed on an ABI 7500 quantitative PCR instrument with the SYBR Premix Ex Taq kits (Takara Bio). The PCR primers were displayed in Table 1. U6 was utilized as the loading control for miR-126-5p. GAPDH was the internal reference of the TRAF6 mRNA. The determination of relative gene levels was realized with the 2 -ΔΔCT method.

| Detection of inflammatory factors and immunological indicators
The levels of interleukin-6 (IL-6) in peripheral blood were examined by ELISA Kits (BD Biosciences, San Diego, CA, USA). Procalcitonin (PCT) was detected by sandwich immunoassay utilizing a mini VIDAS automatic fluorescence immunoassay analyzer (Block Scientific, Bohemia, NY, USA) with VIDAS BRAHMS PCT Quantitative Assay Kit (Block Scientific, Bohemia, NY, USA). T lymphocyte subsets (CD3 + , CD4 + , and CD8 + ) were tested by Coulter Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA). C-reactive protein (CRP) and immunoglobulins (IgA, IgM, IgG) were analyzed by rate nephelometry, and the instruments and reagents were provided by Beckman Coulter (Miami, FL, USA).

| Dual-luciferase reporter gene assay
The StarBase (https://starbase.sysu.edu.cn/) bioinformatics software predicted that miR-126-5p had a complementary binding site to the 3 0 UTR region of TRAF6 gene. To further verify the targeting relationship between miR-126-5p and TRAF6, sequences containing the miR-126-5p binding site on the TRAF6 3 0 UTR were amplified and cloned into the pGL3-basic luciferase plasmids (TaKaRa, Japan) to construct wild type TRAF6 (TRAF6-WT) recombinant plasmids. The miR-126-5p binding site on TRAF6-WT was mutated utilizing a point mutation kit (TaKaRa) to construct mutant TRAF6 (TRAF6-MUT) recombinant plasmids (plasmids were designed and constructed by TaKaRa). The 293T cells (National Collection of Authenticated Cell Cultures) were cultured and inoculated in 24-well plates, and when the cells reached 85% confluence, TRAF6-WT or TRAF6-MUT reporter plasmids were cotransfected with miR-126-5p mimic and mimic NC into 293T cells according to the Lipofectamine 2000 reagent (Invitrogen, USA) procedures. After 48 h, the luciferase activity of the TRAF6-WT or TRAF6-MUT reporter plasmids was measured by implementing the Dual-Luciferase ® Reporter Assay System (Promega, USA). Dual-luciferase reporter assay was performed with three biological replicates, each with three technical replicates.

| Statistics
Data were analyzed utilizing SPSS 21.0 software and GraphPad Prism 6.0 software. The measurement data, in the form of mean ± standard deviation, were analyzed by the t-test or variance of analysis (ANOVA) with Tukey's pairwise post-hoc test. The enumeration data, expressed as numbers (percentage), were compared by the chi-square test. The correlation was analyzed by the Pearson correlation test. Receiver operating characteristic (ROC) curve analysis was performed and the diagnostic accuracy of miR-126-5p for the area under T A B L E 1 Primer sequences for genes utilized in RT-qPCR assay.

| General characteristics of patients with sepsis
Among the 120 patients with sepsis, there were 60 patients with sepsis without ALI (Sepsis, no ALI) and 60 patients with sepsis without ALI (Sepsis and ALI). No significant differences were witnessed in age, gender, body mass index (BMI), chronic kidney failure, and cardiomyopathy, as well as an infected site between the aforesaid two groups (all p > 0.05). However, obvious differences were observed in smoking history, ALI/ARDS score, and FiO2 (%) between the two groups (both p < 0.05; Table 2).

| Inflammatory factors in patients with sepsis-induced ALI
To address the expression of inflammatory factors in sepsis-induced ALI, we tested the levels of CRP, PCT, and IL-6 in patients with sepsis without ALI and patients with ALI. There were high levels of the above-mentioned factors in sepsis-induced ALI in comparison with those without ALI (all p < 0.05; Table 2).

| Comparison of immune-related indicators in patients with sepsisinduced ALI
In order to observe the levels of immune-associated indicators in patients with sepsis-induced ALI, we tested the levels of immunoglobulins (IgA, IgM, and IgG) and T lymphocyte subsets (CD3+, CD4+, and CD8+) The results in Table 3 demonstrated that the levels of immunoglobulins and T lymphocyte subsets in sepsis-induced ALI were lower than those without ALI (all p < 0.05).
3.6 | Genetic validation assay for miR-126-5p and its target gene TRAF6 According to the prediction analysis, miR-126-5p had a binding site with TRAF6 3 0 UTR (Figure 2A). To verify whether miR-126-5p inhibited TRAF6 expression by binding to the 3 0 UTR of TRAF6, the dual-luciferase reporter gene assay was performed. TRAF6-WT or TRAF6-MUT luciferase reporter was transfected into 293T cells together with miR-126-5p mimic or mimic NC. Overexpression of miR-126-5p repressed the luciferase activity of the TRAF6-WT reporter plasmids, but did not affect the luciferase activity of the TRAF6-MUT reporter plasmids ( Figure 2B). Thus, these results suggested that TRAF6 was a direct target of miR-126-5p. We further examined TRAF6 expression in patients with sepsis-induced ALI utilizing RT-qPCR and the findings suggested ( Figure 2C) that plasma TRAF6 expression levels in patients with sepsis-induced ALI were elevated compared with those in patients with sepsis without ALI (p < 0.05). Further analysis of the correlation between TRAF6 and miR-126-5p in patients with sepsis-induced ALI unraveled that miR-126-5p was negatively correlated with TRAF6 in patients with sepsis-induced ALI.

| DISCUSSION
ALI is a primary complication in sepsis, and miRNAs play vital parts in sepsis-associated disorders. 28 The biology of sepsis-induced ALI is multifactorial, which involves the secretion of inflammatory factors along with the damage to the lung microvascular barrier. 29 As reported, miRNAs participate in diverse physiological and pathological processes, including lung injury. 30 In this research, we aimed at finding out a novel method for sepsis-induced ALI by evaluating the expression of miR-126-5p in the plasma of patients with sepsis-induced ALI and its relationship with inflammation and immune function.
Emerging works have highlighted that miRNAs are of significance in regulating oxidative stress and inflammation, and are linked to sepsis-induced ALI. For instance, Jiang et al. have pointed out that miR-155 accelerates macrophage proliferation and aggregates septic lung injury. 31 Wang et al. have supported that miR-326, as an autoimmunity-associated miRNA, could provide a latent molecular basis in sepsis-induced ALI. 32 In our research, we observed a reduced miR-126-5p in the plasma of patients with sepsis and sepsis-induced ALI. Complimentary to this, author X et al, have reported decreased miR-126 expression in sepsis and that reduced serum levels of miR-126 are associated with sepsis severity. 33 Another research has mentioned that miR-126-5p is downregulated in LPS-induced ALI mice, and restoration of miR-126-5p may alleviate ALI by decreasing its downstream gene VEGFA. 30 As stated above, miR-126 might be downregulated in either sepsis or LPS-induced ALI, and our work highlighted that miR-126 expression was lower in patients with sepsis with ALI than that in patients with sepsis without ALI. Besides, most previous articles tested miR-126-5p expression in serum, while this paper tested it in plasma, which is a highlight of this paper because the results of plasma detection will be more accurate.
Some proinflammatory cytokines are implicated in the pathogenesis of sepsis. 34 As previously described, pathogens activate the secretion of inflammatory factors, contributing to sepsis-induced ALI or ARDS. 35 In addition, it is suggested that reducing these cytokines has some protective functions in animal models with acute fulminant infections. 36 In our article, we found that the levels of pro-inflammatory factors were enhanced in sepsis-induced ALI. Meanwhile, plasma miR-126-5p concentration correlated to systemic inflammation in sepsis-induced ALI. In recent years, it is reported that the sepsis-associated severe infection originates from an imbalance between anti-inflammatory and/or inflammatory factors. 37 Meanwhile, miR-126 impairs sepsis inflammation and improves the prognosis of sepsis. 38 Nong et al. in their study have stated that the downregulation of miR-126 suppresses the levels of proinflammatory factors and elevates anti-inflammatory factor levels in septic rats, which could relieve persistent damage resulting from excessive inflammation. 21 A previous study has reported that plasma miR-126 levels are elevated in smokers who had completely quit smoking. 39 In this study, more smokers were found in patients with sepsis with ALI than in patients with sepsis without ALI, and miR-126-5p expression was lower in patients with sepsis with ALI than that in patients with sepsis without ALI, indicating that miR-126-5p expression may be associated with smoking history. Furthermore, we investigated the relationship between miR-126 expression and the levels of inflammatory factors, T lymphocyte subsets, and immunoglobulins in patients with sepsis-induced ALI and determined the diagnostic value of miR-126-5p. The focus of the research was aimed at patients with sepsis-induced ALI. Therefore, we did not analyze the correlation among miR-126-5p expression and the levels of inflammatory factors, T lymphocyte subsets, and immunoglobulins in patients with sepsis without ALI, which is similar to other scholars' research. 40 In addition, we also found that the levels of immunoglobulins and T lymphocyte subsets were decreased in patients with sepsis-induced ALI. Meanwhile, miR-126-5p expression was found to correlate with markers of systemic inflammation and immunological function indicators in patients with sepsis-induced ALI. Similar to our findings, evidence has shown that the reduced plasma levels of endogenous immunoglobulins have a correlation to decreased survival in severe sepsis. 41 Acquired immunosuppression is an important event in sepsis, leading to the hypothesis that stimulating the immune response and/or replacing the components of the individual immune system could be a promising therapeutic approach. 42 T lymphocytes are vital cells in the immune system, both serving as the cell-modulated immune effector cells. 43 As reported, overexpression of miR-126 is capable of regulating the immune response (Th1/Th17), and exerting functions in immune signal transduction and immune cell migration. 44 Additionally, miR-126 impedes the development of the immune response and modulates the differentiation of T lymphocytes with the involvement of Th2 or Tregs. 14 Furthermore, in our paper, ROC analysis showed the miR-126-5p expression for the evaluation of the AUC of sepsis: 0.897; for sepsisinduced ALI: 0.777. These findings reveal that miR-126-5p might be a potential diagnostic agent for sepsisinduced ALI. As previously reported, TRAF6 expression is up-regulated in LPS-induced ALI. 45 Bioinformatics website along with a related study has revealed that TRAF6 is a target gene of miR-126. 25 In this study, miR-126-5p was reduced in sepsis-induced ALI, and it was speculated that the target gene TRAF6 would be elevated in sepsis-induced ALI. We have tested the expression of TRAF6 in patients with sepsis-induced ALI, and the results were as speculated: TRAF6 expression was elevated in sepsis-induced ALI.
In conclusion, this research provides evidence that miR-126-5p is decreased in sepsis-induced ALI, and miR-126-5p correlates to systemic inflammation and immune function indicators. This emphasizes that miR-126-5p might be a potential biomarker for the diagnosis of sepsis-induced ALI. In this study, we did not detect the roles of other miRNAs in sepsis-induced ALI, which could be a limitation of this study, and we would further investigate this aspect in future research.

ACKNOWLEDGMENTS
We would like to give our sincere gratitude to the reviewers for their constructive comments.

CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to declare that are relevant to the content of this article.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICAL STATEMENT
This research was approved by the ethics committee of The Second People's Hospital of Zhuji, and all participants provided informed consent.