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Lnc‐MEG3 acts as a potential biomarker for predicting increased disease risk, systemic inflammation, disease severity, and poor prognosis of sepsis via interacting with miR‐21

Abstract Background This study aimed to investigate the correlations of long non‐coding RNA maternally expressed gene 3 (lnc‐MEG3), microRNA (miR)‐21, and lnc‐MEG3/miR‐21 axis with disease risk, inflammation, disease severity, and 28‐day mortality of sepsis. Methods Totally, 219 sepsis patients and 219 health controls (HCs) were enrolled. Plasma samples were obtained from sepsis patients within 24 hours after admission and from HCs on enrollment to detect lnc‐MEG3 and miR‐21 expressions by real‐time quantitative polymerase chain reaction. Results The lnc‐MEG3 expression and lnc‐MEG3/miR‐21 axis were increased, while miR‐21 expression was decreased in sepsis patients compared with HCs. Lnc‐MEG3 (area under the curve (AUC): 0.887, 95% confidence interval (CI): 0.856‐0.917) and lnc‐MEG3/miR‐21 axis (AUC: 0.934, 95% CI: 0.909‐0.958) had good values for predicting elevated sepsis risk, while miR‐21 (AUC: 0.801, 95% CI: 0.758‐0.844) presented a good predictive value for reduced sepsis risk. Furthermore, lnc‐MEG3 expression and lnc‐MEG3/miR‐21 axis positively correlated with, whereas miR‐21 expression negatively correlated with acute pathologic and chronic health evaluation II, sequential organ failure assessment score, serum creatinine, C‐reactive protein, tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐6, and IL‐17 in sepsis patients. Additionally, lnc‐MEG3 (AUC: 0.704, 95% CI: 0.626‐0.783) and lnc‐MEG3/miR‐21 axis (AUC: 0.669, 95% CI: 0.589‐0.750) exhibited acceptable values in predicting higher 28‐day mortality risk, while miR‐21 (AUC: 0.588, 95% CI: 0.505‐0.672) presented a poor predictive value for lower 28‐day mortality risk in sepsis patients. Conclusion Lnc‐MEG3 might serve as a potential biomarker for the development, progression, and prognosis prediction of sepsis via interacting with miR‐21.


| INTRODUC TI ON
Sepsis, a devastating and life-threatening syndrome, is caused by an aberrant systemic host response to infections, resulting in excessive inflammatory responses and multiple organ failures. 1 Sepsis has emerged as a major public health problem with an increasing incidence worldwide, being the main contributor of death among patient admitted to the intensive care units. 2 Despite the improvements in antibiotic treatments, ventilator management, and resuscitative strategies, the prognosis of sepsis is still unsatisfied due to under-recognition of sepsis, delayed assessment and development of antibiotic resistance. 1,3 Currently, there is still a lack of sensible and efficient disease monitoring and prognostic biomarkers for sepsis in clinical practice. Therefore, it is of great need to explore novel and rapidly measurable biomarkers for assisting on the identification of sepsis and prognosis prediction in sepsis patients.
Long non-coding RNAs (LncRNAs), a class of non-coding RNA, are characterized by non-coding transcripts longer than 200 nucleotides in length with no open reading frame, which function as decoys, scaffolds, guides, and enhancers to regulate DNA, RNA and proteins. 4 Among the identified lncRNAs, long non-coding RNA maternally expressed gene 3 (lnc-MEG3) is reported to be positively associated with inflammation and organ injury. 5,6 For instance, lnc-MEG3 enhance inflammatory response via upregulating the expressions of early growth response protein 1 and Toll-like receptor 4. 5 Another study illuminates that overexpression of lnc-MEG3 upregulates the creatine kinase and lactate dehydrogenase activities, inducing cell apoptosis and myocardial ischemia-reperfusion injury. 6 Furthermore, it is reported that lnc-MEG3 acts as a molecular sponge for several microRNAs such as microRNA (miR)-21, miR-181a, and miR-7-5p to inhibit their downstream signaling pathways. 5,7,8 Among these target miRNAs, miR-21 functions as an important contributor for preventing inflammation and organ dysfunctions such as liver, kidney, and lung in sepsis. [9][10][11] Based on these previous studies, we hypothesized that lnc-MEG3 might be involved in the development and progression of sepsis via interacting with miR-21. Therefore, this study aimed to investigate the correlations of lnc-MEG3, miR-21 and lnc-MEG3/miR-21 axis with disease risk, inflammation, disease severity, and 28-day mortality of sepsis.

| Data and sample collection
After enrollment, all clinical features of sepsis patients were recorded.
Besides, peripheral blood samples of sepsis patients were collected within 24 hours after admission, and peripheral blood samples of HCs were collected on enrollment. Then, the peripheral blood samples were immediately separated for plasma at the condition of 3000 g for 25 minutes (4°C). The levels of TNF-α, IL-1β, IL-6, and IL-17 in plasma were measured by enzyme-linked immunosorbent assay (ELISA), and the levels of lnc-MEG3 and miR-21 in plasma were detected by realtime quantitative polymerase chain reaction (qPCR).

| Severity assessment
The severity of sepsis was assessed using acute pathologic and chronic health evaluation II (APACHE II) score, which included acute physiology score, age score, and chronic health score. The total score of APACHE II ranges from 0 to 71, and the higher score represented more severe disease condition. 13 The severity of organ dysfunction of sepsis patients was evaluated using sequential organ failure assessment (SOFA) score, which included respiration score, coagulation score, liver score, cardiovascular score, central nervous system score, and renal score. The total SOFA score was calculated by summing up each item scores (range 0-24), and higher score represented more severe organ dysfunction. 14

| ELISA
Commercial human ELISA Kits (Thermo Fisher Scientific) were used to measure the level of TNF-α, IL-1β, IL-6, and IL-17 in plasma, and all procedures were performed according to the manufacturer's instruction. In brief, plasma samples were added to the wells of target-specific antibody pre-coated microplate. After incubation, the wells were washed to remove unbound material. The sandwich was formed by the addition of the second antibody, and a tetramethylbenzidine substrate was added to produce measurable signal.
After stop solution was added, the optical density was measured at 450 nm wavelengths on microplate reader (BioTek).

| Real-time quantitative polymerase chain reaction (qPCR)
Firstly, total RNA was extracted from plasma using QIAamp RNA Blood Mini Kit (Qiagen). Then, cDNA was synthesized using iScript ™ Reverse Transcription Supermix (Bio-Rad). Subsequently, qPCR was performed to quantify lnc-MEG3 and miR-21 relative expressions using QuantiNova SYBR Green PCR Kit (Qiagen). Lnc-MEG3 relative expression was calculated by 2 −△△Ct method using GAPDH as internal reference, and miR-21 relative expression was calculated by 2 −△△Ct method using U6 as internal reference. Primers (designed based on a previous study) applied were as follows 15

| Treatment and follow-up
Appropriate treatment regimens were given to the sepsis patients according to guideline of sepsis. 16 All patients were followed up to 28 days after enrollment, and during follow-up, the number of deaths was recorded to calculate 28-day mortality. And based on the survival status in 28-day follow-up, patients were categorized into survivors and deaths.

| Statistical analysis
Statistical analysis was performed with the use of SPSS 24.0 software (IBM), and figure was made using GraphPad Prism 7.01 software (GraphPad Software). Continuous variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR). Categorical variables were expressed as count (percentage).
Comparison of lnc-MEG3 relative expression, miR-21 relative expression, or lnc-MEG3/miR-21 axis between two groups was determined by Wilcoxon rank-sum test. Correlation of lnc-MEG3 relative expression, miR-21 relative expression, or lnc-MEG3/miR-21 axis with APACHE II score, SOFA score, and biochemical indexes was analyzed by Spearman's rank correlation test. The performances of continuous variables in predicting sepsis risk and 28-day mortality risk were performed using receiver operating characteristic (ROC) curve and the area under the curve (AUC) with 95% confidence interval (CI). P value < 0.05 was considered significant.

| The values of lnc-MEG3, miR-21, and lnc-MEG3/miR-21 axis for predicting sepsis risk
The lnc-MEG3 relative expression was elevated in sepsis patients   for predicting sepsis risk, among which lnc-MEG3/miR-21 axis had a numerically superior predictive value for sepsis risk ( Figure 1D).

21, and lnc-MEG3/miR-21 axis with biochemical and inflammatory indexes in sepsis patients
The lnc-MEG3 relative expression was positively correlated with Scr  Table 2). And no correlation of lnc-MEG3/miR-21 axis with WBC was observed in sepsis patients (P = .359, r = .062).

| D ISCUSS I ON
In the present study, we have discovered that: Lnc-MEG3, an imprinted gene, is transcribed from human chromosome 14q32.3 that is implied in the induction of inflammatory response and multiple organ dysfunction such as heart. 8,17,18 For instance, the overexpression of lnc-MEG3 enhances the expressions and secretions of inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α in osteoarthritis. 17 Another study discloses that lnc-MEG3 promotes apoptosis in cardiomyocytes via direct binding with fused in sarcoma protein in myocardial infarction. 18 Besides, it is evident that lnc-MEG3 directly binds to miR-21 and subsequently inhibits its downstream signaling pathways. 7   and alleviated tissue damage as well as organ dysfunctions, thereby, leading to lower levels of inflammation and attenuated disease severity in sepsis patients. 11 Notably, lnc-MEG3/miR-21 axis exhibited relatively higher correlation coefficients with disease severity (APACHE II score and SOFA score) compared with lnc-MEG3 and miR-21 individually, which implied that lnc-MEG3/miR-21 axis was with better clinical value in sepsis progression.  and lnc-MEG3/miR-21 axis were associated with increased inflammation, multiple organ dysfunction and accelerated disease severity, thus, resulting in raised 28-day mortality risk in sepsis patients.
(b) MiR-21 exhibited a dual effect in the regulation of inflammation and organ injuries. From a review, miR-21 suppressed inflammatory responses and alleviated organ injuries in some cases, whereas it promoted inflammatory responses and accelerated organ injuries in other cases. 19 Thus, the value of miR-21 for predicting 28-day mortality risk was poor in sepsis patients. Interestedly, compared with common prognostic factors (such as APACHE II score, SOFA score et al), lnc-MEG3/miR-21 axis exhibited a numerically inferior predictive value for 28-day mortality risk in sepsis. However, the predictive value of lnc-MEG3/miR-21 axis still implied its potential as a biomarker that could be used to assist with disease monitoring and prognosis in sepsis.
There were certain limitations in the present study. Firstly, the sample size was relatively small, which might reduce the statistic power. Secondly, sepsis patients who died within 24 hours after admission were excluded in order to eliminate the extremely severe disease conditions (confounding factor). However, at the same time, the exclusion of sepsis patients who died within 24 hours after admission might cause selection bias and limit the generalizability of the findings.
Lastly, the detailed mechanisms of lnc-MEG3 and miR-21 on the development and progression of sepsis were not investigated.
In conclusion, lnc-MEG3/miR-21 axis predicts higher sepsis risk and correlates with elevated systemic inflammation, disease severity as well as 28-day mortality risk of sepsis.