Potential of long non‐coding RNA KCNQ1OT1 as a biomarker reflecting systemic inflammation, multiple organ dysfunction, and mortality risk in sepsis patients

Abstract Background Long non‐coding RNA potassium voltage‐gated channel subfamily Q member 1 opposite strand 1 (lnc‐KCNQ1OT1) represses inflammation and multiple organ dysfunction, whereas its clinical value in sepsis is unclear. Thus, this study aimed to explore this issue. Methods Lnc‐KCNQ1OT1 from peripheral blood mononuclear cells were detected by RT‐qPCR in 116 sepsis patients and 60 healthy controls (HCs). Moreover, sepsis patients were followed‐up until death or up to 28 days. Results Lnc‐KCNQ1OT1 decreased in patients with sepsis than in HCs (p < 0.001). In sepsis patients, lnc‐KCNQ1OT1 was negatively correlated with sequential organ failure assessment (SOFA) scores (r = −0.344, p < 0.001) and several SOFA subscale scores (including respiratory system, coagulation, liver, and renal systems) (all r < 0, p < 0.05). Furthermore, lnc‐KCNQ1OT1 was negatively correlated with CRP (r = −0.386, p < 0.001), TNF‐α (r = −0.332, p < 0.001), IL‐1β (r = −0.319, p < 0.001), and IL‐6 (r = −0.255, p = 0.006). Additionally, lnc‐KCNQ1OT1 levels were lower in sepsis deaths than in sepsis survivors (p < 0.001), and the receiver operating characteristic curve showed that lnc‐KCNQ1OT1 had an acceptable ability to predict 28‐day mortality (area under the curve: 0.780, 95% confidence interval: 0.678–0.882). Meanwhile, its ability to predict 28‐day mortality risk was higher than that of CRP, TNF‐α, IL‐1β, and IL‐6, but slightly lower than the SOFA score and acute physiology and chronic health evaluation II score. Conclusion Lnc‐KCNQ1OT1 serves as a potential biomarker for monitoring disease severity and prognosis in patients with sepsis.


| INTRODUC TI ON
Sepsis is a life-threatening disease induced by dysfunctional responses to infection. 1 Meanwhile, inflammatory storm and multiple organ dysfunction (such as lung, kidney, liver, cardiac, and nervous system) are hallmarks of sepsis. 2 Furthermore, sepsis affects approximately 18 million people worldwide and continues to be the major contributor to infection-induced death globally (especially in critically ill patients), which results in huge economic and disease burdens. [2][3][4] Considering that the prognosis of sepsis patients is still poor and that mortality continues to climb, the exploration of novel biomarkers to improve the management of sepsis is important. 2,3,5 Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 (KCNQ1) opposite strand 1 (lnc-KCNQ1OT1) has been shown to suppress inflammation and multiple organ dysfunction. [6][7][8][9] For instance, lnc-KCNQ1OT1 is able to inhibit inflammation through nuclear factor kappa B inhibitor alpha (IκBα) and regulating microRNA (miR)-506-3p, 6,8,9 while lnc-KCNQ1OT1 has the capacity to attenuate multiple organ dysfunction (such as cardiomyopathy, liver injury, and sepsis-induced cardiac injury) via several approaches, including regulation of miR-214-3p, caspase-1, miR-122-5p and carboxylesterase 2, as well as miR-192-5p and the X-linked inhibitor of apoptosis protein (XIAP) axis. 7,10,11 Based on this information, we speculated that lnc-KCNQ1OT1 levels might be correlated with inflammation and multiple organ dysfunction in sepsis, while the relevant data are obscured. Therefore, the present study aimed to explore potential correlations involving lnc-KCNQ1OT1 and inflammation, multiple organ dysfunction, and mortality risk among sepsis patients.

| Subjects
A total of 116 sepsis patients treated in our hospital from February 2018 to June 2020 were consecutively enrolled in this prospective study. The enrollment criteria were as follows: (i) diagnosis of sepsis according to the Third International Consensus Definitions for Sepsis 12 ; (ii) aged >18 years; and (iii) were admitted to our department within 24 h after the onset of symptoms. Patients were ineligible for inclusion if they had experienced the following conditions: (i) complications involving carcinomas or blood malignancies, (ii) concomitant autoimmune diseases, (iii) used immunosuppressants before enrollment, (iv) received chemotherapy within 3 months, (v) pregnancy and lactating women, and (vi) poor study compliance.
In addition, 60 healthy subjects who underwent physical examination in our hospital from January 2020 to June 2020 were recruited as healthy controls (HCs). The recruitment criteria for HCs were as follows: (i) age-and sex-matched to sepsis patients, (ii) no history of sepsis or severe infection, and (iii) had normal biochemical index levels. HCs were also excluded from the study if they met the exclusion criteria for sepsis patients. This study was approved by the Institutional Review Board of No. 904th Hospital of The Joint Logistics Support Force of the PLA, and all participants or their relatives signed informed consent forms.

| Data documentation
Demographics, comorbidities, disease characteristics, and biochemical indices were recorded after clinical and laboratory examinations.
Acute Physiology and Chronic Health Evaluation II (APACHE II) score and Sequential Organ Failure Assessment (SOFA) scores were assessed within 24 h of hospitalization to evaluate the disease status of patients. All sepsis patients were closely followed-up until death or for up to 28 days, and deaths within 28 days were recorded.

| Peripheral blood (PB) collection
PB was sampled from sepsis patients immediately upon admission and from HCs after enrollment. Peripheral blood mononuclear cells (PBMCs) and serum were separated from the PB samples by density gradient centrifugation.

| Lnc-KCNQ1OT1 determination
Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay was carried out to analyze the expression of lnc-KCNQ1OT1 in PBMCs. In brief, total RNA was extracted by QIAamp RNA Blood Mini Kit (Qiagen, Duesseldorf, Nordrhein-Westfalen, Germany) and reverse-transcribed using iScript™ cDNA Synthesis Kit with oligo d(T) and random hexamer primers (Bio-Rad, Hercules, California, USA). qPCR was performed using SYBR ® Green Real-time PCR Master Mix (Toyobo, Osaka, Kansai, Japan). Relative expression was calculated by the 2 −ΔΔCt method, and GAPDH was used as an internal reference. Primers used for PCR amplification were designed according to a previous study. 13

| Inflammatory cytokine determination
Tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in sera of patients with sepsis were determined by enzyme-linked immunosorbent assay (ELISA). All ELISA kits were employed to perform statistical analysis and graph plotting, respectively. The Mann-Whitney U test was used to compare lnc-KCNQ1OT1 expression between the two groups. Correlations between lnc-KCNQ1OT1 expression and clinical data were analyzed using Spearman's rank correlation test. The performance of variables in evaluating mortality risk was estimated using receiver operating characteristic (ROC) curve analysis. Statistical significance was determined for p values <0.05 in the corresponding analyses.

| Clinical features
Among 116 sepsis patients, the mean age was 58.4 ± 13.4 years, and 77 (66.4%) were male. In addition, the mean APACHE II score was 12.1 ± 6.5 and the mean SOFA score was 5.5 ± 2.7. In terms of  Table 1.

| Comparison of lnc-KCNQ1OT1 between sepsis patients and HCs
To determine the expression of lnc-KCNQ1OT1 in sepsis patients and HCs, RT-qPCR was performed in the present study. We found that lnc-KCNQ1OT1 levels were lower in sepsis patients (median [in-

| Discrimination of sepsis deaths by lnc-KCNQ1OT1, inflammatory indices, and SOFA and APACHE II scores
Multivariate Cox regression analysis showed that higher lnc-KCNQ1OT1 levels were independently correlated with septic death (p = 0.009, hazard ratio = 0.017) (Table S1). Furthermore, lnc-KCNQ1OT1 abundance was lower in sepsis deaths (median   Figure 4D).

| DISCUSS ION
Several studies have shown that lnc-KCNQ1OT1 is able to inhibit inflammation and multiple organ dysfunction, 6 , SOFA score-respiratory system (B), SOFA score-coagulation (C), SOFA score-liver (D), SOFA score-cardiovascular system (E), SOFA score-nervous system (F), and SOFA score-renal system (G). SOFA, sequential organ failure assessment; lnc-KCNQ1OT1, long non-coding RNA potassium voltage-gated channel subfamily Q member 1 (KCNQ1) opposite strand 1 via miR-30e-3p. 17 Considering that sepsis is often correlated with multiple organ dysfunction, 16 we hypothesized that lnc-KCNQ1OT1 might be correlated with multiple organ dysfunction in sepsis. Thus, we assessed SOFA scores and subscales in sepsis patients, which revealed that lnc-KCNQ1OT1 was negatively correlated with SOFA scores and its partial subscales (respiratory system, coagulation, liver, and renal systems) in sepsis patients.

F I G U R E 4
Ability of lnc-KCNQ1OT1, inflammatory indices, and SOFA and APACHE II scores in predicting mortality risk in sepsis patients. Comparison of lnc-KCNQ1OT1 levels between sepsis survivors and sepsis deaths (A); discriminatory ability of lnc-KCNQ1OT1 (B), CRP, TNFα, IL-1β and IL-6 (C), as well as SOFA score and APACHEII score (D) to distinguish sepsis deaths from sepsis survivors. CRP, C-reactive protein; TNFα, tumor necrosis factor alpha; IL-1β, interleukin-1β; IL-6, interleukin-6; lnc-KCNQ1OT1, long non-coding RNA potassium voltage-gated channel subfamily Q member 1 (KCNQ1) opposite strand 1; SOFA, sequential organ failure assessment; APACHE II, acute physiology and chronic health evaluation II between lnc-KCNQ1OT1 levels and SOFA scores and its partial subscales, suggesting that lnc-KCNQ1OT1 was negatively correlated to multiple organ dysfunction in sepsis.
Previous studies have illustrated that lnc-KCNQ1OT1 can modulate inflammation. 6 In conclusion, lnc-KCNQ1OT1 serves as a potential biomarker for monitoring disease severity and prognosis in sepsis patients, which might consequently improve the management of this disease.

This study was supported by 2021 Research Project of Clinical
Medical Science and Technology Development Fund of Jiangsu University (JLY2021140).

CO N FLI C T S O F I NTE R E S T
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.