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Linkage of lncRNA CRNDE sponging miR-181a-5p with aggravated inflammation underlying sepsis

This investigation was performed to verify whether lncRNA CRNDE sponging miR-181a-5p was involved with sepsis-relevant inflammatory dysfunctions. Aggregately 136 sepsis patients and 151 healthy people were recruited, and their fasting peripheral blood was gathered to detect expressions of CRNDE and miR-181a-5p. In addition, THP-1 cells were transfected with si-CRNDE, miR-181a-5p mimic, pcDNA3.1-TLR4 and si-TLR4, and then sepsis-specific inflammatory cytokines within the cells were quantified. The sponging relationships between CRNDE and miR-181a-5p, as well as between miR-181a-5p and TLR4, were ascertained by means of luciferase reporter gene assay. The experimental results revealed that over-expressed CRNDE and under-expressed miR-181a-5p were associated with shortened lifespan of sepsis patients. Mechanically, si-CRNDE-1 and miR-181a-5p mimic were able to reverse the promoting effects of LPS on production of NF-kB, TNF-α, IL-1β and IL-6 by THP-1 cells. Moreover, the expressional change of miR-181a-5p in THP-1 cells was in part owing to its being sponged by CRNDE. Lastly, TLR4, subjected to targeted modification of miR-181a-5p, was capable of disturbing the contribution of CRNDE and miR-181a-5p to THP-1 cells’ release of NF-kB, TNF-α, IL-1β and IL-6. Collectively, the CRNDE/miR-181a-5p/TLR4 axis seemed to have potential in modifying sepsis-related inflammatory pathogenesis, which offered a direction for sepsis diagnosis and treatment.


Introduction
Sepsis, a systemic inflammatory response syndrome, usually occurs along with severe trauma, burns, shock and severe surgery, and it is clinically featured by massive release of inflammatory mediators into peripheral blood. 1,2 The mortality caused by severe sepsis is expected to achieve 28-50%, 3,4 and more unfortunately, patients with a dysfunctional cardiovascular system are more prone to death when they were complicated by sepsis. [5][6][7] Hence, it is of utmost importance to clarify root causes that explain sepsis onset, and also biomarkers for the progression of sepsis. Acknowledging the fundamental role of proinflammatory cytokines in boosting sepsis development, 8 exploring biomarkers that powerfully modify inflammatory responses might be beneficial to uncovering sepsis etiology.
To date, sepsis-focused research has been mostly related to protein-coding genes, rather than noncoding RNAs, such as long non-coding RNAs (lncRNAs), that fail to encode proteins. 9,10 Nonetheless, the involvement of lncRNAs in immune reactions has been increasingly revealed, [11][12][13] and the immune-modulatory effects of lncRNAs have also been confirmed in various immune cells, such as monocytes and dendritic cells. 14 Furthermore, profiling of lncRNAs showed discrepant patterns during differentiation of Th1, Th2 and Th17 cells, 15 which further highlighted the high-level involvement of lncRNAs in inflammation. Notable, lncRNA colorectal neoplasia differentially expressed (CRNDE), according to results derived from Kyoto Encyclopedia of Genes and Genomes (KEGG) database, was conjectured as an immunity-relevant gene, and a majority of its functions were centered on regulation of immune responses, such as TLR signaling, TNF-a signaling and the cytokinecytokine receptor interaction pathway. 16 The in vitro experiments also supported the role CRNDE played in activation of MyD88-independent TLR signaling and subsequent NF-jB signaling, 16 whose normal functioning appeared fundamental for restraint of sepsis development. [17][18][19][20] As a consequence, it is plausible that CRNDE could play a part in the acceleration of sepsis aggravation. In addition, miR-181a-5p was negatively modified by CRNDE, and the Wnt/b-catenin signaling regulated by it served as a powerful mediator underlying sepsis-correlated inflammation. 21,22 In addition, the activity of NF-jB could be undermined after stimulation of over-expressed miR-181a, 23 even though whether the inflammation-boosting capacity of NF-jB would be weakened by miR-181a remained uncertain. Furthermore, miR-181a-5p was additionally supposed as a modulator of immune responses in dendritic cells via negative modification of TNF-a. 24 In summary, CRNDE and miR-181a-5p were tightly linked with inflammatory disorders inherent in sepsis etiology; nonetheless, whether CRNDE combined with miR-181a-5p indeed promoted or delayed sepsis development was still unknown.
In order to fill this knowledge gap, this investigation was designed to explore the inner linkage of CRNDE with miR-181a-5p in sepsis, which might guide fruitful diagnosis and treatment for sepsis in future.

Inclusion of clinical samples
In total 136 sepsis patients aged between 18 and 76 yr, were recruited from the respiratory department of Chenzhou No.1 People's Hospital, and their confirmed diagnoses were in accordance with the criteria formulated by American College of Chest Physicians and Critical Care Medicine in 2012. 25 Sepsis cases were excluded when they were found with symptoms related with immune system disorders, hematological diseases, chronic organ dysfunctions, seriously impaired hepatorenal functions and cancers. In the meantime, 151 healthy people who took physical examinations in the hospital were incorporated into the control group. The fasting peripheral blood samples collected from patients with sepsis and healthy controls in the morning were centrifuged at 594 g for 10 min, after which the supernatants were stored at -80 C. All procedures required by this investigation have been approved by Chenzhou No.1 People's Hospital and the ethics committee of Chenzhou No.1 People's Hospital. All the participants and their relatives have signed informed consents after being fully notified.

Cell treatment and cell transfection
The THP-1 cell line, a human myeloid leukemia mononuclear cell line, was purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were incubated in the atmosphere of 5% CO 2 and 37 C, and their culture medium was managed as DMEM complete medium that contained 10% FBS. 26 The THP-1 cells at the logarithmic phase were seeded at the concentration of 5.0 Â 10 6 /ml, and cell models of sepsis were built by stimulating the THP-1 cell line with 100 ng/ml LPS for 4 h. 27 Also, the THP-1 cells that grew to 70%-80% confluence were transfected with siRNAs against CRNDE, si-NC, miR-181a-5p mimic, miR-NC and si-TLR4 (Genepharma, China) (Supplemental Table 1). After 48 h, the cells were used for the following experiments.

RT-PCR
The total RNAs were extracted from blood or cells according to instructions of the Trizol kit (Invitrogen, USA), and they were then dissolved within diethylpyrocarbonate (DEPC)-treated distilled water. The concentration and quality of RNAs were measured using a spectrophotometer (model: Nano Drop 2000, Applied Biosystems, USA), and their quality was satisfied when the A260/A280 ratio ranged between 1.9 and 2.1. Afterward, the collected RNAs were reversely transcribed into cDNAs, aided by the PrimeScript TM RT reagent kit (Takara, Japan). Then with primers synthesized by Sangon (Shanghai, China) (Table 1), a PCR reaction was implemented on a PCR instrument (model: Applied Biosystems 7600, ABI, USA), as per the instructions of SYBR Premix ExTaq II Kit (Takara, Japan). The reaction steps were predenaturation at 95 C for 30 s, 40 cycles of denaturation at 95 C for 5 s, annealing at 60 C for 5 s and extension at 72 C for 30 s. Finally, expression of target genes was quantified on the basis of 2 ÀDDCt method, with U6 or GAPDH as the internal reference.

Western blotting
The cells or blood were added with pre-cooled lysis buffer, and the supernatants were taken after 4 C and 9590 g centrifugation for 5 min. The concentration of total protein was detected using the Bradford method, and 40 lg of the protein was used for 10% SDS-PAGE. The samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane via the wet method, and were blocked at 37 C within TBST that contained 5% skim milk powder for 1 h. Subsequently, the samples were incubated within primary Abs (rabbit antihuman, Abcam, USA) against NF-kB (1:1000, Cat. After development and exposure in the dark, the Gene Tools software was applied to measure the expression level of target proteins, with GAPDH as the reference.

Statistical analyses
The data were statistically analyzed using SPSS 13.0 software. The count data expressed as percentage (%) were compared through v 2 test, and the measurement data presented in the form of mean AE standard deviation (SD) were contrasted using student's t-test. Survival curves were plotted utilizing the Kaplan-Meier method, with log-rank test adopted for between-group comparisons. Statistical significance was deemed at P values of < 0.05.

Comparison of baseline clinical characteristics between sepsis patients and healthy controls
The patients with sepsis and the healthy controls were matched with respect to mean age (P ¼ 0.130) and sex ratio (P ¼ 0.204) ( Table 2). However, significantly heightened levels of white blood cells (WBC), C-reactive protein (CRP) and procalcitonin (PCT) were detected among sepsis patients, when compared with healthy controls (P < 0.001). The average MAP, SOFA and APACHE II scores of sepsis patients were 0.34 AE 0.08, 7.64 AE 0.67 and 17.15 AE 1.22, respectively.
Predictive role of CRNDE and miR-181a-5p for prognosis of patients with sepsis Over-expressed CRNDE was associated with poorer overall survival than under-expressed CRNDE (P ¼ 0.001), while on the contrary, under-expressed miR-181a-5p might prolong the lifespan of patients with sepsis in comparison to over-expressed miR-181a-5p (P ¼ 0.002) (Figure 1c). In addition, both CRNDE and miR-181a-5p were identified as significant predictors for the prognosis of sepsis patients, according to results of univariate and multivariate analyses (P < 0.05) ( Table 4). Concurrently, higher  The CRNDE expression was negatively correlated with miR-181a-5p expression among the sepsis patients. (c) The low-expressed CRNDE and high-expressed miR-181a-5p were associated with favorable survival of sepsis patients, with high-expressed CRNDE and low-expressed miR-181a-5p, respectively, as the reference.  SOFA score and APACHE II score were also reflective of poor prognosis of patients with sepsis than lower scores (P < 0.05).

Discussion
Sepsis is a principal trigger of septic shock and multiple organ dysfunction syndrome, with an alarming growth rate of 1.5-9% annually. 28,29 Despite conspicuous advances in anti-infective treatments, the death toll caused by severe sepsis stands high at 30-50%. 29 Since a superfluous expression of pro-inflammatory cytokines, such as TNF-a, IL-1 and IFN-c, are direct contributors to runaway inflammation and thereby sepsis onset, 30 identification of biomarkers that prohibit inflammatory responses seems particularly vital to curb sepsis progression.
A number of pathways have been suggested to be sepsis related, including MAPK signaling, 31 tyrosine kinase-signaling and transcriptional activator signaling (JAK-STAT), 32 phosphatidylinositol 3-kinase/serine threonine protein kinase (PI3K/AKT) signaling 33 and glycogen synthase-3 (GSK-3) signaling. 33 Nevertheless, there has been scant evidence that verified the role of lncRNAs in sepsis, although over-expression of lncRNAs HOTAIR and MALAT1 were discovered in septic mouse models. 34,35 The present study investigated lncRNA CRNDE as a potential modulator of inflammatory responses underlying sepsis pathogenesis. Specifically, the clinical analyses demonstrated that CRNDE was differentially expressed within sepsis patients of distinct prognosis (Figure 1c), and in vitro tests also uncovered that CRNDE could propel expression of sepsis-specific biomarkers, including TNF-a, 36 IL-1b, 37 NF-kB 38 and IL-6 34 ( Figure 2). CRNDE had been previously reported as a modulator of sepsis-initiated inflammatory pathways as mentioned above. For instance, the TLR signaling was disordered when CRNDE was over-expressed, 16 and knock-out of CRNDE could dramatically alter the functions of NF-jB signaling, JAK/STAT signaling and PI3K/AKT signaling. 39,40 Consequently, it was reasonable to speculate CRNDE as a potent biomarker that could greatly influence the inflammatory pathogenesis inherent in sepsis.
In addition, we shed light on the role of miR-181a-5p in CRNDE-caused inflammatory responses, allowing that miR-181a-5p was subjected to targeted modulation of CRNDE (Figure 2f, 2g). In fact, the anti-inflammation function of miR-181a-5p has been documented earlier, such as suppression of NF-jB signaling, 41  blockage of TNF-a expression. 43 Within this study, miR-181a-5p was further demonstrated to impede over-expressed TNF-a, IL-1b, NF-jB and IL-6 that were characteristic for sepsis progression (Figure 2e). Building on these molecular mechanisms, underexpressed miR-181a-5p could serve as a pronounced indicator for poor prognosis of patients with sepsis (Table 3, Figure 1c). Beyond the above, TLR4, whose expression was suppressed under treatments of si-CRNDE and miR-181a-5p mimic (Figure 3d-3f), was discovered to influence the effects of miR-181a-5p on macrophages' secretion of cytokines (Figure 3i). According to the cellular experiments, TLR4-induced inflammation in sepsis cell models was probably mediated by NF-Kb. In fact, there was clear evidence that TLR4 was capable of activating NF-jB, 16 which then entered the nucleus to promote expression of inflammatory cytokines (e.g. TNF-a and IL-1). 44 Intriguingly, the extent to which NF-jB was activated mirrored the prognostic condition of sepsis patients. 45 The linkage of the CRNDE/miR-181a-5p/TLR4 axis and sepsis-related inflammation was thus formed. Summing up the above, the CRNDE/miR-181a-5p/ TLR4 axis was involved in dysfunctional inflammation underlying sepsis onset and development. Nonetheless, a comprehensive understanding of how CRNDE/miR-181a-5p/TLR4 axis plays a part in the underlying sepsis etiology would entail more proof from multiple angles. For instance, CRNDE could sponge more than miR-181a-5p to exert effects on sepsis-centered inflammation, and the miR-181a-5p/TLR4 axis might be influenced by other upstream lncRNAs. As for the clinical part, the included sepsis patients were Chinese-focused, and the derived study results might not be applicable for a sepsis population of other ethnicities. Above all, more convincing evidence is needed to overcome the deficits in experimental design mentioned above.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental material
Supplemental material for this article is available online.