Sepsis is an infection-related condition characterized by a systemic inflammatory reaction, often resulting in extensive tissue damage, such as the development of multi-organ dysfunction, systemic hypotension, renal hypoperfusion and renal ischemia-reperfusion (Singer et al., 2016). About 18 million new cases of sepsis are diagnosed worldwide each year, and the number is increasing year by year (Zhou et al., 2013). According to relevant epidemiological surveys, sepsis’ morbidity and mortality rate has surpassed that of myocardial infarction, and approximately 14,000 people die from sepsis complications every day, making it the leading cause of death among non-cardiac patients in intensive care units (ICUs), posing a great threat to human health (Keppler et al., 2018; Pellegrini et al., 2021).

Although sepsis was treated appropriately, the overall clinical outcome was unsatisfactory. In addition, available studies suggest that early diagnosis is quite difficult due to several complications and the lack of effective predictive techniques (Novosad et al., 2016). Therefore, new biomarkers linked with sepsis are urgently needed for early diagnosis, monitoring, and therapeutic intervention in sepsis. Many researchers have sought to discover novel sepsis biomarkers. Previous studies suggested that triggering receptor expressed on myeloid cells-1 (TREM-1), IL-27, neutrophil CD64, preprotease, and cell-free plasma DNA (cfDNA) were new promising biomarkers for sepsis diagnosis and therapy (Sandquist and Wong, 2014). Newly discovered biomarker classes, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and the human microbiome, are also of widespread interest (Kim and Choi, 2020). Despite the increased number of putative biomarkers, these efforts have yet to provide satisfying findings, necessitating more validation.

MiRNAs are single-stranded ncRNA molecules with a length of 21–24 nucleotides that bind to complementary sequences in the 30 untranslated regions (UTRs) of target messenger RNAs (mRNAs), causing mRNA degradation or inhibition (Sandquist and Wong, 2014). Mirna-186 has been shown to ameliorate renal injury caused by sepsis via the PTEN/PI3K/Akt/p53 pathway (Li et al., 2020). LncRNAs are non-protein-coding RNAs with a length of more than 200 nucleotides that play key roles in biological processes, participating in post-transcriptional regulation, cell-cell signaling, and protein metamorphosis regulation (Mattick and Rinn, 2015). In animal experiments, researchers have found that lncRNA-NEAT1 gene knockout can inhibit TLR2/NF-κB signaling pathway improves myocardial injury induced by sepsis (Wang et al., 2019). Salmena et al. (Salmena et al., 2011) suggested that molecules may have a regulatory function in competing for endogenous RNAs (ceRNA) by competing with the same miRNA response element, and the ceRNA hypothesis indicates that lncRNAs can sponge-bind and inactivate miRNAs, eventually lowering mRNA degradation or suppressing mRNA translation and thereby influencing protein coding. In the background of ceRNA network, there are few comprehensive analyses on the association between ceRNA network and sepsis. The identification of the interaction between ceRNA network and sepsis may provide important enlightenment for us to better understand the pathogenesis of sepsis.

In this work, we utilized bioinformatics approaches to detect differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) in the sepsis gene expression datasets from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database. We created a protein protein interaction (PPI) network and recognized hub genes. Then, in order to further investigated the key biological functions of DEmRNAs, we performed transcription factor (TF) enrichment, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene set enrichment analysis (GSEA) on DEmRNAs. Next, we built a lncRNA-miRNA-mRNA network based on the ceRNA theory to define the functional lncRNAs in sepsis, screen critical lncRNAs substantially linked to the disease, predict their molecular regulation mechanisms, and find new targets for diagnostic and therapy. Finally, using qRT-PCR, key lncRNAs were molecularly confirmed (Figure 1). To the best of our knowledge, this is the first research to analyze the differential expression profiles of the ceRNA network in sepsis using bioinformatics tools.

Sepsis is one of the most serious medical diseases nowadays. Sepsis and severe sepsis account for 30% and 37% of patients in European intensive care units, respectively (Vincent et al., 2006). Gaieski et al. (Gaieski et al., 2013) point out that after heart disease and cancer, severe sepsis is the third leading cause of mortality in the United States. However, the paucity of biomarkers for the diagnosis of sepsis provides a significant hurdle for the early and accurate clinical diagnosis of sepsis. High-throughput biotechnology was developed lately and is now frequently employed in fundamental research (Layton et al., 2019). From a genetic standpoint, it is possible to demonstrate hundreds of genetic differences in illness progression, which may give a biological basis for the early and accurate diagnosis of sepsis (van der Poll et al., 2017). The hub genes of sepsis were obtained through comprehensive analysis and the ceRNA network was constructed, which laid the foundation for future research into the mechanism of sepsis progression.

In the present study, 57 DElncRNAs (32 downregulated and 25 upregulated), 6 DEmiRNAs (3 downregulated and 3 upregulated) and 317 DEmRNAs (128 downregulated and 189 upregulated) were obtained from 7 different datasets in the GEO database. Seven mRNAs (ALPL, ADM, UGCG, CD5, ST6GAL1, DRAM1 and ADAM9) were predicted to interact with drugs among twenty-eight mRNAs in the network. Both PPI and lncRNA-miRNA-mRNA networks confirmed that ETS proto-oncogene 1 (ETS1), C-C motif chemokine receptor 7 (CCR7) and CD5 are key genes of the ceRNA network in sepsis. The Ets family is made up of proteins that share a DNA binding domain (winged helix-turn-helix motif), which enables Ets proteins to interact with GGAA/T-containing DNA elements and play a crucial role in mammalian immune regulation (Wasylyk et al., 1993). ETS1, a member of the family, interacts with AP-1 and NF-B, among other transcription factors, and has a role in the regulation of Tumor necrosis factors, integrins, extracellular proteases, and genes involved in the survival of inflammatory cells (Barthel et al., 2003; Chung et al., 2005). Ryter et al. (Ryter et al., 2002) found that ETS1 is an important regulator of human natural killer (NK) cell development and terminal differentiation. Ruan et al. (Ruan et al., 2019) found that ETS1 plays an important role in the establishment of intercellular crosstalk and contributes to the commitment of cardiac lineage in pluripotent state. Heme oxygenase (HO)-1 is a cytoprotective enzyme that also has anti-inflammatory properties (Ryter et al., 2002). It has been found that EST1 can induce the activity of HO-1 promoter, drive the expression of HO-1 and prevent excessive inflammatory reaction and oxidative tissue damage during endotoxemia (Chung et al., 2005).

The protein encoded by CCR7 belongs to the G protein coupled receptor family, which activates B and T cells and is found in many lymphoid tissues. It has been demonstrated that it can inhibit memory T cell migration to inflammatory tissue while also promoting dendritic cell maturation (Bill et al., 2022). In lymph nodes, the signal mediated by this receptor regulates the homeostasis of T cells and might possibly be involved in T cell activation and polarization, as well as the pathogenesis of chronic inflammation (Bill et al., 2022). Flores-Mejía et al. (Flores-Mejía et al., 2019) examined blood samples from sepsis patients and healthy volunteers and found that CCR7 was overexpressed in NK cells. Yang et al. (Yang et al., 2022) found that the antigen-presenting cell related marker CCR7 on fresh γδ T cells was considerably greater in sepsis patients compared to the control group. In addition, Almansa et al. (Almansa et al., 2015) believe that quantitative analysis of the expression level of CCR7 and other genes is helpful to evaluate the disease severity and immunological changes of sepsis. Yin et al. (Yin et al., 2021) found that astaxanthin can downregulate the expression of CCR7 to avoid the immune dysfunction of dendritic cells, which provides a novel approach for the potential treatment of sepsis. Therefore, we speculate that CCR7 has a critical role in the diagnosis and treatment of sepsis.

The CD5 gene produces a protein that belongs to the scavenger receptor cysteine-rich (SRCR) class and functions as a T cell, B1Mura cell, and B-CLL receptor (Burgueño-Bucio et al., 2019). This receptor has been reported to be a positive and negative regulator of T cell receptor (TCR) signal transduction, as well as a negative regulator of B cell receptor (BCR) signal transduction (Soldevila et al., 2011). Recent studies have shown that CD5 promotes T cell survival by preventing activation-induced cell death in autoreactive T cells, promotes peripheral regulatory T cell induction from the start, regulates Th17 and Th2 differentiation, and modulates immune response via regulating dendritic cell function (Burgueño-Bucio et al., 2019). Because CD5 receptor is an immune checkpoint regulator, it can be exploited as an immunological intervention target in a variety of diseases, including cancer, autoimmune disease, and infection (Freitas et al., 2018). Vera et al. (Vera et al., 2009) found that CD5 lymphocyte receptors can detect the presence of conservative fungal components and support the therapeutic vaule of soluble CD5 forms in the treatment of fungal sepsis. Therefore, CD5 is predicted to become a biomarker and therapeutic target of sepsis.

Transcription factor enrichment analysis, GO analysis and KEGG analysis were performed on 317 DEmRNAs. The functions of transcription factor targets mainly include transcription, immune response and morphological changes of animal organs, which indicates that sepsis is a systemic immune response caused by pathogens, microorganisms or endotoxins, and severe patients have multiple organ failure or even death (Singer et al., 2016). GO analysis revealed that in BP, it was primarily enriched in neutrophil activation, T cell activation and lymphocyte differentiation. Overwhelming evidence suggests that neutrophil activation directly associated to the emergence and progression of sepsis (Ren et al., 2020). Recent evidence suggests that the function and differentiation of T lymphocytes, including the increased differentiation from Th1 (inflammation) to Th2 (anti-inflammation) and Treg cells, are related to the development of immunosuppression during the late stages of sepsis (Mori et al., 2015). Among CC, it is mainly enriched in specific granule and plasma membrane. Maitra et al. (Maitra et al., 2010) pointed out that matrix metalloproteinase-9, which is a crucial effector in acute inflammatory disorders like sepsis, is deposited in the tertiary granules of polymorphonuclear leukocytes. Johar et al. (Rahman et al., 2021) showed that the changes of cell permeability, gas exchange, cytokine migration and protein transport caused by sepsis were closely related to the plasma membrane. In MF, it is mainly enriched in transferase activity and protein serine/threonine kinase activity. Some studies have pointed out that in sepsis patients, glutathione S-transferase A1-1 can be an early indicator of liver dysfunction (Yan et al., 2014). Urinary glutathione S-transferase can be a good early indicator of renal dysfunction in intensive care patients with sepsis (Walshe et al., 2009). Previous research has indicated that serine/threonine kinases play a significant role in sepsis-induced inflammation (Packiriswamy et al., 2016). KEGG pathway analysis showed that it was primarily enriched in fluid shear stress and atherosclerosis pathway, hematopoietic lineage pathway and Th cell differentiation related pathway. No studies have reported the direct relationship between fluid shear stress and atherosclerosis pathway and sepsis, but increasingly evidence show that this pathway has a strong link to vascular oxidative stress, inflammation and atherosclerosis (Jen et al., 2013). Muranski et al. (Muranski and Restifo, 2013) have found that regulating the differentiation pathway of Th17 cells can regulate the expression of interleukin-17 and prevent excessive inflammation, which is very important for the protection of immunity and host cells. Interestingly, allergies can be caused by inappropriate growth of human Th2 cells, while autoimmunity can be caused by an excessive Th1 response, so the balance between Th1/Th2 subsets is regulated through Th1 and Th2 cell differentiation pathways, thus playing an important role in the treatment of sepsis (Amsen et al., 2009). These biological processes discovered by GSEA are obviously similar to the above results. All of the information presented above suggest that our conclusions are quite trustworth and that these DEmRNAs play a key role in sepsis.

During the previous several decades, including lncRNAs and miRNAs, they have attracted more and more attention because of their role in physiological and pathological responses. At present, more and more researches have been conducted to give evidence to support the ceRNA hypothesis that lncRNAs carrying MREs can competitively combine with some miRNAs, thus, at the post-transcriptional stage, controlling miRNA-mediated downstream target gene silencing (Khorkova et al., 2015). For example, Zhang et al. (Zhang et al., 2020) have demonstrated that lncRNA TCONS_00016233 aggravates septic acute renal injury induced by LPS by binding to miR-22-3p and preventing the down-regulation of miR-22-3p-mediated apoptosis inducing factor mitochondria associated 1 (AIFM1); Wang et al. (Wang et al., 2021) found that lncRNA-LUCAT1 can regulate the expression of ROCK1 in H9C2 cells induced by LPS by secreting miR-642a. Knockout of lncRNALUCA T1 can inhibit myocardial injury in sepsis induced by LPS; An et al. (An et al., 2021) believe that LncRNA ZFAS1, as the ceRNA of miR-138-5p, up-regulates the expression of SESN2, thus improving cardiomyocyte scorch death induced by sepsis. Therefore, it is of great significance to investigate the role and regulation mechanism of lncRNAs as ceRNAs in sepsis, as well as their potential role in diagnostics.

In this research, DEmiRNAs was used to predict the combination of lncRNAs and mRNAs of miRNAs to construct a ceRNA network. There were five lncRNAs in the network, including PCED1B-AS1, SATB1-AS1 and LINC01422 down-regulated, BACH1-IT2 and FAM157C up-regulated, but what role these lncRNAs in the progression of sepsis is not clear. It is reported that there is a certain relationship between PCED1B-AS1 and immune cells. Fan et al. (Fan et al., 2021) found that PCED1B-AS1 overexpression in hepatocellular carcinoma cells has been shown to impair the activity of co-cultured T cells and cause immunosuppression; In addition, Li et al. (Li et al., 2019) demonstrated that by directly binding to miR155, PCED1B-AS1 controls macrophage apoptosis and autophagy. Acute myeloid leukemia (AML) is an invasive hematopoietic tumor. Zhou et al. (Zhou et al., 2021a) found that inhibition of SATB1-AS1 can up-regulate miR-580 and down-regulate OAS2, thus increasing the sensitivity of AML cells. The rest of lncRNAs have not been reported before, and we are attempting to clarify their molecular mechanism in the occurrence and progression of sepsis using ceRNA hypothesis. We hypothesize that BACH1-IT2 and FAM157C may influence target gene expression in sepsis through competitive binding to has-miR-3188. To support our prediction, miR-3188 was shown to be downregulated in atherosclerotic patients, which significantly promoted macrophage damage by reducing cell viability, inducing apoptosis and increasing the production of inflammatory cytokines (including IL-1 β, IL-6, MCP-1 and TNF- α) (Li et al., 2017). Furthermore, Zhou et al. (Zhou et al., 2021b) found that miR-3188 can promote cell cycle by up-regulating cyclin cyclind1 and down-regulating p21 protein expression in polycystic ovary syndrome, resulting in abnormal proliferation of granulosa cells. For lncRNA SATB1-AS1-has-miR-4286-mRNA network, studies have shown that has-miR-4286 can improve vascular endothelial cell injury by inhibiting transforming growth factor-β1 (TGF-β1) and reducing apoptosis and inflammation (He et al., 2020). We speculate that PCED1B-AS1 may competitively combine with has-miR-7977 to change the expression of downstream sepsis-related mRNAs. Fichna et al. (Fichna et al., 2021) suggested that miR-7977 is increased in CD4+T cells of autoimmune Eddie’s disease patients and plays a crucial function in autoimmune diseases; Horiguchi et al. (Horiguchi et al., 2016) found that miR-7977 can reduce the hematopoietic support ability of bone marrow CD34⁺ cells. Our results show that LINC01422 can combine not only with has-miR-7977, but also competitively with has-miR-197-3p. Qiao et al. (Qiao et al., 2021) demonstrated that miR-197-3p attenuates apoptosis, inflammation and oxidation of epithelial cells. Similarly, Akkaya-Ulum et al. (Akkaya-Ulum et al., 2021) believe that miR-197-3p combines with the interleukin-1β receptor type I (IL1R1) gene and plays an anti-inflammatory role in monocytes and macrophages. Finally, we utilized qRT-PCR to confirm the expression levels of five important lncRNAs, and all of them were substantially different between sepsis patients and healthy control participants. Future research should focus on the precise function and mechanism of these lncRNAs in the occurrence and progression of sepsis.

Although our current findings have good clinical implications and may serve as a foundation for future study into the mechanism of sepsis, there are certain limitations to be aware of. First, our sample size is relatively small, so future study should investigate a bigger sample size to confirm the veracity of our findings. Secondly, the particular mechanism of lncRNA-miRNA-mRNA network of sepsis has to be researched further for in vivo and in vitro confirmation.

In conclusion, we constructed a sepsis-specific ceRNA network to help further understand the relationship between lncRNAs, miRNAs, and mRNAs, and found that five lncRNAs were closely related to sepsis. Moreover, these discoveries will contribute to a better understanding of the pathogenesis and molecular mechanism of sepsis. We believe that our research will help to the progression of new molecular targets that will enable the early diagnosis and targeted treatment of sepsis patients.