High-throughput transcriptome sequencing RNA gene analysis
Unsupervised hierarchical cluster analysis was used to analyze the expression pattern of lncRNAs in the serum from the sepsis rats at different periods (0- 6- 24 hour). The results showed that there was a significant difference in the lncRNA expression profiles between volcano map (Fig. 1a) and heat map (Fig. 1b). In this study, 63 lncRNAs (29 up-regulated and 34 down-regulated) were identified as significantly differentially expressed between the normal control group (NC) and sepsis group (Supplementary Table S1 online). Here we select the 29 up-regulated ones for confirmatory analysis. Among those up-regulated lncRNAs, we identified the human-rat orthologous sequences by UCSC (University of California, Santa Cruz) Genome Browser and found only lncRNA PKN2-AS1 (LOC108350126, ENST00000645056.1) and AC068888.1 (LOC108351556, ENST00000663863.1) had homologous conserved gene sequences. Other DElncRNAs were mRNA (messenger RNA) sequences or not having homologous gene sequences in the human genome (Supplementary Table S2 online). Neither lncRNA PKN2-AS1 nor AC068888.1 has ever reported in sepsis before. Hence, those two lncRNAs were screened out for clinical verification.
Baseline characteristics
The average age was 57.5(22.0) years in patients with sepsis, and there were 62 (35.2%) females and 114 (64.8%) males. Specific to chronic complications, 12 (6.8%), 32 (18.2%), 9 (5.1%), 1 (0.6%), 31 (17.6%) and 62 (35.2%) sepsis patients had chronic obstructive pulmonary disease (COPD), cardiomyopathy, chronic kidney failure, cirrhosis, diabetes and hypertension disease, respectively. Then, 75 (42.6%), 19 (10.8%), 12 (6.8%), 13 (7.4%), 35 (19.9%), 10 (5.7%) and 12 (6.8%) sepsis patients had a respiratory infection, abdominal infection, skin and soft tissue infection, urinary tract infection, blood stream infection, central nervous system (CNS) infection, and other infections, respectively. In addition, the sepsis patients suffered from multiple organs failure, and 88 (50%), 51 (29%), 77 (43.8%), 40 (22.7%), 63 (35.8%), 163 (92.6%), 151 (85.8%), 68 (38.6%) had heart failure, acute liver failure, acute renal failure, gastrointestinal failure, CNS failure, respiratory failure, circulatory failure, clotting dysfunction, respectively. And the average number of organ failures was 4.0 (2.0). Some sepsis patients were treated with glucocorticoids, such as dexamethasone, methylprednisolone, and hydrocortisone. Here, we converted multiple glucocorticoids into hydrocortisone equivalents, and the final statistical median use of glucocorticoids was 80.0 (1000.0). Besides, the median APACHE II score was 20.0 (8.0) and the median SOFA score was 10.0 (4.0) in sepsis patients. The detailed information regarding other characteristics such as body mass index (BMI) and biochemical indexes were shown in Table 1.
Absolute expression of serum lncRNA PKN2-AS1 and AC068888.1 and their diagnostic value in the sepsis
The absolute expression of serum lncRNA PKN2-AS1 was 329.3 (77.4) gc/mL in patients with sepsis and 203.1 (96.1) gc/mL in the HCs. Meanwhile, the absolute expression of lncRNA AC068888.1 was 312.1 (91.5) gc/mL in patients with sepsis and 175.0 (93.6) gc/mL in the HCs, and further comparison analysis showed that lncRNA PKN2-AS1 and AC068888.1 expression were increased in in patients with sepsis compared with the HCs (P < 0.001) (Fig. 2a, 2b). From the ROC curve, the lncRNA PKN2-AS1 and AC068888.1 were differentiated between the sepsis patients and the HCs with an AUC of 0.879 (95% CI: 0.843-0.915) and 0.842 (95% CI: 0.801-0.884) (Fig. 2c, 2d). The optimal cut-off point for the level of lncRNA PKN2-AS1 that distinguished patients with sepsis from HCs was >238.0, and the specificity and sensitivity were 93.8% and 67.0%, respectively. Correspondingly, the optimal cut-off point for lncRNA AC068888.1 level that distinguished patients with sepsis from HCs was >210.6, and the specificity and sensitivity were 88.6% and 71.0%, respectively. These data suggested that both lncRNA PKN2-AS1 and AC068888.1 might be biomarkers for the sepsis risk. However, the predictive effect of lncRNA PKN2-AS1 was more prominent in comparison (Table 2).
Correlation of the lncRNA PKN2-AS1 and AC068888.1 absolute expression with the conventional evaluation indicators of sepsis, and inflammatory cytokines
Spearman's correlation analysis revealed that the serum absolute expression level of lncRNA PKN2-AS1 exhibited positively correlations with SOFA score (r = 0.427, P < 0.001), APACHE II score (r = 0.303, P < 0.001), Lac level (r = 0.347, P < 0.001), PCT level (r = 0.245, P = 0.001), and CRP level (r = 0.156, P = 0.039) (Fig. 3a-e). Meanwhile, the serum absolute expression level of lncRNA AC068888.1 also exhibited positively correlations with SOFA score (r = 0.492, P < 0.001), APACHE II score (r = 0.317, P < 0.001), Lac level (r = 0.480, P < 0.001), PCT level (r = 0.261, P < 0.001), and CRP level (r = 0.149, P = 0.048) (Fig. 3h-l). As to the inflammatory cytokines, lncRNA PKN2-AS1 absolute expression was positively correlated with IL‐6 (r = 0.422, P < 0.001) and TNF‐α (r = 0.401, P < 0.001) (Fig. 3f-g), and the lncRNA AC068888.1 absolute expression level also showed homogeneously correlations with IL‐6 (r = 0.476, P < 0.001) and TNF‐α (r = 0.516, P < 0.001) (Fig. 3m-n). These data indicated that both lncRNA PKN2-AS1 and AC068888.1 might be biomarkers for disease severity and inflammation in patients with sepsis, however, neither lncRNA PKN2-AS1 nor lncRNA AC068888.1 showed absolute advantages.
Clinical characteristics of septic shock and no septic shock sepsis patients,and risk factors for septic shock
During the 28-day follow-up period, 100 (56.8%) sepsis patients occurred septic shock, and they were grouped as septic shock patients, 76 (43.2%) patients did not occur septic shock, and they were grouped as no septic shock patients. Univariate analysis revealed that the number of organ dysfunction, platelet (PLT), serum creatinine (Scr), PCT, Lac, prothrombin time (PT), activated partial thromboplastin time (APTT), APACHE II score, SOFA score (P < 0.001), other infections (P = 0.021), CRP (P = 0.022), fibrinogen (FIB) (P = 0.016), the absolute expression of lncRNA PKN2-AS1 (P < 0.001, Fig. 4a) and lncRNA AC068888.1 (P < 0.001, Fig. 4b) were significantly higher in patients with septic shock compared with in those without septic shock. The others were not significantly associated with the occurrence of septic shock (Table 3). In addition, likelihood ratio forward stepwise multivariate logistic regression analysis demonstrated that the highly absolute expression of lncRNA AC068888.1 (P < 0.001), Lac level (P = 0.045), SOFA score (P = 0.031), and PT (P = 0.026) were independent risk factors for septic shock, whereas the absolute expression of lncRNA PKN2-AS1 and others exhibited no association with the severity of septic shock (Table 4).
Diagnostic value of the lncRNA PKN2-AS1 and AC068888.1 level for septic shock
The ROC curve analysis exhibited significant predictive value for lncRNA PKN2-AS1 (AUC = 0.704) and AC068888.1 (AUC = 0.812) in distinguishing patients with septic shock from those without septic shock (Fig. 4c). The predictive value of lncRNA AC068888.1 was parallel to Lac (AUC = 0.816), but apparently higher compared with those for APACHE II score (AUC = 0.668), SOFA score (AUC = 0.791) and lncRNA PKN2-AS1. At the optimal cut-off point of >302.3 for the serum absolute expression level of lncRNA AC068888.1, the specificity and sensitivity were 78.0% and 77.6%, respectively. At the optimal cut-off point of >297.6 for the serum absolute expression level of lncRNA PKN2-AS1, the specificity and sensitivity were 85.0% and 51.3%, respectively. At the optimal cut-off point of >3.6 for Lac levels, the specificity and sensitivity were 81.0% and 76.3%, respectively. At the optimal cut‑off point of >9.5 for SOFA score, the specificity and sensitivity were 81.0% and 64.5%, respectively. At the optimal cut‑off point of >20.5 for APACHE II score, the specificity and sensitivity were 56.0% and 72.4%, respectively. Those independent risk factors of septic shock were used to construct the predictive model for septic shock risk in sepsis patients (including lncRNA AC068888.1 score, SOFA score, Lac level), then the following ROC curve analysis manifested that the predictive model exhibited a good value for identifying septic shock risk in sepsis patients (AUC = 0.882) (Fig. 4c and Table 5).
Clinical characteristics of survival sepsis and non-survival sepsis patients, and risk factors for the unfavored prognosis of sepsis
96 (54.6%) sepsis patients survived during the 28-day follow-up period, and they were grouped as survival patients, 80 (45.4%) sepsis patients died, and they were grouped as non-survival patients. As presented in Table 6, univariate analysis revealed that number of organ dysfunction, the levels of PCT and Lac, APACHE II score, SOFA score (P < 0.001), glucocorticoid drugs (P = 0.007), septic shock (P = 0.021), PLT (P = 0.010), albumin (P = 0.030), Scr (P = 0.004), CRP (P = 0.013), PT (P = 0.011) and FIB (P = 0.008) were significantly different between survivors and non-survivors in patients with sepsis. Meanwhile, lncRNA PKN2-AS1 level (P < 0.001, Fig. 5a) and lncRNA AC068888.1 level (P<0.001, Fig. 5b) were significantly increased in non-survivors compared with survivors. The other variables were not significantly different between survivors and non-survivors. In addition, the likelihood ratio forward stepwise multivariate logistic regression analysis demonstrated that APACHE II score (P < 0.001), high PCT level (P = 0.032), number of organ dysfunction (P = 0.001), septic shock (P = 0.008), the highly absolute expression of lncRNA PKN2-AS1 (P = 0.019) and lncRNA AC068888.1 levels (P = 0.006) were independent risk factors for the poor prognosis of sepsis, whereas other variables exhibited no association with prognosis in patients with sepsis (Table 7).
Prognostic value of the lncRNA PKN2-AS1 and AC068888.1 levels for sepsis
The ROC curve analysis exhibited significant predictive value for lncRNA PKN2-AS1 (AUC = 0.747) and AC068888.1 (AUC = 0.717) in distinguishing non-survivors from survivors (Fig. 5c), which were higher compared with its for Lac level (AUC = 0.694), but lower compared with those for APACHE II score (AUC = 0.806) and SOFA score (AUC = 0.778). At the optimal cut-off point of >342.13 for the serum absolute expression of lncRNA PKN2-AS1, the specificity and sensitivity were 60.0% and 81.2%, respectively. At the optimal cut-off point of >330.1 for the serum absolute expression of lncRNA AC068888.1, the specificity and sensitivity were 52.5% and 83.3%, respectively. At the optimal cut-off point of >5.7 for Lac level, the specificity and sensitivity were 45.5% and 88.5%, respectively. At the optimal cut-off point of >12.5 for SOFA score, the specificity and sensitivity were 46.3% and 94.8%, respectively. At the optimal cut‑off point of >19.5 for APACHE II score, the specificity and sensitivity were 78.8% and 67.7%, respectively. The addition of either SOFA score alone (AUC = 0.778) or APACHE II score alone (AUC = 0.806) or both (AUC = 0.825) did not significantly improve the predictive ability to predict prognosis of septic patients. However, if SOFA score and APACHE II score were combined with lncRNA PKN2-AS1 and AC068888.1, the prognosis of sepsis patients was improved to a certain extent (AUC = 0.860) (Fig. 5c and Table 8).
Kaplan–Meier survival analysis
Kaplan–Meier survival analysis was performed to evaluate the clinical outcomes between two subgroups of sepsis patients divided by the absolute expression of lncRNA PKN2-AS1 or AC068888.1. We found that the 28-day survival exhibited much worse in sepsis patients with lncRNA PKN2-AS1 high expression than in sepsis patients with lncRNA PKN2-AS1 low expression (hazard ratio (HR) = 4.089, 95% CI: 2.520 - 6.634, χ2 = 32.529, P < 0.001, Fig. 6a). Likewise, analogous consequence was observed between two subgroups of sepsis patients which was distributed by lncRNA AC068888.1 expression (HR = 3.156, 95% CI: 1.914 - 5.204, χ2 = 30.794, P < 0.001, Fig. 6b).