miRNAs have been used as biomarkers for various type of disease since discovery of circulating miRNAs in human peripheral sera. Our study demonstrated that hsa-let-7d-3p had the potential to be a novel diagnostic biomarker of sepsis, with high sensitivity and specificity. Previous studies have found several miRNA as diagnostic and prognostic biomarkers for sepsis[8]. Yao and colleagues found that miR-25 displayed a superior diagnostic accuracy for sepsis compared to well established markers such as CRP and PCT according to ROC curve analysis in a well characterized cohort of 70 patients with sepsis and 30 patients with non-infectious SIRS[18]. They further found that decreased miRNA-25 level was related to the level of oxidative stress indicators in sepsis patients. Xie et al found that circulating miR-122 as a potential biomarker of critical illness and sepsis in a sample of 214 patients with sepsis (117 survivor and 97 non-survivors), Mir-122 predicted patients’ short and long term survival with high accuracy[19 20]. The hsa-let-7d-3p is one of the tumor suppressive let-7d family members. Let-7d is down regulated in numerous types of cancer, including ovarian cancer and directly targets various oncogenes. Gunal have reported that has-let-7d-3p could down regulated HMGA2 and KRAS gene and the loss of has-let-7d-3p expression led to the progression of epithelial ovarian cancer related to the tumorigenesis, invasion, and metastasis[21]. However, it have not been reported to be associated with the early diagnosis of sepsis. To the best of our knowledge hsa-let-7d-3p was the first time be identified as a biomarker of sepsis.
In our study, gene chip analysis revealed that 11 miRNAs satisfied the screening criteria of p < 0.05, fold change fold change ≥ 2 or < 0.5. Limited data has been presented regarding the relationship of these 11 miRNAs with sepsis because of the following factors: 1) Sepsis was a complicated syndrome with multi organ failure in pathogenic microorganism, severity and comorbidities. The complexity of sepsis lead to the complexity of sepsis biomarker, it is difficult to find a single biomarker of sepsis with high sensitivity and specificity. The miRNAs which can early diagnosis the sepsis various with different studies.
In our study, the number of samples in the gene chip analysis was limited to the serum of 3 patients, but we further verified the results of the gene chip through qRT-PCR analysis to compensate for the limitations of our study. The regulating of the hsa-let-7d-3p was down regulated in the gene chip analysis, and it was up regulated in the further validation in the sepsis patients. The reason of the opposite results may be we only enrolled three samples of patients for screening. However the results of the qRT-PCR validation were more convincing than PCR screening.
There were several advantages of our study. First, we choose self-control comparison to primary validate the different expressed miRNAs between sepsis stage and condition improved stage. Self-control study can maximum reduce the influence factors: such as age, gender, illness severity, comorbidities and so on. So the results of our study were more convincing than other studies. Second, reference gene cel-mirRNA-39-3p was chosen as reference gene in our study as previously reported[13 22]. Currently, there is no consensus on what reference gene should be used[13 22]. Reference gene such as U6, 5S rRNA in the serum or plasma had been used in previous study, however they were found to be unstable in other studies[14 23]. Fabian Benz have reported that levels of U6 was significantly up-regulated in sera of patients with critical illness and sepsis and was correlated with inflammation. Third, the selection of the control group in our study was better. Compared with healthy control in other studies control group in our study included patients with COPD, hypertension, diabetes malignant tumor, trauma and so on. There were no significant difference of comorbidity between sepsis group and control group. The selection of control group in our study were more similar to real world condition. Distinguish sepsis patients from healthy control is easier than distinguish sepsis patient from patients with infectious disease and other comorbidities. So the results of our studies were more convincing than other studies which using normal healthy control.
There were several limitations of our study. First, in the RT-PCR validation of miRNAs, we found that 11 miRNA meeting our screening criteria, however we only enrolled 3 miRNAs for primary validation for shortage of funds. Second, the total number of cases enrolled in the study was limited. Hence, the value of hsa-let-7d-3p in predicting early sepsis should be further evaluated in large samples.