The potential of tRF-21-U0EZY9X1B plasmatic level as a biomarker of children with obstructive sleep apnea-hypopnea syndrome

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a sleep disorder caused by partial or complete obstruction of the upper respiratory tract, accompanied by apnea and insufficient ventilation during sleep [1]. It is a common chronic disease in children. The prevalence of OSAHS in children ranged from 1.0 to 5.7%, with no significant difference between genders [2]. Upper airway obstruction during sleep in children with OSAHS results in decreased blood oxygen saturation and excessive blood carbonic acid content, which not only leads to cognitive impairment, excessive daytime sleepiness and inattention, but also tends to increase the risk of emotional instability and depression, thus affecting their health and leading to growth and development problems in children [3‐5]. Polysomnography (PSG) is the gold standard for the diagnosis of OSAHS [6]. However, the detection rate of children’s PSG is not high, due to the limitations of PSG requiring admission monitoring, long time-consuming, cumbersome operation, high price and low child cooperation [7, 8]. Therefore, it is of great significance to research the biomarkers for screening OSAHS in children.

A novel type of small non-coding RNA, tRNA-derived fragments (tRF), has attracted our attention. tRNA-derived small RNAs fragments (tsRNA) are a class of small non-coding RNAs specifically cleaved from tRNAs with a length of 14–48 nucleotides [9]. Based on length and origin, they are divided into three main categories, namely tRNA fragments, stress-induced tRNA-derived RNA fragments (tiRNA), and toxic small tRNA-derived RNA fragments [10]. As the second most common small ncRNA after micro RNAs (miRNA), more and more data suggest that these ncRNAs have a wide range of biological functions, including cell and tissue stress response, tumorigenesis, immune response and so on [11]. Similar to miRNAs, stable stem loop structure, stable nucleoprotein complex and exosome encapsulation keep tRFs at a high concentration in serum [10]. tRFs in serum is sensitive to acute inflammation, aging, acute kidney disease and tissue injury, and may be a useful non-invasive biomarker [11].

Obesity is one of the important causes of OSAHS [12]. Research shows that the prevalence of OSAHS in obese children is 19% ~ 60% [13]. Like patients suffering from Obesity, the OSAHS patients would seem to be characterized by a chronic systemic inflammatory state [14, 15]. Chronic intermittent hypoxia leads to repeated hypoxia and reoxygenation cycles, enhances systemic oxidative stress, and leads to the production of systemic inflammation related biomarkers. Under specific conditions such as stress and hypoxia, tRNAs will be specifically cleaved into tsRNAs [9]. In recent years, it has been found that some small RNA molecules, including miRNA and circular RNAs (circRNA), are involved in the occurrence and development of OSAHS [16, 17]. According to all aforementioned findings, we assume that particular tRFs could be promising molecular biomarkers in OSAHS.

OSAHS can occur at all ages, but the peak onset period in children is 4 to 7 years old because of the tonsils and adenoids accounting for the largest proportion of airway volume at this time. Therefore, we recruited children between the ages of 4 to 7 for this study. Interestingly, we observed that children with OSAHS had significantly lower height and higher TC and Scr than the control group (Table 1). A case-control study found that underweight children with OSAHS, as compared with those without OSAHS, were more likely to have decreased height and the height was negatively correlated with OSAHS [18]. Obesity is a recognized risk factor for OSAHS. However, in our study, there was no difference in weight and BMI between the two groups, but the TC of children in the OSAHS group was significantly higher than that in the control group. Several studies have shown that the TC of adolescents and middle-aged OSAHS patients was significantly increased, which is the same as our results [19‐21]. OSAHS contributes the impairs of renal function [22, 23]. A study on the relationship between OSAHS and renal function found that there were varying degrees of impairment of renal function in OSAHS patients, but there was no statistically significant difference in Scr between the OSAHS group and the control group [24].

In this study, we first performed high-throughput sequencing to detect the expression profile of plasma tsRNAs in 5 OSAHS children and 5 control children. According to the sequencing data, tRF-21 with the most significant difference was selected for qRT-PCR to verify the authenticity of the profile. The comparison results of the sequencing of products showed that the amplification primers were specific (Fig. 1). Then, we detected the expression level of tRF-21 in the plasma of all 50 children in the case group and 38 children in the control group. The results showed that tRF-21 was significantly down regulated in children with OSAHS compared with healthy controls (Fig. 4). tRF-21 is usually studied and explored as a tumor biomarker, such as pancreatic ductal Adenocarcinoma [25], papillary thyroid cancer [26], cholangiocarcinoma [27] and lung adenocarcinoma [28]. Pan et al. found that tRF-21-VBY9PYKHD, as an inflammatory cytokine–regulated transfer fragment has a tumor-suppressive effect [25]. In addition to tumor inhibition, other effects of tRF-21 have not been understood.

Previous studies have reported that OSAHS is a disease with chronic systemic inflammation and metabolic changes [29, 30]. OSAHS, through intermittent hypoxia and sleep fragmentation, was involved with sympathetic activation, endothelial dysfunction, inflammatory state, and oxidative stress with multi-organ involvement and comorbidities, in particular, such as cardiovascular, cerebrovascular and neoplastic [31]. A research by Santamaria-Martos et al. found that patients with OSAHS exhibit a dysregulated miRNA profile compared to healthy controls [32]. It appears that intermittent hypoxia associated with OSAHS, could lead to differential expression of some hypoxia-induced miRNAs. Another study reported that double-stranded RNA composed of a tRF and its complementary sequence significantly promoted the survival of myocardial cells after hypoxia/reoxygenation in vitro [33]. There are few studies on the relationship between tRF and hypoxia or sleep disorders, which is also one of the future research directions. The relationship between tRFs and hypoxia or sleep disorders needs more researches in the future.

ROC curve showed that the area under the curve of tRF-21 was 0.7734, and the sensitivity and specificity were 85.71% and 63.16% respectively. These results suggest the potential value of tRF-21 in OSAHS screening in children. At present, studies on OSAHS in children have not obtained clear biomarkers, and existing studies also have some limitations, such as lack of specificity and sensitivity analysis [34]. As a screening method, the tRF has the characteristics of easy sample acquisition and convenient laboratory operation, which can be regarded as a new research direction. Of course, it requires further validation of the results in a larger cohort.

The results of correlation analysis showed that the expression level of tRF-21 was linearly correlated with the degree of tonsillar enlargement. It suggests that the level of tRF-21 may be related to the severity of OSAHS. However, there is no significant correlation between the tRF and the OAHI index. In addition, the tRF-21 level is significantly correlated with Hb, MCH, TG and CK-MB, which could be predicted by MCH, TG and CK-MB. At present, the research on the function of tRF-21 is very limited.

In conclusion, our study found that the plasma tRF-21-U0EZY9X1B level of 4 to 6-year-old OSAHS children was down regulated, which was closely related to Hb, MCH, TG and CK-MB, and could be used as a potential biomarker for the diagnosis of OSAHS in children. However, our research has some limitations, including the problem of insufficient sample size. In the future, we will verify this result in a larger cohort.