TsRNA molecule tRF5-30-GlyCCC-2 and application thereof

Abstract

The invention provides a tsRNA (Ribonucleic Acid) molecule tRF5-30-GlyCCC-2 and an application of the tsRNA molecule. Belongs to the technical field of in-vitro diagnosis, and particularly relates to a cDNA (complementary deoxyribonucleic acid) sequence of tRF5-30-GlyCCC-2 provided by the invention as shown in SEQ ID No.1. According to the invention, tRF5-30-GlyCC-2 is used as a diagnostic marker for detection and typing of the brain injury of the premature infant for the first time. The kit realizes non-invasive diagnosis of the brain injury of the premature infant, has the advantages of high sensitivity and good specificity, and effectively overcomes the existing defects of premature infant brain injury imaging examination.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and more specifically, to a tsRNA molecule tRF5-30-GlyCCC-2 and its applications. Background Technology

[0002] The sequelae of preterm birth manifest as continuous neurological dysfunction, with delayed brain development in premature infants compared to full-term babies. Particularly among infants born before 29 weeks of gestation, 55% experience adverse outcomes. Of the numerous complications faced by preterm infants, brain injury is the most severe and devastating neurological sequela. Preterm brain injury refers to central nervous system damage during infancy caused by various perinatal high-risk factors, infections, trauma, accidental asphyxia, poisoning, and cerebrovascular diseases, including intraventricular hemorrhage (IVH), periventricular leukosclerosis (PVL), and hypoxic-ischemic encephalopathy (HIE). Notably, complications such as IVH significantly increase the risk of permanent motor deficits and cerebral palsy. Furthermore, these early injuries can lead to persistent neurodevelopmental disorders, including ADHD, autism spectrum disorder (ASD), and cognitive deficits, which can persist into adulthood, ultimately affecting educational achievement and independent living. Studies have shown that early intervention in infants with brain injuries can effectively improve their developmental outcomes. Therefore, it is urgent to decipher the underlying pathological mechanisms to improve clinical prognosis. Early identification and assessment of brain injury in premature infants can provide the optimal opportunity for early intervention.

[0003] Currently, clinical detection of brain injury in premature infants primarily relies on imaging examinations, including cranial ultrasound (cUS) and magnetic resonance imaging (MRI). cUS offers advantages such as convenience, no need for sedation, and suitability for bedside monitoring; it can be repeated. However, its accuracy is highly dependent on the operator's skill, and cUS has limited resolution for small lesions. While MRI boasts high resolution, deep individual sedation is required before MRI examination, and premature infants' unstable vital signs make them difficult to tolerate. Because the lesion area in mild premature brain injury is small, both cUS and MRI have low detection sensitivity, failing to accurately detect mild premature brain injury. This leads to a lack of early intervention for mild premature brain injury, exacerbating the long-term risk of sequelae.

[0004] Complex processes such as brain development, injury response, and repair are accomplished through intricate interactions of genetic and epigenetic mechanisms, with non-coding RNAs (ncRNAs) playing a crucial regulatory role. Emerging evidence suggests that tRNA-derived small RNAs (tsRNAs), including tRNA-derived fragments (tRFs) and tRNA-derived stress-inducible tRNAs (tiRNAs), play a key role in the pathophysiology of neurological diseases and neurotrauma. In the context of ischemic brain injury, these small RNAs function as dynamic regulators of tissue recovery. For example, deep sequencing of the ischemic rat brain revealed a significant upregulation of tRNA (Val) and tRNA (Gly)-derived tRFs. These fragments are produced through angiopoietin-mediated cleavage and negatively regulate post-ischemic angiogenesis by inhibiting endothelial cell proliferation and tube formation. Beyond their regulatory functions, tRFs are gaining attention as sensitive biomarkers of cellular stress and injury. In vitro studies using PC12 neurons have shown that oxidative stress and oxygen-glucose deprivation (OGD) can trigger the release of tiRNAs before severe morphological damage occurs, suggesting that they may be early indicators of neuronal injury and reperfusion stress. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the detection sensitivity of brain injury in premature infants.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a tsRNA molecule tRF5-30-GlyCCC-2, the cDNA sequence of which is shown in SEQ ID No. 1.

[0007] The second aspect of this invention provides an application of the tsRNA molecule tRF5-30-GlyCCC-2 described in the first aspect, specifically, using tRF5-30-GlyCCC-2 for the detection and typing of brain injury in premature infants.

[0008] Preferably, the tRF5-30-GlyCCC-2 is derived from umbilical cord blood, whole blood, serum, plasma, or cerebrospinal fluid.

[0009] In the technical solution provided by this invention, tRF5-30-GlyCCC-2 is derived from infant body fluids, specifically detectable in infant umbilical cord blood, whole blood, serum, plasma, or cerebrospinal fluid. The content of tRF5-30-GlyCCC-2 in umbilical cord blood can serve as a diagnostic biomarker for brain injury in premature infants. This molecule is a specific cleavage product of glycine tRNA (anticodon GCC). This invention is the first to confirm that this cleavage product can serve as a biomarker for detecting brain injury in premature infants, and it has the advantages of high specificity and high sensitivity.

[0010] A third aspect of the present invention provides a tRF5-30-GlyCCC-2 detection product, wherein the tRF5-30-GlyCCC-2 detection product includes the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No. 3.

[0011] Preferably, the tRF5-30-GlyCCC-2 detection product also includes a control gene detection reagent.

[0012] Preferably, the control gene detection reagent includes a housekeeping gene detection reagent.

[0013] Preferably, the tRF5-30-GlyCCC-2 detection product also includes any one or more of RNA extraction reagents, cDNA synthesis reagents, and PCR premixes.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to confirm that tRF5-30-GlyCCC-2 can be used as a diagnostic marker for brain injury in premature infants. At the same time, the expression level of tRF5-30-GlyCCC-2 is positively correlated with the degree of brain injury. Combined with the matching detection reagents provided by this invention, quantitative classification of brain injury in premature infants can be achieved, providing a new approach for the detection and classification of brain injury in premature infants. 2. The tRF5-30-GlyCCC-2 marker for detecting premature infant brain injury provided by this invention can be found in umbilical cord blood. By detecting the expression level of tRF5-30-GlyCCC-2 in umbilical cord blood, non-invasive detection of premature infant brain injury can be achieved. By detecting the expression level of tRF5-30-GlyCCC-2 in the blood or cerebrospinal fluid of the infant, minimally invasive detection of premature infant brain injury can be achieved. Compared with the sedation method of MRI detection, the detection method of the premature infant brain injury marker provided by this invention is gentler, reducing the detection threshold and minimizing the discomfort of the infant. 3. The brain injury biomarkers for premature infants provided by this invention can be prepared into test kits and other test products. The products have a high degree of integration. While promoting mutual recognition of brain injury detection in premature infants, they can significantly reduce the detection cost compared with MRI examination. 4. This invention reveals for the first time the strong correlation between tRF5-30-GlyCCC-2 and brain injury in premature infants, which opens up a new path for the molecular diagnosis, treatment and prognosis of brain injury in premature infants. Attached Figure Description

[0015] Figure 1 This is the result of the analysis of the relative expression level of tRF5-30-GlyCCC-2 in healthy infants and children with brain injury in Example 1 of the present invention; Figure 2 This is the result of logistic regression combined with ROC analysis in Embodiment 1 of the present invention. Detailed Implementation

[0016] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0017] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] As described in the background section, existing methods for detecting brain injury in premature infants suffer from drawbacks such as insufficient specificity and sensitivity, and significant invasiveness.

[0019] In view of this, a specific embodiment of the present invention provides a tsRNA molecule tRF5-30-GlyCCC-2, the cDNA sequence of which is shown in SEQ ID No. 1.

[0020] More specifically, SEQ ID No.1: GCGCCGCTGGTGTAGTGGTATCATGCAAGA.

[0021] tRF5-30-GlyCCC-2 can be detected in infant fluids such as umbilical cord blood, whole blood, serum, plasma, or cerebrospinal fluid. tRF5-30-GlyCCC-2 can be used as a diagnostic marker for brain injury in premature infants. In particular, non-invasive detection of brain injury in premature infants can be achieved by detecting the level of tRF5-30-GlyCCC-2 in umbilical cord blood.

[0022] The technical solution of the present invention is further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0023] Example 1 Biomarker validation Umbilical cord blood was collected from preterm infants without brain injury (control group) and preterm infants diagnosed with brain injury (case group). After RNA extraction and cDNA synthesis, the obtained cDNA was the tRF5-30-GlyCCC-2 gene.

[0024] Due to errors in RNA concentration quantification and RNA reverse transcription efficiency, the cDNA content of each sample in the same volume is not entirely the same. To correct for this difference, this invention uses the housekeeping gene U6 as an internal control. The relative content of the target gene in the sample is obtained by dividing the value of the internal control in this sample by the value of the target gene in the sample. The primer sequences, annealing temperatures, and product lengths of the U6 gene and the tRF5-30-GlyCCC-2 gene are shown in Table 1. Table 1 Prepare the PCR reaction system according to the system shown in Table 2: Table 2 After mixing the PCR system shown in Table 2, a real-time PCR reaction was performed. The PCR reaction program was (95℃ pre-denaturation, 10 min) × 1 cycle + [(95℃, 10 s) + (60℃ annealing, 60 s)] × 40 cycles. Fluorescence was collected during annealing to obtain the PCR product.

[0025] After electrophoresis confirmed that the PCR product was a single specific amplification band, the concentration of the PCR product was set to 1, and the PCR product was serially diluted 10-fold, with the lowest concentration being 1×10⁻⁶. -9 times.

[0026] Take the diluted PCR product and prepare the Realtime PCR reaction system shown in Table 3: Table 3 The system shown in Table 3 was placed in a 284-PCR plate and subjected to PCR reaction on a Realtime PCR instrument. The PCR reaction program was (95℃ pre-denaturation, 10 min) × 1 cycle + [(95℃, 10 s) + (60℃ annealing, 60 s)] × 40 cycles. Fluorescence was collected during annealing. After amplification, the melting curve generation program was run. The melting curve generation program was: [(95℃ denaturation, 10 s) + (60℃ annealing, 60 s) + (Ramp Rate 0.075℃ / s to 95℃, 15 s)] × n cycles to establish the melting curve.

[0027] The target gene and housekeeping gene for each sample were subjected to Real-time PCR reactions. Based on the plotted serially diluted DNA standard curves, the concentrations of the target gene and housekeeping gene for each sample were directly generated by the machine. The corrected relative content of this gene in each sample was calculated by dividing the target gene concentration by the housekeeping gene concentration.

[0028] The relative expression levels of tRF5-30-GlyCCC-2 in preterm infants without brain injury (control group) and preterm infants diagnosed with brain injury (case group) were analyzed as follows: Figure 1 As shown. By Figure 1 It is evident that the expression of tRF5-30-GlyCCC-2 differed significantly between the case and control groups. The rank mean of the case group (19.37) was significantly higher than that of the control group (11.63). The standardized test statistic was 2.406, the asymptotic significance (two-tailed test) was 0.016, and the exact significance (two-tailed test) was 0.015, all less than the significance level of 0.05. This indicates that the difference in tRF5-30-GlyCCC-2 expression between the two groups was statistically significant, suggesting that the expression level of this molecule differs between the case and control groups. This difference may be related to conditions of interest in research such as neonatal brain injury (if this comparison is from a related study). This reveals that preterm infants with brain injury have unique tRF expression characteristics.

[0029] Figure 2 The results of logistic regression combined with ROC analysis are as follows: Figure 2 It is evident that tRF5-30-GlyCCC-2 demonstrates a certain effectiveness in differentiating neonatal brain injury. Its AUC value of 0.758 indicates a moderate level of discriminatory ability, and the result is statistically significant (P = 0.016). The sensitivity of this indicator reaches 0.867, which can effectively identify newborns with brain injury and reduce missed diagnoses; the specificity is 0.733, indicating a certain ability to exclude newborns without brain injury, with a cutoff value of 0.79.

[0030] Based on the results of Example 1, it is evident that tRF5-30-GlyCCC-2 can be used as a reliable diagnostic biomarker for brain injury in preterm infants in clinical practice.

[0031] Example 2 Mother-infant correlation analysis of tRF5-30-GlyCCC-2 This embodiment aims to explore the potential association between the expression level of tRF5-30-GlyCCC-2 and multiple clinical and physiological parameters of newborns and mothers, in order to comprehensively evaluate the biological significance of this biomarker and its indicative role under different physiological and pathological conditions.

[0032] The correlation analysis results of tRF5-30-GlyCCC-2 expression levels between mothers and infants of preterm infants without brain injury and mothers and infants of preterm infants diagnosed with brain injury and clinical data are shown in Table 4.

[0033] Table 4: Correlation analysis of tRF5-30-GlyCCC-2 expression levels and clinical indicators in preterm infants with and without brain injury. In preterm infants with and without brain injury, the expression of tRF5-30-GlyCCC-2 exhibited different clinical relevance patterns. Specifically, in the brain-injured infant group, the level of tRF5-30-GlyCCC-2 was significantly negatively correlated with the mother's pre-pregnancy weight (r=−0.703, P=0.011) and pre-pregnancy BMI (r=−0.720, P=0.008), meaning that the higher the maternal obesity level, the lower the tRF level in the infant. However, this association of maternal metabolic indicators was not observed in preterm infants without brain injury. Conversely, in non-brain-injured preterm infants, tRF5-30-GlyCCC-2 showed a strong positive correlation with the infants' blood urea nitrogen (BUN) (r=0.787, P=0.002), suggesting that it participates in some homeostatic regulation in normal preterm infants; however, this significant physiological association was not present in the brain-injured group (r=0.298, P=0.403).

[0034] The inventors believe that these significant differences strongly suggest that tRF5-30-GlyCCC-2 is not merely a simple biomarker, but may lie at the intersection of maternal metabolic health and fetal neurodevelopment. Lifestyle factors such as preconception diet and exercise are closely related to preconception weight and BMI. Unhealthy diets and lack of exercise can lead to weight gain and increased BMI, and these lifestyle factors may also affect the body's RNA expression profile, including the expression of tRF5-30-GlyCCC-2. Differential expression levels of tRF5-30-GlyCCC-2 may be related to the development and occurrence of neonatal brain injury. This provides a new perspective for the application of this biomarker in brain injury diagnosis, risk assessment, and research into its potential mechanisms.

[0035] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A tsRNA molecule tRF5-30-GlyCCC-2, characterized in that, The cDNA sequence of tRF5-30-GlyCCC-2 is shown in SEQ ID No.

1.

2. An application of the tsRNA molecule tRF5-30-GlyCCC-2 according to claim 1, characterized in that, The tRF5-30-GlyCCC-2 was used for the detection and classification of brain injury in premature infants.

3. The application as described in claim 2, characterized in that, The tRF5-30-GlyCCC-2 is derived from umbilical cord blood, whole blood, serum, plasma, or cerebrospinal fluid.

4. A tRF5-30-GlyCCC-2 testing product, characterized in that, The tRF5-30-GlyCCC-2 detection product includes the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No.

3.

5. The tRF5-30-GlyCCC-2 testing product as described in claim 4, characterized in that, The tRF5-30-GlyCCC-2 detection product also includes a control gene detection reagent.

6. The tRF5-30-GlyCCC-2 testing product as described in claim 5, characterized in that, The control gene detection reagent includes a housekeeping gene detection reagent.

7. The tRF5-30-GlyCCC-2 testing product as described in claim 5, characterized in that, The tRF5-30-GlyCCC-2 detection product also includes any one or more of RNA extraction reagents, cDNA synthesis reagents, and PCR premixes.

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