TRNA derivative fragment and application thereof

Abstract

The invention discloses a tRNA (transfer ribonucleic acid) derivative fragment and provides a potential functional application of the tRNA derivative fragment in DKD. And a tRNA derivative fragment, the tRNA derivative fragment is tRF3-IleAAT, and the nucleotide sequence of the tRNA derivative fragment is SEQ ID No. 1. The invention discloses an application of tRF3-IleAAT in preparation of a medicine for treating diabetic nephropathy and an application of a preparation for detecting the expression quantity of tRNA derived fragments in preparation of a diagnostic reagent for diagnosing diabetic nephropathy. According to the research, a new insight is provided for the molecular mechanism of the DKD from the perspective of tRFs and ferroptosis deposition, and a theoretical basis is provided for clinical diagnosis and treatment of the DKD.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to a tRNA derivative fragment and application thereof. Background Art

[0002] Diabetic nephropathy (DKD), characterized by massive proteinuria and decreased glomerular filtration rate, is one of the most common complications of diabetes and has become a global public health issue. In most developed countries, DKD is also the main cause of end-stage renal disease. Increasing evidence shows that renal fibrosis is a recognized pathological pathway of DKD. Mesangial cells are important fibrotic effector cells that can be abnormally activated and proliferate in the early stages of DKD, promoting the synthesis of extracellular matrix (ECM), which is a key factor leading to pathological changes in DKD.

[0003] tRNA-derived fragments (tRFs) are a class of small noncoding RNAs that are produced by cleavage of specific nucleases at specific sites on the tRNA loop. The development of next-generation sequencing technology has brought many major discoveries. A series of differentially expressed tRFs were found during the growth, differentiation, and injury of renal mesangial cells and podocytes treated with transforming growth factor-1; therefore, tRFs may play an important regulatory role in the development of chronic kidney disease (CKD). In the applicant's previous study, six differentially expressed tRFs were screened by high-throughput sequencing of the serum of patients with diabetes and DKD, indicating that tRFs may play an important regulatory role in DKD. However, the specific mechanism of action of tRFs in the pathogenesis of DKD remains unclear.

[0004] Ferroptosis is a novel iron-dependent form of programmed cell death characterized by increased production of reactive oxygen species (ROS), altered mitochondria, and a host of genetic and protein changes. Glutathione peroxidase 4 (GPX4) plays a crucial role as an antioxidant enzyme. GPX4 activity is activated by glutathione (GSH), which is produced by solute carrier family 7, member 11 (SLC7A11); when the antioxidant system is unstable, GSH production is affected, and the regulatory core enzyme GPX4 is further reduced and loses its antioxidant capacity. As a result, a unique form of cell death characterized by low GSH levels and high lipid peroxidation occurs. Polyunsaturated fatty acids are important targets of lipid peroxidation, and their binding to phospholipids is an important cause of ferroptosis. This binding is functionally dependent on the participation of acyl-CoA synthetase long-chain family, member 4 (ACSL4); activation of ACSL4 prompts the iron-containing enzyme lipoxygenase to produce lipid oxides, which further promotes ferroptosis. Although ferroptosis was first discovered in cancer cells, a growing body of research suggests that ferroptosis plays an important role in kidney disease. Summary of the invention

[0005] The purpose of the present invention is to provide a tRNA-derived fragment and its potential functional application in DKD.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: a tRNA-derived fragment, wherein the tRNA-derived fragment is tRF3-IleAAT, and the nucleotide sequence is SEQ ID No.1.

[0007] The invention discloses an application of a tRNA-derived fragment in the preparation of a drug for treating diabetic nephropathy. The tRNA-derived fragment is tRF3-IleAAT, and the nucleotide sequence is SEQ ID No.1.

[0008] Further drugs are used to reduce the expression of ZNF281 to inhibit ferroptosis, thereby reducing the synthesis of ECM in mesangial cells and delaying the progression of diabetic nephropathy.

[0009] Use of a reagent for promoting the expression of tRF3-IleAAT in the preparation of a drug for treating diabetic nephropathy, wherein the nucleotide sequence of the tRF3-IleAAT is SEQ ID No. 1.

[0010] A drug for treating diabetic nephropathy, comprising tRF3-IleAAT and / or an agent promoting the expression of tRF3-IleAAT, wherein the nucleotide sequence of the tRF3-IleAAT is tRF3-IleAAT of SEQ ID No. 1.

[0011] The invention discloses an application of a preparation for detecting the expression amount of a tRNA-derived fragment in preparing a diagnostic reagent for diagnosing diabetic nephropathy, wherein the tRNA-derived fragment is tRF3-IleAAT and the nucleotide sequence is SEQ ID No.1.

[0012] Furthermore, the test preparation detects the expression of tRF3-IleAAT in the serum of the subjects, and finds that the expression is significantly reduced compared with the healthy control group, so as to determine whether the subjects have diabetic nephropathy and screen the diabetic nephropathy population.

[0013] A diagnostic reagent for diabetic nephropathy, comprising: a preparation for detecting the expression amount of tRF3-IleAAT, wherein the nucleotide sequence of the tRF3-IleAAT is shown in SEQ ID NO.1.

[0014] A diagnostic kit for diabetic nephropathy, comprising the above-mentioned diagnostic reagent.

[0015] DKD is a progressive disease that 40% of diabetic patients will develop. The rising incidence of diabetes and delayed diagnosis of DKD pose challenges to the public health system; in addition, there is continued pressure to determine the pathogenesis of DKD and develop new targeted therapies. The pathological basis of DKD includes basement membrane thickening, glomerular hypertrophy, deposition of multiple ECM components, and proliferation and hypertrophy of mesangial cells as important fibroblasts. Mesangial cell proliferation, hypertrophy, and ECM deposition largely contribute to the progression of DKD. tRFs, as a new type of regulatory non-coding RNA, are widely present in various organisms and have shown great potential. Supported by next-generation sequencing technology, some studies have discovered a variety of tRFs that play a key role in the development of kidney diseases. In previous studies, the applicant's sequencing results showed that the expression of tRF3-IleAAT in the serum of DKD patients was significantly reduced, as verified by RT-qPCR of serum samples. In further functional analysis, the enrichment of cell death and metabolism attracted the applicant's attention.

[0016] Ferroptosis is a unique form of cell death that is involved in regulating the development of a variety of systemic diseases. Iron accumulation, high lipid oxidation, and mitochondrial changes are the main characteristics of ferroptosis. Iron balance determines the normal function of renal cells, and chronic renal failure is also closely related to oxidative stress and mitochondrial dysfunction. Increasing evidence shows that ferroptosis plays a vital role in kidney disease, which provides new opportunities for the diagnosis and management of DKD. Since the first description of the ferroptosis process, more and more genes or proteins involved in this cell death process have been discovered; these molecules regulate the cellular lipid peroxidation status and iron levels, thereby regulating the iron oxidation process. GPX4 can reduce intracellular lipid peroxidation, and SLC7A11 can promote the production of GSH by transporting cysteine ​​to the cytoplasm, thereby reducing the level of intracellular lipid peroxidation and inhibiting ferroptosis; these are classic pathways for regulating ferroptosis. In the field of kidney disease, high-mobility group box-1 regulates HG-induced mesangial cell ferroptosis through the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Upregulation of Nrf2 can inhibit ferroptosis, thereby delaying the progression of DKD. These findings suggest that ferroptosis plays an important role in kidney disease and that the occurrence of ferroptosis may be regulated by a variety of RNAs or target genes. For example, noncoding RNAs induce ferroptosis in acute kidney injury by directly targeting GPX4 and SLC7A11 and inducing upregulation of miR-182-5p and miR-378-3p, and their potential involvement has been confirmed in acute injury models (33). There are fewer studies on the link between ferroptosis and tRFs, but a recent study reported that alveolar macrophage-derived exosomal tRF-22-8BWS7K092 can induce ferroptosis in acute lung injury by activating the Hippo signaling pathway. The applicant innovatively proposed that tRF3-IleAAT, a tRF with reduced expression in the serum of DM and DKD patients, can inhibit ferroptosis in DKD mesangial cells, thereby reducing ECM synthesis.

[0017] Here, the applicant found that in db / db mice and HG-treated mesangial cells, tRF3-IleAAT expression was reduced and ECM synthesis was increased, and the applicant detected that ferroptosis occurred while the renal function of db / db mice was significantly reduced. The applicant observed that in the DKD model, overexpression of tRF3-IleAAT inhibited ferroptosis, reduced ECM synthesis, and alleviated renal dysfunction in db / db mice, indicating that tRF3-IleAAT is involved in the regulation of ferroptosis and fibrosis in DKD. Fer-1 is a classic ferroptosis inhibitor and is used in the DKD model. In order to further study the key role of ferroptosis in ECM synthesis in renal fibrosis, the applicant conducted ferroptosis inhibition experiments and verified its interference efficiency. The experimental results showed that Fer-1 can effectively inhibit ferroptosis in db / db mice and HG-induced mesangial cells; in addition, renal fibrosis in the DKD model was further alleviated, and ECM and collagen deposition were reduced, confirming the important role of ferroptosis in the progression of DKD. In view of these findings, the applicants preliminarily concluded that tRF3-IleAAT reduced ECM synthesis in DKD mesangial cells by inhibiting ferroptosis.

[0018] The applicant further explored the potential mechanism by which tRF3-IleAAT inhibits ferroptosis in DKD. The mode of action of tRF is similar to that of microRNA, including binding to target genes. Through bioinformatics analysis and expression level verification, the applicant obtained evidence that ZNF281 is the main downstream target gene. Zinc finger proteins are a class of transcription factors containing finger-like domains that can bind to GC-rich regions in the promoters of various genes. These proteins play a vital role in gene regulation, are key regulators of tissue development, and are significantly upregulated in human malignancies, including colon cancer and renal cancer. ZNF281 has been studied and identified as an epithelial-mesenchymal transition-inducing transcription factor (36). Silencing ZNF281 in inflammatory bowel disease can significantly reduce the expression of inflammatory factors and fibrosis genes, and eliminate inflammation-induced increases in extracellular collagen levels and morphological changes (38). The applicant detected a significant increase in the expression of ZNF281 in the DKD model, indicating that ZNF281 may also be associated with DKD renal fibrosis, and its relationship with ferroptosis has not been reported before. Since ZNF281 is a downstream target gene of tRF3-IleAAT, the applicant used ZNF281 as a starting point to conduct an in-depth study of the potential mechanism by which tRF3-IleAAT inhibits ferroptosis in DKD. After knocking down the expression of ZNF281 in HG-treated mesangial cells, the applicant found that ferroptosis was significantly inhibited. After overexpressing tRF3-IleAAT, the expression level of ZNF281 was significantly reduced, and the dual luciferase reporter experiment further verified the targeted binding of tRF3-IleAAT to ZNF281 3'UTR. Co-transfection of tRF3-IleAAT mimetic and ZNF281 plasmids largely offset the inhibitory effect of tRF3-IleAAT on ferroptosis. The applicant's data showed that tRF3-IleAAT inhibits ferroptosis by reducing the expression of ZNF281 in HG-induced mesangial cells.

[0019] This application discloses the role and mechanism of tRF3-IleAAT in DKD animal models and HG-treated mesangial cells. The applicant believes that tRF3-IleAAT inhibits ferroptosis by reducing the expression of ZNF281, thereby reducing the synthesis of ECM in mesangial cells and delaying the progression of DKD. However, the mechanism of action of ZNF281 in ferroptosis is still worthy of further in-depth study. This study has proposed new insights into the molecular mechanism of DKD from the perspective of tRFs and ferroptosis deposition, and provides a theoretical basis for the clinical diagnosis and treatment of DKD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is tRF3-IleAAT that is downregulated in the DKD model.

[0021] Figure 2 is the expression of FN and collagen I in the DKD model.

[0022] Figure 3 Overexpression of tRF3-IleAAT can reduce ECM synthesis in the DKD model.

[0023] Figure 4 Overexpression of tRF3-IleAAT can reduce the body weight, blood glucose, and expression of FN and collagen I in DKD model mice.

[0024] Figure 5 Inhibition of ferroptosis can reduce ECM synthesis in in vivo models.

[0025] Figure 6 Inhibition of ferroptosis can reduce ECM synthesis in in vitro models.

[0026] Figure 7 Overexpression of tRF3-IleAAT alleviates ferroptosis in the DKD model.

[0027] Figure 8 Overexpression of tRF3-IleAAT alleviated the expression of GPX4, SLC7A11, and ACSL4 in both in vivo and in vitro models.

[0028] Fig. 9 Knockdown of ZNF281, a downstream target gene of tRF3-IleAAT, can alleviate HG-induced ferroptosis of mesangial cells.

[0029] Fig.10 The expression of ZNF281 in in vivo and in vitro models and the knockdown of ZNF281 in in vitro experiments alleviated HG-induced ferroptosis.

[0030] Fig.11 It is tRF3-IleAAT that directly targets ZNF281 and negatively regulates its expression.

[0031] Fig.12 It is the expression of ZNF281 after overexpression of tRF3-IleAAT in the DKD model and the expression of ferroptosis-related indicators in the DKD in vivo model.

[0032] Fig.13 The significantly low expression of tRF3-IleAAT was detected in the DKD model established by high-glucose-induced mesangial cells, and the occurrence of ferroptosis was verified, which further promoted the synthesis of ECM of renal mesangial cells. Mechanistically, tRF3-IleAAT directly targets the target gene ZNF281 and negatively regulates its expression level. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0034] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0035] Animal models and therapeutic experiments

[0036] Eight-week-old male db / m and db / db mice for in vivo experiments were purchased from Jicui Yaokang (Nanjing, China). All experiments were performed with the approval of the Laboratory Animal Welfare and Ethics Committee of Nanjing Medical University (No. IACUC-2206034). Blood glucose, body weight, urine volume, and urine protein were measured at necessary time points. The DKD model was successfully established at 12 weeks and AAV intervention as well as corresponding negative control (NC) and ferrostatin-1 (Fer-1) (MCE, USA) intervention were performed (19). In summary, mice were divided into the following groups (n = 6 per group): (1) db / m group, (2) db / db group, (3) db / db+AAV tRF3-IleAAT group, (4) db / db+AAV tRF-NC group, (5) db / db+Fer-1 group, and (6) db / m+Fer-1 group. db / db+AAV tRF3-IleAAT and db / db+AAV tRF-NC mice were injected once via the tail vein with 1×10 12 AAV carrying tRF3-IleAAT or tRF3-IleAAT NC. Starting from 12 weeks of age, db / m+Fer-1 and db / db+Fer-1 mice were injected intraperitoneally with Fer-1 at a dose of 1 mg / kg, once every two days, for a total of 8 weeks. Fer-1 was dissolved in dimethyl sulfoxide (DMSO) (MCE) and further diluted to a final concentration of no more than 0.1%. Mice were anesthetized with sodium barbital (100 mg / kg) and sacrificed at 20 weeks of age, and more samples were collected for subsequent experiments.

[0037] Cell lines and culture conditions

[0038] Mouse glomerular mesangial cells (SV40-MES13) (CL-0470, batch number: BXO5T8XYYI) were purchased from Pronocell Life Science and Technology Co., Ltd. (Wuhan, China) and cultured and adapted in normal glucose (5.5 mM) DMEM containing 10% fetal bovine serum (Gibco). Cells were cultured in DMEM medium containing 10% FBS, and glucose was added to establish the HG group (30 mM), while the mannitol group (5.5 mM + 24.5 mM) was established to exclude the effect of the high osmotic pressure of HG on the cells.

[0039] Cell transfection

[0040] tRF3-IleAAT mimics, ZNF281 small interfering RNA (siRNA), and corresponding NCs were purchased from RiboBio (Guangzhou, China). ZNF281 plasmid was constructed by RiboBio for overexpression. Cells were seeded into 6-well or 12-well plates one day before transfection, and when the cell density reached 50%, transfection was performed using the RiboFect CP transfection kit (RiboBio, Guangzhou, China). The transfection mixture was prepared using specific buffers and reagents with a final concentration of 20 nM and appropriate experimental interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were performed to detect transfection efficiency. The sequences of tRF3-IleAAT and ZNF281 used for transfection are shown in Supplementary Table 1.

[0041] Table 1

[0042]

[0043]

[0044] RT-qPCR

[0045] Total RNA was extracted from kidney tissue or cells using TRIzol reagent (Invitrogen, USA). For tRF3-IleAAT, 1,000 ng of total RNA was reverse transcribed (Vazyme, Nanjing, China). Subsequently, RT-qPCR was performed for tRF3-IleAAT, and the expression level was normalized to that of U6. When measuring mRNA, cDNA synthesis and subsequent RT-qPCR were performed using HIScript III RT SuperMix (Vazyme) and ChamQ Universal SYBR qPCR Master Mix (Vazyme), respectively. β-actin was used as an internal control for normalization. All mRNA primers were purchased from GeneRay Biotechnology Co., Ltd. (Shanghai, China), and the sequences are shown in Supplementary Table 2.

[0046] Table 2

[0047]

[0048] Western blotting

[0049] Total protein from renal tissue and mesangial cells was extracted using lysis buffer (Biyuntian, Shanghai, China). The suspension was collected and lysed on ice, and then centrifuged at 12,000 × g for 30 min at 4°C. Protein concentration was determined using a bicinchoninic acid protein assay kit (Biyuntian). After quantification, 50 μg of total protein was loaded and separated by electrophoresis. The electrophoresis was then transferred to a polyvinylidene fluoride membrane (Millipore, USA). After blocking, the membrane was immersed in the primary antibody and incubated overnight at 4°C. The next day, the membrane was incubated with the secondary antibody and washed, and then the protein bands were revealed. The following primary antibodies were used for immunoblotting analysis: anti-fibronectin (ab2413, Abcam), anti-collagen I (ab34710, Abcam), anti-GPX4 (ab125066, Abcam), anti-SLC7A11 (ab175186, Abcam), anti-ACSL4 (sc-365230, Santa Cruz), and anti-ZNF281 (PAB37901, Bioswamp). The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit antibody and goat anti-mouse antibody, purchased from Abbkine (Wuhan). β-tubulin (AF7011, Affinity) was used as an internal control.

[0050] Serum creatinine (sCr) and 24-hour urine protein

[0051] The collected mouse blood and urine samples were centrifuged and the supernatant was used for further testing. The test was performed according to the instructions of the kit (Jiancheng, Nanjing, China). The blood samples were added to a 96-well plate (including standard wells, blank wells, and test wells) and incubated at 37°C for 5 minutes. The absorbance values ​​of all wells were measured at a wavelength of 546 nm, and the content of sCr in each sample (μmol / L) was calculated according to the formula in the instructions. When measuring urine protein, the 5× reagent was diluted and used, and the samples were added to the 96-well plate in turn. After the samples were added, they were mixed thoroughly and allowed to stand for a few minutes. The absorbance values ​​of all wells were measured at a wavelength of 595 nm. The urine protein concentration (mg / L) was obtained according to the calculation formula provided, and the 24-hour urine protein value was further calculated based on the 24-hour urine volume of the mice corresponding to the samples.

[0052] Immunofluorescence and immunohistochemistry

[0053] 3 μm thick paraffin-embedded renal tissue sections were prepared. The tissue sections were baked, dewaxed, hydrated, and blocked with 5% bovine serum albumin for 2 hours. The immunostained samples were incubated with the corresponding primary antibodies and images were obtained under a confocal microscope (FV1000, Olympus).

[0054] Malondialdehyde (MDA) determination

[0055] MDA content was measured using an MDA microassay kit purchased from Abbkine (Wuhan, China) to assess lipid peroxidation levels. 6 Cells, add 1 ml of pre-cooled extraction buffer. Perform ultrasonic cell disruption, then centrifuge at 13,000×g for 10 minutes at 4°C. Collect the supernatant and prepare the working reagent as needed. The sample was incubated in a 95°C water bath for 30 minutes, then centrifuged at 10,000×g for 10 minutes at room temperature. The supernatant was added to a 96-well plate, and the absorbance value of each well was measured at 532nm and 600nm wavelengths to further calculate the MDA content of each well.

[0056] GSH detection

[0057] GSH is used to help determine the redox state of cells. Collect cells or tissue samples and add them to pre-chilled extraction buffer. After lysing the sample, centrifuge and place the supernatant on ice. Prepare a 200μg / ml GSH standard and set up standard wells, blank wells, and test wells in a 96-well plate according to the kit instructions. Add the sample and measure the absorbance at a wavelength of 412nm. Draw a standard curve to calculate the GSH content in the test wells.

[0058] Ferrous ions (Fe 2+ ) determination

[0059] Fe was performed according to the manufacturer's instructions (Elabscience, Wuhan, China). 2+ Detection. Collect about 1×10 6 Cells, add 0.2 ml buffer and mix. Lyse cells on ice and centrifuge at 15,000 × g for 15 minutes. Take the supernatant for later use. Prepare 100 μmol / L standard and add it to the standard wells at different concentrations. Then add the samples to the control wells and the test wells in turn, mix and incubate. Measure the absorbance of all wells at a wavelength of 593 nm and calculate the Fe of each sample. 2+ concentration.

[0060] ROS detection

[0061] ROS levels were loaded in situ using a fluorescent probe (Biyuntian, Shanghai, China). After setting a negative reference and exploring the optimal probe concentration for the cells, the probe was diluted with serum-free culture medium at a ratio of 1:2000 to a final concentration of 5 μmol / L. After removing the cell culture medium, the probe was added to cover the cells, and then the cells were placed in a 37°C cell culture incubator with shaking for 40 minutes. The cells were washed and resuspended in phosphate-buffered saline, and the ROS levels of the cells were evaluated by flow cytometry.

[0062] Masson staining

[0063] The tissue wax blocks were cut into 3 μm thickness and then baked in an oven overnight. The sections were immersed in xylene and different concentrations of alcohol in a fume hood and then rinsed with ddH2O. The sections were immersed in Masson's solution in sequence using a masson staining kit (Seville, Wuhan, China). The sections were then rinsed and separated with 1% glacial acetic acid and sealed with neutral gum after soaking in anhydrous ethanol and xylene. Finally, microscopic examination was performed and images were collected for analysis.

[0064] Dual luciferase reporter assay

[0065] Since ZNF281 is a predicted target gene of tRF3-IleAAT, the ZNF281 mRNA 3'UTR sequence was chemically synthesized, and wild-type ZNF281 and mutant ZNF281 plasmids were constructed by RiboBio. When the cell density reached 50%, the luciferase reporter plasmid, tRF3-IleAAT mimics, and tRF-IleAAT NC were co-transfected into mesangial cells using Lipofectamine 2000. The cells were collected and lysed, and the luciferase activity was detected using a dual-luciferase reporter kit (Jima, Shanghai, China). The data were then analyzed.

[0066] Statistical analysis

[0067] All measured data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed using SPSS 20 software. Unpaired t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. P < 0.05 indicated statistical significance.

[0068] result

[0069] tRF3-IleAAT is downregulated in the DKD model

[0070] Our previous sequencing results showed that the expression of tRF3-IleAAT was significantly reduced in the serum of DKD patients, and further functional analysis showed that cell death and energy metabolism were related to tRF3-IleAAT. Here, we first verified the downregulation of tRF3-IleAAT in db / db mice ( Figure 1 a). In addition, the 24-hour urine protein and sCr levels of all mice were measured at 20 weeks of age, and it was found that the urine protein and sCr levels of db / db mice were significantly higher than those of db / m mice ( Figure 1 b). The histopathological morphology of the kidneys of the two groups of mice at 20 weeks of age was examined using an optical microscope. The histopathological morphology of the kidneys of the db / db group of mice was abnormal, with increased and significantly expanded glomerular mesangial matrix, increased interstitial collagen deposition and fibrosis ( Figure 1 c and S1a). In addition, the fluorescence of the ECM component fibronectin was significantly enhanced in the glomeruli of db / db mice ( Figure 1 d), indicating that ECM synthesis increased. The expression of fibronectin and collagen I related to ECM synthesis was detected by RT-qPCR and Western blot. The results showed that the expression of fibronectin and collagen I in the db / db group was significantly higher than that in the db / m group ( Figure 1 e, 1f and S1b). The low expression level of tRF3-IleAAT in HG-induced glomerular mesangial cells was verified by RT-qPCR ( Figure 1 g). Fibronectin and collagen I were detected by RT-qPCR and Western blot. The results showed that the expression of fibronectin and collagen I in the HG group was significantly higher than that in the control group, but there was no significant change in the mannitol group ( Figure 1 h, 1i and 2c), indicating that high osmotic pressure did not significantly affect the synthesis of ECM in mesangial cells.

[0071] Overexpression of tRF3-IleAAT attenuates ECM synthesis in DKD models

[0072] Considering that downregulation of tRF3-IleAAT is associated with ECM synthesis in the DKD model, we hypothesized that tRF3-IleAAT may have a therapeutic effect on DKD. At 12 weeks of age, the body weight, blood glucose, urine volume, and 24-hour urine protein level of db / db mice increased significantly, indicating that the model was successfully established ( Figure 4a and 4b). We overexpressed tRF3-IleAAT by tail vein injection of AAV and established the corresponding NC group. When the mice were 20 weeks old, the expression level of tRF3-IleAAT in the mice was detected. Compared with the expression level of tRF3-IleAAT in the kidney tissue of the db / db+AAVtRF-NC group, the expression level of tRF3-IleAAT in the kidney tissue of the db / db+AAV tRF3-IleAAT group was significantly increased, indicating that the overexpression was successful ( Figure 3 a).

[0073] We further evaluated the role of tRF3-IleAAT in ECM synthesis in db / db mice. To examine renal function, we measured 24-hour urine protein and sCr levels, and the results showed that sCr and 24-hour urine protein levels were reduced in the db / db+AAV tRF3-IleAAT group of mice ( Figure 3 b). We tracked the changes in body weight and blood glucose in each group of mice; however, there was no significant difference between the db / db+AAV tRF-NC group and the db / db+AAV tRF3-IleAAT group ( Figure 4 b). In terms of histopathology, Masson staining showed that the area of ​​collagen deposition was reduced in the db / db+AAV tRF3-IleAAT group compared with the db / db+AAV tRF-NC group; combined with periodic acid-Schiff (PAS) staining, this finding indicated a decrease in glomerular dilatation and fibrosis ( Figure 3 c and 4c). Immunofluorescence also showed that the fibronectin fluorescence intensity in the db / db+AAV tRF3-IleAAT group was reduced compared with that in the db / db+AAV tRF-NC group ( Figure 3 d). The mRNA and protein expression levels of the db / db+AAV tRF3-IleAAT group were significantly lower than those of the db / db+AAV tRF-NC group ( Figure 3 e, 3f and 4d). To clarify the effect of tRF3-IleAAT on ECM synthesis of HG-treated mesangial cells, tRF mimetic was used to overexpress tRF3-IleAAT in vitro, and its overexpression was verified by RT-qPCR. The results showed that the expression level of tRF3-IleAAT in HG+tRF3-IleAAT mimetic was significantly higher than that in HG+tRF3-IleAATNC group, indicating that the overexpression was successful ( Figure 3g). On this basis, we further determined the role of tRF3-IleAAT in ECM synthesis. The results showed that the mRNA and protein expression levels of fibronectin and collagen I in the HG+tRF3-IleAAT mimetic group were significantly lower than those in the HG+tRF3-IleAAT NC group ( Figure 3 g, 3h, and 4e). Inhibition of ferroptosis reduces ECM synthesis in vivo and in vitro

[0074] In our previous study, the Kyoto Encyclopedia of Genes and Genomes suggested that tRF3-IleAAT may be closely related to cell death and lipid metabolism, and its function is highly enriched in ferroptosis-related signaling pathways such as mammalian target of rapamycin, erythrocyte oncogene B, and 5'-adenosine monophosphate-activated protein kinase. Here, in order to study the role of ferroptosis in DKD, we evaluated the occurrence of ferroptosis in the DKD model. We used RT-qPCR and Western blot to determine the expression levels of ferroptosis-related indicators in mice. The results showed that in the renal tissue of the db / db group of mice, the expression level of ACSL4 was significantly higher than that of the db / m group ( Figure 5 a, 5b and 6a), while the expression levels of GPX-4 and SLC7A11 were significantly lower than those in the db / m group, confirming the occurrence of ferroptosis. Previous studies have shown that Fer-1 is a potent selective inhibitor of ferroptosis, so we observed the effects of ferroptosis on renal function and ECM synthesis by intraperitoneal injection of Fer-1 into db / db mice. RT-qPCR and Western blot showed that compared with the db / db group, the expression of ACSL4 in the db / db+Fer-1 group was significantly reduced, and the expression of GPX-4 and SLC7A11 was significantly increased ( Figure 5 a, 5b, and 6a). We focused on the key question of how renal fibrosis (including ECM synthesis) would be affected in db / db mice after ferroptosis inhibition. We were surprised to find that the 24-hour urine protein and sCr levels in the db / db+Fer-1 group were significantly lower than those in the db / db group ( Figure 5 c). In terms of histopathology, compared with the db / db group, the db / db+Fer-1 group of mice had reduced collagen deposition, reduced glomerular dilatation, and reduced fibrosis ( Figure 5 d and 6b). Further immunofluorescence showed that the fluorescence intensity of fibronectin was reduced in the db / db+Fer-1 group ( Figure 5 e). In addition, RT-qPCR showed that the expression of renal fibronectin and collagen I in the db / db+Fer-1 group was significantly lower than that in the db / db group ( Figure 5f). The results of Western blotting and RT-qPCR were consistent ( Figure 5 g and 6c).

[0075] In the cell experiment, the expression levels of ferroptosis-related indicators were also measured to evaluate the occurrence of ferroptosis. RT-qPCR and Western blot results showed that the relevant expression levels in the HG group changed significantly compared with the control group, while there was no significant difference in the mannitol group ( Figure 6 d and 6e). In addition, compared with the control group, the Fe 2+ The content and MDA levels increased significantly, while the GSH level showed a downward trend ( Figure 6 f, 6g and 6h). Flow cytometry analysis also showed that ROS levels were significantly increased in the HG group ( Figure 6 i). We further investigated the causal relationship between ECM synthesis and ferroptosis in HG-induced mesangial cells. Compared with the HG group, the changes in the expression levels of ferroptosis-related indicators in the HG+Fer-1 group were reversed, which once again demonstrated the inhibitory effect of Fer-1 on ferroptosis ( Figure 5 h, 5i and 6j). The MDA content and GSH level in the HG+Fer-1 group also changed, further clarifying the role of Fer-1 ( Figure 6 k and 6l). Next, the expression levels of fibronectin and collagen I were detected by RT-qPCR and Western blot to clarify the effect of ferroptosis on ECM synthesis. The two methods obtained the same results: the expression of fibronectin and collagen I in the HG+Fer-1 group was significantly lower than that in the HG group ( Figure 5 h, 5i and 6j). Cell experiments verified that ferroptosis is a key factor in ECM synthesis.

[0076] Overexpression of tRF3-IleAAT alleviates ferroptosis in the DKD model

[0077] It is known that overexpression of tRF3-IleAAT and inhibition of ferroptosis can reduce ECM synthesis in DKD. Here, we hypothesized that tRF3-IleAAT could inhibit ferroptosis and thus reduce ECM synthesis. Therefore, we evaluated the role of tRF3-IleAAT in the occurrence of ferroptosis in db / db mice. The results showed that the expression of GPX4 and SLC7A11 in the db / db+AAV tRF3-IleAAT group was significantly higher than that in the db / db+AAV tRF-NC group, while the expression of ACSL4 was significantly lower than that in the db / db+AAV tRF-NC group ( Figure 7 a, 7b and 8a). The GSH level in the db / db+AAV tRF3-IleAAT group was significantly increased ( Figure 4 c). In vitro, tRF3-IleAAT was first overexpressed in HG-induced mesangial cells, and then RT-qPCR and Western blot were performed to detect the expression levels of ferroptosis-related indicators. The results showed that compared with the HG+tRF3-IleAAT NC group, the expression of GPX-4 and SLC7A11 in the HG+tRF3-IleAAT mimetic group was significantly increased, while the expression of ACSL4 was significantly decreased ( Figure 7 d, 7e and 8b). The ROS level in the HG+tRF3-IleAAT mimetic group was reduced to some extent ( Figure 4 f). Transmission electron microscopy was used to examine mitochondrial morphology. Transmission electron microscopy revealed typical changes in mitochondria in the HG group, including morphological miniaturization, increased membrane density, and reduced cristae. After tRF3-IleAAT mimetic treatment, mitochondrial cristae in mesangial cells were clearly visible, and morphological changes such as atrophy and increased membrane density were alleviated ( Figure 7 g). GSH assay results showed that the GSH level in the HG+tRF3-IleAATmimetic group was significantly higher than that in the HG+tRF3-IleAAT NC group ( Figure 7 h), while Fe 2+ and MDA levels tend to decrease ( Figure 7 i and 7j), further confirming the inhibitory effect of tRF3-IleAAT on ferroptosis in the DKD model.

[0078] Knockdown of tRF3-IleAAT downstream target gene ZNF281 alleviates HG-induced ferroptosis in mesangial cells

[0079] We further explored the possible mechanism by which tRF3-IleAAT reduces ECM synthesis and inhibits ferroptosis in mesangial cells treated with DKD mice and HG. We predicted the downstream target genes of tRF3-IleAAT using the miRDB database (21) and found 26 target genes, among which ZNF281 had the highest target gene score and was highly likely to be involved in tissue cell fibrosis and inflammatory response ( Fig. 9 a). We successfully detected that the expression of ZNF281 in db / db mice was significantly increased by RT-qPCR and Western blot. The results showed that the expression level of ZNF281 was significantly increased in db / db mice ( Fig. 9 b, 9c and 10a). Immunohistochemistry results showed that ZNF281 was highly expressed in the kidney tissue of db / db mice ( Fig. 9 d). The same results were obtained in HG-induced mesangial cells ( Fig. 9e, 9f, and 10b). We speculated that tRF3-IleAAT might target ZNF281 and alleviate ferroptosis in HG-treated mesangial cells. To verify this, we knocked down the expression of ZNF281 in HG-treated mesangial cells using ZNF281 siRNA and verified the efficiency of knockdown by RT-qPCR and Western blot ( Fig. 9 g, 9h and 10c). In addition, RT-qPCR and Western blot were used to detect the expression levels of related indicators of ferroptosis and ECM synthesis to clarify the relationship between ZNF281 and ferroptosis and ECM synthesis. The results showed that the expression of ACSL4, fibronectin and collagen I was significantly reduced, and the expression of GPX4 and SLC7A11 was significantly increased in the HG+ZNF281 siRNA group. Fig. 9 g, 9h and 10c. GSH detection showed that the GSH level in the HG+ZNF281 siRNA group was higher than that in the HG+ZNF281 empty load group ( Fig. 9 i). The same results were observed in the MDA content test ( Fig. 9 j), further elucidating the correlation between ZNF281 and ferroptosis.

[0080] tRF3-IleAAT directly targets ZNF281 and negatively regulates its expression

[0081] To further clarify the interaction between tRF3-IleAAT and the target gene ZNF281, we detected the changes in ZNF281 expression after overexpression of tRF3-IleAAT in the DKD model to evaluate the effect of tRF3-IleAAT on gene expression levels. RT-qPCR and Western blot results showed that the expression of ZNF281 in the db / db+AAV tRF3-IleAAT group was significantly lower than that in the NC group ( Fig.11 a, 11b and 12a). Immunohistochemistry results showed that the expression level of ZNF281 in the db / db+tRF3-IleAATmimetic group was significantly lower than that in the db / db+tRF3-IleAAT NC group ( Fig.11 c). After overexpressing tRF3-IleAAT in HG-treated mesangial cells, we observed the same results as in vivo experiments ( Fig.11 d, 11e and 12b). Combined with the previous bioinformatics analysis results, these results indicate that ZNF281 is an effective downstream target of tRF3-IleAAT. Figure 6As shown in f, there is full base complementarity between tRF3-IleAAT and the 3'UTR of ZNF281, and the sequence is conserved. In order to confirm the targeted binding of tRF3-IleAAT to the 3'UTR of the ZNF281 gene, RiboBio constructed reporter plasmids of the ZNF281 wild-type 3'UTR (ZNF281-3'UTR-WT) and ZNF281 mutant 3'UTR (ZNF281-3'UTR-MUT) and transfected them into HEK-293T cells. These plasmids were verified by dual luciferase reporter experiments. The results showed that compared with the transfected NC (tRF3-IleAAT NC), the luciferase activity of cells was significantly reduced after co-transfection of tRF3-IleAAT mimetic plasmid and ZNF281-3'UTR-WT plasmid; on the contrary, co-transfection of tRF3-IleAAT mimetic and ZNF281-3'UTR-MUT plasmid did not significantly change the luciferase activity in cells ( Fig.11 g). In addition, after verifying the relationship between ZNF281 and ferroptosis, we also determined whether overexpression of ZNF281 could offset the protective effect of tRF3-IleAAT on ferroptosis in mesangial cells. We found that co-transfection of tRF3-IleAAT mimetic and ZNF281 plasmids largely offset the inhibitory effect of tRF3-IleAAT on ferroptosis, as evidenced by significant changes in the levels of GPX4, SLC7A11, ACSL4, fibronectin, and collagen I ( Fig.11 These data suggest that tRF3-IleAAT can inhibit ferroptosis and ECM synthesis in DKD by directly targeting ZNF281.

[0082] Mechanism Fig.13 As shown, significantly low expression of tRF3-IleAAT was detected in the DKD model established by high glucose-induced mesangial cells, and the occurrence of ferroptosis was verified, which further promoted the synthesis of ECM of renal mesangial cells. Mechanistically, tRF3-IleAAT directly targets the target gene ZNF281 and negatively regulates its expression level.

[0083] It should be understood that the present invention is described by way of example only and can be modified within the scope and spirit of the present invention. The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary skills in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A tRNA-derived fragment, characterized in that: The tRNA-derived fragment is tRF3-IleAAT, and the nucleotide sequence is SEQ ID No.

1.

2. Use of tRNA derivative fragments in the preparation of a drug for treating diabetic nephropathy, characterized in that: The tRNA-derived fragment is tRF3-IleAAT, and the nucleotide sequence is SEQ ID No.

1.

3. Use of the tRNA-derived fragment according to claim 2 in the preparation of a drug for treating diabetic nephropathy, characterized in that: The drug is used to reduce the expression of ZNF281 to inhibit ferroptosis, thereby reducing the synthesis of ECM in mesangial cells and delaying the progression of diabetic nephropathy.

4. Use of an agent for promoting tRF3-IleAAT expression in the preparation of a drug for treating diabetic nephropathy, characterized in that: The nucleotide sequence of the tRF3-IleAAT is SEQ ID No.

1.

5. A drug for treating diabetic nephropathy, characterized in that: The drug comprises tRF3-IleAAT and / or an agent promoting the expression of tRF3-IleAAT, and the nucleotide sequence of the tRF3-IleAAT is tRF3-IleAAT of SEQ ID No.

1.

6. Use of a preparation for detecting the expression of tRNA-derived fragments in the preparation of a diagnostic reagent for diagnosing diabetic nephropathy, characterized in that: The tRNA-derived fragment is tRF3-IleAAT, and the nucleotide sequence is SEQ ID No.

1.

7. The use according to claim 1, characterized in that: The test preparation detects the expression of tRF3-IleAAT in the serum of the subjects, and if the expression is significantly reduced compared with the healthy control group, it is judged that the subjects have diabetic nephropathy.

8. A diagnostic reagent for diabetic nephropathy, characterized in that include: A preparation for detecting the expression amount of tRF3-IleAAT, wherein the nucleotide sequence of the tRF3-IleAAT is shown in SEQ ID NO.

1.

9. A diagnostic kit for diabetic nephropathy, characterized in that: The diagnostic kit comprises the diagnostic reagent according to claim 8.

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