Application of tsRNA-20 or its mimics in the preparation of drugs for acute-on-chronic liver failure and / or promoting hepatocyte proliferation

Abstract

This invention relates to the field of liver failure treatment technology, specifically disclosing the application of tsRNA-20 or its mimics in the preparation of drugs for acute-on-chronic liver failure and / or promoting hepatocyte proliferation. This invention confirmed the downregulation of tsRNA expression in plasma samples and liver tissues of patients with acute-on-chronic liver failure through tsRNA sequencing and qRT-PCR. Cellular experiments revealed that tsRNA-20 regulates the ACPH / GRHL1 axis to promote hepatocyte proliferation. Specifically, it was found that tsRNA-20 binds to acyl-acylpeptide hydrolase (ACPH), thereby activating the Ras-ERK pathway and promoting the nuclear translocation of granulomatous transcription factor 1 (GRHL1), ultimately enhancing hepatocyte proliferation. Therefore, in the treatment of acute-on-chronic liver failure, tsRNA-20, its precursor tRNA-GIy-CCC, and its regulatory targets ACPH and GRHL1 can be regulated as targets to promote hepatocyte proliferation, thereby promoting liver regeneration, improving the therapeutic effect of acute-on-chronic liver failure, prolonging the survival of liver disease patients, and potentially becoming a new target for precision medicine, possessing high clinical application value.

Description

Technical Field

[0001] This invention relates to the field of liver failure treatment, and more specifically, to the use of tsRNA-20 or its mimics in the preparation of medicaments for acute-on-chronic liver failure and / or for promoting hepatocyte proliferation. Background Technology

[0002] Acute-on-chronic liver failure (ACLF) is a common end-stage liver disease characterized by a rapid deterioration of liver function within a short period and a very high short-term mortality rate. Currently, there are no effective treatments, making it one of the major diseases threatening the health of Chinese residents. Hepatocyte regeneration and repair remain one of the main pathways for liver recovery during chronic liver injury, and promoting liver regeneration is an effective treatment strategy. Therefore, there is an urgent clinical need to develop non-transplant medicine treatments, including cell-based liver regeneration therapies, to intervene in the progression of liver failure as quickly as possible. However, the molecular basis of ACLF is not yet fully understood, and further research into its molecular mechanisms is needed to identify new diagnostic and therapeutic targets.

[0003] tRNA-derived RNAs (tsRNAs) are a novel class of small non-coding RNAs (sncRNAs) produced through specific cleavage of mature or precursor transfer RNAs, regulating gene expression at both the transcriptional and translational levels. Increasing evidence supports the hypothesis that tsRNAs play diverse roles in numerous regulatory pathways and possess evolutionarily conserved functions in various molecular processes, such as gene regulation, RNA processing, protein biosynthesis, and oncogenic transformation. Some tsRNAs are also associated with the proliferation, migration, and invasion of certain types of cancer cells. Nevertheless, few studies have explored the effects of tsRNAs on liver injury or hepatocyte proliferation, and the functional roles and potential mechanisms of tsRNAs in acute-on-chronic liver failure remain largely unexplored.

[0004] Therefore, in order to overcome the gaps in existing treatment technologies, it is necessary to find new mechanisms and targets for acute-on-chronic liver failure or hepatocyte proliferation in order to improve treatment plans and enhance treatment efficacy. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of at least one of the prior art mentioned above, and provides the application of tsRNA-20 or its mimics in the preparation of drugs for acute-on-chronic liver failure and / or promoting hepatocyte proliferation. By binding to ACPH and regulating its expression in hepatocytes, it promotes the nuclear translocation of GRHL1, enhances hepatocyte proliferation, and improves the therapeutic effect of acute-on-chronic liver failure. Furthermore, based on the relationship between tsRNA-20, ACPH, Ras / ERK, and GRHL1 revealed by this invention, it can provide new targets for liver regeneration therapy of acute-on-chronic liver failure, provide new strategies for promoting hepatocyte proliferation, and fill the gaps in the prior art.

[0006] One object of the present invention is to provide the use of tsRNA-20 or its mimics in the preparation of drugs for acute-on-chronic liver failure and / or promoting hepatocyte proliferation, wherein the nucleotide sequence of tsRNA-20 or its mimics includes GCATT GGTGGTTCAG TGGTA GAATT CTCGC CT. In one embodiment of the present invention, it was experimentally found that synthetically produced tsRNA-20 mimics significantly reduced the cytotoxic effects of exosomes from patients with acute-on-chronic liver failure (ACLF) on cultured human normal hepatocyte L02 cells. Therefore, tsRNA-20 or its mimics can be used in corresponding drugs for acute-on-chronic liver failure and / or promoting hepatocyte proliferation.

[0007] Furthermore, tsRNA-20 or its mimics bind to ACPH and promote GRHL1 nuclear translocation, thereby promoting hepatocyte proliferation. In one or more embodiments of the present invention, the relevant mechanism was experimentally verified, revealing that tsRNA-20 binds to ACPH and regulates its expression in hepatocytes, thereby activating the Ras-ERK pathway and promoting GRHL1 nuclear translocation, ultimately enhancing the proliferation of normal hepatocytes.

[0008] Another object of the present invention is to provide the use of tsRNA-20 promoters, ACPH promoters, and / or GRHL1 promoters in the preparation of drugs for treating acute-on-chronic liver failure and / or promoting hepatocyte proliferation. Based on the above findings, tsRNA-20 promoters have the potential for use in the preparation of ACLF drugs. In the embodiments of the present invention, the expression levels of ACPH and GRHL1 in liver tissue and plasma exosomes of ACLF patients and in ACLF mouse models were significantly lower than those in the corresponding control groups; therefore, ACPH promoters and / or GRHL1 promoters also have the potential for use in the preparation of ACLF drugs.

[0009] Another object of the present invention is to provide the use of human tRNA-GIy-CCC, ACPH, and / or GRHL1 as targets in the preparation of drugs for treating acute-on-chronic liver failure and / or promoting hepatocyte proliferation. Human tRNA-GIy-CCC is a precursor of tsRNA-20, which in the human body is formed by the cleavage of mature tRNA-Gly-CCC.

[0010] Another object of the present invention is to provide a medicament for treating acute-on-chronic liver failure, comprising tsRNA-20, tsRNA-20 mimics, tsRNA-20 promoters, ACPH promoters and / or GRHL1 promoters; wherein the nucleotide sequence of said tsRNA-20 or its mimics comprises GCATT GGTGG TTCAG TGGTA GAATT CTCGC CT.

[0011] Another object of the present invention is to provide a drug for promoting hepatocyte proliferation, characterized in that it comprises tsRNA-20, tsRNA-20 mimics, tsRNA-20 promoters, ACPH promoters and / or GRHL1 promoters; wherein the nucleotide sequence of said tsRNA-20 or its mimics comprises GCATT GGTGG TTCAG TGGTA GAATT CTCGC CT.

[0012] Another object of the present invention is to provide a kit for detecting tsRNA-20, characterized in that it comprises one or more of the following: nucleic acid extraction reagent, RT primers, qPCR primers, and gene-specific probes;

[0013] The sequences of the RT primers include: GTCGT ATCCA GTGCA GGGTC CGAGG TATTC GCACT GGATACGACA GGCGA;

[0014] The sequences of the qPCR primers include: F': TTGGT GGTTC AGTGG TAGAA TTC; R': AGTGCAGGGT CCGAG GTATT;

[0015] The sequences of the gene-specific probes include: Sense: GCAUU GGUGG UUCGU GGUAG AAUUCUCGCC U; Antisense: AGGCG AGAAU UCUAC CACUG AACCA CCAAU GC.

[0016] Since the embodiments of this invention have demonstrated the important role of tsRNA-20 in promoting hepatocyte proliferation, the kit for detecting tsRNA-20 has certain application potential and can be widely used in the study of related signaling pathways and in medical experiments and clinical trials of various liver diseases.

[0017] Another object of the present invention is to provide a shRNA that knocks down tsRNA-20, the nucleotide sequence of which includes CCACCAATGC.

[0018] Another object of the present invention is to provide an ACPH knockdown shRNA, the nucleotide sequence of which includes GAACACTTTG ATGCA AGCCA T.

[0019] In the comparative examples of this invention, shRNAs with knockdown of tsRNA-20 and ACPH were used for experimental verification of the relevant mechanisms. Since the embodiments of this invention have demonstrated the important role of tsRNA-20 in promoting hepatocyte proliferation, shRNAs with knockdown of tsRNA-20 and ACPH have certain application potential and can be widely used in research on related signaling pathways and in medical experiments and clinical trials for various liver diseases.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: it reveals that tsRNA-20 promotes hepatocyte proliferation through the ACPH / GRHL1 regulatory axis, thus providing a new strategy and target for the treatment of acute-on-chronic liver failure. Specifically, it was found that tsRNA-20 binds to ACPH and regulates its expression in hepatocytes, thereby activating the Ras-ERK pathway and promoting the nuclear translocation of GRHL1, ultimately enhancing the proliferation of normal hepatocytes. Therefore, in the treatment of acute-on-chronic liver failure, tsRNA-20, its precursor tRNA-GIy-CCC, and its regulatory targets ACPH / GRHL1 can be regulated as targets to promote hepatocyte proliferation, thereby promoting liver regeneration, improving the therapeutic effect of acute-on-chronic liver failure, and prolonging the survival of liver disease patients, becoming a new target for precision treatment with high clinical application value. Furthermore, the revealed relationship between tsRNA-20, ACPH, and GRHL1 can also provide a research basis for further improving the therapeutic effect of acute-on-chronic liver failure and promote the development of the field of liver disease treatment. Attached Figure Description

[0021] Figure 1 The study showed that tsRNA-20 was downregulated in plasma exosome samples and liver tissue from ACLF patients, and was also downregulated in in vitro and in vivo mouse models of ACLF.

[0022] Figure 2 Overexpression of tsRNA-20 promotes hepatocyte proliferation and restores the vitality of damaged hepatocytes, while knockdown of tsRNA-20 inhibits hepatocyte proliferation.

[0023] Figure 3 The study showed that tsRNA-20 binds to ACPH and regulates its expression in hepatocytes.

[0024] Figure 4 The results showed that ACPH promotes hepatocyte proliferation by activating the Ras pathway, while knocking down ACPH inhibits hepatocyte proliferation.

[0025] Figure 5 The study showed that knocking down tsRNA-20 can inhibit hepatocyte proliferation by reducing GRHL1 levels.

[0026] Figure 6The results show that ACPH interacts with GRHL1, promoting its nuclear translocation in hepatocytes.

[0027] Figure 7 The study showed that tsRNA-20 regulates hepatocyte proliferation through the ACPH / GRHL1 regulatory axis under both normal and ACLF conditions. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. If the specific experimental conditions are not specified in the embodiments, they are generally performed according to conventional conditions or the conditions recommended by the reagent company. Unless otherwise specified, the reagents, consumables, etc. used in the following embodiments are all commercially available.

[0031] 1. Patients and clinical samples

[0032] This invention selected ACLF patients from the Third Affiliated Hospital of Sun Yat-sen University between December 2019 and December 2022.

[0033] Fresh liver tissue specimens from 11 healthy subjects (n=3) and healthy subjects (n=3), as well as plasma samples from 49 ACLF patients and 52 healthy subjects. All specimens were frozen and stored at -80°C immediately after collection.

[0034] For plasma samples, plasma exosomes were isolated using the Total Exosome Isolation Kit (Cat # 4484450, Invitrogen, USA) according to the manufacturer's protocol. Then, 4 × 10 5 L02 cells were grown to 70%-80% confluence and then cultured with exosomes from ACLF patient plasma for 24 hours to construct an in vitro model.

[0035] 2. Cell lines

[0036] Normal human hepatocyte lines L02 and THLE-2 were obtained from the Type Culture Collection Center of the Chinese Academy of Sciences (Shanghai, China). The hepatocyte lines were grown in DMEM (Gibco) medium supplemented with 10% FBS (CellCook, CM1002L). Human kidney epithelial cells (293T) were cultured in DMEM (Gibco) medium supplemented with 10% FBS (CellCook, CM1002L). Cell lines were incubated in a humidified incubator at 37°C and 5% CO2. Cells were seeded at a density of 20%–40% in 6-well plates. After 24 hours, tsRNA-20 mimics or control mimics (Ribobio, Guangzhou, China) (100 nM) were transfected into the cells using Lipofectamine 3000 (Thermo Fisher, USA). The tsRNA mimic sequence was identical to the tsRNA. The siRNAs targeting GRHL1 (HSS120977, HSS120978, and HSS179071) were purchased from Thermo Fisher Scientific (Cat # 1299001), with HSS120977 used to knock down GRHL1. Cell lines were transiently transfected with the specified siRNAs or negative control siRNAs (Thermo Fisher Scientific, Cat # 12935300).

[0037] 3. Plasmid and lentivirus packaging

[0038] The pGreen-sh-tsRNA20 lentiviral plasmid was constructed according to the manufacturer's protocol using a pGreenPuro-shRNA cloning and expression lentiviral vector (Cat#s SI505A-1; System Biosciences, USA). In short, we designed and constructed the sh-tsRNA20 sequence (5′-CCACCAATG-3′) targeting tsRNA-20. A control shRNA (sh-NC) was used as the negative control (5′-TTCTCCGAGGTCACGT-3′). Lentiviral packaging systems for helper plasmids (pMD2.G and psPAX2 plasmids) and target plasmids (pGreen-sh-tsRNA20, pCDH-3×Flag-GRHL1, pCDH-3×HA-ACPH, and pLKO.1-sh-ACPH) were ordered from Igebio. For lentiviral packaging, a mixture of Lipofectamine 3000 (Invitrogen) and 2 μg target plasmid, 1.5 μg envelope-encoding pMD2.G plasmid, and 2 μg packaging vector psPAX2 plasmid was used for transfection at a rate of 6 × 10⁶. 6 293 T cells. Culture supernatant containing the packaged virus was harvested 48 hours post-transfection. For viral infection, hepatocyte (L02 and THLE-2 cells) confluence plates were mixed with 200 μl of virus (2 × 10⁻⁶ cells / mL). 5The transfected cells were incubated with 6 μg / ml puromycin (transduction units / ml) and 6 μg / ml polybrene as a transduction enhancer for 48 hours. After infection, hepatocytes were grown in a medium containing 0.5 μg / ml puromycin, which was changed every three days to eliminate untransfected cells. After 10 days, hepatocytes were grown in a medium without puromycin.

[0039] 4. Construction of ACLF mouse model

[0040] Wild-type (WT) 6-8 week old male C57BL / 6 mice (GemPharmatech, Guangdong, China) were used. Mice were housed in a specific pathogen-free environment with a 12-hour light / dark cycle and free access to standard mouse food and water. To induce liver fibrosis, mice were administered 5 mL / kg CCl4 dissolved in olive oil (v / v, 10%, Sigma-Aldrich) via intraperitoneal injection (ip) twice weekly for 8 weeks. Following this, 72 hours later, mice were injected with a high dose of 5 mL / kg CCl4 (v / v, 50%). Mice injected with the same dose of olive oil served as controls.

[0041] 5. RNA extraction, reverse transcription, and quantitative real-time PCR

[0042] RNA was extracted from cell lines, plasma exosomes, and preserved ACLF tissue using the AG RNAex Pro RNA Trizol regent (Akara, Cat# AG21102) according to the specific instructions. Relatively pure RNA was obtained after treatment with chloroform, isopropanol, and ethanol. The RNA solution was centrifuged several times during this process. RNA concentration was then determined using Nanodrop, with OD values ​​between 1.8 and 2.0 at a 260 / 280 absorbance ratio. tsRNA expression was analyzed using stem-loop qRT-PCR. The stem-loop primers used for reverse transcription and the primers used for qRT-PCR are listed in Table 2. First-strand cDNA was prepared using the PrimeScript™ RT kit (Takara, Cat# RR037A) according to the specific instructions. Three qPCR programs were performed using TB Green® Ex Taq™ II (Takara, Cat# RR820A). RNA expression was normalized to GAPDH or U6, and gene expression was explored using the ΔΔCt method for relative quantification (RQ). Quantitative RT-PCR was performed using SYBR Green dye from Applied Biosystems. The PCR reaction was performed in triplicate.

[0043] 6. RNA-seq analysis

[0044] L02 cells with tsRNA-20 knockdown and negative control cells were cultured in 6 cm culture dishes. When the cells reached near-complete confluence in the culture dishes, they were lysed, centrifuged, and RNA was isolated using TRIzol (Akerui, Cat #AG21102, Changsha, China). RNA samples were analyzed at the Annoroad Genome Institute in Beijing. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (Cat# E7530L, NEB, USA) according to the manufacturer's instructions, and index codes were added to associate sequences with each sample. Differentially expressed genes were identified using SPORT, followed by functional enrichment analysis.

[0045] 7. RNA pull-down analysis and mass spectrometry detection

[0046] Synbio Technologies synthesized biotin-tagged sense and antisense tsRNA-20 probes. First, 50 μL of streptavidin-tagged Dynabeads (Cat#88802; Thermo Fisher Scientific) were washed three times with binding buffer, resuspended in 200 μL buffer, and then incubated at 4°C for 4 h on a shaker with sense and antisense RNA or blank Dynabeads as a negative control. Subsequently, 2 mg of protein lysate from THLE-2 cells was added, and the mixture was incubated overnight at 4°C by rotation. After washing three times with phosphate-buffered saline (PBS) on a magnetic separation rack, the RNA-protein complex was resuspended in PBS and denatured for 10 min at 99°C in SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE using a 10% gel and visualized using a silver staining kit (Cat#24600; Thermo Fisher, USA). Different protein bands were excised and analyzed by mass spectrometry. Fitgene Biotech used MM File Conversion to process the raw spectral data and analyzed protein identities using Mascot software (version 2.6.0) against the Human UniProt database (https: / / www.uniprot.org / ).

[0047] 8. Protein blotting

[0048] Protein extracts were prepared using RIPA lysis buffer (P0013K, Beyotime, Shanghai, China) containing a protease inhibitor (Roche, P8340, Shanghai, China) and a phosphatase inhibitor (Roche, P0044, Mannheim, Germany). The extracts were incubated on ice for 30 min and then centrifuged at 13,000 × g and 4°C for 10 min. Proteins were then quantified using the Pierce® BCA Protein Assay Kit (Thermo Scientific, USA). Cell lysates were then subjected to 10% tricine-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electron blotting on a 0.2 μM nitrocellulose (NC) membrane (Millipore). After incubation with the target and secondary antibody, the membrane was chemiluminescently visualized using a gel imaging system (Syngene G: BOXF3, USA). GRHL1 antibody was purchased from Sigma-Aldrich (HPA005798). ACPH antibody was purchased from Bethyl (A304-808A). ERK1 / 2 pERK1 / 2 antibodies were purchased from Cell Signaling Technology (CST4695, CST4370). The Ras activation assay kit (BK008-S) containing Pan-Ras antibody was purchased from Cytoskeleton. Rabbit anti-β-actin antibody (CST, 4970) and rabbit anti-GAPDH antibody (CST, 5174) were used as endogenous controls.

[0049] 9. Cell counting kit 8 (CCK-8) assay

[0050] Cell viability was measured using a CCK-8 assay kit (Beyotime Biotechnology, C0038, Shanghai, China). Forty-eight hours after transfection, cells were seeded in 96-well plates at a density of approximately 2 × 10³ cells per well. After discarding the original culture medium, 10 μl of CCK-8 solution and 100 μl of complete culture medium were added to each well at fixed intervals. OD values ​​(450 nm) were measured. All measurements were repeated three times.

[0051] 10. EdU detection

[0052] The Cell-Light EdU Apollo in vitro kit (Beyotime, Guangzhou, China) for EdU assay was used to compare growth capabilities. First, L02 and THLE-2 cell lines were transfected with target RNA or plasmids. Subsequently, 4 × 10⁻⁶ cells were... 3One transfected cell was seeded into each well of a 24-well plate. EdU solution was diluted with complete culture medium to a final concentration of 50 μM. This EdU medium was then added to each well, and the cells were incubated for 2 hours. After fixation, permeabilization, and staining, images were captured using a fluorescence microscope.

[0053] 11. Cloning

[0054] For cell proliferation assays involving target gene overexpression or knockdown, cells were stored at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 10% in 5 mL of medium supplemented with 10% fetal bovine serum (FBS). The medium was changed every 3 days. After 10–14 days of culture, the cells were fixed in 4% formaldehyde and stained with 0.1% crystal violet. ImageJ software v1.8 (National Institutes of Health, Bethesda, MD, USA) was used to count the number of colonies.

[0055] Experimental study on the expression levels of tsRNA-20 in ACLF plasma exosomes and liver tissue

[0056] tsRNA sequencing was performed on plasma exosomes from 3 healthy subjects (labeled N) and 5 ACLF patients (labeled L). Figure 1 Sequencing results showed that all 16 differentially expressed tsRNAs were downregulated in ACLF patients (|FC|≥2; P<0.05), among which tsRNA-5' derived from the 5' end of mature tRNA was the most abundant tsRNA. Figure 1 B). Among these candidate tsRNAs, tsRNA-20 (tRNA-Gly-CCC-1-1) met the predetermined threshold for significant expression levels determined in the study. Figure 1C shows a schematic diagram of the predicted tsRNA-20 cleavage from mature tRNA-Gly-CCC. Furthermore, we detected tsRNA-20 expression levels in patient plasma exosomes and liver tissue, confirming that the expression level of tsRNA-20 in ACLF plasma exosomes (n=49) was significantly reduced compared to normal plasma exosome samples (n=52) (P<0.0001). Figure 1 D). We also conducted a retrospective nested case-control study. Initially, three patients were diagnosed with acute hepatitis B virus flare (HBVflare) and developed acute-on-chronic liver failure (ACLF) within 4 weeks. Their plasma samples were collected and analyzed every three days. Our analysis showed that the tsRNA-20 level in plasma exosome samples during the ACLF phase was significantly lower than that in samples collected at the initial stage of HBV flare (P<0.01). Figure 1E). Samples from the same patient at different time points were identified using patient ID. Furthermore, the expression level of tsRNA-20 in ACLF liver tissue (n=11) was significantly lower than that in normal tissue (n=3) (P<0.05). Figure 1 F). Looking at the above experiments, substantial evidence suggests that tsRNA-20 expression is downregulated in acute-on-chronic liver failure (ACLF), consistent with our sequencing results, indicating that it may play a crucial role in the pathogenesis of ACLF. Therefore, tsRNA-20 was chosen as the subject of this study.

[0057] In addition, we established in vitro and in vivo ACLF models. First, L02 hepatocytes were incubated overnight with plasma exosomes from ACLF patients to establish an in vitro ACLF model. The results showed that, compared with the negative control, the level of tsRNA-20 in the ACLF model was significantly reduced (P<0.01). Figure 1 G). Next, mice injected with CCl4 were used as an in vivo ACLF model. This experiment found that the expression level of tsRNA-20 in the exosomes of primary ACLF mouse hepatocytes was significantly lower than that in wild-type (WT) mouse hepatocytes (P<0.0001) (Figure 1H).

[0058] The experiment showed that the 2tsRNA-20 mimic significantly reduced the inhibitory effect of exosomes on L02 cell proliferation in patients with acute-on-chronic liver failure (ACLF).

[0059] To assess the biological function of tsRNA-20 in ACLF, we performed gain-of-function and loss-of-function experiments in hepatocytes. In the loss-of-function experiment, tsRNA-20 sh-RNA and a negative control (sh-NC) lentivirus were synthesized and transfected into L02 and THLE-2 cells, respectively. Transfection efficiency was assessed by qRT-PCR, and the results are shown below. Figure 2 As shown in Figure A, the knockdown is reliable. CCK-8 assays showed that tsRNA-20 knockdown significantly inhibited normal cell proliferation (…). Figure 2 B). Flow cytometry analysis of the cell cycle showed that knockdown of tsRNA-20 arrested the cell cycle at the G0 / G1 phase. Figure 2 C). Gain-of-function experiments using transfected tsRNA-20 mimics showed that EdU and colony formation assays indicated that tsRNA-20 knockout inhibited cell proliferation, while tsRNA-20 overexpression enhanced cell proliferation, with similar results observed in L02 and THLE-2 cells. Figure 2DE). The cell proliferation capacity of tsRNA-20 in an in vitro ACLF cell model was then detected using the EdU assay. Plasma exosomes derived from ACLF patients were incubated with human L02 hepatocytes transfected with and without tsRNA-20 mimics, respectively. The results showed that exosomes from ACLF patients were significantly detrimental to L02 cells, while the introduction of tsRNA-20 mimics greatly reduced the inhibitory effect on cell growth, indicating that tsRNA-20 promotes the recovery of viability in damaged hepatocytes. Figure 2 F).

[0060] The experiment investigated the interaction between 3tsRNA-20 and ACPH, regulating its expression in hepatocytes.

[0061] After synthesizing biotin-labeled tsRNA-20 sequence probes, this invention uses a sense strand probe sequence comprising: rGrCrArUrUrGrGrUrGrGrUrUrCrGrUrGrGrUrArGrArArUrUrCrUrCrGrCrCrUrUr, and an antisense strand probe sequence comprising: rArGrGrCrGrArGrArArUrUrCrUrArCrCrCrArCrCrCrArArUrGrC. RNA pull-down analysis was performed, followed by mass spectrometry protein analysis to identify proteins interacting with tsRNA-20 in L02 and THLE-2 cells. Figure 3 A). This study shows that tsRNA-20 co-binds with multiple RNA-binding proteins, with ACPH exhibiting the highest binding affinity. RNA immunoprecipitation (RIP) experiments also showed that tsRNA-20 was significantly enriched in RNA after ACPH antibody immunoprecipitation (Figure 3B). Subsequently, we assessed the expression level of ACPH in hepatocyte cell lines. After transfecting L02 and THLE-2 cell lines with sh-tsRNA-20 lentiviral plasmid and tsRNA-20 mimic, we observed that tsRNA-20 overexpression led to upregulation of ACPH protein expression, while knockout of tsRNA-20 decreased ACPH protein expression ( ). Figure 3 CD).

[0062] In addition, we evaluated ACPH expression in human plasma exosomes and liver tissue samples from ACLF patients and healthy controls. As expected, ACPH protein levels in all ACLF samples were significantly lower than in control samples. Figure 3 E, F). In vivo ACLF mouse models also showed that, compared to wild-type samples, ACPH expression was significantly reduced in ACLF samples (E, F). Figure 3 G).

[0063] Experiment 4: Overexpression of ACPH activates the Ras pathway and promotes hepatocyte proliferation

[0064] To evaluate the biological function of ACPH in ACLF, we performed gain-and-loss-of-function experiments in hepatocytes. ACPH sh-RNA, ACPH-overexpressing lentivirus, and negative control (sh-NC) lentivirus were synthesized and transfected into L02 and THLE-2 cells, respectively. Transfection efficiencies are shown in [Figure number missing]. Figure 4 AB. EdU and colony formation assays showed that in THLE-2 and L02 cells, ACPH overexpression promoted hepatocyte proliferation, while ACPH knockout inhibited cell proliferation. Figure 4 CF). To further elucidate the role of tsRNA-20 in regulating cell proliferation through its interaction with ACPH, we conducted a rescue experiment. The results showed that overexpression of ACPH significantly reduced the inhibitory effect of sh-tsRNA-20 on THLE-2 cell proliferation (CF). Figure 4 Based on the above results, we believe that the effect of tsRNA-20 on hepatocyte proliferation is at least partially dependent on ACPH.

[0065] To elucidate the potential pathways by which tsRNA-20 plays a role in ACPH-mediated hepatocyte proliferation, we performed RNA-seq analysis. KEGG pathway analysis revealed that the target molecules of downregulated tsRNA-20 were primarily enriched in the MAPK signaling pathway. Ras pull-down assays showed that ACPH overexpression significantly enhanced Ras enzyme activity compared to the negative control, characterized by increased GTP-bound forms and elevated total Ras protein levels. Figure 4 The above results indicate that ACPH can rescue the inhibitory effect of decreased tsRNA-20 on hepatocyte proliferation by activating the Ras signaling pathway.

[0066] Experiment 5 showed that knocking down tsRNA-20 could inhibit hepatocyte proliferation by reducing GRHL1 levels in ACLF.

[0067] To explore target genes regulated by tsRNA-20, we also performed RNA-seq analysis on L02 cells treated with exosomes from ACLF patients. By performing overlap analysis on differentially expressed RNAs in the two groups of L02 cells (control and ACLF exosomes; sh-NC and sh-tsRNA-20), we identified 12 overlapping RNAs as candidate RNAs. Figure 5A), namely GRHL1, FAM106A, SERPINB3, RAD9B, NOG, INHBE, LXN, CREB3L3, IGLON5, AP001029.1, SPRED1, NR1D2, and RPS6KL. Among them, GRHL1 showed the highest downregulation after tsRNA-20 knockout, which was verified by qRT-PCR. Figure 5 B). Flow cytometry was used to detect cell cycle progression, and EdU and colony formation assays were employed to investigate the biological function of GRHL1. First, we transduced lentiviruses to overexpress GRHL1 or transfected GRHL1-targeting siRNA to knock it out in THLE-2 and L02 cell lines. We found that in both cell lines, overexpression of GRHL1 enhanced cell proliferation and increased the number of cells in S phase, while downregulation of GRHL1 inhibited cell proliferation. Figure 5 Furthermore, the expression levels of GRHL1 in liver tissue and plasma exosomes of ACLF patients, as well as in ACLF mouse models, were significantly lower than those in the corresponding control groups (CE). Figure 5 F, G, H). Furthermore, rescue experiments showed that co-transfection of THLE-2 cells with the GRHL1 overexpression vector and sh-tsRNA-20 significantly increased cell proliferation, indicating that the biological function of tsRNA-20 is partially dependent on GRHL1 (F, G, H). Figure 5 I).

[0068] In conclusion, tsRNA-20 gene knockdown-induced GRHL1 gene downregulation may play an important role in the pathogenesis of ACLF.

[0069] Experiment 6 showed that ACPH interacts with GRHL1, promoting its nuclear translocation in hepatocytes.

[0070] To fully elucidate their underlying mechanisms, we performed subcellular isolation assays. The results showed that tsRNA-20 and ACPH were primarily located in the cytoplasm, while GRHL1 was primarily located in the nucleus. Figure 6 A). The results showed that overexpression of ACPH not only increased the expression level of ACPH but also upregulated the expression of GRHL1. Conversely, knockdown of the ACPH gene led to the suppression of both ACPH and GRHL1 expression. Notably, co-transfection of overexpressed ACPH with sh-tsRNA-20 into THLE-2 cells effectively rescued the reduction in GRHL1 caused by tsRNA-20 downregulation. Figure 6 B). To determine whether ACPH directly interacts with GRHL1, we first used protein binding prediction software to predict that they could directly bind (B). Figure 6C, D). Subsequent immunofluorescence analysis showed that ACPH overexpression promoted the translocation of GRHL1 from the cytoplasm to the nucleus in hepatocytes (C, D). Figure 6 These results indicate that ACPH, which activates the Ras / ERK pathway, is an upstream regulator of GRHL1.

[0071] Overall, this embodiment found that tsRNA-20 promotes hepatocyte proliferation by regulating the ACPH / GRHL1 axis, such as Figure 7 As shown in the figure. Mechanistically, the inventors discovered and experimentally verified that tsRNA-20 binds to ACPH and regulates its expression in hepatocytes; GRHL1 is a key target gene in the tsRNA-20-induced transcriptomic changes; furthermore, it was demonstrated that overexpression of ACPH significantly increased Ras-GTPase activity, indicating that ACPH overexpression alleviated the inhibitory effect of sh-tsRNA-20 on hepatocyte proliferation by activating the Ras / ERK signaling pathway; it was also confirmed that the levels of ACPH and GRHL1 were downregulated in liver tissue and plasma exosomes of ACLF patients, while the upregulation of ACPH and GRHL1 levels promoted hepatocyte proliferation. These findings suggest that the downregulation of ACPH and GRHL1 levels caused by tsRNA-20 downregulation plays an important role in the pathogenesis of ACLF, providing new insights into the pathogenesis of ACLF.

[0072] In terms of drug intervention, the introduction of tsRNA-20 mimics in in vitro experiments significantly reduced the toxicity of exosomes from ACLF patients to L02 cells and promoted their proliferation. Therefore, tsRNA-20 or its mimics, as well as tsRNA-20 promoters, can be used in the preparation of ACLF drugs. Furthermore, since the expression levels of ACPH and GRHL1 in liver tissue and plasma exosomes of ACLF patients, as well as in ACLF mouse models, were significantly lower than those in the corresponding control groups, and this invention demonstrates that overexpression of ACPH and GRHL1 can promote the proliferation of normal hepatocytes, ACPH promoters and / or GRHL1 promoters also have the potential for application in the preparation of ACLF drugs.

[0073] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The use of tsRNA-20 or its mimics in the preparation of medicaments for treating acute-on-chronic liver failure, characterized in that, The nucleotide sequence of the tsRNA-20 or its mimic is GCATT GGTGG TTCAG TGGTA GAATT CTCGC CT.

2. The application according to claim 1, characterized in that, tsRNA-20 or its mimics bind to ACPH and promote GRHL1 nuclear translocation, thereby promoting hepatocyte proliferation.

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