YL1-tiRNA for promoting mouse muscle cell injury repair

Abstract

A YL1-tiRNA for promoting mouse muscle cell injury repair belongs to the technical field of molecular biology, the YL1-tiRNA is formed by cutting mature mt-Tv, a phosphate group is added at the 5th terminal, phosphorylation modification is carried out on the sequence, and a section of sequence is added at the 5th terminal without changing the function of the sequence, the YL1-tiRNA is characterized in that the nucleotide sequence of the YL1-tiRNA is shown as Seq ID No: 1, and the nucleotide sequence of the YL1-tiRNA is shown as Seq ID No: 1. Experiments prove that the YL1-tiRNA can promote mouse muscle injury repair by regulating proliferation of C2C12 cells.

Description

Technical Field

[0001] This invention relates to a YL1-tiRNA that promotes the repair of damaged muscle cells in mice, belonging to the field of molecular biology technology. Background Technology

[0002] Skeletal muscle is the largest tissue in mammals and humans (accounting for approximately 40% of the body), influencing movement, metabolism, and homeostasis. Skeletal muscle development involves the proliferation, fusion, and differentiation of myoblasts to form myotubes, which subsequently develop into mature muscle fibers. Postnatal skeletal muscle development depends on satellite cells located beneath the basement membrane. The proliferation and differentiation of skeletal muscle satellite cells is highly complex, involving the expression and network regulation of numerous genes. Besides the regulatory role of myogenic regulatory factors (MRFs), many small non-coding RNAs (ncRNAs), such as miRNAs and tiRNAs, also participate in the regulation of this process. Therefore, research on the regulation of muscle satellite cell proliferation and differentiation can provide a theoretical basis for the study of muscle development mechanisms. tsRNA is a small RNA fragment produced by specific nucleases (such as Dicer and ANG) cleaving into tRNA loops in specific cells / tissues or under specific conditions (such as cellular stress). It is produced from pre-tRNA or mature tRNA and is an RNA with a precise sequence structure and specific biological function. Based on their cleavage site and length, tsRNAs are mainly divided into tRNA-derived stress-inducible RNAs (tiRNAs) and tRNA-derived fragments (tRFs). tRFs and tiRNAs perform various biological functions as sncRNAs. They can act as miRNAs to interfere with RNA;tiRNAs replace the translation initiation factor eIF4G that binds to mRNA, directly inhibiting protein translation. tiRNAs belong to the short non-coding RNA family, with a length of 30-50 nt. tiRNAs are produced by the specific cleavage of the anticodon loop of mature tRNA by angiopoietin (ANG, a stress-activated ribonuclease) under conditions of hypoxia, phosphorus deficiency, amino acid deficiency, ultraviolet radiation, starvation, viral infection, heat shock, or heavy metal stress. Based on the presence of either a 5' or 3' anticodon in the resulting product, tiRNAs can be divided into two subclasses: 5' tiRNAs and 3' tiRNAs. tiRNAs are mainly located in the cytoplasm, with small amounts present in the nucleus and mitochondria, and can also be detected in the human circulatory system. Current research on tiRNAs and tRFs largely focuses on various cancers, with limited research on their role in skeletal muscle proliferation and differentiation. Studies have shown that 5' tiRNA-Gly induces inflammation through the TGF-β signaling pathway, potentially regulating skeletal muscle regeneration. In addition, tiRNAs with high expression abundance in skeletal muscle include mitochondrial tyrosine (mt-Ty), mitochondrial cysteine ​​(mt-Tc), mitochondrial valine (mt-Tv), and cytoplasmic Val / His / Asp / Gln / Glu / Gly / Lys 5' tiRNAs. There are many patents related to short non-coding RNAs. Patents that are somewhat related to this application but not closely related include: a composition, method, and kit for detecting ribonucleic acid (application number CN200980102210.9); a ligation method using eukaryotic tRNA ligase (application number CN201180060308.X); a method for synthesizing short single-stranded deoxyribonucleotide probes (application number CN201310189835.1); small ncRNAs as biomarkers (application number CN201580029331.0); a short non-coding RNA and its application (application number CN201610874030.4); and a method for constructing and applying a sequencing library of circular small non-coding RNA (application number CN202110958847.0). Methods and kits for detecting target nucleic acids by in situ hybridization, application number CN202180035726.7; Methods for predicting prostate cancer and their uses, application number CN202080021149.1; Methods for diagnosing disease states, application number CN201980090997.5; Size selection of RNA using POLY(A) polymerase, application number CN201980063415.4; RNA-based biocontrol methods for protecting plants from pathogenic bacteria and / or promoting the beneficial effects of symbiotic and commensal bacteria, application number CN201980068695.8; RNA-based treatment methods for protecting animals from pathogenic bacteria and / or promoting the beneficial effects of symbiotic and commensal bacteria, application number CN201980068618.2;Methods for preparing cDNA libraries, application number CN201880074741.0; Cancer scoring and response prediction based on biological humor assessment, application number CN201880066565.6; An antagonist of micro-noncoding RNA and its application, application number CN201810892854.3; A method for improving the efficiency of sheep frozen semen application, application number CN202510346829.5; A method for determining and monitoring xenograft rejection by measuring nucleic acids or proteins derived from xenografts, application number CN202380075986.6; A method for detecting target nucleic acids using RNA blocking molecules, application number CN202380064830.8; An RNA-rich anti-aging composition and its use, application number TW112125039; For the treatment of Allogeneic hypoimmunogenic biomimetic nanovesicles for cancer therapy, application number CN202380051866.2; Controlled expression of therapeutically relevant biomolecules effectively loaded in the lumen of biomimetic nanovesicles and exosomes, application number CN202380051075.X; Hypoimmunogenic cells for generating biomimetic nanovesicles, application number CN202380048055.7; Isolation and diagnostic methods using cell type-specific and / or organ-specific extracellular vesicle (EV) markers, application number CN202380048361.0; Active ingredient, pharmaceutical composition and application for treating age-related macular degeneration, application number CN202310271503.1; A small non-coding RNA molecular marker of exosomes and its application, application number CN202111441546.7. All these patents disclose innovative technologies related to small non-coding RNA from different perspectives, but none of them are directly related to this application; they only mention small non-coding RNA and its related applications. Therefore, clarifying how tsRNA participates in the regulation of skeletal muscle development has become a major challenge that urgently needs to be addressed in the treatment of muscle diseases. YL1-tiRNA is derived from mature mt-Val by adding a phosphate group to the 5' end and phosphorylating the sequence. Simultaneously, a sequence is added to the 5' end without altering its function. Its characteristic is that the nucleotide sequence of the YL1-tiRNA is as shown in SeqID No:1. Experiments have shown that YL1-tiRNA can promote the repair of mouse muscle damage by regulating the proliferation of C2C12 cells. Therefore, it is necessary to invent a YL1-tiRNA that promotes the repair of mouse muscle cell damage. Summary of the Invention

[0003] To clarify the challenge of how tsRNA participates in the regulation of skeletal muscle development in the treatment of muscle diseases, this invention provides a YL1-tiRNA that promotes the repair of damaged mouse muscle cells. The nucleotide sequence of this YL1-tiRNA is shown in Seq ID No:1. Experiments have demonstrated that YL1-tiRNA can promote the repair of damaged mouse muscles by regulating the proliferation of C2C12 cells. It can serve as a small non-coding RNA (sncRNA) for studying skeletal muscle development and has important significance in the treatment of muscle diseases.

[0004] The technical solution adopted by this invention to solve its technical problem is:

[0005] This invention discloses a YL1-tiRNA that promotes the repair of damaged mouse muscle cells. The YL1-tiRNA is cleaved from mature mt-Val, with a phosphate group added to the 5' end and the sequence modified by phosphorylation. Simultaneously, a sequence is added to the 5' end without altering its function. The nucleotide sequence of the YL1-tiRNA is shown in Seq ID No:1. Experiments have demonstrated that YL1-tiRNA can promote the repair of damaged mouse muscle cells by regulating the proliferation of C2C12 cells.

[0006] Experimental methods and results:

[0007] I. YL1-tiRNA promotes muscle damage repair in mice

[0008] To clarify the expression pattern of YL1-tiRNA in muscle injury repair in mice, this experiment induced a muscle injury mouse model by injecting 10 μmol / L cardiotoxin (CTX) into the left anterior tibialis muscle of 6-week-old mice. Mice were euthanized by cervical dislocation on the day of CTX injection (D0), day 1 (D1), day 3 (D3), day 5 (D5), day 7 (D7), and day 14 (D14). The hind limb muscles were collected and hematoxylin-eosin (HE) staining was used to detect whether the injury model was successfully established.

[0009] 1. HE staining

[0010] Hematoxylin-eosin staining (HE staining) is a commonly used staining method in paraffin sectioning. Hematoxylin is an alkaline staining solution, primarily coloring the chromatin in the cell nucleus and nucleic acids in the cytoplasm a purplish-blue hue; eosin is an acidic dye, primarily coloring components in the cytoplasm and extracellular matrix red. HE staining is one of the most fundamental and widely used techniques in histological, embryological, and pathological teaching and research.

[0011] Experimental steps

[0012] (1) Dewaxing: Place the paraffin slices in a 65℃ oven and bake for 1 hour, then place them in xylene (5 min) to remove the wax.

[0013] (2) Moisturizing: Remove xylene and use baths (100%, 90%, 80%, 70%) and water to reduce the hydration alcohol concentration of the tissue sections.

[0014] (3) Nuclear staining: Hematoxylin staining for 3 – 5 minutes.

[0015] (4) Differentiation and blueing: Add 1% hydrochloric acid-ethanol differentiation solution, rinse after 2 seconds, add blueing solution to rinse for a few seconds, and rinse again. The time should not exceed 5 minutes.

[0016] (5) Eosin staining: Add eosin dye to the tissue and rinse after 2 minutes.

[0017] (6) Dehydration: The slices were placed in a gradient of alcohol (70%, 80%, 90%, 100%) and xylene for dehydration.

[0018] (7) Mounting: After dehydration, the slides are mounted with neutral resin, air-dried at room temperature, and photographed and recorded under a microscope.

[0019] Experimental results

[0020] HE staining results of mouse skeletal muscle injury repair at different time points are as follows: Figure 1 As shown, three days after CTX injection (D3), the muscle undergoes extensive dissolution and inflammatory infiltration, with a significant increase in the number of cell nuclei (blue); five days after skeletal muscle injury (D5), some smaller muscle fibers regenerate; seven days after injury (D7), a large number of muscle fibers form; and on day 14 (D14), the muscle fiber bundles are tightly packed, indicating that the repair is basically complete, and the skeletal muscle has completed its post-injury regeneration.

[0021] After determining the timeline for muscle injury repair, the expression of YL1-tiRNA and genes related to cell proliferation and differentiation was detected using qRT-PCR.

[0022] 2. Extracting RNA from tissues

[0023] (1) Extraction of total RNA from tissues

[0024] Add 100 mg of tissue to 1 mL of Trizol and a grinding bead, and grind for 30 seconds until the tissue is completely ground. Let stand at room temperature for 5 minutes, then centrifuge at 12000 rpm for 5 minutes. Collect the supernatant. Add 0.2 mL of pre-chilled chloroform, shake the cells vigorously, and degrade on ice for 10 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Transfer the supernatant to a new EP tube (RNase-free), add 0.5 mL of pre-chilled isopropanol, invert and mix well. Let stand on ice for 10 minutes, then centrifuge at 12000 rpm for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol (DEPC preparation) to each tube to wash the precipitate, centrifuge at 12000 rpm for 5 minutes at 4°C, discard the supernatant, and reflux. Evaporate the ethanol at room temperature. Add 10-30 μL of the precipitate and measure the OD260 and OD280 values ​​to check the RNA purity and concentration. Store at -80°C for later use.

[0025] (2) Reverse transcription

[0026] According to the Cisco reverse transcription reagent instructions

[0027]

[0028] Incubate at 42°C for 2 minutes, place on ice, and then add the following to the PCR tube:

[0029]

[0030] Reaction conditions: Incubate at 50°C for 15 min, heat at 85°C for 5 sec, and cool on ice.

[0031] (3) qRT-PCR

[0032] Using the cDNA obtained in step (2) as a template, real-time quantitative PCR was performed using the fluorescent dye SYBR Green I. The reaction system is shown in the table below.

[0033]

[0034] Reaction conditions: Standard two-step PCR amplification procedure:

[0035] Pre-denaturation stage: 94℃ for 2-3 minutes

[0036] Reaction phases: 94℃ for 5-10 seconds, 60℃ for 30 seconds, 40 cycles.

[0037] Experimental results

[0038] The results showed that YL1-tiRNA expression gradually increased with the increase of damage repair time, reaching its maximum on day 5 (D5). Figure 2A); The expression levels of the skeletal muscle satellite cell marker genes PAX7 and MyoD1 mRNA first increased and then decreased, reaching their maximum on day 3 (D3). Figure 2 B, D), indicating that quiescent satellite cells were activated, and the expression of the proliferation marker gene PCNA was consistent with this. Figure 2 C), indicating that skeletal muscle satellite cells have begun to proliferate. The expression of the differentiation marker gene MyoG mRNA began to increase on day 5 (D5) and continued until day 14 (D14). Figure 2 E), demonstrating that satellite cells are activated and thus enter the myogenic differentiation process. This is consistent with the results of HE staining. Therefore, it is speculated that YL1-tiRNA may be involved in the skeletal muscle injury repair process.

[0039] 3. YL1-tiRNA promotes skeletal muscle damage repair in mice

[0040] To investigate the role of YL1-tiRNA in muscle tissue repair, YL1-tiRNA-NC and YL1-tiRNA-mimic were injected into the muscle tissue of injured mice. The expression level of YL1-tiRNA in the injured muscle tissue was detected using qRT-PCR. The results showed that its expression level significantly increased on day 7 (D7), being 3.3 times that of the control group. Figure 3 A) After confirming YL1-tiRNA overexpression, HE staining was used to observe changes in muscle fibers during injury repair, in order to detect the repair status of mouse skeletal muscle tissue. The results are as follows: Figure 3 As shown, at 3 days (D3) of skeletal muscle injury, the repair in the control group was not obvious. Compared with the control group, the number of muscle fibers in the injured tissue increased after overexpression of YL1-tiRNA. At 7 days (D7), the muscle fiber bundles were tightly arranged. At 14 days, there was no significant difference in muscle fibers between the experimental group and the control group.

[0041] The repair process of skeletal muscle injury in mice involves satellite cell proliferation and differentiation. Therefore, qRT-PCR was used to detect the mRNA expression of proliferation and differentiation marker genes during the repair process. The results are as follows: Figure 4 As shown, compared with the control group, after overexpression of YL1-tiRNA, PAX7 expression was significantly upregulated on day 3 of muscle injury repair; the expression of the proliferation marker gene PCNA increased on day 3 (D3) and gradually decreased with increasing repair time; the expression of the MyoD1 gene gradually increased with repair time; MyoG expression was higher than that of the control group on days 3 (D3) and 7 (D7), while MyoG expression decreased on day 14 (D14). These results suggest that YL1-tiRNA can promote the activation of muscle satellite cells in vivo, thereby promoting myoblastic differentiation and skeletal muscle injury repair.

[0042] II. Effects on promoting C2C12 cell proliferation

[0043] To investigate the mechanism by which YL1-tiRNA promotes the repair of skeletal muscle damage in mice, subsequent experiments were conducted using the mouse skeletal muscle satellite cell line C2C12.

[0044] 1. Cell Culture

[0045] Mouse C2C12 myoblasts were purchased from Pricella (Wuhan). The proliferation medium (GM) was DMEM containing 10% FBS. C2C12 cells were cultured in the proliferation medium for 1-3 days. Cells were then cultured in a 37°C, 5% CO2 incubator. The differentiation medium (DM) consisted of DMEM supplemented with 2% horse serum. When cell confluence reached 80%, the differentiation medium was changed to induce C2C12 cell differentiation for 1-3 days.

[0046] 2. During the proliferation and differentiation of C2C12 cells, the expression level of YL1-tiRNA was detected by stem-loop quantitative RT-PCR.

[0047] C2C12 cells in the logarithmic growth phase were selected, passaged into 6-well plates, and cultured in proliferation and differentiation media for 24, 48, and 72 hours, respectively. Cells were then collected, and RNA was extracted to detect the expression level of YL1-tiRNA during proliferation and differentiation.

[0048] Stem-loop fluorescence quantitative RT-PCR detection method:

[0049] (1) RNA extraction

[0050] Discard the culture medium, wash 2-3 times with PBS, add 1 ml of Trizol to each well, and collect cells by gently scraping the bottom of the culture dish with a pipette tip.

[0051] Add 0.2 ml of pre-cooled chloroform, shake the cells vigorously, degrade on ice for 10 min, and centrifuge at 12000 r / min for 15 min at 4℃.

[0052] Transfer the supernatant to a new EP tube (RNase-free), add 0.5 ml of pre-chilled isopropanol, invert and mix well, incubate on ice for 10 min, and centrifuge at 12000 r / min for 10 min at 4℃.

[0053] Discard the supernatant, add 1 ml of 75% ethanol (DEPC preparation) to each tube to wash the precipitate, centrifuge at 12000 r / min for 5 min at 4℃, discard the supernatant, aspirate the reflux, and evaporate the ethanol at room temperature.

[0054] Add 10-30 μL of the precipitate and measure the OD260 and OD280 values ​​to verify the RNA purity and concentration. Store at -80℃ for later use.

[0055] (2) Reverse transcription

[0056] According to the Cisco reverse transcription reagent instructions

[0057]

[0058] Incubate at 42°C for 2 minutes, place on ice, and then add the following to the PCR tube:

[0059]

[0060] Reaction conditions: Incubate at 50°C for 15 min, heat at 85°C for 5 sec, and cool on ice.

[0061] (3) qRT-PCR

[0062] Using the cDNA obtained in step (2) as a template, real-time quantitative PCR was performed using the fluorescent dye SYBR Green I. The reaction system is shown in the table below.

[0063]

[0064] Reaction conditions: Standard two-step PCR amplification procedure:

[0065] Pre-denaturation stage: 94℃ for 2-3 minutes

[0066] Reaction phases: 94℃ for 5-10 seconds, 60℃ for 30 seconds, 40 cycles.

[0067] Results of stem-loop quantitative RT-PCR detection of YL1-tiRNA expression levels

[0068] Experimental results are as follows Figure 5 As shown, the expression level of YL1-tiRNA was significantly increased during the proliferation of mouse C2C12 cells, indicating that YL1-tiRNA plays a certain role in regulating the proliferation and differentiation of C2C12 cells.

[0069] 3. Steps for cell transfection using the PEI method

[0070] (1) C2C12 cells were seeded into 6-well cell culture plates with growth medium without antibiotics and transfected when the cell confluence rate was about 70%.

[0071] (2) The experiment was divided into one experimental group and one control group, with three wells in each group, i.e., three repeated experiments. 4 μg of plasmid and 6 μl of PEI were added to each well in both the experimental group and the control group.

[0072] (3) Take two sterile 1.5 ml EP tubes and add 100 μl of Opti-MEM incubation solution to each tube. Add 4 μg of plasmid to each EP tube containing the incubation solution, and add 6 μl of PEI to the other two tubes respectively. Incubate for 5 min.

[0073] (4) Mix 100 μl of incubation solution containing PEI with 100 μl of incubation solution containing plasmid evenly, gently blow and aspirate to mix (30-50 times), and then incubate at room temperature for 15 min.

[0074] (5) Remove the culture medium from the 6-well plate during static incubation. Wash the cells twice with serum-free and antibiotic-free DMEM medium. Add 1.8 mL of serum-free and antibiotic-free medium to each well of both the experimental and control groups. After incubation, add 200 μl of the Opti-MEM incubation solution containing PEI and plasmid complex to each well and gently shake to mix.

[0075] (6) After culturing in a 37°C, 5% CO2 cell culture incubator for 6 hours, the culture medium was replaced with C2C12 cell culture medium for continued culture and cell proliferation was detected.

[0076] 4. Effect of CCK8 assay on the viability of YL1-tiRNA on C2C12 cells

[0077] Cell Counting Kit-8, or CCK-8 kit for short, is a rapid and highly sensitive assay kit based on WST-8 that is widely used for the detection of cell proliferation and cytotoxicity.

[0078] This kit can be used to detect cell proliferation induced by cytokines, as well as to detect cytotoxicity induced by toxic agents such as anticancer drugs, or to detect cell growth inhibition induced by some drugs.

[0079] This kit offers exceptional convenience. It contains only one tube of pre-prepared CCK-8 solution containing WST-8, eliminating the need for any further preparation. No isotopes are required; all assays are performed within a single 96-well plate. Cell washing, cell collection, and the need for additional steps to dissolve formazan are unnecessary. It can be used for large-scale sample analysis. Phenol red and serum have no significant impact on the assay. WST-8 exhibits no significant cytotoxicity. After adding the CCK-8 solution for color development, the plate can be repeatedly read using a microplate reader at different times, allowing for greater flexibility in assay timing and facilitating the identification of the optimal assay time.

[0080] C2C12 cells in the logarithmic growth phase were selected and passaged into 24-well plates. After cell attachment, mimic NC and YL1-tiRNA mimic were transfected into C2C12 cells using the PEI method. Cell proliferation was detected by the CCK-8 assay at 24, 48, and 72 hours.

[0081] Detection of cell viability

[0082] (1) Culture the transfected cells in a 96-well plate for 48 hours.

[0083] (2) Add 10 μL of CCK-8 solution to each well (be careful not to generate bubbles in the wells, as they will affect the OD reading).

[0084] (3) Incubate the culture plate in the incubator for 1-4 hours.

[0085] (4) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0086] Results of CCK-8 assay for cell viability

[0087] CCK-8 test results ( Figure 6 The results showed that overexpression of YL1-tiRNA significantly improved the viability of C2C12 cells, with the most significant effect observed at 48 hours. Therefore, subsequent experiments were conducted at 48 hours.

[0088] 5. EdU assay to detect the effect of YL1-tiRNA on C1C12 cell proliferation

[0089] EdU is a thymine nucleoside analogue with an alkyne group rarely found in natural compounds. It can replace thymine (T) and infiltrate into the synthesizing DNA molecule during DNA replication. DNA replication activity can be directly and accurately detected by the specific reaction between Apollo® fluorescent dye and EdU. It is widely used in research on cell proliferation, cell differentiation, growth and development, DNA repair, and viral replication, and is especially suitable for cell proliferation screening experiments of siRNA, miRNA, small molecule compounds, and drugs.

[0090] This study used EdU to detect the effect of YL1-tiRNA on the proliferation of C2C12 cells. C2C12 cells in the logarithmic growth phase were selected, passaged into 24-well plates, and transfected with YL1-tiRNA mimic using the PEI method after cell adhesion. The proliferation was detected by EdU after 48 hours.

[0091] 5.1 Detection of cell proliferation

[0092] (1) EdU labeling: Dilute EdU solution (reagent A) with cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU culture medium; add 300 μL of 50 μM EdU culture medium to each well and incubate for 2 hours, then discard the culture medium; wash cells with PBS 1-2 times, 5 min each time.

[0093] (2) Cell immobilization: Add 250 μL of cell fixation solution (i.e., PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min, then discard the fixation solution; add 250 μL of 2 mg / mL glycine to each well, incubate on a shaker for 5 min, then discard the glycine solution; add 250 μL of PBS to each well, wash on a shaker for 5 min, then discard the PBS; (enhanced) add 250 μL of permeabilizer (PBS containing 0.5% Triton X-100) to each well, incubate on a shaker for 10 min, wash once with PBS for 5 min.

[0094] (3) Apollo staining: Add 250 μL of 1×Apollo® staining reaction solution to each well, protect from light, incubate at room temperature on a decolorizing shaker for 30 min, and discard the staining reaction solution; add 250 μL of penetrant (0.5% Triton X-100 in PBS) and decolorize on a shaker for 2-3 times, 10 min each time, and discard the penetrant; (enhanced) add 250 μL of methanol to each well and wash 1-2 times, 5 min each time, and wash once with PBS for 5 min each time.

[0095] (4) DNA staining: Dilute reagent F with deionized water at a ratio of 100:1 to prepare an appropriate amount of 1×Hoechst33342 reaction solution and store it in the dark; add 250μL of 1×Hoechst33342 reaction solution to each well, incubate in the dark, at room temperature, and then incubate on a decolorizing shaker for 30min, and discard the staining reaction solution; add 250μL of PBS to each well to wash 1~3 times each time.

[0096] (5) Image acquisition and analysis: Observation is carried out immediately after staining. (If conditions are limited, store in the dark at 4°C with moisture until analysis, but it should not exceed 3 days).

[0097] 5.2 Results of EdU detection of cell proliferation

[0098] EdU results ( Figure 7 The results showed that the number of EdU-positive cells increased significantly after YL1-tiRNA overexpression, proving that YL1-tiRNA can significantly promote the proliferation of C2C12 cells.

[0099] 6. RT-PCR and Western Blot were used to detect the expression of proliferation-related genes (PCNA and CCND1) after YL1-tiRNA overexpression.

[0100] 6.1 RT-PCR

[0101] C2C12 cells in the logarithmic growth phase were selected and passaged into 6-well plates. After cell adhesion, mimic NC and YL1-tiRNA mimic were transfected into C2C12 cells using the PEI method. After 48 hours, quantitative real-time RT-PCR was used to detect important proliferation-related genes such as PCNA (Proliferating Cell Nuclear Antigen, found only in normal proliferating cells and tumor cells; studies have shown that PCNA is closely related to cellular DNA synthesis and plays an important role in the initiation of cell proliferation, making it a good indicator of cell proliferation status) and CCND1 (Cyclin D1, a G1 / S-specific cyclin-D1 protein encoded by the human CCND1 gene. The main function of cyclin D1 is to promote cell proliferation. Cyclin D1 binds to and activates the G1 phase-specific cyclin-dependent kinase CDK4, phosphorylating the G1 phase cell repressor protein (Rb). The phosphorylated Rb protein is released from its bound E2F... Transcription factors dissociate, and E2F transcription factors initiate the transcription of genes involved in the living cell cycle, thereby propelling the cell cycle from the G1 phase to the S phase.

[0102] The method for detecting PCNA and CCND1 expression levels by quantitative real-time RT-PCR is the same as the method for detecting YL1-tiRNA by quantitative real-time RT-PCR in the stem-loop region described above.

[0103] Results of quantitative RT-PCR detection of PCNA and CCND1 expression levels ( Figure 8 The results showed that after YL1-tiRNA overexpression, the expression levels of proliferation-related genes PCNA and CCND1 mRNA increased significantly, proving that YL1-tiRNA can promote the proliferation of C2C12 cells.

[0104] 6.2 Western Blot

[0105] Western blotting (also known as protein immunoblotting) is a technique commonly used in research to isolate and identify proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only the antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning.

[0106] Because antibodies bind only to the target protein, only a single, clear band is typically observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of a specific reaction, information about the expression of the target protein in a given cell or tissue homogenate can be obtained.

[0107] Western blot experimental procedures

[0108] (1) Discard the cell culture medium, wash 3 times with 1×PBS (to remove serum from the culture medium), and add 100uL of protein lysis buffer to each well (6-well plate). Quickly scrape the cells off with a cell scraper and transfer them to a 1.5ml tube and place on ice.

[0109] (2) Dissolve on a shaker at 4℃ for 30 min, centrifuge at 12,000 r / min at 4℃ for 15 min, and collect the supernatant into another 1.5 mL tube.

[0110] (3) Take 10 μL and add it to 40 μL of dd H2O and mix well. Take 25 μL and mix it with 200 μL of the mixture (protein concentration determination solution A: solution B = 50:1). Incubate at 37℃ for 30 min. Detect the OD value at 562 nm using a Spark 10M microplate reader. Add 6× Loading Buffer (1:5) to the remaining samples, boil at 95℃ for 10 min, and store at -20℃.

[0111] (4) SDS-PAGE polyacrylamide gel electrophoresis: Prepare separating gel (5ml / gel) and stacking gel and let stand. Insert comb and wait for the gel to solidify. Then put the gel into the electrophoresis tank and add 1× electrophoresis buffer to both the upper and lower tanks.

[0112] (5) Slowly pull out the comb, slowly add 30uL of protein sample to each well, and turn on the power.

[0113] (6) Start with a constant voltage of 90V. After running through the stacking gel, increase the current to 120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein should be run to two-thirds of the position of the separating gel.

[0114] (7) Transfer: Cut the gel according to the marker and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0115] (8) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it along with 4 sheets of 3mm filter paper and a sponge in the transfer buffer. Follow the order below: fiber pad -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber pad (ensure there are no air bubbles, and the transfer time depends on the size of the protein).

[0116] (9) One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0117] (10) Blocking: After the transfer, add blocking solution (prepare 5% blocking solution with skim milk powder) and block for 1 hour.

[0118] (11) Wash the membrane with 1×TBST 3 times, 15 minutes each time.

[0119] (12) Add the diluted antibody and incubate overnight at 4°C on a shaker.

[0120] (13) Recover the primary antibody and wash the membrane 3 times with 1×TBST for 15 minutes each time.

[0121] (14) Add secondary antibody, incubate at room temperature for 1 hour, and wash the membrane 3 times with 1×TBST for 15 minutes each time.

[0122] (15) Sweeping film

[0123] Western blot results

[0124] Test results ( Figure 9 The results showed that after YL1-tiRNA overexpression, the protein expression levels of proliferation-related genes PCNA and CCND1 increased significantly, proving that YL1-tiRNA can promote the proliferation of C2C12 cells.

[0125] The beneficial effects of this invention are as follows: the nucleotide sequence of YL1-tiRNA, which promotes the repair of damaged mouse muscle cells, is shown in Seq ID No:1. Experiments have shown that YL1-tiRNA can promote the repair of damaged mouse muscle by regulating the proliferation of C2C12 cells. It can be used as a small non-coding RNA (sncRNA) for studying skeletal muscle development and has important significance in the treatment of muscle diseases. Attached Figure Description

[0126] The invention will be further described below with reference to the accompanying drawings.

[0127] Figure 1 This image shows the HE staining of YL1-tiRNA, a gene that promotes the repair of damaged mouse muscle cells, as described in this invention, to assess the repair of skeletal muscle damage in mice. Note: Scale bar: 100 μm.

[0128] Figure 2 This is a graph showing the expression changes of related genes during the mouse muscle injury repair process of YL1-tiRNA, which promotes the repair of mouse muscle cell damage according to the present invention. Note: *: P<0.05, **: P<0.01, ns: no significant difference.

[0129] Figure 3 This is a diagram illustrating the effect of YL1-tiRNA on the repair of mouse muscle cell damage, as described in this invention. Note: **: P<0.01, ***: P<0.001, ns: no significant difference. Scale bar: 100 μm.

[0130] Figure 4 This is a diagram showing the effect of YL1-tiRNA, a YL1-tiRNA that promotes the repair of damaged mouse muscle cells, on the expression of related genes during the repair of skeletal muscle damage in mice. Note: *: P<0.05, **: P<0.01, ***: P<0.001, ns: no significant difference.

[0131] Figure 5 This is a diagram showing the expression of YL1-tiRNA, a YL1-tiRNA that promotes the repair of damaged mouse muscle cells, during the proliferation of C2C12 cells. Note: **: P<0.01, ***: P<0.001.

[0132] Figure 6 This figure shows the effect of overexpression of YL1-tiRNA, a YL1-tiRNA that promotes the repair of damaged mouse muscle cells, on the viability of C2C12 cells.

[0133] Figure 7 The figure shows the effect of overexpression of YL1-tiRNA, a YL1-tiRNA that promotes the repair of damaged mouse muscle cells, on the proliferation of C2C12 cells. Note: *: P<0.05.

[0134] Figure 8 This is a graph showing the effect of overexpression of YL1-tiRNA, a gene that promotes the repair of damaged mouse muscle cells, on related proliferation genes in C2C12 cells. Note: **: P<0.01.

[0135] Figure 9 This is a graph showing the effect of overexpression of YL1-tiRNA, a gene that promotes the repair of damaged mouse muscle cells, on genes related to the proliferation of C2C12 cells. Note: **: P<0.01. Note: *: P<0.05, ***: P<0.001. Detailed Implementation

[0136] Example 1:

[0137] As shown in the figure, the YL1-tiRNA in the present invention that promotes the repair of mouse muscle cell damage is obtained by cutting mature mt-Val, adding a phosphate group at the 5' end and modifying the sequence by phosphorylation, and adding a sequence at the 5' end without changing its function.

[0138] sequence synthesis

[0139] Currently, primer synthesis primarily employs the solid-phase phosphoramide-γ-triester method. This method is characterized by high efficiency, rapid coupling, and relatively stable starting reactants. It mainly involves immobilizing DNA on a solid support to complete DNA strand synthesis, extending the primer from the 3' end to the 5' end, with adjacent nucleotides linked by 3'→5' phosphodiester bonds.

[0140] The specific steps for synthesizing primers using the solid-phase phosphoramidite-triester method are as follows:

[0141] 1) The protecting group DMT of the 5'-hydroxyl group on the solid support was removed by trichloroacetic acid to obtain free 5'-hydroxyl groups;

[0142] 2) The phosphorous amide-protected nucleotide monomer is mixed with the activator tetrazolium to obtain a nucleoside phosphorous acid activated intermediate, which undergoes a condensation reaction with the free 5'-hydroxyl group;

[0143] 3) Since it is impossible to guarantee that 100% of the 5'-hydroxyl groups will participate in the condensation, a very small number of 5'-hydroxyl groups may not participate in the reaction (less than 2%). The synthesis can be terminated by acetic anhydride and 1-methylimidazole reagent. This short fragment can be separated during purification.

[0144] 4) Under the action of oxidants, the phosphorous form is converted into a more stable phosphate triester, making the DNA phosphate backbone more stable.

[0145] After the above four steps, a deoxynucleotide is ligated onto the solid support. Repeat these steps until all required bases are ligated. The color of the removed protecting group (DMT) can be monitored during the synthesis process to preliminarily determine the synthesis efficiency.

[0146] The synthesized primers have a hydroxyl group at the 5' end and no phosphate group. We performed phosphorylation modification on the 5' end of the YL1-tiRNA sequence.

[0147] Sequence purification methods

[0148] Currently, commonly used primer purification methods include: HAP purification, ULTRAPAGE purification, HPLC-CE (IVD) purification, PAGE purification, and HPLC purification. We chose HPLC purification.

[0149] High-performance liquid chromatography (HPLC): Utilizing the principles of high-performance liquid chromatography, this is a highly effective purification method for primer DNA, achieving very high purity and sensitivity. After purification, the primer purity can exceed 90%. It is particularly suitable for purifying short-chain (less than 40 mer) primers and modified primers. Advantages: Especially effective for purifying short-chain primers (<40 mer). Disadvantages: High cost and low efficiency in large-scale production.

[0150] HPLC purification is suitable for conventional PCR / multiplex PCR / reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR) / digital PCR (dPCR), first-generation sequencing (Sanger), high-throughput sequencing (NGS), whole gene synthesis, subcloning / point mutation, gene construction / RNA interference, PCR products for cloning and expression studies or gene recombination, modification or labeling primers, etc.

[0151] Table 1. Sequence of YL1-tiRNA

[0152]

[0153] The specific experimental methods and results are as follows:

[0154] I. YL1-tiRNA promotes muscle damage repair in mice

[0155] To clarify the expression pattern of YL1-tiRNA in muscle injury repair in mice, this experiment induced a muscle injury mouse model by injecting 10 μmol / L cardiotoxin (CTX) into the left anterior tibialis muscle of 6-week-old mice. Mice were euthanized by cervical dislocation on the day of CTX injection (D0), day 1 (D1), day 3 (D3), day 5 (D5), day 7 (D7), and day 14 (D14). The hind limb muscles were collected and hematoxylin-eosin (HE) staining was used to detect whether the injury model was successfully established.

[0156] 1. HE staining

[0157] Hematoxylin-eosin staining (HE staining) is a commonly used staining method in paraffin sectioning. Hematoxylin is an alkaline staining solution, primarily coloring the chromatin in the cell nucleus and nucleic acids in the cytoplasm a purplish-blue hue; eosin is an acidic dye, primarily coloring components in the cytoplasm and extracellular matrix red. HE staining is one of the most fundamental and widely used techniques in histological, embryological, and pathological teaching and research.

[0158] Experimental steps

[0159] (1) Dewaxing: Place the paraffin slices in a 65℃ oven and bake for 1 hour, then place them in xylene (5 min) to remove the wax.

[0160] (2) Moisturizing: Remove xylene and use baths (100%, 90%, 80%, 70%) and water to reduce the hydration alcohol concentration of the tissue sections.

[0161] (3) Nuclear staining: Hematoxylin staining for 3 – 5 minutes.

[0162] (4) Differentiation and blueing: Add 1% hydrochloric acid-ethanol differentiation solution, rinse after 2 seconds, add blueing solution to rinse for a few seconds, and rinse again. The time should not exceed 5 minutes.

[0163] (5) Eosin staining: Add eosin dye to the tissue and rinse after 2 minutes.

[0164] (6) Dehydration: The slices were placed in a gradient of alcohol (70%, 80%, 90%, 100%) and xylene for dehydration.

[0165] (7) Mounting: After dehydration, the slides are mounted with neutral resin, air-dried at room temperature, and photographed and recorded under a microscope.

[0166] Experimental results

[0167] HE staining results of mouse skeletal muscle injury repair at different time points are as follows: Figure 1 As shown, three days after CTX injection (D3), the muscle undergoes extensive dissolution and inflammatory infiltration, with a significant increase in the number of cell nuclei (blue); five days after skeletal muscle injury (D5), some smaller muscle fibers regenerate; seven days after injury (D7), a large number of muscle fibers form; and on day 14 (D14), the muscle fiber bundles are tightly packed, indicating that the repair is basically complete, and the skeletal muscle has completed its post-injury regeneration.

[0168] After determining the timeline for muscle injury repair, the expression of YL1-tiRNA and genes related to cell proliferation and differentiation was detected using qRT-PCR.

[0169] 2. Extracting RNA from tissues

[0170] (1) Extraction of total RNA from tissues

[0171] Add 100 mg of tissue to 1 mL of Trizol and a grinding bead, and grind for 30 seconds until the tissue is completely ground. Let stand at room temperature for 5 minutes, then centrifuge at 12000 rpm for 5 minutes. Collect the supernatant. Add 0.2 mL of pre-chilled chloroform, shake the cells vigorously, and degrade on ice for 10 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Transfer the supernatant to a new EP tube (RNase-free), add 0.5 mL of pre-chilled isopropanol, invert and mix well. Let stand on ice for 10 minutes, then centrifuge at 12000 rpm for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol (DEPC preparation) to each tube to wash the precipitate, centrifuge at 12000 rpm for 5 minutes at 4°C, discard the supernatant, and reflux. Evaporate the ethanol at room temperature. Add 10-30 μL of the precipitate and measure the OD260 and OD280 values ​​to check the RNA purity and concentration. Store at -80°C for later use.

[0172] (2) Reverse transcription

[0173] According to the Cisco reverse transcription reagent instructions

[0174]

[0175] Incubate at 42°C for 2 minutes, place on ice, and then add the following to the PCR tube:

[0176]

[0177] Reaction conditions: Incubate at 50°C for 15 min, heat at 85°C for 5 sec, and cool on ice.

[0178] (3) qRT-PCR

[0179] Using the cDNA obtained in step (2) as a template, real-time quantitative PCR was performed using the fluorescent dye SYBR Green I. The reaction system is shown in the table below.

[0180]

[0181] Reaction conditions: Standard two-step PCR amplification procedure:

[0182] Pre-denaturation stage: 94℃ for 2-3 minutes

[0183] Reaction phases: 94℃ for 5-10 seconds, 60℃ for 30 seconds, 40 cycles.

[0184] Experimental results

[0185] The results showed that YL1-tiRNA expression gradually increased with the increase of damage repair time, reaching its maximum on day 5 (D5). Figure 2A); The expression levels of the skeletal muscle satellite cell marker genes PAX7 and MyoD1 mRNA first increased and then decreased, reaching their maximum on day 3 (D3). Figure 2 B, D), indicating that quiescent satellite cells were activated, and the expression of the proliferation marker gene PCNA was consistent with this. Figure 2 C), indicating that skeletal muscle satellite cells have begun to proliferate. The expression of the differentiation marker gene MyoG mRNA began to increase on day 5 (D5) and continued until day 14 (D14). Figure 2 E), demonstrating that satellite cells are activated and thus enter the myogenic differentiation process. This is consistent with the results of HE staining. Therefore, it is speculated that YL1-tiRNA may be involved in the skeletal muscle injury repair process.

[0186] 3. YL1-tiRNA promotes skeletal muscle damage repair in mice

[0187] To investigate the role of YL1-tiRNA in muscle tissue repair, YL1-tiRNA-NC and YL1-tiRNA-mimic were injected into the muscle tissue of injured mice. The expression level of YL1-tiRNA in the injured muscle tissue was detected using qRT-PCR. The results showed that its expression level significantly increased on day 7 (D7), being 3.3 times that of the control group. Figure 3 A) After confirming YL1-tiRNA overexpression, HE staining was used to observe changes in muscle fibers during injury repair, in order to detect the repair status of mouse skeletal muscle tissue. The results are as follows: Figure 3 As shown, at 3 days (D3) of skeletal muscle injury, the repair in the control group was not obvious. Compared with the control group, the number of muscle fibers in the injured tissue increased after overexpression of YL1-tiRNA. At 7 days (D7), the muscle fiber bundles were tightly arranged. At 14 days, there was no significant difference in muscle fibers between the experimental group and the control group.

[0188] The repair process of skeletal muscle injury in mice involves satellite cell proliferation and differentiation. Therefore, qRT-PCR was used to detect the mRNA expression of proliferation and differentiation marker genes during the repair process. The results are as follows: Figure 4 As shown, compared with the control group, after overexpression of YL1-tiRNA, PAX7 expression was significantly upregulated on day 3 of muscle injury repair; the expression of the proliferation marker gene PCNA increased on day 3 (D3) and gradually decreased with increasing repair time; the expression of the MyoD1 gene gradually increased with repair time; MyoG expression was higher than that of the control group on days 3 (D3) and 7 (D7), while MyoG expression decreased on day 14 (D14). These results suggest that YL1-tiRNA can promote the activation of muscle satellite cells in vivo, thereby promoting myoblastic differentiation and skeletal muscle injury repair.

[0189] II. Effects on promoting C2C12 cell proliferation

[0190] To investigate the mechanism by which YL1-tiRNA promotes the repair of skeletal muscle damage in mice, subsequent experiments were conducted using the mouse skeletal muscle satellite cell line C2C12.

[0191] 1. Cell Culture

[0192] Mouse C2C12 myoblasts were purchased from Pricella (Wuhan). The proliferation medium (GM) was DMEM containing 10% FBS. C2C12 cells were cultured in the proliferation medium for 1-3 days. Cells were then cultured in a 37°C, 5% CO2 incubator. The differentiation medium (DM) consisted of DMEM supplemented with 2% horse serum. When cell confluence reached 80%, the differentiation medium was changed to induce C2C12 cell differentiation for 1-3 days.

[0193] 2. During the proliferation and differentiation of C2C12 cells, the expression level of YL1-tiRNA was detected by stem-loop quantitative RT-PCR.

[0194] C2C12 cells in the logarithmic growth phase were selected, passaged into 6-well plates, and cultured in proliferation and differentiation media for 24, 48, and 72 hours, respectively. Cells were then collected, and RNA was extracted to detect the expression level of YL1-tiRNA during proliferation and differentiation.

[0195] Stem-loop fluorescence quantitative RT-PCR detection method:

[0196] (1) RNA extraction

[0197] Discard the culture medium, wash 2-3 times with PBS, add 1 ml of Trizol to each well, and collect cells by gently scraping the bottom of the culture dish with a pipette tip.

[0198] Add 0.2 ml of pre-cooled chloroform, shake the cells vigorously, degrade on ice for 10 min, and centrifuge at 12000 r / min for 15 min at 4℃.

[0199] Transfer the supernatant to a new EP tube (RNase-free), add 0.5 ml of pre-chilled isopropanol, invert and mix well, incubate on ice for 10 min, and centrifuge at 12000 r / min for 10 min at 4℃.

[0200] Discard the supernatant, add 1 ml of 75% ethanol (DEPC preparation) to each tube to wash the precipitate, centrifuge at 12000 r / min for 5 min at 4℃, discard the supernatant, aspirate the reflux, and evaporate the ethanol at room temperature.

[0201] Add 10-30 μL of the precipitate and measure the OD260 and OD280 values ​​to verify the RNA purity and concentration. Store at -80℃ for later use.

[0202] (2) Reverse transcription

[0203] According to the Cisco reverse transcription reagent instructions

[0204]

[0205] Incubate at 42°C for 2 minutes, place on ice, and then add the following to the PCR tube:

[0206]

[0207] Reaction conditions: Incubate at 50°C for 15 min, heat at 85°C for 5 sec, and cool on ice.

[0208] (3) qRT-PCR

[0209] Using the cDNA obtained in step (2) as a template, real-time quantitative PCR was performed using the fluorescent dye SYBR Green I. The reaction system is shown in the table below.

[0210]

[0211] Reaction conditions: Standard two-step PCR amplification procedure:

[0212] Pre-denaturation stage: 94℃ for 2-3 minutes

[0213] Reaction phases: 94℃ for 5-10 seconds, 60℃ for 30 seconds, 40 cycles.

[0214] Results of stem-loop quantitative RT-PCR detection of YL1-tiRNA expression levels

[0215] Experimental results are as follows Figure 5 As shown, the expression level of YL1-tiRNA was significantly increased during the proliferation of mouse C2C12 cells, indicating that YL1-tiRNA plays a certain role in regulating the proliferation and differentiation of C2C12 cells.

[0216] 3. Steps for cell transfection using the PEI method

[0217] (1) C2C12 cells were seeded into 6-well cell culture plates with growth medium without antibiotics and transfected when the cell confluence rate was about 70%.

[0218] (2) The experiment was divided into one experimental group and one control group, with three wells in each group, i.e., three repeated experiments. 4 μg of plasmid and 6 μl of PEI were added to each well in both the experimental group and the control group.

[0219] (3) Take two sterile 1.5 ml EP tubes and add 100 μl of Opti-MEM incubation solution to each tube. Add 4 μg of plasmid to each EP tube containing the incubation solution, and add 6 μl of PEI to the other two tubes respectively. Incubate for 5 min.

[0220] (4) Mix 100 μl of incubation solution containing PEI with 100 μl of incubation solution containing plasmid evenly, gently blow and aspirate to mix (30-50 times), and then incubate at room temperature for 15 min.

[0221] (5) Remove the culture medium from the 6-well plate during static incubation. Wash the cells twice with serum-free and antibiotic-free DMEM medium. Add 1.8 mL of serum-free and antibiotic-free medium to each well of both the experimental and control groups. After incubation, add 200 μl of the Opti-MEM incubation solution containing PEI and plasmid complex to each well and gently shake to mix.

[0222] (6) After culturing in a 37°C, 5% CO2 cell culture incubator for 6 hours, the culture medium was replaced with C2C12 cell culture medium for continued culture and cell proliferation was detected.

[0223] 4. Effect of CCK8 assay on the viability of YL1-tiRNA in C2C12 cells

[0224] Cell Counting Kit-8, or CCK-8 kit for short, is a rapid and highly sensitive assay kit based on WST-8 that is widely used for the detection of cell proliferation and cytotoxicity.

[0225] This kit can be used to detect cell proliferation induced by cytokines, as well as to detect cytotoxicity induced by toxic agents such as anticancer drugs, or to detect cell growth inhibition induced by some drugs.

[0226] This kit offers exceptional convenience. It contains only one tube of pre-prepared CCK-8 solution containing WST-8, eliminating the need for any further preparation. No isotopes are required; all assays are performed within a single 96-well plate. Cell washing, cell collection, and the need for additional steps to dissolve formazan are unnecessary. It can be used for large-scale sample analysis. Phenol red and serum have no significant impact on the assay. WST-8 exhibits no significant cytotoxicity. After adding the CCK-8 solution for color development, the plate can be repeatedly read using a microplate reader at different times, allowing for greater flexibility in assay timing and facilitating the identification of the optimal assay time.

[0227] C2C12 cells in the logarithmic growth phase were selected and passaged into 24-well plates. After cell attachment, mimic NC and YL1-tiRNA mimic were transfected into C2C12 cells using the PEI method. Cell proliferation was detected by the CCK-8 assay at 24, 48, and 72 hours.

[0228] Detection of cell viability

[0229] (1) Culture the transfected cells in a 96-well plate for 48 hours.

[0230] (2) Add 10 μL of CCK-8 solution to each well (be careful not to generate bubbles in the wells, as they will affect the OD reading).

[0231] (3) Incubate the culture plate in the incubator for 1-4 hours.

[0232] (4) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0233] Results of CCK-8 assay for cell viability

[0234] CCK-8 test results ( Figure 6 The results showed that overexpression of YL1-tiRNA significantly improved the viability of C2C12 cells, with the most significant effect observed at 48 hours. Therefore, subsequent experiments were conducted at 48 hours.

[0235] 5. EdU assay to detect the effect of YL1-tiRNA on C1C12 cell proliferation

[0236] EdU is a thymine nucleoside analogue with an alkyne group rarely found in natural compounds. It can replace thymine (T) and infiltrate into the synthesizing DNA molecule during DNA replication. DNA replication activity can be directly and accurately detected by the specific reaction between Apollo® fluorescent dye and EdU. It is widely used in research on cell proliferation, cell differentiation, growth and development, DNA repair, and viral replication, and is especially suitable for cell proliferation screening experiments of siRNA, miRNA, small molecule compounds, and drugs.

[0237] This study used EdU to detect the effect of YL1-tiRNA on the proliferation of C2C12 cells. C2C12 cells in the logarithmic growth phase were selected, passaged into 24-well plates, and transfected with YL1-tiRNA mimic using the PEI method after cell adhesion. The proliferation was detected by EdU after 48 hours.

[0238] 5.1 Detection of cell proliferation

[0239] (1) EdU labeling: Dilute EdU solution (reagent A) with cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU culture medium; add 300 μL of 50 μM EdU culture medium to each well and incubate for 2 hours, then discard the culture medium; wash cells with PBS 1-2 times, 5 min each time.

[0240] (2) Cell immobilization: Add 250 μL of cell fixation solution (i.e., PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min, then discard the fixation solution; add 250 μL of 2 mg / mL glycine to each well, incubate on a shaker for 5 min, then discard the glycine solution; add 250 μL of PBS to each well, wash on a shaker for 5 min, then discard the PBS; (enhanced) add 250 μL of permeabilizer (PBS containing 0.5% Triton X-100) to each well, incubate on a shaker for 10 min, wash once with PBS for 5 min.

[0241] (3) Apollo staining: Add 250 μL of 1×Apollo® staining reaction solution to each well, protect from light, incubate at room temperature on a decolorizing shaker for 30 min, and discard the staining reaction solution; add 250 μL of penetrant (0.5% Triton X-100 in PBS) and decolorize on a shaker for 2-3 times, 10 min each time, and discard the penetrant; (enhanced) add 250 μL of methanol to each well and wash 1-2 times, 5 min each time, and wash once with PBS for 5 min each time.

[0242] (4) DNA staining: Dilute reagent F with deionized water at a ratio of 100:1 to prepare an appropriate amount of 1×Hoechst33342 reaction solution and store it in the dark; add 250μL of 1×Hoechst33342 reaction solution to each well, incubate in the dark, at room temperature, and then incubate on a decolorizing shaker for 30min, and discard the staining reaction solution; add 250μL of PBS to each well to wash 1~3 times each time.

[0243] (5) Image acquisition and analysis: Observation is carried out immediately after staining. (If conditions are limited, store in the dark at 4°C with moisture until analysis, but it should not exceed 3 days).

[0244] 5.2 Results of EdU detection of cell proliferation

[0245] EdU results ( Figure 7 The results showed that the number of EdU-positive cells increased significantly after YL1-tiRNA overexpression, proving that YL1-tiRNA can significantly promote the proliferation of C2C12 cells.

[0246] 6. RT-PCR and Western Blot were used to detect the expression of proliferation-related genes (PCNA and CCND1) after YL1-tiRNA overexpression.

[0247] 6.1 RT-PCR

[0248] C2C12 cells in the logarithmic growth phase were selected and passaged into 6-well plates. After cell adhesion, mimic NC and YL1-tiRNA mimic were transfected into C2C12 cells using the PEI method. After 48 hours, quantitative real-time RT-PCR was used to detect important proliferation-related genes such as PCNA (Proliferating Cell Nuclear Antigen, found only in normal proliferating cells and tumor cells; studies have shown that PCNA is closely related to cellular DNA synthesis and plays an important role in the initiation of cell proliferation, making it a good indicator of cell proliferation status) and CCND1 (Cyclin D1, a G1 / S-specific cyclin-D1 protein encoded by the human CCND1 gene. The main function of cyclin D1 is to promote cell proliferation. Cyclin D1 binds to and activates the G1 phase-specific cyclin-dependent kinase CDK4, phosphorylating the G1 phase cell repressor protein (Rb). The phosphorylated Rb protein is released from its bound E2F... Transcription factors dissociate, and E2F transcription factors initiate the transcription of genes involved in the living cell cycle, thereby propelling the cell cycle from the G1 phase to the S phase.

[0249] The method for detecting PCNA and CCND1 expression levels by quantitative real-time RT-PCR is the same as the method for detecting YL1-tiRNA by quantitative real-time RT-PCR in the stem-loop region described above.

[0250] Results of quantitative RT-PCR detection of PCNA and CCND1 expression levels ( Figure 8 The results showed that after YL1-tiRNA overexpression, the expression levels of proliferation-related genes PCNA and CCND1 mRNA increased significantly, proving that YL1-tiRNA can promote the proliferation of C2C12 cells.

[0251] 6.2 Western Blot

[0252] Western blotting (also known as protein immunoblotting) is a technique commonly used in research to isolate and identify proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only the antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning.

[0253] Because antibodies bind only to the target protein, only a single, clear band is typically observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of a specific reaction, information about the expression of the target protein in a given cell or tissue homogenate can be obtained.

[0254] Western blot experimental procedures

[0255] (1) Discard the cell culture medium, wash 3 times with 1×PBS (to remove serum from the culture medium), and add 100uL of protein lysis buffer to each well (6-well plate). Quickly scrape the cells off with a cell scraper and transfer them to a 1.5ml tube and place on ice.

[0256] (2) Dissolve on a shaker at 4℃ for 30 min, centrifuge at 12,000 r / min at 4℃ for 15 min, and collect the supernatant into another 1.5 mL tube.

[0257] (3) Take 10 μL and add it to 40 μL of dd H2O and mix well. Take 25 μL and mix it with 200 μL of the mixture (protein concentration determination solution A: solution B = 50:1). Incubate at 37℃ for 30 min. Detect the OD value at 562 nm using a Spark 10M microplate reader. Add 6× Loading Buffer (1:5) to the remaining samples, boil at 95℃ for 10 min, and store at -20℃.

[0258] (4) SDS-PAGE polyacrylamide gel electrophoresis: Prepare separating gel (5ml / gel) and stacking gel and let stand. Insert comb and wait for the gel to solidify. Then put the gel into the electrophoresis tank and add 1× electrophoresis buffer to both the upper and lower tanks.

[0259] (5) Slowly pull out the comb, slowly add 30uL of protein sample to each well, and turn on the power.

[0260] (6) Start with a constant voltage of 90V. After running through the stacking gel, increase the current to 120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein should be run to two-thirds of the position of the separating gel.

[0261] (7) Transfer: Cut the gel according to the marker and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0262] (8) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it along with 4 sheets of 3mm filter paper and a sponge in the transfer buffer. Follow the order below: fiber pad -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber pad (ensure there are no air bubbles, and the transfer time depends on the size of the protein).

[0263] (9) One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0264] (10) Blocking: After the transfer, add blocking solution (prepare 5% blocking solution with skim milk powder) and block for 1 hour.

[0265] (11) Wash the membrane with 1×TBST 3 times, 15 minutes each time.

[0266] (12) Add the diluted antibody and incubate overnight at 4°C on a shaker.

[0267] (13) Recover the primary antibody and wash the membrane 3 times with 1×TBST for 15 minutes each time.

[0268] (14) Add secondary antibody, incubate at room temperature for 1 hour, and wash the membrane 3 times with 1×TBST for 15 minutes each time.

[0269] (15) Sweeping film

[0270] Western blot results

[0271] Test results ( Figure 9 The results showed that after YL1-tiRNA overexpression, the protein expression levels of proliferation-related genes PCNA and CCND1 increased significantly, proving that YL1-tiRNA can promote the proliferation of C2C12 cells.

[0272] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as defined by the appended claims and their equivalents.

[0273] Nucleotide and / or amino acid sequence listing

[0274] <110> Qiqihar University

[0275] <120> YL1-tiNA promotes the repair of damaged muscle cells in mice

[0276] <160> 1

[0277] <170> SIPOSequenceListing 1.0

[0278] <210> 1

[0279] <211> 39

[0280] <212> RNA

[0281] <213> YL1-tiNA sequence

[0282] <400> 1

[0283] ggtccauaguguagcuuaauauuaaagcaucuggcccuac 39

Claims

1. A YL1-tiRNA that promotes the repair of damaged mouse muscle cells, wherein the YL1-tiRNA is cleaved from mature mt-Tv, a phosphate group is added to the 5' end and the sequence is phosphorylated, and a sequence is added to the 5' end without changing its function, characterized in that: The nucleotide sequence of the YL1-tiRNA is shown in Seq ID No:

1.

2. The YL1-tiRNA according to claim 1, characterized in that: The 5' end exogenously added sequence is the rGrGrTrCr sequence.

3. The YL1-tiRNA according to claim 1, characterized in that: The 5' end exogenous added sequence is 1-10 nucleotides in length.

4. The application of YL1-tiRNA according to claim 1 in the preparation of muscle damage repair drugs, characterized in that: The application achieves tissue repair by regulating the proliferation of mammalian myoblasts.

5. The application according to claim 4, characterized in that: The muscle injury is either a mechanical injury or a degenerative injury to skeletal muscle.

6. A method for promoting muscle cell repair in vitro, characterized in that... include: The YL1-tiRNA as described in any one of claims 1-3 was introduced into the damaged C2C12 cell line; Promotes cell proliferation and damage repair under culture conditions of 37℃ and 5% CO2.

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