Application of MSTN (myostatin) and downstream target gene thereof as target spot for detecting and treating liver aging

Abstract

The invention discloses an application of MSTN (myostatin) and a downstream target gene thereof as a target spot for detecting and treating liver aging, and belongs to the technical field of biological medicines. The downstream target gene disclosed by the invention is an Slc7a11 gene. The research finds that in mouse primary hepatocytes treated by MSTN recombinant protein for 24 hours, the expression of senescence markers p53, p21, p16 and gamma-H2AX is remarkably up-regulated, and the activity of beta-galactosidase is synchronously enhanced. RNA sequencing analysis shows that cell senescence related gene sets in an MSTN treatment group are remarkably enriched in GO function annotation and GSEA analysis. Differential expression gene heat map analysis further reveals that Slc7a11 is the most significant target gene for down-regulation. Therefore, the MSTN and the downstream target gene Slc7a11 thereof can be used for preparing a reagent and a kit for detecting liver aging; the composition is used for preparing medicines for treating liver aging.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of MSTN and its downstream target genes as targets for detecting and treating liver aging. Background Art

[0002] Aging is a complex, multi-stage, and gradual process that occurs throughout life. With age, organs and muscles gradually age, and certain diseases, including cancer and cardiovascular and cerebrovascular diseases, also occur with aging. The liver is one of the most resilient organs in the body. A healthy liver has a greater regenerative capacity than any other vital organ, but it is susceptible to damage from stress and aging, leading to fatty degeneration, inflammation, fibrosis, and even failure.

[0003] As the population ages, the proportion of people aged 65 and over is increasing significantly, projected to reach 29.8% by 2050. The prevention and treatment of chronic diseases associated with aging is a critical issue that the country urgently needs to address. Sarcopenia, which increases with age, is a key hallmark of aging. It is estimated to affect 5-10% of people over 65, a proportion significantly higher among those with chronic diseases.

[0004] Myostatin (MSTN) is a secreted protein of the transforming growth factor-β (TGF-β) superfamily. It is primarily produced by skeletal muscle fibers and secreted into the circulation, playing a key role in regulating muscle mass. MSTN is synthesized as an inactive precursor protein and cleaved by proteases during secretion to produce three main components: a signal peptide, a propeptide, and a mature peptide. The mature peptide binds to the activin type II receptor (ActRIIB) on the cell membrane through its C-terminal domain, thereby activating the SMAD2 / 3 signaling pathway and inhibiting muscle cell proliferation and differentiation.

[0005] It is not clear whether MSTN secreted by skeletal muscle causes liver aging and whether MSTN can directly "regulate (talk)" to the liver.

[0006] At present, no study has reported the mechanism by which MSTN can regulate liver aging through its downstream target gene Slc7a11. Summary of the Invention

[0007] Purpose of the Invention: This invention aims to provide a method for detecting and treating liver aging using MSTN and its downstream target genes. By targeting MSTN and its downstream target genes, secreted by muscle cells, this invention aims to develop novel intervention strategies against liver aging, including drugs, gene editing, or biologics.

[0008] The present study found that Slc7a11 is an important target gene of MSTN in regulating liver aging. This discovery is of great significance for revealing the potential role of Slc7a11 in the pathogenesis of liver aging, and helps to provide a new research perspective for solving the treatment problem of liver aging.

[0009] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0010] The present invention provides the use of MSTN and its downstream target genes as immune-related prediction biomarkers for liver aging and immunotherapy responsiveness.

[0011] The downstream target gene is the Slc7a11 gene.

[0012] The present invention, through a series of experiments, reveals that MSTN is a key molecule in regulating liver aging. First, it was found that in primary mouse hepatocytes treated with recombinant MSTN protein for 24 hours, the expression of aging markers p53, p21, p16, and γ-H2AX was significantly upregulated, and β-galactosidase activity was simultaneously enhanced. RNA sequencing analysis revealed that the cellular senescence-related gene set in the MSTN-treated group was significantly enriched in both GO functional annotation and GSEA analysis. Heat map analysis of differentially expressed genes further revealed that solute carrier family 7, member 11 (Slc7a11), a key amino acid transport gene, was the most significantly downregulated target gene.

[0013] The present invention discovered and verified that myostatin (MSTN) and its downstream target gene Slc7a11 derived from muscle cells can be used as detection and treatment targets for liver aging.

[0014] The present invention also provides the use of MSTN and / or its downstream target gene Slc7a11 in preparing reagents and kits for detecting liver aging.

[0015] The reagents for detecting liver aging include reagents for enzyme-linked immunosorbent assay, reagents for mass analysis, reagents for protein microarray, reagents for immunoblotting, or reagents for immunoprecipitation analysis.

[0016] The reagent and kit for detecting liver aging are used to detect MSTN in muscle fibers and its downstream target gene Slc7a11 in liver cells.

[0017] The reagents and kits contain the sequences shown in SEQ ID NO.1 to SEQ ID NO.4.

[0018] MSTN nucleotide sequence (SEQ ID NO.1):

[0019]

[0020]

[0021]

[0022] MSTN amino acid sequence (SEQ ID NO. 2):

[0023] Slc7a11 nucleotide sequence (SEQ ID NO.3):

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] Slc7a11 amino acid sequence (SEQ ID NO.4):

[0030]

[0031] The present invention also provides a liver aging detection reagent and kit, which include reagents for detecting the expression levels of MSTN and / or Slc7a11 genes.

[0032] The present invention also provides the use of MSTN and / or its downstream target gene Slc7a11 in preparing a drug for treating liver aging.

[0033] The drug blocks liver aging by reducing MSTN expression in muscle fibers.

[0034] Furthermore, the drug promotes the expression level of the Slc7a11 gene.

[0035] Beneficial effects:

[0036] The present invention provides evidence that MSTN is a key factor in regulating liver aging. Therefore, any method and reagent that can detect MSTN expression levels can be used to screen for the severity of liver aging. Therefore, using MSTN and its downstream target gene, Slc7a11, as detection and treatment targets, can be used to prepare reagents and kits for detecting liver aging, as well as drugs for treating liver aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1Schematic diagram of MSTN-treated mouse primary hepatocytes.

[0038] Figure 2 This is the mRNA expression of senescence-related genes in primary mouse hepatocytes after treatment with MSTN recombinant protein.

[0039] Figure 3 This is the protein expression of senescence-related genes in primary mouse hepatocytes after treatment with MSTN recombinant protein.

[0040] Figure 4 The results of β-galactosidase staining of primary mouse hepatocytes after treatment with MSTN recombinant protein.

[0041] Figure 5 This is the PCA plot of RNA-seq sequencing samples of mouse primary hepatocytes treated with MSTN recombinant protein.

[0042] Figure 6 GO functional annotation and GSEA analysis of RNA-seq sequencing of primary hepatocytes from mice treated with MSTN recombinant protein.

[0043] Figure 7 Heat map of differentially expressed genes in primary hepatocytes from mice treated with MSTN recombinant protein by RNA-seq sequencing. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0045] Example 1 Treatment of primary mouse hepatocytes with MSTN recombinant protein

[0046] To evaluate the effect of MSTN on hepatocyte senescence, this experiment treated mouse primary hepatocytes with 100 ng / ml MSTN recombinant protein (MCE, HY-P72632) for 24 h for subsequent detection.

[0047] Schematic diagram of MSTN recombinant protein treatment of mouse primary hepatocytes is shown in Figure 1 Mouse primary hepatocytes were extracted by a two-step perfusion method and placed in a 37°C, 5% CO2 incubator to allow them to adhere to the wall. 100 ng / ml MSTN recombinant protein working solution was used to act on the mouse primary hepatocytes for 24 hours.

[0048] Primary hepatocytes from wild-type mice were isolated using a two-step perfusion method. The specific method is as follows:

[0049] (1) Preparation

[0050] Prepare digestion solution I (360 ml ddH2O + 2 ml 0.1 mol / L EGTA + 40 ml 10× D-Hanks + 4 ml double antibody (penicillin-streptomycin solution) + 2 ml 200 g / L glucose);

[0051] Prepare digestion II solution (360 ml ddH2O + 40 ml 10× D-Hanks + 4 ml double antibody (penicillin-streptomycin solution) + 2 ml 200 g / L glucose + 3 ml 0.2 M CaCl2 + 0.08 g type IV collagenase).

[0052] The peristaltic pump was disinfected with alcohol and disinfected with ultraviolet light for 30 minutes.

[0053] (2) Anesthetize 6-8 week old C57BL / 6 mice weighing 20-22 g (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) and secure them on a foam board. Cut open the abdomen, separate the organs, and locate the portal vein. Cannulate the portal vein and immediately perfuse it with Digestive Solution I. When the liver turns from red to white, disconnect the inferior vena cava and perfuse approximately 40 ml of Digestive Solution I. Clamp the inferior vena cava for 30 seconds every 1-2 minutes to allow the digestive solution to remain in the liver.

[0054] (3) Perfuse approximately 60 ml of Digestion II solution and stop perfusing if the liver softens or cracks appear.

[0055] (4) The digested liver was placed in a P100 culture dish containing 10 ml of DMEM high-glucose medium containing 1% penicillin-streptomycin. The liver was minced with tweezers, filtered through a 100 μm filter, and centrifuged in a 50 ml centrifuge tube at 400 rpm for 8 min.

[0056] (5) After pouring out the supernatant, the lower layer is the primary hepatocytes. Add 5 ml of SIP (Percoll cell separation solution and 10×PBS, mixed at a ratio of 9:1) and 5 ml of DMEM high-glucose medium containing 1% penicillin-streptomycin, centrifuge at 1200 rpm for 3 min, and pour out the supernatant.

[0057] (6) Add 10 ml of DMEM high-glucose medium containing 1% penicillin-streptomycin and centrifuge at 400 rpm for 5 min. After centrifugation, discard the supernatant and resuspend the remaining hepatocytes. Plant them in a six-well plate and culture the cells in a 37°C, 5% CO2 incubator.

[0058] (7) After 4 hours, the cells were rinsed three times with DMEM high-glucose medium containing 1% penicillin-streptomycin and the medium was replaced to obtain primary mouse hepatocytes.

[0059] Example 2 PCR experiments to verify the mRNA expression levels of p53, p21, p16 and β-gal

[0060] RNA extraction and PCR experiments of primary mouse hepatocytes treated with and without MSTN recombinant protein:

[0061] Total RNA was extracted from cells using the RNAiso Plus extraction kit from TAKARA, Japan. RNA concentration and quality were measured using a Thermo Nanodrop One spectrophotometer. RNA samples from the control group (primary mouse hepatocytes not treated with MSTN recombinant protein) and the experimental group (primary mouse hepatocytes treated with MSTN recombinant protein) were adjusted to a similar concentration (5 ng / ml) for use. Reverse transcription was performed using a Veriti 96-Well Thermal Cycler PCR instrument and HiScript IIQ RT SuperMix for QPCR (Vazyme #R222-01).

[0062] Specific primers were further designed for circRNA, and the PCR reaction program was set up as follows using the ThermoFisher QuantStudio 5 real-time fluorescence quantitative PCR system:

[0063] Step 1: Pre-denaturation at 95°C for 20 seconds;

[0064] Step 2: Cycle reaction at 95°C for 10 seconds and 60°C for 30 seconds;

[0065] Step 3: Melting curve.

[0066] PCR reaction system: 2.5 μl of substrate (DNA), 5 μl of ChamQ SYBR Color qPCR Master Mix (Vazyme #Q411-02), 2 μl of DEPC, and 0.5 μl of primers for q-RT-PCR analysis. Table 1 lists the primer sequences used.

[0067] Table 1 Primer sequence information

[0068]

[0069]

[0070] The above primers were purchased from Anhui General Biological.

[0071] The experimental results are shown in Figure 2 .

[0072] As can be seen from the figure, the RNA results showed that the expression of cell cycle arrest-related genes p53, p21, p16 and β-gal, which are important indicators reflecting liver cell aging, increased in the MSTN recombinant protein-treated group, indicating that liver cells aged after treatment with MSTN recombinant protein.

[0073] Example 3 Western blot experiments to verify the protein expression levels of p53, p21, p16 and γ-H2AX

[0074] Experimental methods:

[0075] (1) Total protein extraction from primary mouse hepatocytes treated with or without MSTN recombinant protein was performed using a total protein extraction kit (Keygen Biotechnology, Nanjing, Jiangsu). The specific method was referred to the kit instructions.

[0076] (2) The total protein content was determined using a BCA assay kit. The specific method was referred to the kit instructions.

[0077] (3) Western blot procedure

[0078] ① Clean the glass plate: rinse the glass plate with tap water, wipe it with 75% alcohol, rinse it with distilled water, and dry it for later use.

[0079] ② Glue filling and sample loading: Align the glass plate and place it in the clamp, secure it vertically on the rack, add an appropriate amount of ultrapure water (ddH2O), and observe for 30 minutes. Assess the liquid level and level, and check for any significant leakage and adjust accordingly. Prepare the separation gel, shake well, and then fill the gel. When pouring the gel, pipette the gel slowly along the glass until the gel surface rises to the middle of the red band. Next, add ddH2O to the gel until it reaches the edge of the glass plate. Place it on a flat surface and wait for gelation, while observing for any leakage. When a refractive index line appears between the ddH2O and the separation gel, the separation gel has solidified. Pour off the ddH2O on top of the separation gel and blot dry with absorbent paper. Set aside. Prepare 10% stacking gel and fill the space above the separation gel in the gel plate with the prepared stacking gel. Immediately insert a lane comb into the stacking gel. Once the stacking gel has solidified, remove the lane comb and rinse the gel wells with ddH2O before placing it in the electrophoresis tank. Total protein from mouse primary hepatocytes treated with MSTN recombinant protein (rMSTN group) and untreated with MSTN recombinant protein (control group) was added to 0.5 ml centrifuge tubes. 5× Loading Buffer was added to a final concentration of 1× and the proteins were denatured by boiling in water for 10 minutes. The sample to be tested was then slowly added to the sample wells.

[0080] ③ Electrophoresis: The constant voltage used for the stacking gel layer is 80V. When the sample enters the dividing line between the stacking gel and the separation gel, change the voltage to 120V for electrophoresis. Stop electrophoresis when the bromophenol blue runs to the bottom of the glass plate and the marker bands are separated enough.

[0081] ④ Transfer: Prepare transfer buffer in advance. Cut PVDF membranes of appropriate size based on the number of samples to be tested and the size of the gel. Activate the PVDF membrane in methanol for 5 minutes. Equilibrate with ddH2O, then place it in transfer buffer for 15 minutes. Pry open the glass plywood to remove the gel, and soak the separation gel in transfer buffer. Stack the gel in the following order: negative electrode → sponge pad → filter paper → gel → PVDF membrane → filter paper → sponge pad → positive electrode, ensuring that the cut PVDF membrane completely covers the gel corresponding to the target band. Connect the electrodes correctly, place the membrane in an ice bath, and transfer the membrane at 200mA for 55 minutes.

[0082] ⑤ Blocking: Prepare blocking solution (5% skim milk powder TBST solution) in advance; after transfer, move the membrane to an incubation box containing blocking solution and shake on a decolorization shaker at room temperature for 2 hours.

[0083] ⑥ Primary Antibody Incubation: Discard the blocking solution and incubate the blocked membrane in the primary antibody (antibodies should be diluted in primary antibody diluent; see Table 2 for antibodies and dilution ratios) overnight at 4°C. Recover the primary antibody the next day and wash the membrane three times with TBST on a shaker for 10 minutes each.

[0084] Table 2 Antibody information

[0085]

[0086] ⑦ Secondary antibody incubation: Use TBST to prepare secondary antibody dilution solution (Biyuntian, 1:10000), incubate at room temperature for 2 hours, and then wash the membrane three times with TBST on a shaker at room temperature for 10 minutes each time.

[0087] ⑧ Development and Imaging: Chemiluminescence was performed using Thermo ECL. In a darkroom, equal volumes of developer solution A and solution B were mixed (1:1, approximately 100 μl). The PVDF membrane was slightly dried with filter paper and then placed on a glass plate. An appropriate amount of developer solution was then added dropwise to the PVDF membrane. The gel was developed and imaged using a Tanon gel imager. Band grayscale values were analyzed using Quantity One software.

[0088] The experimental results are shown in Figure 3 .

[0089] As can be seen from the figure, WB results show that the important indicators reflecting liver cell aging, cell cycle arrest-related genes p53, p21, p16 and DNA damage-related gene γ-H2AX were significantly increased in the MSTN recombinant protein treatment group, which once again proves from the protein perspective that MSTN can promote the aging of primary liver cells.

[0090] Example 4 β-galactosidase staining experiment

[0091] β-galactosidase staining was performed using the Beyotime C0602 kit. The experimental steps are as follows:

[0092] (1) After treating mouse primary hepatocytes with MSTN recombinant protein for 24 h, the cell culture medium was aspirated, washed once with PBS, and 1 ml of β-galactosidase staining fixative was added and fixed at room temperature for 15 min.

[0093] (2) Aspirate the cell fixative and wash the cells three times with PBS, each time for 3 minutes;

[0094] (3) Aspirate PBS and add 1 ml of staining solution to each well. The staining solution contains: 10 μL of β-galactosidase staining solution A, 10 μL of β-galactosidase staining solution B, 930 μL of β-galactosidase staining solution C, and 50 μL of X-Gal solution.

[0095] (4) Incubate at 37°C overnight and seal the 6-well plate with plastic wrap to prevent evaporation;

[0096] (5) Observe under an ordinary optical microscope.

[0097] The results of β-galactosidase staining experiments of primary mouse hepatocytes treated with MSTN recombinant protein (rMSTN group) and without MSTN recombinant protein treatment (control group) are shown in Figure 4 .

[0098] As can be seen from the figure, β-galactosidase staining showed that the β-galactosidase activity of mouse primary hepatocytes treated with MSTN recombinant protein was significantly enhanced, which once again confirmed that MSTN recombinant protein can promote the aging of primary hepatocytes.

[0099] Example 5: RNA-seq of primary mouse hepatocytes treated with MSTN recombinant protein

[0100] The PCA diagram of RNA-seq sequencing samples is shown in Figure 5 .

[0101] The PCA results suggested that the clustering effect between the two groups [MSTN group (MSTN) and control group (Con)] was good, and subsequent analysis was suitable.

[0102] The GO functional annotation and GSEA analysis of RNA-seq sequencing are shown in Figure 6 .

[0103] The results showed that the gene sets related to cell aging were significantly enriched in the experimental groups in three control samples (Con1, Con2, Con3, mouse primary hepatocytes not treated with MSTN recombinant protein) and three MSTN recombinant protein groups (MSTN1, MSTN2, MSTN3, primary hepatocytes treated with 100 ng / ml MSTN recombinant protein for 24 hours).

[0104] Heat map of RNA-seq sequencing samples is shown in Figure 7 .

[0105] The heat map shows the significantly differentially upregulated and downregulated genes in the three control samples (Con1, Con2, and Con3) and the three MSTN recombinant protein groups (MSTN1, MSTN2, and MSTN3). The figure shows that the Slc7a11 gene is significantly downregulated after the addition of MSTN recombinant protein, indicating that Slc7a11 is a downstream gene that causes senescence in primary hepatocytes by MSTN recombinant protein.

[0106] Core features of liver cell aging include:

[0107] (1) Disturbance of cell cycle regulation, manifested by G1 phase arrest and upregulation of the expression of related regulatory factors p53, p21, and p16;

[0108] (2) Enhanced DNA damage response, characterized by DNA double-strand breaks and γ-H2AX accumulation;

[0109] (3) Abnormal lysosomal function can be detected by increased aging-related β-galactosidase activity.

[0110] The present invention, through a series of experiments, reveals that MSTN is a key molecule in regulating liver aging. First, it was found that in primary mouse hepatocytes treated with recombinant MSTN protein for 24 hours, the expression of aging markers p53, p21, p16, and γ-H2AX was significantly upregulated, and β-galactosidase activity was simultaneously enhanced. RNA sequencing analysis revealed that the cellular senescence-related gene set in the MSTN-treated group was significantly enriched in both GO functional annotation and GSEA analysis. Heat map analysis of differentially expressed genes further revealed that solute carrier family 7, member 11 (Slc7a11), a key amino acid transport gene, was the most significantly downregulated target gene.

[0111] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Application of MSTN and its downstream target genes as predictive biomarkers for immune-related prediction of liver aging and immunotherapy responsiveness.

2. The use according to claim 1, characterized in that The downstream target gene is the Slc7a11 gene.

3. Use of MSTN and / or its downstream target gene Slc7a11 in the preparation of reagents and kits for detecting liver aging.

4. The use according to claim 3, characterized in that The reagents for detecting liver aging include reagents for enzyme-linked immunosorbent assay, reagents for mass analysis, reagents for protein microarray, reagents for immunoblotting, or reagents for immunoprecipitation analysis.

5. The use according to claim 3, characterized in that The reagent and kit for detecting liver aging are used to detect MSTN in muscle fibers and its downstream target gene Slc7a11 in liver cells.

6. The use according to claim 3, characterized in that The reagents and kits contain the sequences shown in SEQ ID NO.1 to SEQ ID NO.

4.

7. A liver aging detection reagent or kit, characterized in that: It includes reagents for detecting the expression levels of MSTN and / or Slc7a11 genes.

8. Use of MSTN and / or its downstream target gene Slc7a11 in the preparation of drugs for treating liver aging.

9. The use according to claim 8, characterized in that The drug blocks liver aging by reducing MSTN expression in muscle fibers.

10. The use according to claim 8, characterized in that The drug promotes the expression level of the Slc7a11 gene.

Images