A polypeptide for treating vascular calcification

Abstract

This invention provides a polypeptide for treating vascular calcification, belonging to the field of biotechnology. By analyzing lncRNA sequencing results from patients with chronic kidney disease exhibiting varying degrees of vascular calcification, this invention reveals for the first time a long non-coding RNA (lncRNA)—LINK-A—highly expressed in these patients. Furthermore, it confirms that LINK-A endogenously expresses a 40-amino acid-residue polypeptide—PKM2-associated polypeptide (PKM2AP)—in its second ORF. Subsequently, in vitro and in vivo experiments validated the important role of this polypeptide in inhibiting vascular calcification. This invention not only provides a novel target (LINK-A) for vascular calcification but also offers a potential drug (PKM2AP) for treating vascular calcification.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a polypeptide for treating vascular calcification. Background Technology

[0002] Currently, vascular calcification (VC) is a common pathological manifestation in cardiovascular diseases, atherosclerosis, hypertension, diabetic vascular complications, vascular injury, chronic kidney disease, and aging. It mainly manifests as increased stiffness and decreased compliance of the blood vessel walls, easily leading to myocardial ischemia, left ventricular hypertrophy, and heart failure, triggering thrombosis and plaque rupture. VC is not only an important marker of cardiovascular events caused by atherosclerosis, stroke, and peripheral vascular disease, but also a significant factor contributing to the high incidence and mortality of cardiovascular and cerebrovascular diseases, severely impacting patients' quality of life and prognosis. While existing treatments can alleviate symptoms to some extent, effective therapeutic drugs are still lacking. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a polypeptide for treating vascular calcification, belonging to the field of biotechnology. This invention, through analysis of lncRNA sequencing results from patients with chronic kidney disease exhibiting varying degrees of vascular calcification, reveals for the first time a long non-coding RNA (lncRNA)—LINK-A—highly expressed in these patients. Furthermore, it confirms that its second ORF endogenously expresses a 40-amino acid-residue polypeptide—PKM2-associated polypeptide (PKM2AP). Subsequently, in vitro and in vivo experiments validated the important role of this polypeptide in inhibiting vascular calcification. This invention not only provides a novel target (LINK-A) for vascular calcification but also offers a potential drug (PKM2AP) for treating vascular calcification.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] On one hand, the present invention provides a polypeptide for treating vascular calcification, the polypeptide comprising the amino acid residue sequence shown in SEQ ID NO:8, namely the PKM2 AP polypeptide described in the present invention.

[0006] Furthermore, the concentration of the polypeptide used is 5 μg / g to 100 μg / g, preferably 8 μg / g to 10 μg / g.

[0007] On the other hand, the present invention provides a drug for inhibiting vascular calcification, the drug comprising the polypeptides described above.

[0008] Furthermore, the administration methods of the drug include, but are not limited to, oral, injection, and spray.

[0009] On the other hand, the present invention provides the use of a polypeptide for preparing an inhibitor of vascular calcification, the polypeptide comprising the amino acid residue sequence shown in SEQ ID NO:8.

[0010] Furthermore, the vascular calcification was caused by chronic kidney disease.

[0011] On the other hand, the present invention provides the use of a polypeptide for preparing an agent to inhibit calcium deposition and / or calcium nodule formation, said polypeptide comprising the amino acid residue sequence shown in SEQ ID NO:8.

[0012] On the other hand, the present invention provides the use of a polypeptide for preparing an agent to treat vascular calcification by inhibiting the expression of RUNX2 and / or BMP2 proteins, said polypeptide comprising the amino acid residue sequence shown in SEQ ID NO:8.

[0013] On the other hand, the present invention provides the use of RNA for preparing a polypeptide that inhibits vascular calcification, the polypeptide being encoded by the RNA, the RNA comprising the nucleotide sequence shown in SEQ ID NO:4, namely the ORF2 of LINC01139(LINK-A) of the present invention.

[0014] Furthermore, the RNA comprises the nucleotide sequence shown in SEQ ID NO:1.

[0015] The beneficial effects of this invention include:

[0016] 1. By analyzing the lncRNA sequencing results of patients with chronic kidney disease and vascular calcification, we discovered for the first time that LINK-A (LINC01139, SEQ ID NO:1) is associated with vascular calcification, providing a new target for the treatment of vascular calcification;

[0017] 2. For the first time, it was confirmed that the second ORF (SEQ ID NO:4) of LINK-A has encoding activity, which can encode a polypeptide (PKM2 AP, SEQ ID NO:8) composed of 40 amino acid residues in vivo. Furthermore, it was demonstrated in vitro and in vivo that this peptide affects the glycolysis pathway and lactate production by targeting the ubiquitination and degradation of PKM2 protein, thereby achieving the effect of treating vascular calcification.

[0018] 3. The PKM2 AP peptide is characterized by its small size, high specificity, and low cytotoxicity, and has the potential to be used as a therapeutic drug. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Differentially expressed genes in patients with varying degrees of vascular calcification and chronic kidney disease (volcano plot);

[0021] Figure 2 Schematic diagram of the ORFs distribution of LINC01139 and DANT2;

[0022] Figure 3 : Validation of the translation potential of LINC01139 and DANT2 ORFs, with NC as an empty vector and serving as a negative control;

[0023] Figure 4 : Partial schematic diagram of the recombinant vector LINK-A Flag and Mut LINK-A Flag;

[0024] Figure 5 Functional verification of the ORF2 start codon (LINC01139); Figure A shows the Western blot results, where actin is the internal reference and NC is the negative control transfected with the empty vector; Figure B shows the immunofluorescence staining results, where EV is the empty vector as the negative control, and the scale bar is 20 μm.

[0025] Figure 6 : Validation of endogenous expression of PKM2 AP; Scale bar = 20 μm;

[0026] Figure 7 Figure 1 shows the in vivo validation of PKM2 AP function; Figure A is the flowchart of mouse treatment; Figure B shows the Von Kossa staining results of intact mouse arteries (n=6), with the CTL group representing blood vessels grown under normal conditions, the 1,25(OH)2VitD3 group representing blood vessels treated with 1,25(OH)2VitD3, and the Vehicle group serving as a negative control for the PKM2 AP group; Figure C shows the HE, Alizarin Red S, and Von Kossa staining results of mouse arterial pathological sections (n=6); Figure D shows the detection results of Ca content in mouse blood vessels (n=6); Figure E shows the detection of changes in RUNX2 and BMP2 protein content (n=6); Figure F shows the quantitative results of Figure E, where relative protein expression level refers to the ratio of RUNX2 or BMP2 to GAPDH;

[0027] Figure 8Figure 1 shows the in vitro validation of PKM2 AP function; Figure A shows the results of Alizarin Red staining of human aortic vascular smooth muscle cells (HAVSMCs) (n=3). The CTL group and β-GP group are HAVSMCs grown under normal conditions and HAVSMCs treated with β-GP, respectively. The NC group is the negative control of the PKM2 AP group, that is, no other therapeutic substances were added to this group; Figure B shows the detection results of Ca content in HAVSMCs (n=3); Figures C and D show the detection of RUNX2 and BMP2 gene expression levels (n=3); Figure E shows the immunofluorescence staining results of RUNX2 and BMP2 proteins (n=3); Figure F shows the quantitative results of Figure E. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified. For reagents with specific sources mentioned, those skilled in the art can perform the relevant experiments according to the reagent instructions provided by the manufacturer.

[0030] Example 1: Obtaining a polypeptide for treating vascular calcification

[0031] To identify lncRNAs associated with vascular calcification in chronic kidney disease (CKD), this invention collected abdominal wall artery samples (n=5 per group) from representative dialysis patients with mild and severe vascular calcification. Equal volumes of samples from each group (mild CKD or severe CKD) were mixed thoroughly, and RNA was extracted from the mixed samples from the different groups (mild and severe) according to the "steps for extracting total RNA from arterial tissue" described in the TRIzol extraction reagent (Invitrogen, 15596018) instructions. lncRNA sequencing (Euronext) was then performed. Subsequently, differential expression analysis of the gene expression matrix was performed on the lncRNA sequencing results using the edgeR software package (version 3.4.4). Specifically, before differential gene expression analysis, for each sequencing library, the edgeR package adjusted the read count using a scaling normalization factor, and then used edgeR... The R software package (3.18.1) was used to perform differential expression analysis on lncRNA sequencing results. The Benjamini and Hochberg methods were used to adjust the P-value. The corrected P-value was 0.05 and the absolute fold change was 2. P-value < 0.05 and absolute fold change > 2 were set as the thresholds for significant differential expression.

[0032] The results showed that 2,200 lncRNAs with significant differential expression in patients with different degrees of vascular calcification were identified using the above method. Among them, 588 lncRNAs were upregulated, while 581 were downregulated. Figure 1 The HMPA database (http: / / hmpa.zju.edu.cn / ) and the NCBI website were used to compare the top 20 genes with the largest fold changes in expression (from largest to smallest) (see Table 1). Only LINC01139 (corresponding to ENSG00000215808.4 in Table 1, SEQ ID NO:1) and DANT2 (SEQ ID NO:2) were found to have potential open reading frames (ORFs) (see Table 1). Figure 2 Among them, LINC01139 is an annotated human lncRNA named LINK-A, which has 3 potential ORFs (SEQ ID NO: 3-5); while DANT2 has 2 (SEQ ID NO: 6-7).

[0033] Table 1. Summary of the top 20 lncRNAs by fold change in expression.

[0034]

[0035] To verify the translation potential of the aforementioned ORFs, this embodiment commissioned Wiegand Biotech to synthesize vector pcDNA3.1 containing the aforementioned five ORFs (SEQ ID NO: 3-7) and tagged with 3×Flag, the 3×Flag tags being located at the C-terminus of the ORFs. Next, the five recombinant vectors were transfected into 293T cells (ATCC) using Lipofectamine 2000 (Invitrogen) reagent (transfection steps are described in the Lipofectamine 2000 reagent instructions). After 48 hours of transfection, cells were collected and washed with phosphate-buffered saline (pH = 7.4) for further extraction of whole-cell proteins. An appropriate amount (preferably 200 μL) of 1 mM... 293T cells in 6-well plates were lysed using PMSF (ST506, Beyotime) RIPA lysis buffer (pH 7.4, P0013B, Beyotime). After lysis, the crude protein concentration was first determined using a BCA protein assay kit (Beyotime, P0012S), followed by SDS-PAGE to separate the protein samples. It is important to note that the loading volume needs to be determined based on the protein concentration. The separated samples were then transferred to a PVDF membrane and incubated with 5% nonfat milk for 1 hour. Next, the membrane was incubated overnight at 4°C with primary antibodies of the following types and sources: anti-BMP2 (#5591, Abcam), anti-RUNX2 (ab81371, Abcam), anti-GAPDH (60004-1-Ig, Proteintesh), and anti-FLAG (20543-1-AP, Proteintech). The membrane was then incubated for 1 hour with secondary antibodies conjugated with horseradish peroxidase (A0216, A0208, beyotime). Finally, the proteins were visualized using an enhanced chemiluminescence assay kit (Beyotime, ECL P0018S), and the protein bands were observed using a chemiluminescence scanning membrane instrument.

[0036] Depend on Figure 3 The results showed that the GAPDH protein bands in the control group (NC) and each experimental group were clearly visible, and the gray levels of the bands were relatively consistent between groups, which suggests that the total protein content in each group was the same. Only the LINK-AORF2 group showed a single, clear band of 15 kDa in the lane, and the size of this band was as expected, indicating that only LINK-A (LINC01139) ORF2 (SEQ ID NO:4) can encode a peptide.

[0037] To determine whether the ORF2 start codon in LINK-A was valid, this embodiment constructed LINK-A ORF2 (SEQ ID NO:4) containing either an ATG start codon or an ATT mutant start codon into a pcDNA3.1 vector with a 3×Flag tag (construction steps as described above), obtaining the recombinant vectors LINK-A Flag and Mut LINK-AFlag (SEQ ID NO:4). Figure 4 The 3×Flag tag is located at the C-terminus of LINK-AORF2; and the expression of Flag fusion proteins driven by different start codons was detected by Western blot and immunofluorescence staining experiments. The experimental steps of Western blot were the same as described above, and the specific steps of immunofluorescence staining were as follows: human embryonic kidney cells (HEK293T, ATCC) in 12-well plates (Corning, 3006) were fixed overnight at 4°C with 4% paraformaldehyde; and 0.5% paraformaldehyde was used for staining. Cells were punctured with Triton-X (ST1723, beyotime) and 2% bovine serum albumin (ST025, beyotime) to fix them. The fixed cells were incubated overnight with the primary antibody at room temperature or at 4°C (preferably 4°C in this embodiment). The primary antibody was anti-FLAG with a dilution of 1:500. The primary antibody was detected by appropriate FITC-labeled goat anti-mouse IgG (H+L) (A0568, beyotime) and 4',6-diamidinyl-2-phenylindole (Mpbio, 157574). The secondary antibody had a dilution of 1:500. Imaging was performed using a fluorescence microscope (Zeiss, Axio Vert A1, or Leica TCS-SP8 SR), and analysis was performed using CellProfiler software or PerkinElmer Opera Phenix.

[0038] Depend on Figure 5 As shown in A, under normal circumstances, LINK-AORF2 (LINK-AFlag group) can encode a polypeptide with a molecular weight of approximately 15 kDa. However, after mutating the start codon of ORF2, the target fragment cannot be detected; at the same time, the corresponding fluorescent signal can only be detected when the start codon is not mutated. Figure 5 B). In summary, these experimental results demonstrate that the start codon of LINC01139 ORF2 is functionally recognized; specifically, the start codon is recognized by ribosomes, and the ORF is subsequently translated into a polypeptide.

[0039] LINK-A's ORF2 encodes a 40-aa polypeptide, the specific sequence of which is MGSHSSSLCTPFKCILNPWDSFKKCLLFSFPLLCLFFTDR (SEQ ID NO:8). Homologous base sequences to LINK-A ORF2 in different species (mainly mammals) were searched on the UniProt online website (https: / / www.uniprot.org / ). Based on codon encoding principles, the corresponding amino acid residue sequences were generated and sequence alignment was performed on the UniProt website. This search yielded 40 sequences homologous to LINK-A ORF2. Multiple sequence alignment results showed that the polypeptide encoded by LINK-A ORF2 is highly conserved in higher mammals. However, no known domains / motifs were matched among the 40 retrieved polypeptides, indicating that the peptide was not characterized. Therefore, based on its function and mechanism of action, it is named pyruvate kinase 2-related peptide (PKM2AP, SEQ ID NO:8).

[0040] To confirm that PKM2 AP is expressed endogenously, this invention prepared an antibody against PKM2 AP. First, the specificity of the PKM2 AP antibody was determined, and then the expression of endogenous PKM2 AP was detected by immunofluorescence staining (the experimental steps are the same as described above, and the primary antibody used here is PKM2 AP antibody (GenScript Biotech)). Figure 6 ), and PKM2 AP is evenly distributed in the extranuclear region (cytoplasm).

[0041] Example 2: In vivo functional verification of PKM2 AP

[0042] To verify the role of PKM2 AP in vascular calcification, this embodiment administered 1,25(OH)2VitD3 (1,25-dihydroxyvitamin D3, Sigma) to 6-8 week old C57 / BL6 mice (n=50) at a dose of 32 μg / g / Day for 6 consecutive days. 1,25(OH)2VitD3 promotes calcium absorption in mice, mimicking the process of vascular calcification. Simultaneously, PKM2 AP (SEQ ID NO:8, Hangzhou Zhuntai Biotechnology) was injected via tail vein every two days to treat mice (n=25). An equal volume of physiological saline was injected as a negative control (Vehicle group, n=25). Vascular samples were then collected (see details in [link to procedure]). Figure 7 A) and uses staining to quantitatively analyze calcium deposition in blood vessels and to conduct histological evaluation of vascular pathology. The staining includes Von Kossa staining, hematoxylin-eosin (HE) staining, and Alizarin Red S staining.

[0043] 2.1 Von Kossa staining

[0044] Von Kossa staining primarily utilizes the reducing properties of silver salts. Acidic silver sulfite reacts with calcium salts in the tissue to form black silver deposits, which can then be clearly observed under a microscope to define the distribution of calcium salts. The specific staining steps are as follows: Paraffin sections of the thoracic aorta of mice after modeling (i.e., treatment with 1,25(OH)2VitD3 for 6 days) are prepared with a thickness of 7 μm. The sections are incubated in 5% silver nitrate (AgNO3, Solarebo) solution and exposed to ultraviolet light for 15-30 minutes until staining is complete. The sections are then washed with 5% sodium thiosulfate (Na2S2O3, Solarebo) to remove any residual AgNO3 solution.

[0045] 2.2 Hematoxylin-eosin (HE) staining

[0046] Paraffin sections (7 μm thick) of the thoracic aorta of mice after modeling (treatment with 1,25(OH)₂VitD₃ for 6 days) were prepared. After dewaxing, the sections were stained with hematoxylin (C0105M, Beyotime) for 5-10 minutes (adjusting the time according to the staining results and requirements). Excess staining solution was rinsed off with tap water for about 10 minutes, followed by a second wash with distilled water (for a few seconds). Finally, the sections were dehydrated, cleared, and mounted with neutral resin or other mounting medium. Under a microscope, the cell nuclei appeared blue, while the cytoplasm appeared pink or red.

[0047] 2.3 Alizarin Red Staining

[0048] Paraffin sections of the thoracic aorta from mice after modeling were prepared, with a thickness of 7 μm. The sections were soaked overnight in 95% ethanol solution (Anjie Gaoke), then transferred to 1% KOH (Aladdin) containing 0.004% Alizarin Red (Beyotime) and stained overnight on a shaker. The tissue was then washed twice with 2% KOH to remove any unbound contaminants.

[0049] 2.4 Detection of Ca content in mouse blood vessels

[0050] A mouse model of vascular calcification was dissected to obtain its aorta. The aorta was cut into small fragments and thoroughly lysed in RIPA lysis buffer (pH 7.4, P0013B, Beyotime) containing 1 mM PMSF (ST506, Beyotime). The fragments were then centrifuged at 10,000-14,000 g for 3-5 minutes at 4°C, and the supernatant was collected. The calcium content in the supernatant was determined using a chemiluminescence immunoassay. In this example, the calcium content detection kit (S1063S) from Beyotime Biotechnology Co., Ltd. was used. For standardization, the Bradford assay (Bio-Rad Laboratories) was used to determine the total protein in the sample.

[0051] from Figure 7 As shown in B and C, under normal conditions (CTL group), there was no significant difference in the blood vessels of mice injected with PKM2 AP compared to those injected with saline, indicating that PKM2 AP does not affect vascular morphology. After treatment with 1,25(OH)2VitD3, compared to the control (CTL group), regardless of whether PKM2 AP treatment was used, the blood vessels of mice in the 1,25(OH)2VitD3 group showed significantly darker staining, indicating a significant increase in vascular calcification nodules and calcium deposition, demonstrating the effectiveness of 1,25(OH)2VitD3 treatment. Furthermore, compared to the control, PKM2 AP injection significantly alleviated calcium deposition, thereby alleviating arteriosclerosis, and the results of intravascular Ca content measurement also corroborated this conclusion. Figure 7 D).

[0052] To preliminarily explore the mechanism of action of PKM2 AP, this embodiment used Western blot to detect the content of RUNX2 and BMP2 proteins in samples (n=6). Among them, osteogenic transcription factor RUNX2 is a member of the Runxx-related factor family and is an important protein that regulates the differentiation of mesenchymal stem cells into osteoblasts, playing an important role in osteoblast differentiation and bone formation. Bone morphogenetic protein 2 (BMP2) is a member of the BMP family and has strong osteogenic activity. It is highly expressed in atherosclerotic endothelial cells and is widely involved in the growth and differentiation of osteoblasts. It can regulate the expression of its downstream Runx2 transcription factor, thereby regulating osteoblast transdifferentiation. Except for the primary antibodies used, which were anti-RUNX2 (dilution ratio 1:1000, abcam, ab76956), anti-BMP2 (dilution ratio 1:1000, abcam, ab214821), and anti-GAPDH (dilution ratio 1:5000, Proteintec, 1E6D9), the remaining steps of the protein immunoblotting were the same as described in Example 1, and the protein bands were quantified using ImageJ and Graphpad software.

[0053] Depend on Figure 7 E and Figure 7 As shown in F, 1,25(OH)2VitD3 induces the expression of RUNX2 and BMP2 proteins, which leads to the transdifferentiation of smooth muscle cells. The transformation of vascular smooth muscle cells into osteoblast-like phenotypes is the essence of vascular calcification, which leads to thickening of the vascular wall, narrowing of the lumen, and vascular sclerosis and remodeling. PKM2 AP can significantly inhibit the expression levels of these two proteins, thereby alleviating calcium deposition in blood vessels.

[0054] In summary, PKM2 AP aims to alleviate the symptoms of vascular calcification by reducing the levels of RUNX2 and BMP2 proteins.

[0055] Example 3: In vitro functional validation of PKM2 AP

[0056] Example 2 demonstrated that PKM2 AP could alleviate vascular calcification symptoms in mice. To further confirm the physiological function of PKM2 AP, this example used 40 mmol / L β-glycerophosphate (β-GP) (Hangzhou Zhuntai Biotechnology Co., Ltd.) to treat human aortic vascular smooth muscle cells (HAVSMCs) for 5 days. β-GP is used as an inducer in bone tissue engineering to promote osteogenic differentiation of cells and is often used to study bone formation and mineralization processes. The treated cells were then subjected to relevant index detection, including calcium deposition (Alizarin Red and Alkaline Phosphatase (ALP) staining, Ca content determination), and the expression of RUNX2 and BMP2 genes at the RNA and protein levels (qRT-PCR and immunofluorescence staining). The ALP staining and qRT-PCR experimental steps are as follows, and the experimental steps of the other detection methods are the same as those described in Examples 1 and 2.

[0057] 3.1 Alkaline phosphatase (ALP) staining

[0058] HAVSMC cell samples for detecting endogenous alkaline phosphatase were fixed with 4% paraformaldehyde (P0099, Beyotime) for 15–20 min. The cells were then washed 3–5 times with PBS (G4202, ServiceBio) for 3–5 minutes each time. After the final wash, an appropriate amount of BCIP / NBT staining working solution (buffer:BCIP:NBT = 30:1:2, Beyotime) was added to ensure adequate coverage of the sample. The cells were incubated at room temperature in the dark for 5–30 minutes or longer (up to 24 hours) (preferably 30 min in the dark in this example) until brown cobalt sulfide was formed and the staining site was located within the cytoplasm. The cells were then washed 1–2 times with distilled water to remove the BCIP / NBT staining working solution, thus terminating the colorimetric reaction.

[0059] 3.2 qRT-PCR

[0060] 3.2.1 Extraction of total RNA

[0061] This example uses the SteadyPure Rapid RNA Extraction Kit (catalog number: AG21023) from Acrel to extract RNA from cells after different treatments. The specific procedures are as follows:

[0062] (1) Aspirate the culture medium from the 6-well plate, wash the cells with 1×PBS buffer, discard the PBS buffer, add 350 μL of Buffer QLS lysis buffer to each well, gently shake the culture plate to ensure the lysis buffer is evenly distributed on the cell surface, repeatedly pipette the cells to detach them, and then transfer them to 1.5 mL centrifuge tubes. Pipette the tubes for 30 seconds to 2 minutes until the solution is clear and non-sticky.

[0063] (2) Centrifuge at 12,000 rpm and 4°C for 5 min;

[0064] (3) Carefully aspirate the supernatant into a new 1.5 mL RNase-free centrifuge tube;

[0065] (4) Add 350 μL of 100% ethanol to the above homogenate and mix well by pipetting.

[0066] (5) Immediately transfer all of the above mixture to Quick RNAmini Columns, centrifuge at 12,000 rpm at room temperature for 2 min, and discard the filtrate;

[0067] (6) Add 700 μL of Buffer QWB to Quick RNAmini Columns, centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate;

[0068] (7) Place the Quick RNAMini Columns adsorption column on a new 2.0 mL Collection Tube and centrifuge at 12,000 rpm for 2 min at room temperature.

[0069] (8) Place the Quick RNA Mini Columns on a new RNase Free Tube, add 30 μL of RNase Free Water to the center of the membrane, let stand at room temperature for 3 min, then centrifuge at 12,000 rpm at room temperature for 2 min to elute the RNA, and store the eluted RNA at -80℃.

[0070] 3.2.2 Reverse transcription reaction

[0071] (1) Use a micro spectrophotometer to quantify RNA and measure its concentration;

[0072] (2) RNA was reverse transcribed into cDNA using the Aike Rui Bio Reverse Transcription Premixed Kit (catalog number: AG11728) (cDNA was used as a template for qRT-PCR). The specific steps are as follows: First, gDNA was removed. The reaction system for removing gDNA included 2 μL of gDNAClean Reaction Mix Ver.2 and 1 μg of Total RNA, and the volume of the system was brought up to 10 μL with RNase-free water. The reaction conditions were 42℃ for 2 min. Then, the reverse transcription reaction was performed. The reverse transcription reaction system included 10 μL of the reaction system for removing gDNA, 4 μL of 5×Evo M-MLV RT Reaction Mix Ver.2 and 6 μL of RNase-free water. The reaction program was 37℃ for 15 min - 85℃ for 5 sec, and cDNA was obtained.

[0073] 3.2.3 Real-time fluorescence quantification

[0074] qRT-PCR experiments were performed using ChamQ™ SYBR@qPCR Master Mix (Vazyme) (see product manual for detailed experimental procedures). Specifically, all reactions were performed on a CFX96™ TouchReal-Time PCR system (Bio-Rad) with three mechanical replicates; primer sequences are shown in Table 2 and were synthesized by ChamQ Technology Co., Ltd.; relative expression levels were calculated using the comparative CT (2-ΔΔCT) method.

[0075] Table 2 Primers used in qRT-PCR experiments

[0076] H-GAPDH_F CCTCAACGACCACTTTGTCA SEQ ID NO:9 H-GAPDH_R TTACTCCTTGGAGGCCATGT SEQ ID NO:10 H-Runx2-F ttacccctcctacctgagcc SEQ ID NO:11 H-Runx2-R ttccatcagcgtcaacacca SEQ ID NO:12 H-BMP2-F ttcggcctgaaacagagacc SEQ ID NO:13 H-BMP2-R ccacttccaccacgaatcca SEQ ID NO:14

[0077] 3.3 Immunofluorescence staining

[0078] The primary antibodies used for immunofluorescence staining were: anti-RUNX2 (dilution ratio 1:1000, abcam, ab76956), anti-BMP2 (dilution ratio 1:1000, abcam, ab214821), and anti-GAPDH (dilution ratio 1:5000, Proteintec, 1E6D9). The remaining experimental reagents and procedures were the same as those described in Examples 1 and 2.

[0079] Depend on Figure 8 A and Figure 8As shown in Figure B, under normal conditions (CTL), there was no significant difference between HAVSMC cells treated with PKM2 AP and those in the control group (NC), indicating that PKM2 AP does not affect normal cell growth. After β-GP treatment, HAVSMC cells in different groups showed varying degrees of calcium deposition. Compared with the NC control group, PKM2 AP significantly inhibited the formation of calcium nodules, a result consistent with in vivo validation. Figure 7 B~ Figure 7 D), further confirming the physiological function of PKM2 AP; simultaneously, combined with Figure 7 E~ Figure 7 The results of F, RT-qPCR and immunofluorescence staining experiments ( Figure 8 C~ Figure 8 F) further confirmed that PKM2 AP can downregulate the expression of RUNX2 and BMP2 genes.

[0080] In summary, this invention demonstrates through in vitro and in vivo experiments that PKM2 AP can inhibit the absorption and accumulation of calcium by the body / cells by suppressing the expression of RUNX2 and BMP2 genes, thereby achieving the therapeutic effect on vascular calcification.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0082] SEQ ID NO:1

[0083] LINC01139

[0084]

[0085] SEQ ID NO:2

[0086] DANT2

[0087]

[0088] SEQ ID NO:3

[0089] LINC01139-ORF1

[0090] CTGGCCTGTGCACTAGGGTGGAACCTCAGGAAGTTGATGACATTTGTAGCTGGGAGCAGCCTGGCCCCTCCTCTTCCTGTGTGGAACCTGAAATTCCACCGGCTTGGCAGGAAGCGCTCTAGCAGGGACTCCGGCTTTGCAGAGCATCCCTGTTTCCCCGTTTTTCCCTTTTCACCCAATAAAACCCTGCTTCACTCACCCTTCAAACCATCTGCAAGCCTAAATTTTTGTGGCTGTGGGACGGACAAAGATGCCTTTAGC

[0091] SEQ ID NO:4

[0092] LINC01139-ORF2

[0093] ATGGGTAGCCATTCATCTTCACTCTGTACCCCTTTCAAATGCATCCTGAATCCCTGGGACTCCTTTAAAAAATGCCTTCTCTTTTCCTTTCCCCTTCTCTGTCTTTTCTTCACAGATAGG

[0094] SEQ ID NO:5

[0095] LINC01139-ORF3

[0096] ATGGGAATAAATAGAATGTCCATCACTAGCAAGTTTCCTTTTTGGGAGAGTAAGACCAAGAGCCCTAACCAAAGCCAAGCACCACGCACCGAAACCCTAGCAGGCATAACCATAGCCACCAGTTATCTGGGTGTGTCACAAGACATCCTTTTCTCTCCCTTGTTGGAAGAGGACTCACTTCCACAGCTTTACCTTAGCATTCAGCTTATCATAAGGAGTCCATGCAATCCCAAGCTTTGGGTCAAACTGGATCTGAGGGATCCGGAGGCAGACAATAATGGAAGT

[0097] SEQ ID NO:6

[0098] DANT2-ORF1CTGGGGGTGGCCAACACAGAGATTCATTTCTTATTTATGAAGAAGCTG

[0099] SEQ ID NO:7

[0100] DANT2-ORF2GTGTATTTGTGTACTAATTTTTCTGTGTTTATAAAATCATTCATATATTTATTAATAACATGTTTAGAAGAAATCTCTGCATTAACCTCAGGGCACACAGCTGTCTTTGATCGCCCCCAACTCCTCAGAATGTCCTGCCTCCATTTCACTCTCCCACAGCTGACTTTCTCCCATGCT

[0101] SEQ ID NO:8

[0102] PKM2 AP

[0103] MGSHSSSLCTPFKCILNPWDSFKKCLLFSFPLLCLFFTDR

[0104] SEQ ID NO:9

[0105] H-GAPDH_F

[0106] CCTCAACGACCACTTTGTCA

[0107] SEQ ID NO:10

[0108] H-GAPDH_R

[0109] TTACTCCTTGGAGGCCATGT

[0110] SEQ ID NO:11

[0111] H-Runx2-F

[0112] TTACCCCTCCTACCTGAGCC

[0113] SEQ ID NO:12

[0114] H-Runx2-R

[0115] TTCCATCAGCGTCAACACCA

[0116] SEQ ID NO:13

[0117] H-BMP2-F

[0118] TTCGGCCTGAAACAGAGACC

[0119] SEQ ID NO:14

[0120] H-BMP2-R

[0121] CCACTTCCACCACGAATCCA。

Claims

1. A polypeptide for treating vascular calcification, characterized in that, The polypeptide contains the amino acid residue sequence shown in SEQ ID NO:

8.

2. The polypeptide according to claim 1, characterized in that, The concentration of the polypeptide used is 5 μg / g to 100 μg / g.

3. A drug for inhibiting vascular calcification, characterized in that, The drug comprises the polypeptide as described in any one of claims 1 to 2.

4. The drug as described in claim 3, characterized in that, The administration methods of the drug include, but are not limited to, oral, injection, and spray.

5. The use of a polypeptide in the preparation of an inhibitor of vascular calcification, characterized in that, The polypeptide contains the amino acid residue sequence shown in SEQ ID NO:

8.

6. The use as described in claim 5, characterized in that, The vascular calcification was caused by chronic kidney disease.

7. Use of a polypeptide in the preparation of an agent for inhibiting calcium deposition and / or calcium nodule formation, characterized in that, The polypeptide contains the amino acid residue sequence shown in SEQ ID NO:

8.

8. Use of a polypeptide in the preparation of an agent for treating vascular calcification by inhibiting the expression of RUNX2 and / or BMP2 proteins, characterized in that, The polypeptide contains the amino acid residue sequence shown in SEQ ID NO:

8.

9. The use of RNA in the preparation of polypeptides that inhibit vascular calcification, characterized in that, The polypeptide is encoded by the RNA, which contains the nucleotide sequence shown in SEQ ID NO:

4.

10. The use as described in claim 9, characterized in that, The RNA contains the nucleotide sequence shown in the sequence listing SEQ ID NO:1.

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