CN116574798A - Application of piR-mmu-57256903 in preparation of medicines for preventing and treating heart diseases caused by anticancer medicines - Google Patents
Application of piR-mmu-57256903 in preparation of medicines for preventing and treating heart diseases caused by anticancer medicines Download PDFInfo
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- CN116574798A CN116574798A CN202310473199.9A CN202310473199A CN116574798A CN 116574798 A CN116574798 A CN 116574798A CN 202310473199 A CN202310473199 A CN 202310473199A CN 116574798 A CN116574798 A CN 116574798A
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- A61P9/00—Drugs for disorders of the cardiovascular system
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- Virology (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of piR-mmu-57256903 in preparation of medicines for preventing and treating heart diseases caused by anticancer medicines. The piR-mmu-57256903 can regulate histone methylation by regulating the activity of methylase/demethylase so as to influence the expression of apoptosis-related proteins, and plays an important role in regulating oxidative stress and apoptosis-related pathological processes; based on the principle, the invention takes piR-mmu-57256903 as a target spot to prevent and treat various cardiac diseases caused by anticancer drug administration, improves cardiac function, provides cardiac protection for tumor patients after anticancer drug administration, and simultaneously provides a new drug development path and drug action target spot for diagnosis and treatment of heart failure or myocardial injury after tumor patients drug treatment, thereby having very important medicinal value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of piR-mmu-57256903 in preparation of medicines for preventing and treating heart diseases caused by anticancer medicines.
Background
Doxorubicin (Doxorubicin, dox) is a powerful anticancer chemotherapeutic drug, and is widely used for treating various hematological malignancies, solid sarcomas, and the like. However, there is increasing evidence that the use of Dox in the clinic also produces non-negligible cardiotoxicity in a dose-dependent manner, e.g. heart failure, dilated cardiomyopathy. The molecular mechanisms involved in the cardiotoxicity of Dox are multifactorial, including increased reactive oxygen species, DNA damage, and apoptosis. Since cancer patients are at an increased risk of developing cardiotoxicity following doxorubicin therapy, there is an urgent need to explore new drugs and strategies to prevent Dox-induced cardiomyopathy.
PIWI-interacting RNA (piRNA) is a newly discovered non-coding small RNA which acts with Piwi protein and has the length of about 24-30 nt. piRNA plays a very important role in maintaining reproductive system and stem cell function.
Disclosure of Invention
The invention aims to provide an application of piR-mmu-57256903 serving as a target spot in preparing a medicament for preventing and treating heart diseases caused by administration of anticancer medicaments, and increase medical application of piR-mmu-57256903, and provide a heart protection medicament for clinical tumor treatment.
The invention provides a method for preparing a drug for preventing and treating heart diseases caused by anticancer drug administration by taking piR-mmu-57256903 as a target spot.
Preferably, the anticancer drug comprises doxorubicin, epirubicin or mitoxantrone.
Preferably, the heart disease comprises myocardial injury and/or heart failure.
Preferably, agents that overexpress piR-mmu-57256903 are included in the medicament.
Preferably, the agent that overexpresses piR-mmu-57256903 comprises piR-mmu-57256903 overexpression vector.
Preferably, the initial vector of the piR-mmu-57256903 overexpression vector comprises an adeno-associated viral or lentiviral vector.
Preferably, the adeno-associated virus comprises AAV9; the lentiviral vector includes GV229.
Preferably, the piR-mmu-57256903 overexpression vector comprises piR-mmu-57256903 recombinant adeno-associated virus or piR-mmu-57256903 enriched extracellular vesicles;
the titer of the piR-mmu-57256903 recombinant adeno-associated virus is 1X 10 11 ～1×10 13 Individual viral genomes/mL;
the concentration of extracellular vesicles enriched in piR-mmu-57256903 was 40. Mu.g/mL.
The invention also provides a medicine for preventing and treating heart diseases caused by administration of the anticancer medicine, which comprises a reagent for over-expressing piR-mmu-57256903 and pharmaceutically acceptable auxiliary materials.
Preferably, the agent for overexpressing piR-mmu-57256903 comprises piR-mmu-57256903 recombinant adeno-associated virus or piR-mmu-57256903 enriched extracellular vesicles, the titer of the piR-mmu-57256903 recombinant adeno-associated virus being 1X 10 11 ～1×10 13 Individual viral genomes/mL; the concentration of extracellular vesicles enriched in piR-mmu-57256903 was 40. Mu.g/mL.
The beneficial effects are that:
the invention provides piR-mmu-57256903 serving as a target point for preparing and preventing heart diseases caused by anticancer drug administration, wherein piR-mmu-57256903 can cause transcriptional activation of a downstream target gene by regulating and controlling expression and activity of histone demethylase so as to influence expression of antigenic protein and play an important role in promoting pathological processes related to oxidative stress and apoptosis; based on the principle, the invention takes piR-mmu-57256903 as a target spot to prevent and treat various cardiac diseases caused by anticancer drug administration, improves cardiac function, provides cardiac protection for tumor patients after anticancer drug administration, and simultaneously provides a new drug development path and drug action target spot for diagnosis and treatment of heart failure or myocardial injury after tumor patients drug treatment, thereby having very important medicinal value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of the preparation of a drug for preventing and treating heart diseases caused by administration of anticancer drugs in example 2, wherein piR-mmu-57256903 is used as a target point;
FIG. 2 is a graph showing the results of an echocardiographic test of the cardiac function of a Dox-administered mice with overexpression piR-mmu-57256903 in test example 1;
FIG. 3 is a graph showing results of RT-qPCR animal level verification of AAV9-piR6903 efficiency in test example 2;
FIG. 4 is a GV229 vector map in example 3;
FIG. 5 is a graph showing the process of enriching piR-mmu-57256903 extracellular vesicles in example 3 and the associated characterization of enriched piR-mmu-57256903 extracellular vesicles in test example 3;
FIG. 6 shows the effect of immunofluorescent Tunel staining on the detection of the extracellular vesicles enriched in piR-6903 on Dox-induced apoptosis of cardiomyocytes in test example 3.
Detailed Description
The invention provides a method for preparing a drug for preventing and treating heart diseases caused by anticancer drug administration by taking piR-mmu-57256903 as a target spot.
In the present invention, the sequence of piR-mmu-57256903 is preferably as shown in SEQ ID NO.1, specifically 5'-TGCTGGTGGTGGGGAGTAGCTCCTTCTTCT-3'.
The anticancer drug of the present invention preferably comprises doxorubicin, epirubicin or mitoxantrone, more preferably doxorubicin. The heart disease according to the invention preferably comprises myocardial damage and/or heart failure, more preferably myocardial damage and heart failure. The myocardial damage according to the invention preferably comprises cardiac fibrosis and/or myocardial apoptosis.
In the present invention, agents that overexpress piR-mmu-57256903 are included in the medicament. The agent for overexpressing piR-mmu-57256903 of the present invention preferably comprises piR-mmu-57256903 overexpression vector. The piR-mmu-57256903 is a newly discovered non-coding small RNA, the biological formation process is unique, and the promoter adopted in the over-expression strategy is a non-U6 promoter.
The initial vector in the piR-mmu-57256903 overexpression vector of the invention preferably comprises an adeno-associated viral or lentiviral vector.
The adeno-associated virus of the invention preferably comprises AAV9, more preferably pHBAAV-cTNT-MCS-no light vector. The invention preferably inserts the piR-mmu-57256903 between EcoRI and HindIII of the pHBAAV-cTNT-MCS-no light vector. In the preparation of the piR-mmu-57256903 recombinant adeno-associated virus according to the invention, it is preferred that a promoter and a transcription enhancing element are added upstream of piR-mmu-5725690, the nucleotide sequence of piR-mmu-5725690 of the added promoter and transcription enhancing element preferably being 5'-AAGGTATATTGCTGTTGACAGTGAGCGTGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTAGTGAAGCCACAGATGTAAGAAGAAGGAGCTACTCCCCACCACCAGCATGCCTACTGCCTCG-3' (SEQ ID NO. 2).
When the initial vector is an adeno-associated virus, the piR-mmu-57256903 overexpression vector is piR-mmu-57256903 recombinant adeno-associated virus. The titer of the piR-mmu-57256903 recombinant adeno-associated virus of the invention is preferably 1X 10 11 ～1×10 13 More preferably 1X 10 GC/mL 13 GC/mL. The packaging process of the piR-mmu-57256903 recombinant adeno-associated virus is not particularly limited, and a conventional packaging mode in the field can be adopted. The source of the piR-mmu-57256903 is not particularly limited, and the piR-mmu-57256903 can be obtained by chemical synthesis or biological metabolism.
The lentiviral vector of the invention preferably comprises GV229, and the sequence of elements on GV229 is preferably H1-MCS-CMV-Puromycin. Preferably, the present invention inserts piR-mmu-57256903 between AgeI and EcoRI of GV229 to produce piR-mmu-57256903 recombinant lentivirus containing piR-mmu-57256903.
When the initial vector is a lentiviral vector, the piR-mmu-57256903 overexpression vector of the invention is an extracellular vesicle enriched in piR-mmu-57256903. The extracellular vesicles enriched with piR-mmu-57256903 contain piR-mmu-57256903. The concentration of the enriched piR-mmu-57256903 extracellular vesicles according to the invention is preferably 40. Mu.g/mL, more preferably 10 10 And each mL.
The invention preferably constructs 293T cell lines stably expressing the piR-57256903 cells and the enriched piR-57256903 extracellular vesicles are obtained by isolating the supernatant.
The invention also provides a medicine for preventing and treating heart diseases caused by adriamycin administration, which comprises a reagent for over-expressing piR-mmu-57256903 and pharmaceutically acceptable auxiliary materials.
The agent of the invention that overexpresses piR-mmu-57256903 preferably comprises piR-mmu-57256903 recombinant adeno-associated virus or piR-mmu-57256903 enriched extracellular vesicles. The titer of the piR-mmu-57256903 recombinant adeno-associated virus of the invention is preferably 1X 10 11 ～1×10 13 More preferably 1X 10 GC/mL 13 GC/mL. The concentration of the enriched piR-mmu-57256903 extracellular vesicles according to the invention is preferably 40. Mu.g/mL, more preferably 10 10 And each mL. The agent for overexpressing piR-mmu-57256903 in the agent of the present invention is preferably used in an amount of 1X 10 12 GC。
The auxiliary materials preferably comprise one or more of buffering agents, encapsulating agents, filling agents, binders, transdermal absorbents, wetting agents, disintegrating agents, absorption promoters, surfactants, colorants, flavoring agents and adsorption carriers, and the corresponding auxiliary materials are more preferably selected according to the dosage form of the medicament. The dosage forms of the medicine preferably comprise tablets, powder, granules, capsules, decoction, oral liquid, injection or suppositories, and more preferably comprise injection.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Construction of piR-mmu-57256903 recombinant adeno-associated virus, the procedure is as follows:
a synthetic fragment of piR-mmu-57256903 (hereinafter piR6903,6903) with a promoter and a transcription enhancing element, the specific sequence of which is 5'-AAGGTATATTGCTGTTGACAGTGAGCGTGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTAGTGAAGCCACAGATGTAAGAAGAAGGAGCTACTCCCCACCACCAGCATGCCTACTGCCTCG-3' (SEQ ID NO. 2), was prepared.
1) Initial vector cleavage
Mixing pHBAAV-cTNT-MCS-matt carrier according to the system shown in Table 1, gently sucking and beating, mixing, and placing in a 37 ℃ water bath kettle for reaction for 1-2h; agarose gel electrophoresis is carried out after enzyme digestion is finished, and target fragments are recovered;
TABLE 1 vector cleavage System
2) Acquisition of synthetic fragments of piR6903
Nucleotide sequence of the forward primer AAV-piR603-F of piR 6903: 5'-ACAgaattcAAGGTATATTGCTGTTGACAGTGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTAGTGAAACA-3' (SEQ ID NO. 3); the nucleotide sequence of the reverse primer AAV-piR-R: 5'-ACAaagcttCGAGGCGGCATGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTACATCTGTGGCTTCACTAAGAAGAA-3' (SEQ ID NO. 4). The amplification system is shown in Table 2, the template DNA in Table 2 is derived from a mouse cell or organ, and the amplification procedure is shown in Table 3.
TABLE 2 PCR amplification System for preparing a synthetic fragment of piR6903
| Component name | Volume (mu L) |
| 2×PCR Buffer | 25 |
| dNTPs mix(10mM each) | 1 |
| Positive strand nucleotide (10. Mu.M) | 2 |
| Reverse strand nucleotide (10. Mu.M) | 2 |
| Template DNA (200 ng/. Mu.L) | 1 |
| ddH 2 O | 18 |
| Phanta Super-Fidelity DNA polymerase | 1 |
| Total volume of | 50 |
TABLE 3 PCR amplification procedure for the preparation of piR6903
3) Ligation of the fragment of interest with the vector
Preparing a connection system and connection by referring to the HB infusion TM one-step cloning kit specification of Hanheng biological company;
then, E.coli transformation is carried out, target strains are screened by using an LB culture medium without resistance after transformation, and then the target strains are sent to the engine company for sequencing, and plasmid extraction is carried out by using a nucleic acid extraction kit, so as to obtain piR6903 recombinant adenovirus (hereinafter referred to as AAV9-piR 6903). Sequencing, the sequencing result is consistent with the target sequence, which shows that the recombinant adenovirus AAV9-piR6903 is successfully constructed, and the titer of the constructed AAV9-piR6903 is 1.0x10 13 CG/mL。
Example 2
1. Animal grouping and model building
40 adult male mice purchased from Beijing Veitz laboratory animal technology Co., ltd were equally divided into an doxorubicin administration group (Dox) group (experimental group) and a physiological saline group (control group);
experimental group: dox powder was prepared as a stock solution of 0.5mg/mL using physiological saline, and was continuously injected 4-5 times at a dose of 5 mg/kg/week using a sterile syringe, and the injection amount was accumulated at 25mg/kg, and after completion, cardiac ultrasonic dynamic ultrasonic test was performed to evaluate cardiac function.
Control group: equal volumes of physiological saline were injected and the other procedures were consistent with the experimental group.
2. Tail vein injection AAV9
The mice of the control group and the experimental group are randomly divided into two groups, which are respectively marked as a group of saline+AAV9-control (AAV 9-Ctr), a group of saline+AAV9-piR6903, a group of Dox+AAV9-Ctr and a group of Dox+AAV9-piR 6903. One week before the treatment of step 1, the mice of each group were placed on a tail vein injector, the tail of the mice was sterilized with alcohol, and groups of SALINE+AAV9-piR6903 and Dox+AAV9-piR6903 were injected by tail vein injection using an insulin injector in a 1X 10 manner 13 mu.g/mL of AAV9-piR6903 obtained in example 1 was injected at a dose of 100. Mu.L, the schematic diagram of which is shown in FIG. 1;
the saline+AAV9-Ctr and Dox+AAV9-Ctr groups were injected by tail vein using insulin syringes at 1X 10 13 AAV9-Ctr (pHBAAV-cTNT-MCS-AAV 9 virus without light vector) was injected at a dose of μg/mL, and after the injection, the mice of the experimental group were subjected to the treatment in step 1 as described above using absorbent cotton for hemostasis and then placed in a cage.
Test example 1
Example 2 after the treatment, mice were anesthetized with 1.5% to 2% isoflurane by mass concentration and cardiac function was assessed in the 4 groups of mice of example 2 using Visual sonic 2100 small animal ultrasound imaging system at a frequency of 30 MHZ. B-mode and M-mode images may be acquired and the systolic function index cardiac ultrasound Ejection Fraction (EF) and the cardiac ultrasound shortening Fraction (FS) measured to assess the systolic function of the mice. Each index was measured three times and averaged to assess mouse cardiac function, the results are shown in figure 2. Wherein, a in fig. 2 is a representative graph of mouse heart ultrasound; b is the EF statistical result of the mice, and from left to right, the results are respectively taken as a sample+AAV 9-Ctr, a sample+AAV 9-piR6903, a Dox+AAV9-Ctr, a Dox+AAV9-piR6903 group, and the ejection fraction of each group is 60.0%,63.8%,44.4% and 63.0% in sequence; c is the FS statistical result of the mice, and from left to right, the results are respectively a sample+AAV 9-Ctr, a sample+AAV 9-piR6903, a Dox+AAV9-Ctr and a Dox+AAV9-piR6903, the shortening fraction of each group is 31.5%,33.8%,21.2% and 32.8% in sequence, and p is less than 0.001.
From fig. 2, it can be derived that: overexpression of piR-mmu-57256903 significantly improves cardiac function following doxorubicin administration.
Test example 2
Example 2 after the end of the treatment, the tissue RNA of the mice of the experimental group was extracted by Trizol method and total RNA was reverse transcribed using RevertAi First Strand cDNA Synthesis Kit (Thermo Scientific #K1622). cDNA was quantified by real-time fluorescent quantitative polymerase chain reaction (qPCR) in LightCycler480II (Roche) using the stem-loop method using iTaq Universal SYBR Green Supermix (Bio-Rad #1725121/-20 ℃) and the primers used were purchased from Bose organisms. The relative expression level of piR-mmu-57256903 was examined using 5S (purchased from Ruibo organism) as an internal control. All qPCR reactions were 5 replicates and signals were collected at the end of each cycle. With 2 -ΔΔCt The relative expression level was calculated by the method. The statistical graph of the detection results is shown in fig. 3.
From fig. 3, it can be derived that the relative expression level of piR-6903 in heart tissue of mice in AAV9-piR6903 group is increased 2.4-fold compared to AAV9-Ctr group, indicating that AAV9-piR6903 effectively promotes heart expression of piRNA, representing p <0.001.
Example 3
1.piR 6903 recombinant adenovirus was constructed as follows:
piR6903 recombinant adenovirus was constructed by Shanghai Ji Kai Gene technologies Co., ltd, wherein the vector information is as follows:
1) Carrier name: GV229; element sequence: H1-MCS-CMV-Puromycin; cloning site: age I/EcoR I; the control is the insertion sequence: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO. 5) Scramble is empty; the vector map is shown in FIG. 4.
2) Acquisition of a synthetic fragment of piR-mmu-57256903 (hereinafter piR 6903)
Nucleotide sequence of the forward primer piR6903 (64188-1) -P1 of piR 6903: 5'-ccggTG CTGGTGGTGGGGAGTAGCTCCTTCTTCTttttttg-3' (SEQ ID NO. 6); reverse primer piR-mmu-57256903 (64188-1) -P2 nucleotide sequence: 5'-aattcaaaaaaAGAAGAAGGAGCT ACTCCCCACCACCAGCA-3' (SEQ ID NO. 7).
The primer contains exchange pairing base and enzyme cutting site, and contains 5' -end part sequence of target gene for PCR fishing target gene, and piR6903 (64188-1) -P1 and piR6903 (64188-1) -P2 are used for annealing.
2) Sequencing results and result analysis of positive recombinant clones
Sequencing results: 5' -CAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCA GTTTGGTTAGTACCGGGCCCGCTCTAGACTCGAGATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTCACCGGTGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTTTTTTGAATTCGGATCCATTAGGCGGCCGCGTGGATAACCGTATTACCGCCATGCATTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT-3’(SEQ ID NO.8)。
The comparison result is correct, namely the piR6903 recombinant adenovirus (hereafter piR-6903 plasmid) is constructed.
2. Preparation of enriched piR6903 extracellular vesicles the construction procedure is as follows, the construction scheme is shown as a in figure 5.
1) Host cells 293T are passaged into six-hole plates, and the cell density is cultured to 70% -80%;
2) Preparing a cell transfection reagent A solution: adding piR-6903 plasmid with a concentration of 4 μg into 500 μl of serum-free culture medium, gently mixing, and standing for 5min;
3) Preparing a cell transfection reagent B solution: adding 8 mu L of Lipo2000 transfection reagent into 500 mu L of serum-free culture medium, gently mixing, and standing for 5min;
4) Gently mixing the cell transfection A solution and the cell transfection B solution, and standing at room temperature for 15-20min;
5) Removing old cell culture medium from the cell plate, adding 1mL of serum-free culture medium into each hole, and adding 1mL of uniformly mixed plasmid transfection reagent after standing;
6) After 6-8 hours, changing the normal culture medium;
7) Puromycin (Puro, 1. Mu.g/mL) was added 48h later to remove cells not transformed with plasmid, and the cell state was observed 24h later;
8) Maintaining proper Puro concentration until floating dead cells are not found and cells grow normally after Puro is added, and obtaining a 293T stable transgenic strain which is stably over-expressed piR-6903 and is marked as 293T-EPPIR;
9) Expanding the cells in the six-hole plate to a culture dish, and continuously culturing the cells in a normal culture medium containing Puro.
10 293T stable transgenic strain of the stable over-expression piR-6903 prepared in the step 9), collecting supernatant of cell culture thereof, and performing ultracentrifugation, wherein the method comprises the following specific steps: centrifuging for 5min at 500 g; centrifuging for 10min at 3000 g; centrifuging for 45min at 12000 g; filtering the supernatant with 0.22 μm filter, centrifuging with 100000g for 70min at 4deg.C to obtain enriched piR-mmu-57256903 extracellular vesicles, denoted as EVs-EPPIR-OE, and the specific process is shown in figure 5A.
Test example 3
1. The 293T stable transgenic strain of the stable transgenic control plasmid is used as a control group, and fluorescent quantitative PCR detection is carried out on the 293T stable transgenic strain of the stable over-expression piR-6903 prepared in the step 9) in the example 3 by adopting fluorescent quantitative PCR, wherein the reference gene is 5s, and the steps are as follows:
1) A fluorescent quantitative PCR reaction system was prepared according to the following reaction system, and fluorescent quantitative PCR detection was performed as shown in table 4 (each sample was repeated twice in parallel); the sequence of the specific sense primer was 5'-TGCTG GTGGTGGGGAGTAGCTCCTT-3' (SEQ ID NO. 9).
TABLE 4 fluorescent quantitative PCR reaction System
2) After the configuration is completed, the fluorescent quantitative PCR reaction can be performed according to the following reaction, and the reaction procedure is shown in Table 5;
TABLE 5 fluorescent quantitative PCR reaction procedure
5) The experimental analysis adopts a relative quantitative method to analyze, can reflect the relative expression quantity of target genes of each experimental group relative to a control group, and uses 2 after parallel repeated averaging -ΔΔCt Calculations were performed where Δct=target gene Ct value-internal reference Ct value, ΔΔct=individual group Δct values including control group-control group Δct average. The calculation result is shown in fig. 5B.
From B in fig. 5, it can be derived that: stable transfer cell line construction success, stable over-expression piR-6903
2. The enrichment efficiency of the enriched piR-6903 extracellular vesicles prepared in example 3 was detected by using a real-time fluorescence quantitative PCR method with 293T stable transgenic strain of stable transgenic control plasmid as control group (Ctr), and the internal reference gene was U6, and the result is shown as C in FIG. 5;
from fig. 5C, it can be derived that: extracellular vesicles isolated from stable transformants were successfully enriched for piR-6903.
3. The size and concentration of the enriched piR-6903 extracellular vesicles were detected by nanoparticle tracking analysis technique, and the results are shown as D in fig. 5:
from fig. 5C to D, it can be derived that: this example successfully obtained stable enriched extracellular vesicles of piR-6903
4. Immunofluorescence Tunel staining detects the effect of enriched piR-6903 extracellular vesicles on Dox-induced cardiomyocyte apoptosis, as follows:
(1) Construction of a cell model: primary milk mouse cardiomyocytes were obtained, after overnight culture, doxorubicin (final concentration 0.3 uM) was added and treated in the dark for 24h.
(2) Tunel staining the cell model in step (1) according to the following procedure
1) Cell removal medium, washing with 1 XPBS;
2) Fixing 4% paraformaldehyde at room temperature for 30min, and cleaning with 1×PBS for 3 times and 5min;
3) 0.5% TritonX-100 membrane rupture 20min,1 XPBS 3 times, 5min;
4) Blocking for 1h at room temperature with 5% BSA;
5) Incubating primary antibodies: 5% BSA formulation 1:200 alpha-actinin, 4 ℃ slow shaking table overnight;
6) Cell plates were washed 3 times with 1 XPBS for 5min;
7) Incubating a secondary antibody: 5% BSA formulation 1:200CY3 mouse secondary antibody, and a slow shaking table is kept away from light for 2 hours at room temperature;
8) Cell plates were protected from light, washed 3 times with 1 XPBS for 5min;
9) Cell balance: with ddH 2 O5X Equilibration Buffer was diluted 1X Equilibration Buffer, 100. Mu.L per well, equilibrated at room temperature for 10-30min;
10 Light-shielding preparation of Tunel reaction solution as shown in table 6:
table 6 Tunel reaction liquid formulation
50 mu L of each hole and carrying out constant-temperature light-shielding reaction for 1h at 37 ℃;
11 Light-shielding cell plate, washing with 1×PBS for 3 times and 5min;
12 Incubation of nuclear dye: 5% BSA formulation 1:2000Hoechst, incubating for 20min at room temperature in dark;
13 Light-shielding cell plate, washing with 1×PBS for 3 times and 5min;
14 Immunofluorescence photography was performed.
The results are shown in FIG. 6.
From fig. 6, it can be derived that: the extracellular vesicles enriched in piR-6903 can effectively reduce myocardial apoptosis caused by doxorubicin.
From the above embodiments it can be derived that: the agent for over-expression piR-mmu-57256903 can improve cardiac function after administration of doxorubicin, reduce cardiac fibrosis after administration of doxorubicin, inhibit myocardial apoptosis after administration of doxorubicin, and provide a new prevention and treatment drug development path and drug action target for diagnosis and treatment of cardiac injury and/or heart failure caused by doxorubicin.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
- The pir-mmu-57256903 is used as a target spot in the preparation of medicaments for preventing and treating heart diseases caused by the administration of anticancer medicaments.
- 2. The use according to claim 1, wherein the anticancer drug comprises doxorubicin, epirubicin or mitoxantrone.
- 3. The use according to claim 1, wherein the heart disease comprises myocardial injury and/or heart failure.
- 4. Use according to any one of claims 1 to 3, wherein the medicament comprises an agent which overexpresses piR-mmu-57256903.
- 5. The use of claim 4, wherein the agent that overexpresses piR-mmu-57256903 comprises a piR-mmu-57256903 overexpression vector.
- 6. The use according to claim 5, wherein the initial vector of the piR-mmu-57256903 overexpression vector comprises an adeno-associated virus or lentiviral vector.
- 7. The use of claim 6, wherein the adeno-associated virus comprises AAV9; the lentiviral vector includes GV229.
- 8. The use according to claim 6 or 7, wherein the piR-mmu-57256903 overexpression vector comprises piR-mmu-57256903 recombinant adeno-associated virus or piR-mmu-57256903 enriched extracellular vesicles;the titer of the piR-mmu-57256903 recombinant adeno-associated virus is 1X 10 11 ～1×10 13 Individual viral genomes/mL;the concentration of extracellular vesicles enriched in piR-mmu-57256903 was 40. Mu.g/mL.
- 9. A medicament for preventing and treating heart diseases caused by anticancer medicament administration, which is characterized by comprising an agent for overexpressing piR-mmu-57256903 and pharmaceutically acceptable auxiliary materials.
- 10. The medicament of claim 9, wherein the agent that overexpresses piR-mmu-57256903 comprises piR-mmu-57256903 recombinant adeno-associated virus or piR-mmu-57256903 enriched extracellular vesicles; the titer of the piR-mmu-57256903 recombinant adeno-associated virus is 1X 10 11 ～1×10 13 Individual viral genomes/mL; the concentration of extracellular vesicles enriched in piR-mmu-57256903 was 40. Mu.g/mL.
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