CN107303394A - Purposes of the miR-125a in the medicine for promoting angiogenesis is prepared - Google Patents

Abstract

The present invention relates to the use of miR-125a in the preparation of drugs for promoting angiogenesis. miR-125a can promote the expression of angiogenesis-related genes in endothelial cells, increase the length and number of branches of the tube network in vitro, and increase the number of new blood vessels in vivo, indicating that miR-125a promotes angiogenesis.

Description

Use of miR-125a in the preparation of drugs for promoting angiogenesis

technical field

This field relates to the field of regenerative medicine, in particular to the use of miRNA in the preparation of drugs for promoting angiogenesis.

Background technique

Angiogenesis refers to the process in which endothelial cells proliferate, migrate, and remodel on the basis of existing blood vessels by budding to form new blood vessels [1]. Angiogenesis is closely related to the occurrence and development of various physiological and pathological processes such as growth and development, inflammation, tissue damage repair, tissue ischemia, and tumor growth[2,3].

Angiogenesis is a complex and orderly process, mainly including multiple steps such as endothelial cell activation, proliferation, endothelial cell migration and invasion, endothelial cell budding, basement membrane formation, and new vessel maturation[4-6]. During this process, the microenvironment composed of various cytokines, signaling pathways and stromal cells can maintain the angiogenesis within the physiological range by regulating the balance of numerous positive and negative angiogenesis regulators[7].

For diseases such as tumor growth and diabetic retinopathy, inhibiting angiogenesis can slow down the development of the disease. However, when certain reasons lead to organ or tissue ischemia, and in the process of tissue damage repair, promoting angiogenesis and improving local blood supply through exogenous means can effectively improve tissue ischemia and promote tissue repair[8,9] .

miRNA is a kind of highly conserved endogenous non-coding small molecule RNA with a length of 20-24nt. It mainly regulates gene expression at the post-transcriptional level by fully or partially complementary binding to the 3'-UTR region of homologous mRNA, resulting in the degradation or translational repression of the target mRNA. More than 1000 miRNAs have been confirmed so far (Sanger miRBase). It plays an important regulatory role in various physiological and pathological processes such as growth and development, cell proliferation and differentiation, morphogenesis, and tissue metabolism [10]. Research on the regulation mechanism of angiogenesis also shows that miRNA can affect the formation of blood vessels by regulating multiple steps in the process of angiogenesis [11-14].

In recent years, studies have found that microRNA, a small molecule regulatory substance, can participate in the regulation of endothelial cells and blood vessel functions by regulating the expression of these genes closely related to endothelial cell functions. In particular, some vascular endothelial cell-specific and highly expressed microRNAs play important regulatory functions in the physiological and pathological processes of blood vessels. miR-125 is the currently reported microRNA that is closely related to the function of vascular endothelial cells. The miR-125 family has two members, miR-125a and miR-125b. In vessels with endothelial hyperplasia caused by balloon injury, the expression of miR-125 was significantly reduced, while in monocyte-macrophages, miR-125a could be induced by oxidized low-density lipoprotein stimulation, and could inhibit monocyte proliferation. The uptake of lipids by macrophages and the secretion of inflammatory factors suggest that miR-125a and miR-125b are involved in the vascular inflammatory response of atherosclerotic diseases, vascular endothelial injury and intimal hyperplasia and other vascular pathological processes [15].

Peng Che et al. reported in 2014 that miR-125a-5p caused damage to endothelial cell angiogenesis in aged mice by down-regulating RTEF-1 [16]. Dai J et al. 2015 reported that miR-125a regulates the paracrine of VEGF-A in gastric cancer, thereby inhibiting angiogenesis in tumors [17].

However, there have been no reports of miR-125a therapeutically by promoting angiogenesis.

Contents of the invention

According to some embodiments, the present disclosure provides the use of miR-125a in the preparation of a drug for promoting angiogenesis.

According to some embodiments, the present disclosure provides the use of miR-125a in the preparation of a medicament for improving tissue ischemia or tissue damage. In particular, the use of miR-125a in drugs for the treatment of tissue ischemic diseases. In some embodiments, the tissue ischemic disease is selected from: ischemic heart disease, arteriosclerosis obliterans, ischemic stroke and diabetic foot.

In some embodiments, the treatment of disease by miR-125a refers to the promotion of angiogenesis in ischemic tissue or damaged tissue.

In some specific embodiments, the treatment or improvement refers to improving at least one selected from the following: the number of tip cells of endothelial cells increases, the number of branches of the pipe network increases, the length of the pipe network increases, the neonatal The number of blood vessels increases.

In this specification, blood vessels include arteries, veins, and microvessels (also referred to as capillaries).

In some embodiments, miR-125a is a mature form of miRNA selected from miR-125a-5p and miR-125a-3p; more preferably, miR-125a is miR-125a-5p.

In some specific embodiments, the nucleotide sequence of miR-125a is shown in SEQ ID No.1. SEQ ID No.1 is ucccugagacccuuuaaccuguga, its accession number is miRbaseAccession number MIMAT0000443, see webpage: http://www.mirbase.org/cgi-bin/mature.pl? mature_acc=MIMAT0000443.

In some embodiments, miR-125a is formulated as a gene drug. The skilled person knows that any existing and future forms of gene medicine suitable for introducing nucleic acid molecules into target cells can be used to practice the present application. In some specific embodiments, the gene drug is a transfection gene drug, thus such drug comprises a transfection reagent. In some preferred embodiments, the transfection reagent is a liposome, including but not limited to Lipofectamine 2000.

In some embodiments, the transfection concentration of miR-125a ranges from 50 nmol/L to 200 nmol/L; preferably 100 nmol/L.

Description of drawings

Figure 1A: CD31 staining of human umbilical vein endothelial cells. Scale bar represents 50 μm.

Figure 1B: Hoechst staining of human umbilical vein endothelial cells. Scale bar represents 50 μm.

Figure 1C: Human umbilical vein endothelial cells CD31 staining overlaid with Hoechst staining. Scale bar represents 50 μm.

Figure 2: Human umbilical vein endothelial cells form a tube network. Scale bar represents 200 μm.

Figure 3A: Expression levels of Ang1 and Flk1 after transfection of miR-125a and NC. □: NC; ■: miR-125a.

Figure 3B: Expression levels of Vash1 and TSP1 after transfection of miR-125a and NC. □: NC; ■: miR-125a.

Figure 4A: Tube network formed by human umbilical vein endothelial cells after transfection of NC control.

Figure 4B: After transfection of miR-125a, the tube network structure formed by human umbilical vein endothelial cells.

Figures 5A, 5B and 5C: Human umbilical vein endothelial cells transfected with NC control. 5A: CD34 staining, 5B: Hoechst staining, 5C: CD34 and Hoechst overlay. Scale bar represents 50 μm.

Figures 5D, 5E and 5F: Human umbilical vein endothelial cells transfected with miR-125a. 5D: CD34 staining, 5E: Hoechst staining, 5F: CD34 and Hoechst superimposed bars represent 50 μm.

detailed description

Specific materials and their sources used in embodiments of the present disclosure are provided below. However, it should be understood that these are exemplary only and are not intended to limit the present invention, and materials that are the same or similar to the type, model, quality, property or function of the following tissues, cells, reagents and instruments can be used for implementation this invention.

Example 1: Isolation and culture of umbilical vein endothelial cells

Under sterile conditions, take the umbilical cord of a newborn with a length of 15cm to 20cm (with informed consent), immediately subtract the hemostatic clamp marks on the two sections of the umbilical cord in the ultra-clean workbench and trim the section to find that the tube wall is thinner and the official cavity is larger of the umbilical vein.

Gently insert a syringe into the vein cavity, fix it with tweezers, and wash the vein cavity repeatedly with PBS until the outflowing liquid is transparent.

Inject 0.1% collagenase P into the vein cavity with a syringe until the umbilical vein is full, clamp both ends with hemostats, and digest at 37°C for 12 minutes.

Remove the hemostat and flush the umbilical vein with DMEM/F12 containing 10% FBS.

Collect the culture base in a centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, add M200 medium (purchased from GIBCO; M-200-500), inoculate the cells in a 10cm culture dish, and culture the cells at 37°C, 5% CO ₂ box culture.

Change fresh M200 medium after 24 hours, and then change fresh M200 medium every 3 to 4 days until the cells grow to 80% confluent state.

Example 2: Phenotype and function identification of endothelial cells

1. Immunofluorescence identification:

When the cells in Example 1 grew to 80% confluence, the cells were washed 3 times with PBS. Cells were fixed with pre-cooled 4% paraformaldehyde for 10 min, washed with PBS 3 times, 5 min each time. 10% sheep serum was blocked at room temperature (22 to 28° C.) for 30 min, and CD31 antibody (dilution 1:100, purchased from: BD) was added. In the control group, CD31 antibody was replaced by PBS, and incubated at room temperature for 2 hours. Wash 4 times with PBS, 5min each time. Add FITC-labeled goat anti-rabbit IgG secondary antibody (dilution 1:100), and incubate at 37°C for 30min in the dark. Wash 4 times with PBS, 10min each time. Observe and collect pictures under a fluorescent inverted microscope.

2. Matrigel tube formation experiment in vitro: Add 200 μl of matrigel gel (purchased from BD) to a pre-cooled 24-well plate without air bubbles; incubate the 24-well plate at 37°C for 15 minutes, and the number of cells in each well is about 1 ×10 ⁵ cells, incubated at 37°C for 8 to 10 hours, observed and photographed under an inverted microscope.

Example 3: Transfection of miRNA

1. Reagents:

(1) The transfection reagent is Lipofectamine 2000 (Invitrogen);

(2) miRNA to be transfected:

miR-125a powder was synthesized by American Life Technologies;

An irrelevant sequence (SEQ ID No. 2: UUCUUCGAACGUGUCACGUTT) similar in length to miR-125a was synthesized using known techniques as an NC control.

2. Configuration of miRNA stock solution to be transfected:

The tube containing the miRNA to be transfected was centrifuged at low speed for 5 min, and Opti-MEM was added to dissolve the miRNA according to the instructions of Lipofectamine2000 (Invitrogen), shaken and centrifuged briefly to prepare a 20 μM stock solution.

3. Cell preparation:

Human umbilical vein endothelial cells of the third generation in logarithmic growth phase and 70%-80% confluent in Example 1 were used for transfection.

4. Transfection process:

Dilute the miRNA and Lipofectamine 2000 to be transfected with Opti-MEM, mix gently and place at room temperature for 5 minutes;

Slowly add Lipofectamine 2000 dilution to the miRNA dilution to be transfected drop by drop, mix gently to obtain a mixture of miRNA and Lipofectamine 2000, and incubate at room temperature for 20 minutes;

Take out the cells to be transfected from the incubator, suck out the culture medium, wash 3 times with DPBS solution (Duchener's phosphate buffer solution), and add 1.5ml Opti-MEM to each well after absorbing the washing solution;

Slowly drop the incubated miRNA-Lipofectamine 2000 mixture into the culture dish containing the cells to be transfected. Transfection amount: the final concentration of miRNA is 100nmol/L, shake gently and mix well, then place in the incubator to continue culturing;

After 6 hours of transfection, remove the transfection working solution and wash 3 times with DPBS;

Replace the M200 medium; after 24 hours, remove the medium, wash 3 times with DPBS, and then perform angiogenesis detection.

Example 4: miR-125a promotes angiogenesis in human umbilical vein endothelial cells

The third passage of human umbilical vein endothelial cells (not transfected) in Example 1, the cells obtained in Example 3 (transfected with miR-125a, or transfected with NC control) were respectively taken at 2×10 ⁴ cells/ The density of cm ² was inoculated in T25 culture flasks, and after 48 hours of adherent culture, the cells were collected after observing that the cells reached 70%-80% confluence.

1. Detection of angiogenesis-related genes by real-time fluorescent quantitative PCR

According to the conditions recommended by Takara's M-MLV product manual: Synthesize cDNA, add: 2μg total RNA, 2μl Oligo(dT)18 (500μg/ml), 3μl dNTP (10mmol/l) into a sterile enzyme-free 0.2ml Ep tube , 1 μl RNase inhibitor (40U/μl), 1 μl M-MLV, 6 μl 5×M-MLV reverse transcriptase buffer (containing DTT), DEPC water to 30 μl;

After mixing, reverse transcription at 42°C for 60 minutes, inactivate at 75°C for 10 minutes, and store at -20°C for later use;

According to the instructions of the SYBR Premix Ex TaqTM kit from Takara Company, add: 10 μl 2×SYBR Green I Master Mix, 1 μl cDNA template, 0.8 μl upstream primer F, 0.8 μl downstream primer R (for the primer sequence, see Table 1), 0.4 μl ROX correction dye, DEPC water supplemented to 20 μl;

After mixing, paste the corresponding optical reaction film and run the Real-time PCR reaction on the StepOne PlusTM real-time quantitative PCR instrument. The reaction conditions are: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 60°C, extension for 30 sec, cycle 40 Second-rate.

The primers used in the experiment spanned introns, and their specificity was verified by Blast. The primer sequences are shown in Table 1 below. The specificity of the product was confirmed according to the results of melting curve analysis. The reaction of each pair of primers included a no-template control .

Table 1. RT-PCR primers

2. Detection of the ability of human umbilical vein endothelial cells to form a tube network in vitro

Add 200 μl of matrigel per well to a pre-cooled 24-well plate without air bubbles; incubate the 24-well plate at 37°C for 15 minutes, the number of cells per well is about 1×10 ⁵ , incubate at 37°C for 8 to 10 hours, and incubate at 37°C for 15 minutes. Observe and take pictures with an inverted microscope.

3. Results:

3.1. Human umbilical vein endothelial cells (untransfected) stained positively for CD31 (Figure 1A), and can form a tube network in vitro (Figure 2). This result indicates that human umbilical vein endothelial cells can form new blood vessels in a three-dimensional structure in vitro.

3.2. miR-125a promotes angiogenesis in human umbilical vein endothelial cells

(1) Figures 3A and 3B are qRT-PCR detection of angiogenesis-related genes (Figure 3A is angiogenesis-promoting-related genes, and Figure 3B is angiogenesis-inhibiting-related genes).

Specifically, it can be seen from FIG. 3A that after human umbilical vein endothelial cells were transfected with miR-125a, the expressions of Ang1 and FLK1 related to angiogenesis were increased. Fig. 3B shows that after human umbilical vein endothelial cells were transfected with miR-125a, the expressions of angiogenesis-related genes VASH1 and TSP1 were reduced. This difference suggested that miR-125a could promote angiogenesis in human umbilical vein endothelial cells compared with NC.

(2) Figures 4A and 4B show the results of angiogenesis in human umbilical vein endothelial cells transfected with miR-125a (Figure 4A is the NC control group, Figure 4B is the miR-125a group). Specifically, it can be seen from Figure 4A and Figure 4B that after human umbilical vein endothelial cells were transfected with miR-125a, the number and length of the formed tube network structure increased significantly. This result again demonstrates that miR-125a can promote angiogenesis in human umbilical vein endothelial cells compared with NC.

(3) After miR-125a was transfected into human umbilical vein endothelial cells, the proportion of tip cells in endothelial cells was significantly increased (Fig. 5A to Fig. 5F). This result indicated that miR-125a could promote tip cell formation and further promote endothelial cell angiogenesis compared with NC.

In summary, the above results confirmed that the angiogenesis ability of human umbilical vein endothelial cells increased after miR-125a transfection.

references:

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Claims (8)

1. Use of miR-125a in the preparation of medicines for treating tissue ischemic diseases or tissue damage. 2. The use according to claim 1, said miR-125a is miR-125a-5p. 3. The use according to claim 1, the nucleotide sequence of said miR-125a is shown in SEQ ID No.1. 4. The use according to claim 1, wherein said treatment refers to promoting angiogenesis in ischemic tissue or damaged tissue. 5. The use according to claim 4, wherein said treatment refers to improving at least one selected from the following: the number of tip cells of endothelial cells, the number of branches of the pipe network, the length of the pipe network, the neovascularization Number of. 6. The use according to claim 1, wherein the tissue ischemic disease is selected from the group consisting of ischemic heart disease, arteriosclerosis obliterans, ischemic stroke and diabetic foot. 7. The use according to claim 1, wherein the medicine is a gene medicine. 8. The use according to claim 7, wherein the medicament comprises a transfection reagent, preferably the transfection reagent is a liposome.