CN114250292A

CN114250292A - MicroRNA for regulating VEGF gene expression and application thereof

Info

Publication number: CN114250292A
Application number: CN202111500446.7A
Authority: CN
Inventors: 李鲁远; 赵环宇
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-03-29

Abstract

The invention provides microRNA for regulating VEGF gene expression and application thereof, and discloses the function of miR-374b-5p in VEGF gene expression induced by MCP-1, so that miR-374b-5p is a bridge for connecting the miR-374b-5p and the VEGF gene expression, the function of MCP-1 on VEGF is relieved by exogenously supplementing miR-374b-5p, and the rapid growth and transfer of tumors caused by MCP-1 are inhibited. Therefore, the miR-374b-5p also has potential value in treating VEGF related diseases. On the other hand, when high activity of VEGF is needed to promote angiogenesis under the condition of ischemia related to coronary artery diseases and the like, miR-374b-5p can also be used as a therapeutic target to relieve the inhibition of VEGF gene expression, so that angiogenesis is promoted to improve the ischemic state and the like.

Description

MicroRNA for regulating VEGF gene expression and application thereof

Technical Field

The invention relates to the field of biomedicine, and relates to microRNA for regulating VEGF gene expression and application thereof.

Background

Angiogenesis refers to the process of new blood vessel generation or capillary formation from existing blood vessels, and mainly includes an early angiogenesis stage in which vascular endothelial progenitor cells form the original embryonic vascular network, and a mature angiogenesis stage in which existing blood vessels are regenerated in a budding manner. The growth of the tumor is changed from a slow growth stage without blood vessels to a rapid proliferation stage with blood vessels, and meanwhile, the new blood vessels can be used as channels to mediate tumor cells to transfer to a host, thereby promoting the tumor to continue growing in other parts of a body. Tumor growth is therefore dependent on angiogenesis. Tumor angiogenesis is influenced by various factors, such as hypoxia, inflammation, gene change, bidirectional paracrine action between tumor cells and vascular endothelial cells, and the like, wherein Vascular Endothelial Growth Factor (VEGF) plays an irreplaceable core role in regulating and controlling tumor angiogenesis and is one of the targets of the current anti-cancer treatment. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) is a member of the CC chemokine family, and is primarily one of the key chemokines that regulate monocyte/macrophage migration and infiltration. Cancer cells and tumor-infiltrating monocytes/macrophages produce large amounts of MCP-1, thereby promoting VEGF production by cancer cells and endothelial cells. In addition, MCP-1 can promote cancer cell metastasis by enhancing permeability of tumor vasculature, which is also an important activity of VEGF. MCP-1 is reported to significantly promote increased expression of VEGF in wound healing. However, the molecular mechanism by which MCP-1 regulates VEGF gene expression has not been known so far. Therefore, the search of a mechanism involved in the regulation of VEGF gene expression by MCP-1 is not only beneficial to establishing a connection bridge between MCP-1 and VEGF, but also has important significance for the prevention and treatment of tumor angiogenesis.

Disclosure of Invention

In view of the above, the invention aims to provide the application of miR-374b-5p as a target point for regulating the growth of blood vessels.

Preferably, the medicament is a medicament for inhibiting miR-374b-5p expression so as to inhibit blood vessel growth.

Preferably, the medicament is a medicament for up-regulating miR-374b-5p expression so as to promote blood vessel growth.

Preferably, the medicament regulates the growth of blood vessels by controlling the expression of VEGF gene by regulating the expression of miR-374b-5 p.

Preferably, the VEGF gene expression is MCP-1 mediated VEGF gene expression.

In a second aspect of the invention, a medicament for regulating blood vessel growth is provided, and the medicament contains miR-374b-5 p.

In a third aspect of the invention, the application of miR-374b-5p as a drug treatment target in preparing a drug for treating ischemic coronary artery diseases is provided.

In a fourth aspect of the invention, the application of miR-374b-5p as a drug treatment target in preparing a drug for treating breast cancer is provided.

Compared with the prior art, the invention has the following advantages:

the invention provides the function of miR-374b-5p in VEGF gene expression induced by MCP-1, and defines that miR-374b-5p is a bridge for connecting the miR-374b-5p and the VEGF gene expression. In addition, the inventors found that MCP-1 increases the acetylation level of stat3 and that subsequent binding of the ac-stat3/DNMT1/EZH2 protein complex promotes the binding of DNMT1 to the miR-374b-5p promoter, which is the reason for the inhibition of miR-374b-5p by MCP-1. This series of processes provides a key link for the molecular mechanism of MCP-1 to stimulate VEGF gene expression. These findings not only help to better understand the role of MCP-1 and VEGF in tumor angiogenesis and tumor growth, but also provide a potential therapeutic approach. The action of MCP-1 on VEGF is relieved by exogenous miR-374b-5p supplementation, and the rapid growth and metastasis of tumors caused by MCP-1 are inhibited. The key of the blocking-unblocking mechanism is that the large amount of VEGF gene expression can be blocked by the addition of miR-374b-5p, and the blocking is released by the action of MCP-1. This mechanism is of interest for the regulation of essential multifunctional growth factors (such as VEGF), and therefore miR-374b-5p is also of potential value in the treatment of VEGF related diseases. On the other hand, when high activity of VEGF is needed to promote angiogenesis under the condition of ischemia related to coronary artery diseases and the like, miR-374b-5p can also be used as a therapeutic target to relieve the inhibition of VEGF gene expression, so that angiogenesis is promoted to improve the ischemic state and the like.

Drawings

FIG. 1 is a graph showing the effect of MCP-1 on angiogenesis in mice.

FIG. 2 shows the effect of MCP-1 on VEGF mRNA and protein expression in matrigel in mice. Wherein a is the mRNA expression level of VEGF; b is the protein expression level of VEGF.

FIG. 3 is a graph showing the effect of MCP-1 on the breast tumor volume in mice.

FIG. 4 shows the effect of MCP-1 on angiogenesis in a breast tumor in mice.

FIG. 5 is a graph showing the effect of MCP-1 on VEGF expression in breast tumors in mice. Wherein a is the mRNA expression level of VEGF; b is the protein expression level of VEGF.

FIG. 6 shows the effect of MCP-1 on the expression of miR-374b-5p in matrigel and breast tumor in mice. Wherein a is in a matrigel; b is inside a breast tumor.

FIG. 7 is a graph showing the effect of adding miR-374b-5p on VEGF expression in HUVEC cells. Wherein a is the mRNA expression level of VEGF; b is the protein expression level of VEGF.

FIG. 8 shows the effect of miR-374b-5p injection in matrigel on MCP-1-induced angiogenesis and VEGF in mice. Wherein a is the matrigel angiogenesis; b. c is the mRNA and protein expression of VEGF in the matrigel, respectively.

FIG. 9 is a graph of the effect on MCP-1 induced tumor growth after miR-374b-5p injection in mouse subcutaneous breast tumors. Wherein a is a tumor picture, growth volume change and mouse weight change; b is the angiogenesis inside the tumor.

FIG. 10 is a graph of the effect of miR-374b-5p injection on intratumoral VEGF expression in mouse subcutaneous breast tumors. Wherein a and b are respectively mRNA and protein expression changes of VEGF.

FIG. 11 is a schematic diagram of miR-374b-5p promoter recombinant plasmid design.

FIG. 12 shows luciferase activity assay of miR-374b-5p promoter recombinant plasmid.

FIG. 13 is a graph of the effect of 5-aza on miR-374b-5p expression in HUVEC cells.

FIG. 14 is a graph of MCP-1 increasing DNMT1 binding to the miR-374b-5p promoter in HUVEC cells.

FIG. 15 is a graph of the effect of MCP-1 on stat 3-related protein expression in matrigel and breast tumors in mice. Wherein a is in a matrigel; b is inside a breast tumor.

FIG. 16 is a graph of the effect of PF-CBP1 on miR-374b-5p expression in HUVEC cells.

FIG. 17 shows the analysis of ac-stat3/DNMT1/EZH2 interaction by co-immunoprecipitation in HUVEC cells. Wherein a is the promoting effect of MCP-1 on the combination of the complex; b is the inhibitory effect of PF-CBP1 on the increased binding of MCP-1-induced complex.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 cell culture and stimulation

HUVEC cells and bEnd.3 cells were cultured in 1640 or DMEM medium containing 10% fetal bovine serum, 1% streptomycin (double antibody), respectively, at 37 ℃ with 5% CO₂Culturing in an incubator; the experiment was performed using log phase cells with good growth status. MCP-1(PeproTech)100ng/mL stimulates cells for 48h to establish an in vitro model; mu.M PF-CBP1 hydrochloride (MedChemexpress), 4. mu.M 5-aza-2' -deoxycytidine (Jena bioscience) pretreated cells for 2h and then co-stimulated with MCP-1 for 48 h.

Example 2 in vivo angiogenesis assay

The bEnd 3. cell digestions were counted, every 5X 10⁵Adding 100 μ L matrigel into cells, mixing with corresponding reagent (MCP-130 ng, miR-Ctrl/miR-374b-5p 100n μm), and shaking at 4 deg.C for 4 hr to thoroughly mix the cells with matrigel solution; 100 μ L of the suspension was injected subcutaneously into the abdomen of female C57BL/6J mice, the reagents were supplemented in the gel on

days

3 and 5, and the mice were euthanized on day 10, then the matrigel was taken out for photography and subsequent experiments were performed.

Example 3 mouse breast cancer 4T1 cell allograft tumor model

2 x 10 to⁶4T1 cells were injected subcutaneously into the back of female BALB/c mice when tumors reached approximately 100mm³Time is divided randomly and recorded as

day

0, 100 mu L of corresponding reagent (MCP-1300n, miR-Ctrl/miR-374b-5p 100nM) is injected into tumor every 3 days; mice were euthanized at day 15 and tumors were photographed and post-analyzed.

Example 4 Dual luciferase reporter Gene assays

HEK-293T cells are transfected by the constructed recombinant plasmid of the miR-374b-5p promoter, and the main region of the miR-374b-5p promoter is evaluated by detecting the fluorescence value of the cells by using a dual-luciferase reporter gene detection kit (assist in san Living creature) 24h later.

Example 5RNA extraction and qPCR detection

Total RNA and microRNA were extracted with TRIzol reagent (Invitrogen) and miRNeasy Mini Kit (Qiagen), respectively; reverse transcription and amplification were performed using the First-strand cDNA synthesis SuperMix kit and Top Green qPCR SuperMix kit (all gold). The primer sequence is as follows: h-VEGF,5 '-CGCAAGAAAT CCCGGTATAA and 5' -AAATGCTTTC TCCGCTCTGA; h-beta-actin, 5 '-GCGAGAGATG ACCCAGATC and 5' -CCAGTGGTAC GGCCAGAGG. miRNA primers were purchased from rubo bio ltd, guangzhou. The relative expression of the relevant mRNA was analyzed by the 2- Δ Δ Ct method.

Example 6 chromatin immunoprecipitation assay (ChIP)

Experiments were performed using the ChIP assay kit (bi yun tian). Carrying out 37% formaldehyde crosslinking, cell lysate cracking and ultrasonic cracking on cells, adding a corresponding antibody into the supernatant, incubating overnight at 4 ℃, adding Protein G magnetic beads to precipitate an antibody-DNA compound, eluting, decrosslinking, extracting and purifying DNA; carrying out amplification analysis on the DNA fragment by using a PCR kit; the primer sequences of the miR-374b-5p promoter are 5'-GGTCATTTCA CCTTTTATTT CTCTG and 5' -AGAGGAAGAA ACTGAAGTTC AACAA. The resulting product was subjected to agarose gel electrophoresis.

Example 7 Co-immunoprecipitation (co-IP) and Western blotting

Lysing the cells on ice with NP-40 lysis buffer (Biyuntian), centrifuging and collecting the supernatant for use; adding 200 μ L diluted antibody (EZH2, DNMT1, IgG) solution into 50 μ L protein A/G magnetic beads (Bimake), mixing at room temperature, turning for 15min, and removing supernatant by magnetic adsorption; adding the lysate into magnetic beads combined with the antibody, and turning over for 1h at room temperature to form a protein-antibody-magnetic compound; boiling the sample buffer solution with the volume of 1 multiplied by the volume of the sample buffer solution to elute the protein, and discarding the magnetic beads; samples were analyzed by SDS-PAGE immunoblotting.

The test results of examples 1-7 are discussed below:

MCP-1 promotes VEGF expression and tumor angiogenesis

The invention evaluates the effect of MCP-1 on VEGF-induced tumor angiogenesis in animal models. In a mouse angiogenesis model (matrigel experiment), the number of blood vessels formed by bEnd.3 cells in the matrigel injected with MCP-1 is about 2 times that of the control group (figure 1), and the mRNA and protein expression level of VEGF is also obviously improved compared with the control group (figure 2). In a tumor model formed by subcutaneous implantation of mouse breast cancer 4T1 cells in BALB/c mice, we found that the intratumoral injection of MCP-1 resulted in an approximately 2-fold increase in tumor volume compared to the control group (FIG. 3), and immunofluorescence staining with the endothelial cell marker CD31 after tumor sectioning revealed that the intratumoral vascular density of MCP-1 in the intratumoral injection group was more than 2-fold higher than that in the control group (FIG. 4), accompanied by an increase in intratumoral VEGF expression (FIG. 5). These data indicate that MCP-1 promotes VEGF expression as well as tumor angiogenesis.

2, miR-374b-5p inhibits high expression of VEGF gene caused by MCP-1

Compared with the control group, the expression level of miR-374b-5p in MCP-1 stimulated matrigel and breast tumor is obviously reduced (figure 6). When miR-374b-5p is added into HUVEC cells, the mRNA and protein expression of VEGF in cells of the miR-374b-5p group is found to be obviously reduced compared with that of the miR-Ctrl group; moreover, the addition of miR-374b-5p can prevent the high expression of VEGF caused by MCP-1 (FIG. 7). These findings suggest that miR-374b-5p may play a key role in the regulation of VEGF expression by MCP-1, and the expression of VEGF can be inhibited by adding exogenous miR-374b-5 p.

miR-374b-5p inhibits angiogenesis and tumor growth caused by MCP-1

Injection of miR-374b-5p in mouse matrigel reduced in-gel angiogenesis by MCP-1 stimulation by about 60% (FIG. 8a), and addition of miR-374b-5p inhibited VEGF production by MCP-1 stimulation in matrigel (FIG. 8b, c). Similarly, the miR-374b-5p can obviously inhibit the growth of tumors by intratumoral injection, does not influence the weight of a mouse, and shows that the miR-374b-5p has no toxicity to the mouse; and reduced the mean tumor volume under MCP-1 stimulation by about half (FIG. 9 a); the angiogenic density of the blood vessels inside the tumor was greatly inhibited (fig. 9 b). The expression level of VEGF in the tumor is also reduced due to the addition of exogenous miR-374b-5p (FIG. 10). These findings indicate that the addition of miR-374b-5p can obviously inhibit angiogenesis and tumor growth in tumors, and simultaneously prevent MCP-1 from promoting angiogenesis and tumor growth.

MCP-1 increases the binding of DNMT1 and miR-374b-5p promoter, and the expression of VEGF gene can be blocked by the added miR-374b-5p, so that the effect of miR-374b-5p blocking is required when MCP-1 induces the expression of VEGF. The inventor constructs a plurality of recombinant plasmids (figure 11) according to the miR-374b-5p promoter region sequence (shown as SEQ ID NO: 1), namely a sequence 2000bp upstream of the miR-374b transcription initiation site in the human genome (chrX:74217547-74219546), divides and selects: promoter1 (P1): chrX 74218937 and 74219546(610 bp); promoter2 (P2): 74218937-; promoter3 (P3): the plasmid was inserted into pGL3-Basic-Vector plasmid to obtain three recombinant plasmids, chrX:74219229-74219546(318 bp). By measuring the luciferase activity of the recombinant plasmid, the inventors found that the functional region of the miR-374b-5P promoter is mainly concentrated on chrX:74218937-74219228(P2, FIG. 12). When the DNA methyltransferase inhibitor 5-Aza-2' -deoxycytidine (5-Aza) was used, 5-Aza blocked the down-regulation of miR-374b-5p expression by MCP-1 in HUVEC cells (FIG. 13). Since DNMT1 is a key gene for DNA methylation in epigenetic modification of mammalian genomes, the inventors next investigated whether DNMT1 is associated with changes in miR-374b-5p expression. DNMT1 was found to bind to the miR-374b-5p promoter by chromatin co-precipitation (ChIP) analysis, and MCP-1 promoted the binding of both (FIG. 14). In conclusion, DNMT1 is involved in the regulation of miR-374b-5p expression by MCP-1, and the expression of miR-374b-5p is hindered by increasing the binding with miR-374b-5p promoter.

MCP-1 promotes binding of DNMT1 to miR-374b-5p promoter by increasing formation of ac-stat3/DNMT1/EZH2 complex

It is known that a complex of acetylated stat3(ac-stat3) and DNMT1 can synergistically methylate a number of tumor suppressor genes, while DNMT1/EZH2 can collectively catalyze miRNA methylation. By examining the changes in mouse matrigel and stat3 in breast tumors in vivo, MCP-1 stimulation did not affect changes in mouse matrigel and in the total amount of intratumoral stat3 phosphorylation (active form) and stat3, but increased the acetylation level of stat3 at Lys685 site (fig. 15). We further found that the addition of PF-CBP1 (a specific inhibitor of the acetylated form of stat3) was effective in relieving the inhibitory effect of MCP-1 on miR-374b-5p expression (FIG. 16). Furthermore, co-immunoprecipitation analysis showed that MCP-1 promotes the interaction of the ac-stat3/EZH2/DNMT1 complex in HUVEC cells, while PF-CBP1 inhibits these interactions (FIG. 17). These findings indicate that MCP-1 promotes formation of the ac-stat3/EZH2/DNMT1 complex by increasing acetylation level of stat3 at the Lys685 site, thereby leading to increased binding of DNMT1 to the miR-374b-5p promoter, and ultimately inhibiting expression of miR-374b-5 p.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of southern kayak

<120> microRNA for regulating VEGF gene expression and application thereof

<141> 2021-12-07

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Claims

Use of miR-374b-5p as a drug treatment target in preparation of a drug for regulating blood vessel growth.
2. Use according to claim 1, characterized in that: the medicine is a medicine for inhibiting miR-374b-5p expression so as to inhibit blood vessel growth.
3. Use according to claim 1, characterized in that: the medicine is a medicine for promoting the growth of blood vessels by up-regulating miR-374b-5p expression.
4. Use according to claim 1, characterized in that: the drug regulates the growth of blood vessels by controlling the expression of VEGF gene through regulating the expression of miR-374b-5 p.
5. Use according to claim 4, characterized in that: the VEGF gene expression is MCP-1 mediated VEGF gene expression.
6. Use according to claim 1, characterized in that: the medicine contains miR-374b-5 p.
Application of miR-374b-5p as a drug treatment target in preparation of drugs for treating ischemic coronary artery diseases.
Application of miR-374b-5p as a drug treatment target in preparation of drugs for treating breast cancer.