CN113930428B

CN113930428B - miRNA for treating CNV and preparation method and application thereof

Info

Publication number: CN113930428B
Application number: CN202111222559.5A
Authority: CN
Inventors: 吕洋; 李红; 苏芳菊; 杨芳芳; 赵颖英; 李静
Original assignee: 904th Hospital of the Joint Logistics Support Force of PLA
Current assignee: 904th Hospital of the Joint Logistics Support Force of PLA
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2023-04-25
Anticipated expiration: 2041-10-20
Also published as: CN113930428A

Abstract

The invention relates to miRNA for treating CNV, a preparation method and application thereof. The sequence of miRNA for treating CNV is shown in a sequence table SEQ ID No. 1; the preparation method comprises the steps of firstly treating a sample to obtain a sample cell, extracting total RNA in the sample cell, then carrying out reverse transcription to obtain a reverse transcription product, and carrying out real-time fluorescent quantitative PCR detection on the obtained reverse transcription product to obtain the miRNA for treating CNV. The invention also provides a slow virus expression vector and a pharmaceutical preparation containing the miRNA, and application of the miRNA and the slow virus expression vector containing the miRNA in pharmaceutical preparation. The invention utilizes the miRNA to regulate and control the biological characteristics of ECs to express one-to-many, achieves the aim of efficiently treating CNV at multiple angles, and provides a brand new thought and strategy for treating CNV by gene therapy.

Description

miRNA for treating CNV and preparation method and application thereof

Technical Field

The invention belongs to the technical field of gene medicines, and particularly relates to miRNA for treating CNV, and a preparation method and application thereof.

Background

Choroidal Neovascularization (CNV) refers to a series of pathological changes that result from the proliferation of pathologic neovascularization of the choroid and ingrowth through the Bruch's membrane, invasion of the subretinal space, followed by exudation, hemorrhage, and scarring. Is associated with at least over 40 ocular diseases, including degeneration, inflammation, trauma, and the like. Such as age-related maculopathy, pathologic myopia macular degeneration, idiopathic CNV, ocular histoplasmosis syndrome, ocular trauma, etc. CNV is common sign of common ocular neovascular diseases, which is the main cause of blindness in people older than or equal to 50 years, and should be discovered and treated in time. CNV formation includes two forms of vascular Endothelial Cell (ECs) proliferation in situ, transitional angiogenesis, and angiogenesis involving bone marrow-derived vascular Endothelial Progenitor Cells (EPCs), with later vascular remodeling/fibrosis being closely related to ECs endothelial mesenchymal transition (EndoMT). At present, medicines for resisting vascular endothelial growth factor (vascular endothelial growth factor, VEGF) are mainly represented as first-line treatment schemes for clinically treating CNV diseases. The proposal aims at inhibiting the formation of new blood vessels, and obviously improves the life quality of patients, but has the advantages of limited clinical curative effect, high recurrence rate and even aggravated risk of fibrous scar formation.

miRcoRNAs are important non-coding genes that regulate 30% of the protein-coding genes, specifically inhibit the expression of the target gene by complementarily binding to the 3' utr region of its target mRNA molecule. The human miR-655 gene is located on chromosome 14 and belongs to the miR-154 family.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides miRNA for treating CNV, a preparation method thereof, a slow virus expression vector containing the miRNA, a pharmaceutical preparation containing the miRNA, and application of the miRNA and the slow virus expression vector containing the miRNA in preparation of a medicine for treating CNV. The miRNA regulates and controls the biological characteristics of ECs to express one-to-many, achieves the aim of efficiently treating CNV at multiple angles, and provides a brand new thought and strategy for treating CNV by gene therapy.

At present, there are two forms of CNV: angiogenesis (vasculogenesis) and vasculogenesis). The former refers to the new lumen structure formed by the sprouting, proliferation and migration of the original vascular endothelial cells (Endothelial Cells, ECs); the latter is the construction of bone marrow-derived vascular endothelial progenitor cells (endothelial progenitor cells, EPCs) that are activated in specific environments and recruited, induced to differentiate, and participate in neovasculature. Previous domestic and foreign researches focus on revealing early CNV generation mechanism, making important progress, and converting into a first-line treatment scheme for clinically treating CNV diseases represented by anti-vascular endothelial growth factor (vascular endothelial growth factor, VEGF) medicines. The proposal can inhibit angiogenesis, but can not inhibit pathological angiogenesis mediated by EPCs from bone marrow, which results in limited curative effect, high recurrence rate and even risk of aggravating fibrous scar formation, and the reason is related to important links such as vascular remodeling or fibrosis which can not inhibit two key processes of angiogenesis and vasculogenesis at the same time and does not relate to the later stage of angiogenesis.

Inflammation, hypoxia and oxidative stress can all become causes of ECs to generate endoMT, transforming growth factor-beta (transforming growth factor-beta, TGF-beta), snail transcription factor, FOXC1, bone morphogenic protein (bone morphogenetic protein, BMP) and other signal molecules play an important role in the regulation and control channel of endoMT. TGF-beta mediated signaling is widely demonstrated to induce endoMTs of tumor cells and ocular vascular ECs. In vitro and in vivo experiments demonstrated that blocking TGF-beta can inhibit EndomT in the above cells. TGF-beta can regulate downstream gene expression both by the Smads protein family, both dependent and independent. Smads family proteins play a critical role in the transmission of TGF- β signals from cell surface receptors to the nucleus. TGF-beta activates Smads complex to promote transcriptional expression of Snai 1. The members of the Snail family Snail 1 (Snail), snail 2 (Slug) and part of the basic helix-loop-helix family of proteins bind to the promoter region of genes involved in intercellular adhesion, thereby inducing endoMT to occur. Snai1 can bind to the E-box sequence of the promoter region of epithelial cadherin (epithelial cadherin, E-cadherin) and vascular endothelial cadherin (vascular endothelial cadherin, VE-cadherin) via the carboxy-terminus of its zinc finger domain, down-regulate its expression, while up-regulating vitronectin (vitronectin), fibronectin (fibronectin), and neural cadherin (N-cadherin), etc., to promote the transformation of epithelial cells/ECs to mesenchymal cells. Numerous studies suggest that TGF- β/Smads/Snai1 mediated signaling is probably the most predominant molecular mechanism of EndoMT in CNV to induce ECs. Our earlier experiments showed that ECs decreased in CNV and fibroblasts increased; expression of Snai1 gradually increases in the late stages of CNV. In addition, FOXC1 and BMP mediated signaling might also play an important role for EndoMT of ECs in CNV. FOXC1 is considered an important regulator for induction of EndoMT in many tumor cells. Over-expression of FOXC1 in various cancer cells results in down-regulation of a range of genes involved in EndoMT, such as E-cadherin, VE-cadherin, CD31, etc., while up-regulation of mesenchymal related gene products, such as N-cadherin, vitronectin, fibronectin, etc. Recent studies have found that FOXC1 is involved in the formation of corneal neovascularization. Both FOXC1 and BMP may promote the function of the Snai1 family of proteins. Thus, endoMT of ECs regulates a variety of factors, playing an important role in vascular remodeling/fibrosis to participate in CNV formation.

Expression of CXCR4 on human/murine EPCs and expression of FOXC1, BMPR2, smad5, samd12 and Snai2 on human HRMEC cells play a key role in regulating EPCs cell migration and HRMEC cell endoMT. Modulation of the expression levels of the above proteins can affect the migration of EPCs cells and EndoMT of HRMEC cells. The inventor researches the relation of miRNA-655-3p participating in CNV formation and vascular remodeling/fibrosis through methods such as a luciferase reporting system, a lentivirus over-expression/interference technology, an in-vivo fluorescence imaging method and the like, and discovers that after the expression level of miRNA-655-3p is up-regulated, the expression of FOXC1, BMPR2, smad5, samd12 and Snai2 in HRMEC cells can be down-regulated in turn, so that endoMT of the cells is inhibited; meanwhile, the expression of CXCR4 in the EPCs in the choroidal blood vessel can be down regulated, and the mobilization, migration, adhesion and differentiation of the EPCs in the choroid can be inhibited; miRNA-655-3p can regulate the expression of CXCR4 in EPCs to regulate chemotaxis of the EPCs, thereby affecting angiogenesis; simultaneously modulating the expression of EndoMT related molecules FOXO1, BMPR2, smad5, samd12 and Snai2 in ECs affects CNV vascular remodeling/fibrosis. The molecular mechanism of miRNA-655-3p in the occurrence and development of CNV is obtained, the therapeutic potential of miRNA-655-3p in CNV is clarified, and a new strategy and a new target point are provided for the treatment of CNV.

The technical scheme adopted by the invention is as follows:

miRNA for treating CNV, specifically miRNA-655-3p, has a nucleotide sequence shown in SEQ ID NO: 1: UUUCUCCAAUUGGUACAUAAUA.

A lentiviral expression vector comprising the miRNA-655-3 p.

A pharmaceutical formulation comprising the miRNA-655-3 p.

A method of preparing miRNA for treating CNV, comprising the steps of:

(1) Taking a sample for treatment to obtain sample cells;

(2) Extracting total RNA in the sample cells to obtain a reverse transcription template;

(3) Performing reverse transcription on the reverse transcription template to obtain a reverse transcription product;

(4) And carrying out real-time fluorescence quantitative PCR detection on the reverse transcription product to obtain the miRNA.

Further, in step (1), the sample is a femur or tibia of a human or mouse.

Further, in the step (1), the processing procedure is as follows: taking femur and tibia of a human or a mouse, cutting off two ends of the femur, flushing bone marrow cells in a bone marrow cavity by using sterile PBS, centrifugally separating mononuclear cells by using a density gradient, and inoculating the mononuclear cells into a incubator, wherein the culture solution is DMEM containing 15% fetal bovine serum; after 3 days, the non-adherent cells were removed by washing with PBS, and the remaining monolayer cells were cultured in fresh medium until confluence; passaging with 0.25% pancreatin+0.1% edta after 6-7 days; analyzing cell surface antigens by using flow cytometry, and performing in vitro differentiation induction experiments; after cell ablation, flow cytometry analysis was performed with FITC-labeled anti-CD 34/VEGFR2 antibodies to obtain sample cells.

Further, in the step (2), the process of extracting total RNA in the sample cells is as follows: lysing the sample cells by using 700ul QIAzol, standing at room temperature for 10min, adding 140ul chloroform, shaking for 15s, standing at room temperature for 2-3min, and centrifuging at 12000G and 4deg.C for 15min to obtain supernatant; adding 525ul of 100% ethanol into the supernatant, and uniformly mixing to obtain a mixed solution; taking 700ul of the mixed solution, centrifuging for 15s at 8000G at normal temperature, adding the rest mixed solution, centrifuging for 15s at 8000G at normal temperature, and discarding the supernatant to obtain a precipitate; adding 40ul of RNase-free water into the precipitate, mixing well, centrifuging at 8000G for 1min, and preserving at-70deg.C.

Further, in the step (3), the sequence of the reverse transcription primer is shown as SEQ ID NO. 2; the reverse transcription procedure is: denaturation at 37℃for 60min, extension at 95℃for 5min; the reverse transcription product was stored at 4 ℃.

Further, in the step (4), the real-time fluorescence quantitative PCR detection amplification procedure is that the temperature is 98 ℃ for 3min, the temperature is 98 ℃ for 15s, the temperature is 55 ℃ for 15s, the temperature is 72 ℃ for 10min, and the total time is 40 cycles; setting an internal reference, and analyzing to obtain the relative expression quantity of miRNA.

The application of the miRNA in preparing a medicine for treating CNV.

The slow virus expression vector containing miRNA-655-3p is applied to the preparation of medicines for treating CNV.

The beneficial effects of the invention are as follows:

the sequence of miRNA for treating CNV is shown in a sequence table SEQ ID No. 1; the preparation method comprises the steps of firstly treating a sample to obtain a sample cell, extracting total RNA in the sample cell, then carrying out reverse transcription to obtain a reverse transcription product, and carrying out real-time fluorescent quantitative PCR detection on the obtained reverse transcription product to obtain the miRNA for treating CNV. The invention also provides a slow virus expression vector and a pharmaceutical preparation containing the miRNA, and application of the miRNA and the slow virus expression vector containing the miRNA in pharmaceutical preparation.

Expression of CXCR4 on human/murine EPCs and expression of FOXC1, BMPR2, smad5, samd12 and Snai2 on human HRMEC cells play a key role in regulating EPCs cell migration and HRMEC cell endoMT. Modulation of the expression levels of the above proteins can affect the migration of EPCs cells and EndoMT of HRMEC cells. The inventor obtains the relationship of miRNA-655-3p participating in CNV formation and vascular remodeling/fibrosis through a luciferase reporting system, a slow virus over-expression/interference technology, an in-vivo fluorescence imaging method and the like.

(1) The expression level of miRNA-655-3p can be up-regulated, and then the expression of FOXC1, BMPR2, smad5, samd12 and Snai2 in HRMEC cells can be down-regulated, so that the endoMT of the cells can be inhibited.

(2) The miRNA-655-3p can down regulate the expression of CXCR4 in EPCs cells in choroidal blood vessels after the expression level is up-regulated, and inhibit the mobilization, migration, adhesion and differentiation of the EPCs cells. The miRNA-655-3p acts on an SDF-1/CXC4-CNV signal path, CXCR4 high expression activates a PI3K/AKT signal path in bone marrow-derived EPCs, induces the differentiation of the bone marrow-derived EPCs to ECs, and promotes the angiogenesis of CNV (Yang Lv, et al, exp Eye Res.2020). When the factors in the signal path are high expressed, VEGF is high expressed, and TGF-beta is high expressed; when the inhibition in the whole signal path is reduced, VEGF is still expressed in high degree, and TGF-beta expression is reduced; that is, it is shown that the signal path has no necessary relation with VEGF, but has relation with TGF-beta. Thus, part of reasons for limited clinical efficacy against VEGF and high recurrence rate in some patients are explained. The miRNA-655-3p acts on SDF-1/CXCR4 in the signal path firstly, so that the expression of the path is regulated downwards, and TGF-beta expression is inhibited, thereby reducing the molecular mechanism of angiogenesis.

In conclusion, the miRNA-655-3p targets and inhibits the expression of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2, influences the differentiation of ECs EndomT and the migration and the colonisation of bone marrow-derived EPCs to the damaged part of the choroid, namely, the miRNA-655-3p influences important links such as angiogenesis and later vascular remodeling or fibrosis, so as to obtain a multi-molecule regulation mechanism of the miRNA-655-3p in the occurrence and development of CNV, clarify the therapeutic potential of the miRNA-655-3p in the CNV, and provide a new strategy and a new target for the treatment of the CNV; the therapeutic significance of the miRNA-655-3p in treating CNV null patients with partial VEGF is also demonstrated. The invention utilizes the biological characteristics of miRNA for regulating and controlling ECs to express one-to-many, achieves the aim of efficiently treating CNV at multiple angles, and provides brand-new thought, strategy and target point for treating CNV by gene therapy.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a cell flow assay CD34/VEGFR2 co-labeling results;

FIG. 2 is a potential target gene for the miRNA-655-3 p;

FIG. 3 is the expression of miR-655-3p in ECs at various time points after hypoxia;

FIG. 4 is the expression of miR-655-3p in CNV at various time points after laser photocoagulation of mice;

FIG. 5 is the expression levels of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 in ECs under normoxic and hypoxic conditions;

FIG. 6 is a graph showing that CXCR4, FOXO1, BMPR2, smad5, smad12 and Snai2 are expressed in the mouse CNV region at day 7 after laser induction of mouse CNV;

FIG. 7 is a correlation analysis result of in vitro experiments;

FIG. 8 is a correlation analysis result of in vivo experiments;

FIG. 9 is the results of a CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai 2' UTR dual luciferase reporter assay;

FIG. 10 is the results of mRNA expression of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 in ECs in miR-655-3p negative regulation anaerobic environment;

FIG. 11 is an illustration of the regulation of TGF-beta signaling by miRNA-655-3 p;

FIG. 12 is an illustration of the regulation of human and mouse stem cell differentiation by miRNA-655-3 p;

FIG. 13 is an illustration of the regulation of mouse stem cell differentiation by miRNA-655-3 p;

FIG. 14 is a diagram of the design of a test concept for verifying miRNA-655-3p multi-molecule targeted modulation CNV;

FIG. 15 is an animal experiment demonstrating that SDF-1/CXCR4 can promote the aggregation of more BMCs-derived EPCs into ocular CNV regions and exacerbate CNV disease;

FIG. 16 is a laser scanning confocal microscope of the co-localization of each group of CXCR4 and endothelial cells/BMCs in the local expression of mouse CNV;

FIG. 17 is the severity of CNV in SDF-1/CXCR4 aggravated mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Example 1

The embodiment provides miRNA for treating CNV, wherein the miRNA is miRNA-655-3p, and the nucleotide sequence is shown as SEQ ID NO. 1.

The embodiment also provides a method for preparing the miRNA, which comprises the following steps:

(1) Taking human femur, shearing off two ends of the femur, flushing out bone marrow cells in a bone marrow cavity by using sterile PBS, centrifugally separating mononuclear cells by using a density gradient, and inoculating the mononuclear cells into a incubator, wherein the culture solution is DMEM containing 15% fetal calf serum; after 3 days, the non-adherent cells were removed by washing with PBS, and the remaining monolayer cells were cultured in fresh medium until confluence; passaging with 0.25% pancreatin+0.1% edta after 6-7 days; analyzing cell surface antigens by using flow cytometry, and performing in vitro differentiation induction experiments; after cell ablation, carrying out flow cytometry analysis (shown in figure 1) with FITC-labeled anti-CD 34/VEGFR2 antibody, and sorting to obtain sample cells;

(2) Extracting total RNA in sample cells: lysing the sample cells by using 700ul QIAzol, standing at room temperature for 10min, adding 140ul chloroform, shaking for 15s, standing at room temperature for 2-3min, and centrifuging at 12000G and 4deg.C for 15min to obtain supernatant; adding 525ul of 100% ethanol into the supernatant, and uniformly mixing to obtain a mixed solution; taking 700ul of the mixed solution, centrifuging for 15s at 8000G at normal temperature, adding the rest mixed solution, centrifuging for 15s at 8000G at normal temperature, and discarding the supernatant to obtain a precipitate; adding 40ul of RNase-free water into the precipitate, mixing uniformly, centrifuging at 8000G for 1min, and preserving at-70deg.C to obtain reverse transcription template;

(3) The sequence of the reverse transcription primer is shown as SEQ ID NO. 2; denaturation at 37 ℃ for 60min, extension at 95 ℃ for 5min for reverse transcription to obtain a reverse transcription product; the reverse transcription product is stored at 4 ℃;

(4) Detecting the reverse transcription product by using real-time fluorescence quantitative PCR, uniformly mixing in a dark place, and detecting on a machine; the amplification procedure was 98℃pre-denaturation for 3min,98℃denaturation for 15s,55℃annealing for 15s,72℃extension for 10min for a total of 40 cycles; setting an internal reference, and analyzing to obtain the relative expression quantity of miRNA to obtain the miRNA.

Example 2

The difference between this example and example 1 is only that the sample used in this example was the tibia of the mouse, and the other operations were the same as in example 1.

Example 3

The difference between this example and example 1 is only that the sample used in this example was human tibia, and the other operations were the same as in example 1.

Example 4

The difference between this example and example 1 is only that the sample used in this example was a femur of a mouse, and the other operations were the same as in example 1.

Experimental example

A first part: verification of over-expression miR-655-3p for inhibiting occurrence and development of CNV

I. Description of key technology

Assuming successful validation, it was critical whether miRNA-655-3p could target modulation of CXCR4 expression on human/murine EPCs, FOXC1, BMPR2, smad5, samd12 and Snai2 expression on human HRMEC. Multiple bioassay software analyses resulted in miRNA-655-3p with stable binding sites at the 3' UTR end of these genes. Depending on the molecular mechanism of the miRNA, up-regulation of miRNA-655-3p could theoretically down-regulate the expression of the above molecules in cells.

It was also necessary to continue to verify whether miRNA-655-3p could inhibit the migration of human/murine EPCs, and the endoMTs of HRMEC cells, by specifically targeting miRNA-655-3p to modulate the expression of CXCR4 on human/murine EPCs and the expression of FOXC1, BMPR2, smad5, samd12 and Snai2 on human HRMEC cells. There is a great deal of literature currently demonstrating the critical role of the above proteins in the regulation of EPCs cell migration and HRMEC cell EndoMT. Theoretically, regulating the expression level of the above proteins can affect the migration of EPCs cells and EndoMT of HRMEC cells.

After the physiological effects of miRNA-655-3p are clarified, it is further verified whether the mouse CNV progression is affected by the intravitreal injection of lentiviral-coated miRNA-655-3p overexpression vector. The expression level of miRNA-655-3p in local cells can be up-regulated theoretically by injecting miRNA-655-3p over-expression vector wrapped by slow virus into the vitreous body. The up-regulation of the expression level of miRNA-655-3p can in turn down-regulate the expression of FOXC1, BMPR2, smad5, samd12 and Snai2 in HRMEC cells, thereby inhibiting endoMT of the cells; meanwhile, the expression of CXCR4 in the EPCs in the choroidal blood vessel can be down-regulated, and the adhesion and the colonization of the EPCs in the choroid can be inhibited.

II. Feasibility analysis

1. The theory is feasible: a large body of literature suggests that EPCs are involved in the formation of CNV. SDF-1 expressed on choroidal tissues can bind to CXCR4 expressed on EPCs, thus recruiting EPCs to aggregate toward the injured choroid. EPCs colonize the choroidal tissues and differentiate into vascular endothelial cells to promote the formation of new blood vessels. Targeting miRNA-655-3p to inhibit CXCR4 expression on EPCs in this experiment theoretically could inhibit chemotaxis of EPCs to choroidal tissues.

2. Studies have shown that vascular endothelial cells EndoMT are also involved in CNV formation. FOXC1, BMPR2, smad5, samd12 and Snai2 play a key role in vascular endothelial cell EndoMT processes. In theory, miRNA-655-3p targeting inhibition of FOXC1, BMPR2, smad5, samd12 and Snai2 expression can inhibit vascular endothelial cell EndomT.

III, verification scheme

1. Verification of the Regulation of the target Gene by miRNA-655-3p

1.miRNA target gene prediction

The binding site of miRNA-655-3p at the 3' UTR of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 was predicted using targetscan (http:// www.targetscan.org/vert_72 /) software, while the RNA22 software analysis showed that it had multiple target genes such as: sites within 3' UTRs of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2mRNA are complementary to miR655-3p (shown in FIG. 1); functional cluster analysis results indicate that CXCR4, FOXC1, BMPR2, smad5, samd12, and Snai2 affect neovascularization. Thus, CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 are target genes for miR655-3p, as shown in FIG. 2.

(1) The gene sequence query websites for CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 are as follows:

CXCR4

Human：Gene ID: 7852https://www.ncbi.nlm.nih.gov/gene/7852

Mouse:Gene ID: 12767https://www.ncbi.nlm.nih.gov/gene/12767

FOXC1：

Human：Gene ID: 2296https://www.ncbi.nlm.nih.gov/gene/2296

Mouse：Gene ID: 17300https://www.ncbi.nlm.nih.gov/gene/17300

BMPR2

Human：Gene ID: 659https://www.ncbi.nlm.nih.gov/gene/659

Mouse：Gene ID: 12168https://www.ncbi.nlm.nih.gov/gene/12168

SMAD5

Human：Gene ID: 4090https://www.ncbi.nlm.nih.gov/gene/4090

Mouse：Gene ID: 17129https://www.ncbi.nlm.nih.gov/gene/17129

SNAI2

Human：Gene ID: 6591https://www.ncbi.nlm.nih.gov/gene/6591

Mouse：Gene ID: 20583https://www.ncbi.nlm.nih.gov/gene/20583

SAMD12

Human：Gene ID: 401474https://www.ncbi.nlm.nih.gov/gene/401474

Mouse：Gene ID: 320679https://www.ncbi.nlm.nih.gov/gene/320679

(2) The primer sequences for designing CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 are shown in SEQ ID No. 3 to SEQ ID No. 14:

the CXCR4 primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4;

the FOXC1 primer sequences are shown as SEQ ID No. 5 and SEQ ID No. 6;

the BMPR2 primer sequences are shown as SEQ ID No. 7 and SEQ ID No. 8;

smad5 primer sequences are shown in SEQ ID No. 9 and SEQ ID No. 10;

the sequence of the Snai2 primer is shown as SEQ ID No. 11 and SEQ ID No. 12;

the Samd12 primer sequences are shown as SEQ ID No. 13 and SEQ ID No. 14.

2. qRT-PCR detection

Extraction and quantification of RNA: culturing RF/6A cell line and RPE cell line with low sugar DMEM containing double antibody, amplifying and passaging, taking 9 dishes after cell density reaches about 80%, and incubating in low oxygen incubator (O) ₂ 1% of CO ₂ 5% and N ₂ 94%) and taking out after 6h, 12h and 24h of hypoxia respectively; meanwhile, taking 3 dishes of cells which are not subjected to hypoxia treatment as a normal control group; pouring out low-sugar DMEM culture solution in 12 cell dishes, buckling the cell dishes on paper to suck the culture solution, pre-cooling 1xPBS for 2 times at 4 ℃ to wash the cells in the dishes so as to wash out the culture solution, pouring out 2ml of 1xPBS each time, and placing the dishes on ice after the PBS is completely poured out; 1ml Trizol was added for cell lysis, and after sufficient cell lysis, the cells were transferred into EP tubes, followed by RNA extraction steps. After total smallRNA is extracted, quantification is carried out;

qRT-PCR reaction

(1) Reaction system

MicroRNA Prime Script RT Enzyme Mix2μL

Total RNA 1. Mu.g

2*MicroRNA Reaction Buffer Mix(for Real Time)10μL

0.1%BSA2μL

RNase Free dH ₂ O is topped up to 20 mu L

(2) The reaction conditions are that the temperature is 37 ℃ and the application time is 30min, and the time is 10s at 85 DEG C

(3) The sample is stored at-80 ℃ for subsequent experiments;

C. real-time quantitative PCR: firstly, preparing a PCR reaction system, uniformly mixing in a dark place, and detecting on a machine. The reaction conditions were as follows: amplification procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 15s, annealing at 55℃for 15s, extension at 72℃for 10min for a total of 40 cycles. There were 3 duplicate amplification tubes per sample;

D. analysis of experimental results: beta-actin is used as an internal reference, and the relative expression quantity of mRNA is analyzed by software.

IV, test results

1. CXCR4, FOXC1, BMPR2, smad5, samd12, and Snai2 are expressed localized in the mouse CNV region and affect the severity of CNV.

2. The change in miR-655-3p expression in ECs under physical hypoxia conditions was detected by a real-time quantitative PCR method, and the miR-655-3p expression in ECs at different hypoxia time points (6, 12, 24 and 48 h) was compared with that in normoxic group (0 h) (FIG. 3). As a result, it was found that hypoxia resulted in decreased miR-655-3p expression in ECs, and that the expression level gradually decreased as the period of hypoxia increased within 24 hours of hypoxia. At 24h hypoxia, miR-655-3p expression in MSCs is lowest, and is reduced by about 6 times compared with miR-655-3p expression in normoxic ECs (hypoxia 24hvs. Normoxic: 0.15+ -0.05 vs.1.00+ -0.12, P < 0.001).

3. In early development of CNV, red fluorescence expressed miR-655-3p gradually decreases to the valley at 7 days, and then gradually rises back, as shown in FIG. 4 a; the miR-655-3p expression quantity is semi-quantitatively analyzed by taking Relative Fluorescence Intensity (RFI) as a unit, and the result shows that: after photocoagulation, the expression level of miR-655-3p was reduced by 50% on day 7 compared with day 3, as shown in FIG. 4 b.

4. The differences in CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 expression up-regulated in ECs under hypoxic conditions (#p < 0.05) were statistically significant, as shown in figure 5.

5. Co-expression of CXCR4, FOXO1, BMPR2, smad5, smad12 and Snai/CD 31 in each group was observed on day 7 post-CNV induction in mice. Red fluorescence represents each sub-molecule and green fluorescence represents the endothelial cell specific marker CD31. Part of the red and green confocal in the CNV region suggests: CXCR4, FOXO1, BMPR2, smad5, smad12, and Snai2 expressed on EPCs were localized in the mouse CNV region (as shown in fig. 6).

6. miR-655-3p has a negative correlation with expression levels of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai 2; FIGS. 7 and 8 show the results of correlation analysis of miR-655-3p and CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 in vitro and in vivo experiments.

7. The negative correlation between miR-655-3p and CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 expression levels was followed by the selection of whether miR-655-3p binds directly to the 3' UTR of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 using a well-established dual luciferase reporter gene system. The results show that: compared with transfected mimics NC, miR-655-3p mimics has obvious downregulation effect on the report fluorescence of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai 2' UTR wild type vectors, and the report fluorescence in the mutant vectors is not changed obviously after mutation of predicted target sites. Similarly, miR-655-53P micrometers had similar effects on the reported fluorescence of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 'UTR wild-type and mutant vectors (CXCR 4, FOXC1, BMPR2, smad5, samd12 and Snai2: P < 0.0001, as shown in FIG. 9 (WT: wild type; mut: mutant), miR-655-3P micrometers had a significant downregulating effect on the reported fluorescence of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai 2' UTR wild-type vectors, but no significant effect on the reported fluorescence of mutant vectors. The above results indicate that mouse miR-655-3P can regulate expression of the corresponding genes through the corresponding sites on CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 'UTR 3'.

8. miR-655-3p negatively regulates CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 expression. mimics and inhibitor work over-express and interfere miR-655-3p in the cell, and observation under a fluorescence microscope shows that: this green fluorescence was detected 12h after FAM-labeled mimos and inhibitor transfection. The positive ECs transfected successfully showed weaker fluorescence and scattered in the cytoplasm. The real-time quantitative PCR detection shows that the miR-655-3p expression in the ECs of the micrometers is greatly increased compared with that of the ECs of the micrometers, the miR-655-3p expression in the inhibitor is reduced compared with that of the NC of the inhibitor, and further the miR-655-3p micrometers and the inhibitor can be effectively over-expressed and interfere with the miR-655-3p expression. miR-655-3P negatively regulates expression of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 in ECs in an anoxic environment, miR-655-3P mimics is adopted to overexpress mRNA and protein expression of miR-655-3P in ECs in the anoxic environment, CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 are reduced (P is less than 0.001); whereas the inhibition of miR-655-3P in ECs in hypoxic conditions using inhibitor showed a slight increase in mRNA and protein expression of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2, but no statistical difference (P > 0.05), which may be associated with a lesser expression of miR-655-3P in ECs in hypoxic conditions, as shown in FIG. 10 (real-time quantitative PCR assay results indicate that mRNA of CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 slightly increased but no statistical difference after inhibition of miR-655-3P in ECs in hypoxic conditions, whereas mRNA of CXCR4, FOXC1, BMPR2, smad12 and Snai2 significantly decreased after mimics overexpressed miR-655-3P), these results suggest that in hypoxic conditions, CXCR4, FOXC1, BMPR2, samd12 and Snad 2 can negatively regulate mRNA expression of miR-655-3P in hypoxic conditions.

9. Analyzing all target genes of miRNA-655 by using targetscan, and then analyzing signal molecules in TGF-beta signals in the target genes and the influence of the signal molecules on the TGF-beta signals by using R language (as shown in figure 11); furthermore, the effect of the signal molecules regulating the differentiation of human and mouse stem cells on stem cell differentiation in the target gene was analyzed by using the R language (see FIGS. 12 and 13).

From the above test results, through strict multi-level verification test design (as shown in fig. 14), through methods such as a luciferase reporting system, a lentivirus over-expression/interference technology, in vivo fluorescence imaging and the like, the relationship of miRNA-655-3p in CNV formation and vascular remodeling/fibrosis is explored, the molecular mechanism of miRNA-655-3p in CNV occurrence and development is clarified, the negative regulation mechanism of miRNA-655-3p on target genes CXCR4, FOXC1, BMPR2, smad5, samd12 and Snai2 expression is obtained, and a series of pathological changes of miRNA-655-3p in CNV microenvironment are clarified, so that the molecular mechanism of the pathogenesis of CNV is further clarified, namely the miRNA-655-3p can regulate the expression of CXCR4 in EPCs to regulate the chemotaxis of EPCs, and further influence angiogenesis; simultaneous modulation of the expression of the endoMT-related molecules FOXC1, BMPR2, smad5, samd12 and Snai2 in ECs affects CNV vascular remodeling/fibrosis. Therefore, the biological characteristics of 'one-to-many' ECs expression can be regulated and controlled by miRNA, the aim of efficiently treating CNV at multiple angles is fulfilled, and a brand new thought and strategy for treating CNV by gene therapy are provided.

A second part: SDF-1/CXCR4 promotes the recruitment of more BMCs-derived EPCs to the mouse CNV and exacerbates disease progression

1. Experimental procedure

1. Antibodies and reagents

CXCR4 (Abcam, 1670), CD34 (BioLegend, 119301), VEGF/bFGF (Peprotech), CD31 (Abcam, 28364); anti-beta-actin antibodies were obtained from Santa Cruz Biotechnology (CA, USA), recombinant human secondary antibodies, including goat anti-rabbit/mouse/sheep antibodies conjugated to Alexa Fluor 594/CY3 or Alexa Fluor 488/FITC, purchased from Beijing Rehabilitation (beijin), SYBR Premix Ex Taq II and Multiscript RT purchased from Takara (chinese company), mouse CXCL12/SDF-1 protein purchased from Sino Biological inc. (50025-MNAE), AMD3100 purchased from Selleckchem (cat.s8030)

2. Animal experiment

GFP transgenic adult mice were purchased from the fourth army university of medical neurological biology line and bone marrow was obtained from bone. After counting the cells in the washed out cell suspension, the suspension is adjusted to 1X 10 ⁷ Individual cells/ml. Recipient mice were exposed to 8gy Co 60 radiation to kill their naive bone marrow cells, and then BMCs prepared with GFP transgenic mice were transplanted. One month later, analysis of bone marrow transplant chimeras was performed. Successful bone marrow transplantation was confirmed by flow cytometry examination, 85% of the cells were GFP positive, and conditioned mice were included for subsequent experiments. For laser induction in CNV mice, the recipient mice were administered with a fringing eye drop, followed by 532n wavelengthm, a three-way mirror with the power of 90-110mW is used for laser photocoagulation at the front part of the eyes of the mice. The exposure time around the optic disc was 0.1 seconds, the spot diameter was 75 μm and the radius was 1.5mm. The Pupillary Distance (PD) is 4-6 light freezing points. The lack of air bubbles and bleeding is considered to be an effective finding.

3. Cell culture

Rhesus monkey chorioretinal EC (RF/6A) were obtained from a cell bank purchased from the national academy of sciences and cultured in Dulbecco's Modified Eagle's medium (DMEM; invitrogen, carlsbad, calif., USA) containing 15% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin at 37℃and 5% CO ₂ Culturing in the environment.

4. Western blot analysis

Tissue or cell lysates were prepared in lysis buffer and protein concentration was determined using BCA protein assay kit. Equal amounts of protein were electrophoresed on a 10% SDS polyacrylamide gel and transferred to a 0.22-mm PVDF membrane (Millipore; bedford, mass., USA). Membranes were incubated with primary antibodies overnight at 4 ℃, then they were washed and incubated with secondary antibodies for 30 minutes. The secondary antibody is selected based on the type of primary antibody. After washing the membrane three times in TBST, protein bands were detected using an enhanced chemiluminescent system (Millipore). The density of the bands was normalized to that of β -actin. Beta-actin levels were used to normalize protein mass, and all experiments were repeated at least three times.

5. Immunofluorescent staining

After 7 days of treatment, mice were anesthetized with sodium pentobarbital and perfused sequentially with PBS and 4% paraformaldehyde through the left ventricle. After the sacrifice, eyes of each animal were removed and fixed in frozen 4% paraformaldehyde in 0.1M Phosphate Buffer (PB) for 2 hours. Then, the anterior segment and vitreous were excised and the posterior eye shield was cryoprotected in a graded series of sucrose solutions (20% and 30% in PB). The eye shields were embedded, sectioned vertically, 8 μm thick, permeabilized for 10 minutes, blocked for 30 minutes, and then incubated overnight with primary antibodies at 4 ℃. The sections were then incubated for 1 hour with a combination of secondary antibodies and stained with diamino-2-phenyl-indole (DAPI). Immunofluorescent staining was observed using confocal microscopy.

6. Quantitative real-time reverse transcription PCR

Quantitative reverse transcription RT-PCR (qRT-PCR) was performed. Total RNA was obtained from choroidal tissues and RPE cells were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was prepared using RT systems (Takara Dalia, china). Quantitative real-time PCR was performed in triplicate using the kit (SYBR Premix EX Taq; takara) and the ABI PRISM 7500 real-time PCR system, with beta-actin serving as an internal control. The PCR primer sequences are shown as SEQ ID No. 15 and SEQ ID No. 16.

7. Hematoxylin and Eosin (HE) staining

Histopathological analysis was performed as described previously (3 eyes per group, 6 eyes per eye), and the four treatment groups described above were analyzed 7 days after CNV induction, and anesthetized mice were first transdermally perfused with 0.9% saline and then transdermally perfused with 4% paraformaldehyde. Fixed tissues were embedded in paraffin, serially sectioned at 3mm thickness, and stained with Hematoxylin and Eosin (HE). Sequential sections through each CNV sample were examined and CNV thickness was measured vertically from adjacent RPE layers to the top of the CNV region using an optical microscope. The length of the CNV area (in μm) was measured using Image-Pro Plus (IPP) 6.0 software and recorded as the maximum horizontal distance of the CNV.

8. Fluorescent Angiography (FFA)

Conjunctival FFA was performed using confocal scanning laser ophthalmoscopes. Pupils were separated after regular anesthesia of the mice. After injection of 2% sodium fluorescein (0.15 ml), a timer was started. Recording was started after 2 minutes and the later phase was observed 6-8 minutes after injection. At least three ophthalmologists evaluated the results in a double blind manner. All fluorescence penetration intensities were scored using software as follows: 0, no leakage, 1, slight leakage, 2, moderate leakage, 3, significant leakage. The Mann-Whitney U test was used to analyze scoring results.

9. OCT detection

Using "TruTrack TM active eye tracking" and "Automatic Real Time (ART)" techniques, and successive images are from the same lesion; the thickness of the bump in the CNV RPE layer was quantified using software provided by 4D-ISOCT (OptoProbe Research, canada). Four to six spots in each eye were selected for analysis. CNV is defined as the spindle-shaped subretinal hyperreflective region above the RPE layer. The thickness of each mouse CNV lesion was selected to be assessed by "geometric" lesion center scan analysis.

10. Statistical analysis

All statistical analyses used SPSS 17.0 statistical software. Experimental data are normally distributed, expressed as mean ± SD, and comparisons between the two groups are made with t-test. One-way analysis of variance is used to compare the mean between multiple sample groups. The Student-Newmann-Keuls (SNK) test was used for any pairwise comparison between groups. The Mann-Whitney U test was used for FFA detection. p <0.05 is considered statistically significant.

2. Experimental results

1. SDF-1 induced BMC cells were recruited into laser photocoagulation-induced murine CNV lesions

Endothelial Cells (ECs) play a key role in CNV. To determine whether BMCs-derived ECs are involved in neovascularization and its underlying mechanisms, we used GFP mice that were successfully bone marrow transplanted and performed laser photocoagulation-induced mouse CNV models. SDF-1 is involved in CNV pathogenesis and plays an important role as a typical chemokine. Thus, we propose that SDF-1 may promote rapid recruitment of BMCs to the mouse ocular CNV region. First, we found that SDF-1 mRNA levels were significantly elevated on day 3 after laser CNV in mice, and this increase was maintained until day 7 after laser injury. However, on day 3 of laser-induced mouse CNV SDF-1 mRNA levels peaked, while SDF-1 protein levels gradually increased to day 7 (FIG. 15A). HE staining showed that in the CNV animal model of GFP-BMCs transplantation, the choroid thickness at CNV increased significantly, whereas the choroid thickness in CNV region increased significantly after intravitreal injection of SDF-1 (fig. 15B). We also observed chemotaxis of GFP-BMCs by in vivo fluorescence imaging, and found that more green fluorescence-labeled BMCs accumulated in the CNV lesions of the mice than in mice without CNV lesions, suggesting that more BMCs were promoted to the choroidal lesions following SDF-1 (fig. 15C). Furthermore, CNV tissues were strongly labeled with GFP, which increased significantly after stimulation with SDF-1, compared to normal choroidal tissues, indicating that GFP-labeled BMC and BMC-derived cells migrate and aggregate in the CNV region (FIG. 15D).

2. SDF-1/CXCR4 signaling aids BMC recruitment and differentiation of CNV regions

Co-expression localization of CXCR4 and CD31/BMCs of each group 7d after CNV induction, CXCR4 is red fluorescence, BMCs are green fluorescence, and CD31 is blue fluorescence. CXCR4 is highly expressed locally in CNV, and the expression level of SDF-1 group is obviously higher than that of the pure CNV group and SDF-1+AMD3100 group (figure 16). NS indicates that there is no statistical significance for the differences between groups, #p <0.05, and that there is statistical significance for the differences.

Specific: 7d after laser photocoagulation, laser confocal showed the presence of BMCs under the retina at the visible laser lesions after three different treatment groups. CNV was locally blue fluorescent stained with VEC marker (CD 31), suggesting: there is neovascularization in the lesion area of CNV. There is a large number of red fluorescent expressed CXCR4 localized on CD31/BMCs, suggesting that a large number of CXCR4 are expressed on CD31 and BMCs. The expression of CXCR4 chemotactic for the SDF-1 group to CNV was significantly increased (Con: 76.88 + -33.26 vs SDF-1: 155.88 + -50.79, P < 0.05) and the ratio of CXCR4 number/CD 31 number/BMCs number was significantly increased (Con: 0.17+ -0.06 vs SDF-1: 0.34+ -0.10, P < 0.05) compared to the CNV alone group, suggesting that SDF-1 promotes differentiation of BMCs into VECs to express CXCR4 and the proportion occupied was increased. The number of CXCR4 chemotactic to CNV was significantly reduced in the SDF-1+AMD3100 group compared to the SDF-1 group (SDF-1+AMD3100:111.38.+ -. 27.53, P < 0.05) and the CXCR4 number/CD 31 number/BMCs number ratio was significantly reduced (SDF-1+AMD3100:0.26.+ -. 0.10, P < 0.05), suggesting: functional inhibitors of CXCR4 can inhibit BMCs from differentiating into VECs expressing CXCR4. There was no statistical difference in the ratio of CXCR4 number/CD 31 number/BMCs number between the SDF-1+amd3100 group chemotactic to CNV compared to the CNV alone, P >0.05. Prompting: SDF-1 promotes the production of more CXCR4, VEC and BMCs involved in CNV, and CXCR4 expressed on VEC and BMCs is increased under the mediation of SDF-1.

3. Severity of CNV in SDF-1/CXCR4 aggravated mice

Based on previous effects on SDF-1/CXCR4 inducing more BMCs recruitment to the mouse CNV region and involved in its differentiation, we next assessed the effect of SDF-1/CXCR4 on CNV disease severity, using HE staining, optical Coherence Tomography (OCT), FFA, choroidal flatmount, HE to observe the effect of different treatment conditions on mouse CNV. Fig. 17A shows that the normal mice, the vitreous cavity-injected SDF-1 group after CNV and the vitreous cavity-injected SDF-1+amd3100 group after CNV were OCT-observed, the RPE layer ridge height of CNV was highest on day 7 after CNV, significantly higher than 3d and 14d after CNV, and the RPE layer ridge height of CNV was significantly higher than that of the simple CNV group and SDF-1+amd3100 group. Compared with the SDF-1 group, the elevation of the RPE layer of the CNV of the SDF-1+AMD3100 group is obviously reduced; FIG. 17B is a graph showing leakage of three sets of FFA evaluation CNV for different treatments; fig. 17C is the area and volume of CNV observed for three sets of choroidal flatmount of different treatments; * P <0.01, P <0.05, the difference is statistically significant. FIG. 17D shows three HE staining groups treated differently to observe CNV length, at day 7 post-CNV induction, intravitreal injection of SDF-1 enhanced CNV length (347.25 + -15.30 μm vs 244.45 + -14.03 μm) with P <0.05, the difference being statistically significant. Thus, the inhibitor AMD3100 of CXCR4 can effectively reduce the progression and severity of CNV.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

SEQUENCE LISTING

<110> Chinese people liberation army allied oneself with guard army ninth four-good hospital

<120> miRNA for treating CNV, preparation method and application thereof

<130> 2010

<160> 16

<170> PatentIn version 3.3

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Claims

The application of miRNA in preparing a medicine for treating CNV is characterized in that the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1.
2. Use of a lentiviral expression vector comprising the miRNA of claim 1 in the preparation of a medicament for treating CNV.