CN116656674A

CN116656674A - Antisense nucleotide of targeting circRNA and application of antisense nucleotide in resisting pulmonary arterial hypertension

Info

Publication number: CN116656674A
Application number: CN202210146579.7A
Authority: CN
Inventors: 孙岚; 张天泰; 张子睿; 吕婷婷; 周禹; 李尚�; 李尚为
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2022-02-17
Filing date: 2022-02-17
Publication date: 2023-08-29

Abstract

The application belongs to the technical field of medical biology, and discloses a targeting circRNA antisense nucleotide hsa-circRNA-026272-siRNA, and a pharmaceutically acceptable carrier or a viral carrier and auxiliary materials thereof; for but not limited to low oxygen environments, chronic thromboembolism, drug induction, and pulmonary hypertension secondary to congenital heart disease. The application discovers that hsa-circRNA-026272-siRNA can obviously inhibit proliferation and migration of pulmonary artery smooth muscle cells and improve cardiac and pulmonary hemodynamic changes and cardiac and pulmonary vascular remodeling induced by monocrotaline. The application provides a safe, effective and economic solution for treating and preventing pulmonary arterial hypertension and complications thereof.

Description

Antisense nucleotide of targeting circRNA and application of antisense nucleotide in resisting pulmonary arterial hypertension

Technical Field

The application belongs to the technical field of biological medicines, relates to a novel nucleic acid medicine targeting endogenous circRNA and application thereof, and in particular relates to application of an siRNA nucleotide pharmaceutical composition containing targeting hsa-circRNA-0026272 in preventing or treating pulmonary hypertension and complications thereof.

Background

Pulmonary arterial hypertension (Pulmonary artery hypertension, PAH) is known as "cancer of the cardiovascular system", a rare disease that is difficult to diagnose and treat. The conservation of PAH patients in China is estimated to be about 500 ten thousand to 800 ten thousand, the average age of onset is only 36 years, the median survival period is only 2.8 years, and the survival quality is extremely poor in the survival period with diseases. Although new drugs are continuously introduced in recent years, the survival rate of PAH patients in 5 years is still only 57%, and how to further prolong the service life of the patients and improve the survival quality of the patients is a great challenge.

At present, the pathogenesis of pulmonary arterial hypertension is not clearly concluded, and vascular occlusion caused by primary hyperplasia of pulmonary arterioles is probably the main cause of the pathogenesis. Pulmonary revascularization is a cellular phenotypic transformation of pulmonary artery smooth muscle cells (Pulmonary artery smooth muscle cells, PASMCs) to accommodate changes in the internal environment, primarily manifested by smooth muscle cell contractile dysfunction, loss of phenotype-associated proteins, migration and proliferation of cells under the intimal wall across damaged endothelial cell barriers, exhibiting "cancer or neoplastic growth", causing intimal thickening in the intima, neointimal formation and progressive occlusion of the lumen of the blood vessel, increased vascular resistance and hemodynamic changes, ultimately leading to right and even full heart failure. Because of the complex course of PAH and low early diagnosis rate, severe and irreversible pulmonary vascular remodeling has occurred in most patients diagnosed with PAH. At present, the pathogenesis of pulmonary hypertension is not completely clear, and the prevention, diagnosis and treatment of pulmonary hypertension cannot achieve satisfactory effects, so that the development of a novel technical method for effectively inhibiting pulmonary arterial hypertension of pulmonary vascular remodeling is an important and urgent task.

siRNA is a small RNA molecule (about 21-25 nucleotides) that is a powerful and powerful biological tool for silencing genes, and can induce silencing of sequence-specific target genes, rapidly blocking gene activity. In 2001, researchers find that the synthesized siRNA can induce RNAi effect in mammals, inhibit the activity of target genes, and has great application value. Gene knockdown by transfection of exogenous siRNA is generally unsatisfactory because the effect is only transient, particularly in rapidly dividing cells. This can be overcome by expression vectors that produce siRNA. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is a short hairpin RNA (shRNA) that can be processed into functional siRNA by Dicer in its usual manner. Recent studies have found that circular RNA (circRNA) also plays an important role in organisms. CircRNA is a specific class of non-coding RNA molecules, usually produced by specific selective cleavage, present in large amounts in the cytoplasm of eukaryotic cells. The circRNA molecule is in a closed loop structure, can be combined with miRNA endogenous to cells, and plays a role of miRNA sponge (miRNA spike) in the cells, so that the expression of miRNA is regulated to perform corresponding functions.

Research shows that the circRNA regulates the expression of key molecules for reconstructing various pulmonary arterial hypertension blood vessels, and is a pulmonary arterial hypertension therapeutic molecule and target with great development prospect. Revealing the function of the circRNA in the pulmonary artery high pressure vascular reconstruction process, searching the molecule of the specific expression of the pulmonary artery high pressure, clarifying the action mechanism, and having great significance and broad prospects for developing the application of the siRNA targeting the circRNA in the aspects of diagnosing and treating the pulmonary artery high pressure.

However, no report is available on research on hsa_circRNA_0026272, and no report is available on the role of antisense nucleotide shRNA of targeting the molecule and a pharmaceutical composition thereof in pulmonary arterial hypertension treatment.

Disclosure of Invention

The technical problem solved by the application is to overcome the defects existing in the prior art, and develop a novel pharmaceutical composition for preventing and treating pulmonary arterial hypertension according to hsa_circRNA_0026272siRNA capable of regulating and controlling pulmonary arterial smooth muscle cell abnormal proliferation and migration and application of hsa_circRNA_0026272siRNA in pulmonary arterial pressure elevation induced by Monocrotoline (MCT), pulmonary arterial vascular remodeling, right heart failure, right heart remodeling, myocardial hypertrophy, myocardial fibrosis and other pulmonary arterial hypertension complications.

For this purpose, the application provides the following technical scheme:

the application provides an application of hsa-circRNA-0026272-siRNA in preparing an anti-pulmonary hypertension drug.

A circRNA antisense nucleotide for use in the treatment of pulmonary hypertension disease, wherein the circRNA is derived from the hsa_circrna_0026272 gene.

Further, the hsa_circRNA_0026272 is homologous to rat Rno_circRNA_017665, and the sequence is shown in SEQ ID NO 1-3.

Further, the hsa_circRNA_0026272siRNA comprises one or more mixed nucleotides as shown in SEQ ID NO 4-6.

Further, the hsa_circRNA_0026272 targeting formulation composition includes, including, an infection titer of 10 ⁶ -10 ¹⁶ PFU over-expresses hsa_circrna_0026272siRNA, a virus or a combination of adjuvants. The virus vector is one or more of adenovirus vector, slow virus vector and retrovirus vector. The carrier is cholesterol, nano particles or liposome; the viral vector is one or more of adenovirus vector, slow virus vector and retrovirus vector; the auxiliary materials are one or more of mannitol, phosphate buffer solution and physiological saline; the medicine is one or more of oral preparation, injection preparation, tablet and dry powder, preferably small water injection or freeze-dried powder injection.

Furthermore, the pharmaceutical composition is prepared into various preparations of anti-pulmonary arterial hypertension medicines according to a conventional preparation method.

Still further, the pulmonary hypertension includes, but is not limited to, low oxygen environments, chronic thromboembolism, drug induction, and pulmonary hypertension caused by secondary congenital heart disease.

The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.

All combinations of items to which the term "and/or" is attached "in this description shall be taken to mean that the respective combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

The "circRNA" is a special non-coding RNA molecule with a closed annular structure, is not influenced by RNA exonuclease, is more stable in expression and is not easy to degrade.

The siRNA is a small RNA molecule (about 21-25 nucleotides), is a very powerful biological tool for silencing genes, and can induce the silencing of sequence-specific target genes and rapidly block the gene activity.

The shRNA, namely short hairpin RNA, (a small hairpin RNA or short hairpin RNA, shRNA) is an RNA sequence with a tight hairpin loop (tight hairpin turn), and is commonly used for RNA interference silencing target gene expression. That is, the siRNA sequences SEQ ID4-6 were cloned as "short hairpin" into a plasmid vector. When delivered to an animal, the hairpin sequence is expressed to form a "double stranded RNA" (dsRNA).

In the application, the abbreviations and the full names are compared with each other: circRNA: circular RNA, circular RNA.

ncRNA: non-coding RNA, non-coding RNA.

qRT-PCR: quantitative real time polymerase chain reaction, real-time quantitative PCR.

MCT: monocrototaline, monocrototaline.

RNase R: riboclearer, a 3'-5' ribonuclease derived from the RNR superfamily of e.

HE staining: hematoxylin-eosin staining.

PA-SMC, pulmonary artery smooth muscle cells;

PFU: plaque forming units (plaque forming unit), abbreviated pfu. Under standard conditions, 1 plaque forming unit corresponds generally to 1 viral particle. Is used to describe the number of viruses that have infectious capacity.

MOI: multiplicity of infection, the ratio of viral and cellular numbers.

The beneficial effects of the application include, but are not limited to: the application adopts rat pulmonary artery smooth muscle cells (PA-SMC) to examine the effect of hsa-circRNA-0026272-siRNA in inhibiting PA-SMC proliferation and migration, and the result shows that the hsa-circRNA-0026272-siRNA 3 sequences of the over-expression recombinant adenovirus comprises the following steps: SEQ ID NO4, SEQ ID NO5, SEQ ID NO6 can significantly inhibit TGF-beta induced cell growth and migration viability. The application adopts a monocrotaline-induced rat pulmonary artery high-pressure model to examine the effects of hsa-circRNA-0026272-siRNA over-expression recombinant adenovirus seed sequence on improving pulmonary blood flow, reducing pulmonary artery pressure, improving right heart failure, right heart fibrosis and myocardial hypertrophy, and the results show that the tail vein injection step and other specified components or steps can not be substantially influenced.

ncRNA: non-coding RNA, non-coding RNA.

MCT: monocrototaline, monocrototaline.

HE staining: hematoxylin-eosin staining.

PA-SMC, pulmonary artery smooth muscle cells;

MOI: multiplicity of infection, the ratio of viral and cellular numbers.

The beneficial effects of the application include, but are not limited to: the application adopts rat pulmonary artery smooth muscle cells (PA-SMC) to examine the effect of hsa_circRNA_0026272siRNA in inhibiting PA-SMC proliferation and migration, and the result shows that the hsa_circRNA_00262728 siRNA 3 sequences of the over-expression recombinant adenovirus comprises the following steps: SEQ ID NO4, SEQ ID NO5, SEQ ID NO6 can significantly inhibit TGF-beta induced cell growth and migration viability. The application adopts a monocrotaline induction rat pulmonary hypertension model, examines the effects of improving pulmonary blood flow, reducing pulmonary artery pressure, improving right heart failure, right heart fibrosis and myocardial hypertrophy of hsa_circRNA_0026272siRNA over-expression recombinant adenovirus seed sequence, and shows that tail vein injection hsa_circRNA_0026272siRNA obviously inhibits pulmonary artery pressure and right ventricular pressure rise caused by MCT induction, can obviously improve and reduce right heart index, reduce pulmonary vascular lumen stenosis and reduce right heart failure marker NT-proBNP plasma level, thereby providing a safe, effective and economic solution for pulmonary hypertension and complications prevention and treatment.

Drawings

FIG. 1 shows a map of the interference vector pADV U6 shRNA CMV EGFP vector selected for the experiment.

FIG. 2 effect of hsa_circRNA_00262728 siRNA on proliferation rate of rat pulmonary smooth muscle cells (PA-SMCs). Mean±sd, n=4.

Figure 3. Hsa_circrna_00262728 sirna affects mean±sd, n=4 on rat pulmonary smooth muscle cell migration activity.

FIG. 4. Hsa_circRNA_00262728 siRNA improves the mean systolic pressure (mPAP) increase in rat pulmonary arteries induced by monocrotaline. Mean±sd, n=10

Figure 5 hsa_circrna_00262728 sirna ameliorates the rise in Right Ventricular Systolic Pressure (RVSP) in rats induced by monocrotaline. Mean±sd, n=10

FIG. 6. Hsa_circRNA_00262728 siRNA improves cardiac organ coefficients in rats induced by monocrotaline. Mean±sd, n=10.

FIG. 7 shows that hsa_circRNA_00262728 siRNA reduces the increase in right heart index in rats induced by monocrotaline. Mean±sd, n=5.

FIG. 8 hsa_circRNA_00262728 siRNA inhibits monocrotaline induced stenosis of pulmonary and pulmonary arteriole lumens in rats. Mean±sd, n=10.

Fig. 9. Hsa_circrna_00262728 sirna inhibited monocrotaline-induced right heart failure in rats. Mean±sd, n=10.

Detailed Description

The present application will be described in further detail with reference to the accompanying drawings, in order to make the objects, technical solutions, and advantages of the present application more apparent.

hsa_circ_0026272 (circBase number: hsa_circ_0026272, chromosomal position: chr12: 51108177-51112818) was aligned with the Blast (https:// Blast. Ncbi. Lm. Nih. Gov/Blast. Cgi) sequence and homologous to transcript XM_039080375.1 of the rat gene, dip2 b. The specific embodiment of the application adopts the in-vivo and in-vitro research of pulmonary arterial hypertension rats, and based on the in-vivo and in-vitro research, shRNA interference fragments are designed aiming at rat homologous sequences. The sequence of the fragment is shown as SEQ ID NO 7-10. The interference fragment is constructed into an adenovirus vector by a molecular biological means, the vector can realize the interference of hsa_circ_0026272 genes, besides the direct transient transfection of cells for the interference of hsa_circ_0026272 genes, and the vector can further pack adenovirus to interfere the expression of hsa_circ_0026272 genes at animal level.

1. Principle of experiment

RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA double stranded RNA (dsRNA) induced, highly efficient and specific degradation of homologous mRNA during evolution. Since the expression of a specific gene can be specifically reduced or shut down using RNAi technology, the technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. shRNA is a method for RNA interference, which is to transcribe short interfering RNA (RNA double strand of siRNA 19-21 nucleotides) DNA molecules by using RNA polymerase pol III promoters such as U6 and H1 of human and mouse sources. Intracellular transcribed shRNA is processed and incorporated into RNA-induced silencing complexes (RNA inducedsilencing complex RISC), directing nucleases to degrade target RNA. The shRNA expression vector has the advantage that it can be studied for a longer period of time, and the vector with antibiotic markers can continuously inhibit the expression of the target gene in cells for several weeks or even longer. The viral vector can also be used for shRNA expression, and has the advantages of being capable of directly and efficiently infecting cells for gene silencing research, avoiding various inconveniences caused by low plasmid transfection efficiency, and ensuring more stable transfection effect. The map of the selected interference vector pADV U6 shRNA CMV EGFP vector is shown in figure 1, and the shRNA sequence to be constructed is inserted.

siRNA targets were designed based on transcripts of the rat DiP2b gene, and primer synthesis was arranged. Annealing the single-stranded primer into a double-stranded oligo sequence, and connecting the double-stranded primer into a double-enzyme tangential RNA interference vector to replace the original ccdB toxicity gene. Transformants were screened for colony PCR and the screened positive clones were sequenced. Sequencing confirmed the correct clone and high purity plasmid extraction was performed. The experiment is divided into 8 main steps:

1. interference target design and primer synthesis:

and designing siRNA targets according to the general principle of shRNA design.

Viral vector construction frameworks are shown in Table 1

TABLE 1 viral vector sequences

2. Primer annealing to form a cohesive end double-stranded fragment:

the synthesized oligo was dissolved in 20. Mu.M by oligo annealing buffer, and 30. Mu.l of each complementary single strand was mixed. And heating the oligo mixture in a water bath at 95 ℃ for 5min, opening the water bath, standing at room temperature, and naturally cooling to room temperature to form double-chain oligo fragments. Mu.l was taken for subsequent ligation reactions and the rest was stored at 20 ℃.

Primer fragments are as in table 2:

TABLE 2 primer sequences

3. Preparation of linearized expression vector:

the restriction enzyme is used for carrying out enzyme digestion on the expression vector, and the enzyme digestion reaction system is as follows: plasmid 2. Mu.g 10 Xreaction Buffer 5. Mu.l, restriction enzymes 1. Mu.l each, deionized water make up 50. Mu.l, and incubated in a 37℃water bath for more than 2 h. And (3) detecting the enzyme digestion effect by agarose gel electrophoresis of the enzyme digestion product, cutting the target carrier band from the gel after agarose gel electrophoresis, and recycling the gel by using TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver.3.0.

4. The interference fragment is connected into an expression vector:

the ligation was carried out overnight at 16 ℃. The annealed double-stranded oligo added to the positive control is a previously annealed fragment for verification, and has the same length as the annealed double-stranded oligo added to the ligation set, but is independent of the target sequence.

5. Transformation of competent cells:

transformation of DH 5. Alpha. Competent cells is described in detail in the guidelines for experiments in well-defined molecular biology.

6. Colony PCR identification of positive transformants:

transformants grown on plates were picked up and resuspended in 10. Mu.l LB medium, and 1. Mu.l template was used for colony PCR identification. The reaction system and PCR cycle conditions were as follows:

PCR reaction conditions

PCR reaction solution composition

7. Sequencing positive clones:

and (5) identifying the obtained positive clone by colony, and sending the positive clone to a sequencing company for sequencing verification.

8. Plasmid small extracts:

and (3) verifying correct positive clones through sequencing, and carrying out plasmid miniprep.

2. Experimental procedure

Example 1 effect of hsa_circ_00262728 sirna on proliferation activity of rat pulmonary artery smooth muscle cells (PA-SMCs).

The method comprises the following steps:

SD rat pulmonary artery smooth muscle cells (PA-SMC) were primary cultured and cell growth was observed, i.e., passaged by digestion with 0.25% trypsin, and the passaged medium was DMEM medium containing 10% FBS. Observing the state of the cells by a microscope, culturing until the 3 rd to 8 th generation, observing the state of the cells by a microscope, and selecting the cells with good growth state for experiments.

TGF-beta cytokine-induced cell proliferation was detected using CCK-8 kit (Cell Counting Kit-8). Taking 5000 PASMCs in logarithmic growth phase, planting the PASMCs in 96-well plates, when cells grow to 80% and fuse at night, infecting recombinant adenoviruses with different sequences of has_circ_0026272siRNA according to the dosage of final concentration 10MOI, wherein the corresponding sequences are SEQ ID NO4, SEQ ID NO5 and SEQ ID NO6, incubating the recombinant adenoviruses in an incubator for 24 hours, adding TGF-beta to each group of cells except a control group and a blank group to the final concentration of 5ng/mL, adding CCK-8 to measure the absorbance of OD450 after 48 hours, and obtaining the cell proliferation rate= (test group OD 450-blank OD 450)/(control group OD 450-blank group OD 450).

The results are shown in FIG. 2 and Table 3, where hsa-circRNA-026272-siRNA significantly inhibited PASMCs cell proliferation (FIG. 2).

TABLE 3 influence of hsa_circ_00262728 siRNA recombinant adenovirus on proliferation Rate of rat pulmonary artery smooth muscle cells

Example 2 influence of hsa_circ_00262728 siRNA on rat pulmonary artery smooth muscle cell migration Activity

The method comprises the following steps:

cell migration viability was measured using a scratch Assay (work-sealing Assay). Taking PA-SMC in logarithmic phase 10 ⁵ The recombinant adenovirus which is infected with hsa_circ_0026272siRNA with different sequences is planted in 24 hole plates at a density of 500 mu L of cell suspension in each hole, and when 80% of cells are fused, the recombinant adenovirus is infected with hsa_circ_0026272siRNA with different sequences as SEQ ID NO4, SEQ ID NO5 and SEQ ID NO6 (corresponding to the groups of SEQ ID NO4, SEQ ID NO5 and SEQ ID NO 6) according to the dosage of 10MOI of the final concentration. The cells were scored with a 200 μl gun head perpendicular to the well plate, ensuring as much as possible consistent individual scratch widths. The cell culture solution was aspirated, and the well plate was washed three times with physiological saline to wash away cell debris generated by the scratches. Except for the control group and the blank group, TGF-beta was added to the cells of each group to a final concentration of 5ng/mL, and culture was continued. After 24 hours, the recordings were photographed under an inverted microscope and migrated with each groupThe ratio of the area of (c) to the migration area of the control group was used to evaluate the migration capacity of cells:

results: as shown in fig. 3 and table 4, the different sequences hsa_circ_0026272siRNA recombinant adenovirus significantly inhibited smooth muscle cell migration capacity, manifested as a decrease in cell fusion area.

TABLE 4 influence of hsa_circ_00262728 siRNA recombinant adenovirus on rat pulmonary artery smooth muscle cell mobility

Note that: the data in the table are the migration areas (expressed as pixel) before and after 24 hours of each group of cells scratch, i.e., cell migration area = cell fusion area (pixel) 24 hours after scratch) -cell fusion area (pixel) at (0 h) scratch.

Example 3. Establishment of rat pulmonary artery high pressure model.

The method comprises the following steps:

pulmonary hypertension is caused by a variety of causes, and pulmonary vascular remodeling is the primary pathological cause. The Monocrotaline (MCT) is injected once, and the pulmonary arterial hypertension model is formed by progressively increasing the pulmonary dynamic pressure caused by changes such as delayed progressive vascular endothelial cell injury of the pulmonary artery of the rat, platelet thrombosis of the pulmonary artery, abnormal proliferation of smooth muscle cells and the like. 3 days before model establishment, the tail vein was given a different sequence of has-circRNA-026272-siRNA recombinant adenovirus, followed by intraperitoneal injection of MCT 60mg/Kg body weight. In the embodiment, the systolic pressures of the pulmonary artery and the right ventricle of the rat are measured, the thickening degree of the right ventricle, the serum pulmonary artery high pressure marker and the histomorphology change are measured, and the protection effect of the Bexarotene on the pulmonary artery high pressure of the rat and the application of the Bexarotene in preparation of the medicine for preventing and treating the pulmonary artery high pressure are judged.

Example 50 SD rats were selected, weighing about 200g, and randomly divided into 5 groups of 10 animals each: normal Control (Control), pulmonary arterial high pressure Model Control (Model), hsa-circ_0026272siRNA SEQ ID NO4 recombinant adenovirus (SEQ ID NO 4), hsa-circ_0026272siRNA SEQ ID NO5 recombinant adenovirus (SEQ ID NO 5) and hsa-circ_0026272siRNA SEQ ID NO6 recombinant adenovirus (SEQ ID NO 6). Preparing the monocrotaline into a 1% concentration solution by using physiological saline, uniformly mixing the solution under ultrasonic, and regulating the pH value to 7.2-7.4; normal control group was given an equal dose of physiological saline and other rats were given a single intraperitoneal injection of 60mg/kg MCT to prepare a rat pulmonary arterial hypertension model. Right heart catheter manometry and aortic catheterization were performed on day 28 of molding, and right ventricular systolic pressure, pulmonary artery systolic pressure, and aortic systolic pressure were measured in rats; the rats were perfused with physiological saline, and pulmonary arteries and lung tissues were taken for formaldehyde fixation, paraffin embedding, serial sectioning, HE staining and pathological observation.

The rats were anesthetized with pentobarbital sodium solution (30 mg/kg) and fixed on an operating table, and surrounding tissues were blunt-separated near the collarbone, and the right external jugular vein was separated about 1cm long, and 1 line was buried. A v-shaped incision is made at the proximal end by using Alice scissors, a bent PE catheter pre-filled with heparin physiological saline is inserted into a vein, and the catheter is slightly fixed with the blood vessel by using a suture line to reduce bleeding. The catheter is slowly advanced, inserted down and slowly advanced. Insertion 1-2cm into the superior vena cava, 2-3cm into the right atrium, 3-4cm into the right ventricle, and 4-6cm into the pulmonary artery. And judging the position reached by the catheter according to the pressure value and the pressure waveform shown by the monitor.

Results: as shown in fig. 4 and 5, the right ventricular pressure (RVSP) and pulmonary arterial pressure (mPAP) of the rat after the molding of the monocrotaline were significantly increased, and the different sequences hsa_circ_0026272siRNA recombinant adenovirus (set of SEQ id no4, SEQ id no5, and SEQ id no 6) significantly reduced the right ventricular mean systolic pressure and the pulmonary mean systolic pressure of the rat with pulmonary arterial hypertension.

Example 4: hsa_circ_0026272siRNA effect method for improving right heart hypertrophy index of pulmonary arterial hypertension rat:

after measuring the pressure, the whole body is irrigated by normal saline after blood is taken out, the chest is dissected to take out the rat heart, and atrial tissues are sheared off. The whole heart was weighed, heart organ coefficients (heart weight/rat weight×100) were calculated, and the right ventricle free wall, left ventricle and ventricular septum (Left Ventricular plus Septum, lv+s) were cut out. The water on the upper surface is sucked by filter paper and weighed respectively, the right ventricular hypertrophy index [ RV/(LV+S) ] is calculated, and the right ventricular hypertrophy degree is judged.

Results: as shown in figures 6 and 7, the hsa_circ_0026272siRNA recombinant adenovirus comprises SEQ ID NO4, SEQ ID NO5 and SEQ ID NO6, and can obviously improve cardiac hypertrophy and right ventricular hypertrophy caused by pulmonary arterial hypertension.

Example 5: effect of hsa_circ_0026272siRNA recombinant adenovirus on pulmonary small vessel reconstruction

The method comprises the following steps:

after normal saline systemic perfusion, pulmonary artery and left lung were fixed in 4% neutral paraformaldehyde solution, paraffin embedded, serial sections and HE staining. Five fields are randomly selected for each sample, the wall thickness of the pulmonary artery blood vessel is detected, and the stenosis of the pulmonary artery is observed. 5 pulmonary arteriole vessels with diameters of 100+/-50 mu m are randomly selected, and the proportion WA% = (TA-IA)/TA of the cross section area of the vessel wall to the area of the vessel cavity is counted by adopting IPP 6.0 (Image-Pro-Plus 6.0) Image processing software and is used for evaluating the reconstruction condition of the pulmonary arteriole vessels.

The results showed (FIG. 8) that the hsa_circ_0026272siRNASEQ ID NO4, SEQ ID NO5, SEQ ID NO6 groups improved pulmonary small vessel remodeling to some extent, as evidenced by inhibition of MCT-induced pulmonary small vessel lumen stenosis (percent decrease in lumen area) and rat pulmonary small vessel lumen area ratio approaching that of the normal group rats.

Example 6: hsa_circ_0026272siRNA reduces NT-proBNP content in serum of pulmonary arterial hypertension rat

The method comprises the following steps:

serum was obtained as in example 4, and the content of NT-proBNP in the serum was measured by ELISA. NT-proBNP means: the amino terminal brain natriuretic peptide B precursor (N terminal pro B type natriuretic peptide, NT-proBNP) is currently the most important cardiac function biomarker. Compared with proBNP, NT-proBNP is more stable, easy to track and simple and efficient to use, so that NT-proBNP has become a more sensitive and widely used index for representing pulmonary arterial hypertension.

The present example uses ELISA to detect NT-proBNP in serum of rats with pulmonary hypertension. The concentration of this substance in serum was calculated by measuring the absorbance (OD value) at a wavelength of 450nm using an enzyme-labeled instrument.

The results show (figure 9) that the content of NT-proBNP in the serum of the pulmonary artery high pressure model animal is obviously increased, and the hsa_circ_0026272siRNASEQ ID NO4, SEQ ID NO5 and SEQ ID NO6 groups obviously reduce the release of NT-proBNP, thus proving that the hsa_circ_0026272siRNA has the prevention and treatment effect on the pulmonary artery high pressure concurrent right heart failure.

In conclusion, hsa_circ_0026272siRNA recombinant adenovirus SEQ ID NO4, SEQ ID NO5 and SEQ ID NO6 have the effects of preventing, relieving and/or treating pulmonary arterial hypertension; the pharmacological action mechanism is to inhibit pulmonary vascular remodeling. The application provides an ideal novel nucleotide for treating pulmonary arterial hypertension and complications thereof, which can be applied to preparing products for preventing, relieving and/or treating various etiologies and primary pulmonary arterial hypertension.

Finally, it is noted that the above embodiments are merely for illustrating the technical solution of the present application and not for limiting the same, and that although the present application has been described by reference to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes in implementation and details may be made therein without departing from the spirit and scope of the present application as defined by the appended description.

Sequence listing

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Claims

1. An antisense nucleotide targeted to a circRNA, wherein the circRNA is derived from the hsa_circrna_0026272 gene.

2. The hsa_circrna_0026272 gene sequence according to claim 1 comprising SEQ ID NO: 1. SEQ ID NO:2 and/or SEQ ID NO:3, or comprises a sequence identical to SEQ ID NO: 1. SEQ ID NO:2 and/or SEQ ID NO:3 having a nucleotide sequence having a homology of 80% or more.

3. An antisense nucleotide, wherein the antisense nucleotide is an siRNA targeting a circular RNA, i.e., hsa_circrna_0026272 siRNA; preferably, the hsa_circrna_0026272siRNA sequence comprises SEQ ID NO: 4. SEQ ID NO:5 and/or SEQ ID NO:6, or comprises a sequence identical to SEQ ID NO: 4. SEQ ID NO:5 and/or SEQ ID NO:6 having a nucleotide sequence having a homology of 80% or more.

4. A pharmaceutical composition comprising as an active ingredient the antisense nucleotide of any one of claims 1-3 and/or a carrier and an adjuvant thereof.

5. An antisense nucleotide pharmaceutical composition for treating pulmonary hypertension, characterized in that the pharmaceutical composition comprises an infectious titer of 10 ⁶ -10 ¹⁶ Vector, virus or auxiliary material of PFU over-expression hsa_circRNA_0026272 siRNA; the carrier is one or more of chitosan, cholesterol, nano particles and liposome; the viral vector is one or more of adenovirus vector, slow virus vector and retrovirus vector; the auxiliary materials are one or more of mannitol, phosphate buffer solution and physiological saline.

6. The antisense nucleotide pharmaceutical composition for treating pulmonary hypertension according to claim 4, wherein hsa_circrna_0026272siRNA further comprises a pharmaceutically acceptable carrier; preferably, the vector is one or more of an adenovirus vector, a chronic virus vector, an adeno-associated virus, a retrovirus vector; more preferably, the vector is an adenovirus vector.

7. The antisense nucleotide pharmaceutical composition for treating pulmonary artery disease according to claim 4, wherein the hsa_circrna_0026272siRNA recombinant adenovirus further comprises pharmaceutically acceptable excipients; preferably, the auxiliary materials are one or more of mannitol, phosphate buffer solution and physiological saline; preferably, the auxiliary material is phosphate buffer.

8. The antisense nucleotide pharmaceutical composition for treating pulmonary artery disease according to claim 4, wherein the dosage form of the pharmaceutical composition is one or more of oral preparation, injection preparation, tablet, and dry powder dosage form.

9. An antisense nucleotide pharmaceutical composition for treating pulmonary arterial hypertension, which is characterized in that the antisense nucleotide pharmaceutical composition is orally taken or injected for preventing and treating pulmonary arterial hypertension, wherein the pulmonary arterial hypertension is pulmonary arterial hypertension caused by hypoxia environment, chronic thromboembolism, drug induction and congenital heart disease secondary.

10. Use of an antisense nucleotide according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention or treatment of pulmonary hypertension.