Pharmaceutical use of RP11-544D21.2, use in preparing anti-heart failure drugs, drugs and preparation methods

Abstract

The present invention "Pharmaceutical use of RP11‑544D21.2, use in the preparation of anti-heart failure drugs, drugs and preparation methods" belongs to the field of biomedicine. The present invention provides the pharmaceutical use of RP11‑544D21.2, and the use of RP11‑544D21.2 in the preparation of anti-heart failure drugs or the preparation of heart failure animal models. The present invention further provides a new anti-heart failure drug targeting RP11‑544D21.2. Based on RP11‑544D21.2, the present invention also provides a screening method and a preparation method for anti-heart failure drugs, as well as a method for preparing a heart failure animal model. The experiments of the present invention confirm that knocking down RP11‑544D21.2 can inhibit the proliferation of fibroblasts and alleviate angiotensin II-induced differentiation into myofibroblasts. Animal experiments suggest that it is expected to improve cardiac function and alleviate cardiac remodeling.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the pharmaceutical use of RP11-544D21.2, its use in preparing anti-heart failure drugs, and the drugs and preparation methods. Background Art

[0002] Chronic heart failure is a clinical syndrome in which different cardiovascular diseases develop to the terminal stage. Its main pathophysiological characteristics are impaired ventricular filling and / or ejection capacity, which ultimately leads to poor ventricular pumping function. It has a poor prognosis and high mortality rate. It is one of the main causes that threaten human health and increase the medical burden. Due to the aging population, the incidence of cardiovascular disease-related risk factors such as hypertension, hyperlipidemia, and diabetes remains high, and the prevalence of heart failure is also increasing year by year. It is reported that the 5-year mortality rate of heart failure is as high as 50%, which also brings a heavy medical burden to the economy and society. Therefore, there is an urgent need to find new and more effective therapeutic targets for heart failure.

[0003] Long non-coding RNA is a type of RNA with a length of more than 200 nucleotides and no coding ability. In the past, long non-coding RNA was considered to be the dark matter of genome transcription. In recent years, more and more studies have shown that long non-coding RNA can play an important regulatory role in growth and development as well as the occurrence and development of diseases. Unlike microRNA, which has classic pathways and modes of action, the mechanism of action of long non-coding RNA is very complex and diverse. It can interact with proteins, RNA and DNA molecules, and can play a regulatory role in the entire process of gene expression, including chromatin remodeling, transcriptional activation or inhibition, variable splicing, post-translational modification, etc. At present, the role of long non-coding RNA in heart failure, especially cardiac fibrosis, is still unclear.

[0004] RP11-544D21.2 is a reported long non-coding RNA, which was disclosed in the article "Identification of cardiac long non-coding RNA profile in human dilated cardiomyopathy". However, the pharmaceutical use of RP11-544D21.2 has not been reported in the existing technology in this field, especially its pharmaceutical use for treating heart failure. Summary of the invention

[0005] In view of the above-mentioned gaps in the prior art in the art, the present invention provides the use of RP11-544D21.2 in preparing anti-heart failure drugs, and the drugs and preparation methods thereof.

[0006] The technical solution of the present invention is as follows:

[0007] Pharmaceutical uses of RP11-544D21.2.

[0008] Use of RP11-544D21.2 in preparing anti-heart failure drugs or preparing heart failure animal models.

[0009] Fighting heart failure by inhibiting, silencing, knocking down, knocking out, or down-regulating the expression of RP11-544D21.2, and / or binding to or degrading RP11-544D21.2;

[0010] Preferably, the anti-heart failure means: inhibiting the proliferation or differentiation of fibroblasts, and / or increasing the cardiac ejection fraction, and / or improving the cardiac contractile function, and / or improving the cardiac diastolic function.

[0011] An anti-heart failure drug, characterized in that its drug targets include: RP11-544D21.2.

[0012] The pharmacologically active ingredients of the anti-heart failure drug include: substances that inhibit or silence or knock down or knock out or down-regulate the expression of RP11-544D21.2, and / or substances that bind to or degrade RP11-544D21.2;

[0013] Preferably, the substance that inhibits or silences or knocks down or knocks out or downregulates the expression of RP11-544D21.2, or the substance that binds to or degrades RP11-544D21.2 comprises: an antisense complementary fragment of RP11-544D21.2;

[0014] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0015] Preferably, the antisense complementary fragment of RP11-544D21.2 is selected from the group consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, and GapmeR-FIRL;

[0016] The target sequence of ASO1 is shown in SEQ ID NO.2;

[0017] The target sequence of ASO2 is shown in SEQ ID NO.3;

[0018] The target sequence of ASO3 is shown in SEQ ID NO.4;

[0019] The target sequence of the siRNA4 is shown in SEQ ID NO.5;

[0020] The target sequence of the siRNA5 is shown in SEQ ID NO.6;

[0021] The target sequence of the siRNA6 is shown in SEQ ID NO.7;

[0022] The sequence of the GapmeR-FIRL is shown in SEQ ID NO.8;

[0023] More preferably, the antisense complementary fragment of RP11-544D21.2 is selected from: a mixture consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, or GapmeR-FIRL;

[0024] GapmeR-FIRL can be particularly used to alleviate the symptoms of heart failure in mice or to prepare anti-heart failure drugs in mice.

[0025] Preferably, the medicament further comprises pharmaceutically acceptable excipients, and / or reagents for buffering, synthesizing, and / or purifying the antisense complementary fragment of RP11-544D21.2.

[0026] A method for screening anti-heart failure drugs, characterized in that substances that can bind to or degrade RP11-544D21.2 and / or substances that can inhibit, silence, knock down, knock out or down-regulate the expression of RP11-544D21.2 are screened from candidate substances.

[0027] A method for preparing an anti-heart failure drug, characterized in that it includes: using a substance that binds to or degrades RP11-544D21.2, and / or a substance that inhibits, silences, knocks down, knocks out, or down-regulates the expression of RP11-544D21.2 as the pharmacologically active ingredient of the anti-heart failure drug.

[0028] Preferably, the substance that binds to or degrades RP11-544D21.2, or, the substance that inhibits or silences or knocks down or knocks out or down-regulates the expression of RP11-544D21.2 comprises: an antisense complementary fragment of RP11-544D21.2;

[0029] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0030] Preferably, the antisense complementary fragment of RP11-544D21.2 is selected from one or more of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, and GapmeR-FIRL;

[0031] The target sequence of ASO1 is shown in SEQ ID NO.2;

[0032] The target sequence of ASO2 is shown in SEQ ID NO.3;

[0033] The target sequence of ASO3 is shown in SEQ ID NO.4;

[0034] The target sequence of the siRNA4 is shown in SEQ ID NO.5;

[0035] The target sequence of the siRNA5 is shown in SEQ ID NO.6;

[0036] The target sequence of the siRNA6 is shown in SEQ ID NO.7;

[0037] The sequence of the GapmeR-FIRL is shown in SEQ ID NO.8;

[0038] More preferably, the antisense complementary fragment of RP11-544D21.2 is selected from: a mixture consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, or GapmeR-FIRL;

[0039] Preferably, pharmaceutically acceptable excipients are added to the anti-heart failure drug, and / or reagents for buffering, synthesizing, and / or purifying the antisense complementary fragment of RP11-544D21.2.

[0040] A method for establishing an animal model of heart failure, characterized by overexpressing RP11-544D21.2 in the animal.

[0041] The overexpression of RP11-544D21.2 refers to: infecting animals with a recombinant adeno-associated virus expression vector connected with the RP11-544D21.2 sequence;

[0042] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0043] Preferably, the animal is selected from the group consisting of mice, rabbits and monkeys.

[0044] Under the premise that the patent laws of some countries and regions permit, the present invention also requests protection for the use of RP11-544D21.2 in preventing and treating heart failure.

[0045] One object of the present invention is to provide a drug for treating heart failure, wherein the active ingredient of the drug includes RP11-544D21.2 as a drug target, and the drug has the effect of treating heart failure by binding to, degrading, and / or downregulating the expression of the RP11-544D21.2.

[0046] Binding: that is, the expression of RP11-544D21.2 can be reduced by binding a certain substance to it, such as GapmeR, ASO, smartsilence, siRNA, microRNA, etc.

[0047] Degradation: that is, RP11-544D21.2 can be induced to degrade by nucleases through certain substances, such as GapmeR, ASO, smartsilence, siRNA, microRNA, etc., which can bind to the single strand of RP11-544D21.2 in a base pairing manner to form a double-stranded structure, which is easily recognized and degraded by nucleases;

[0048] Down-regulation: that is, the production of RP11-544D21.2 is reduced by a certain substance;

[0049] Any of these three methods can play a role in reducing the expression level of RP11-544D21.2. Any behavior of packaging the above substances into a product package marked with anti-heart failure use for commercial purposes on any scale falls within the scope of protection claimed by the present invention.

[0050] Furthermore, the pharmacological ingredients of the drug include substances that downregulate the expression of RP11-544D21.2.

[0051] Furthermore, the drug also includes pharmaceutically acceptable excipients and / or reagents for buffering, synthesizing, and / or purifying the antisense complementary sequence fragment RP11-544D21.2-antisense nucleic acid. Those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-heart failure drug of the present invention according to objective needs to prepare various dosage forms for easy sales or promotion.

[0052] Another object of the present invention is to provide a method for screening drugs for treating heart failure, by detecting whether the candidate substance can bind to, degrade, and / or downregulate the expression of RP11-544D21.2; thereby, screening out substances that can inhibit the expression of RP11-544D21.2.

[0053] The third object of the present invention is to provide a method for preparing a drug for treating heart failure, comprising: using a substance that can bind to, degrade, and / or down-regulate the expression of the RP11-544D21.2 as an active ingredient of the drug for treating heart failure.

[0054] The present invention found that RP11-544D21.2 is localized in the nucleus of cardiac fibroblasts and is upregulated in heart failure, and developed a drug for treating heart failure with RP11-544D21.2 as the target. Subsequently, cell experiments confirmed that downregulating RP11-544D21.2 can inhibit the proliferation and differentiation of fibroblasts, and knocking down the homologous gene GM26861 in mice found that inhibiting the expression of GM26861 can inhibit cardiac fibrosis and improve cardiac function, suggesting that treatment targeting RP11-544D21.2 can improve heart failure.

[0055] The present invention has conducted an in-depth study on the molecular mechanism of RP11-544D21.2 and named it FIRL according to its function. According to the sequence of RP11-544D21.2 (FIRL), the applicant designed RP11-544D21.2 smart silencer to interfere with the expression of RP11-544D21.2 in cells (si-FIRL). The RP11-544D21.2 smart silencer is selected from one or more of 3 siRNAs and / or 3 ASOs, preferably 3 siRNAs and 3 ASOs. The sequences of the 3 siRNAs and 3 ASOs are shown in SEQ ID NO.2-7. In addition, the applicant packaged adenovirus to overexpress RP11-544D21.2 in cells (Ad-FIRL). The results of the study show that interfering with the expression of RP11-544D21.2 (si-FIRL) can inhibit the proliferation of fibroblasts and reduce the expression of differentiation markers (ACAT2, POSTN, FN1) induced by angiotensin II. Overexpression of RP11-544D21.2 (Ad-FIRL) can promote the proliferation of fibroblasts and further increase the expression of angiotensin II-induced differentiation markers (ACAT2, POSTN, FN1). According to the location of RP11-544D21.2 (FIRL) in the human genome, the present invention found the homologous gene GM26861 in the mouse genome, and knocked down the expression of GM26861 through GapmeR (gapmeR-FIRL, the specific sequence is SEQ ID NO.3). The results showed that inhibiting the expression of FIRL can alleviate TAC-induced cardiac function damage, inhibit cardiac fibrosis, and improve myocardial remodeling.

[0056] The beneficial effects of the present invention are: the present invention proposes for the first time the pharmaceutical use of RP11-544D21.2, especially its use in the preparation of anti-heart failure drugs, and based on the pharmaceutical use, proposes a new anti-heart failure drug with RP11-544D21.2 as a drug target. The active ingredients of the drug for treating heart failure of the present invention include substances that use RP11-544D21.2 as a drug target and bind, degrade, and / or downregulate the expression of the RP11-544D21.2; the present invention can significantly inhibit the proliferation and differentiation of cardiac fibroblasts by using RP11-544D21.2 as a drug target and downregulating the expression of the RP11-544D21.2 molecule, potentially alleviating myocardial remodeling and improving cardiac function. As a new tool for gene expression regulation, antisense nucleic acid is a new type of targeted molecular therapeutic drug. Different from traditional therapeutic drugs, it has the advantages of being easy to synthesize, easy to detect, accurate in quantification, and able to increase its stability and affinity after chemical modification, and can be effectively located to the target organ through a special drug delivery system. The present invention uses RP11-544D21.2 as a therapeutic target, and can inhibit the proliferation of fibroblasts and relieve angiotensin II-induced differentiation into myofibroblasts through transfection of antisense nucleic acids. Animal experiments suggest that it is expected to improve cardiac function and relieve cardiac remodeling. The successful development of this drug will open up a new situation for the treatment of heart failure and provide a reference for the development of therapeutic drugs for other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0058] Figure 1 Expression and subcellular localization of RP11-544D21.2 (FIRL) in cardiac tissues of patients with heart failure and controls. A, qPCR detection of RP11-544D21.2 (FIRL) expression in cardiac tissues of patients with heart failure and controls; B, fluorescence in situ hybridization detection of RP11-544D21.2 (FIRL) expression and subcellular localization in cardiac tissue sections of patients with heart failure and controls. FIRL was co-stained with fibroblasts (Col1a1), cardiomyocytes (cTNT), endothelial cells (CD31), and immune cells (CD45), respectively. The scale bar is 20 μm.

[0059] Figure 2Effects of RP11-544D21.2 (FIRL) on human cardiac fibroblasts (HCF). AB, EdU detection of HCF proliferation after knocking down the expression of RP11-544D21.2 (FIRL) in HCF (si-FIRL), representative staining figures (A) and statistical figures (B); CD, EdU detection of HCF proliferation after overexpressing RP11-544D21.2 (FIRL) in HCF (Ad-FIRL), representative staining figures (C) and statistical figures (D); EF, under basal conditions or under the condition of angiotensin II (AngII, 1 μM) intervention, the expression of RP11-544D21.2 (FIRL) in HCF was knocked down (si-FIRL) (E) or RP11-544D21.2 (FIRL) was overexpressed in HCF (Ad-FIRL), and the expression of fibroblast differentiation-related markers (ACTA2, POSTN, FN1) was detected by qPCR, and the scale bar is 100 μm.

[0060] Figure 3 Results of cardiac function tests after knocking down the RP11-544D21.2 homologous gene GM26861 in mice. A, Ultrasound results showed that knocking down the expression of FIRL (gapmeR-FIRL) can improve the cardiac ejection fraction (Ejection Fraction) and improve cardiac function damage; B, Heart Miller catheter hemodynamics test found that knocking down the expression of FIRL (gapmeR-FIRL) can improve the systolic and diastolic function of the heart. DETAILED DESCRIPTION

[0061] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Reagents and consumables

[0063] The sources of the instruments, equipment, reagents, consumables and biological materials involved in Experimental Examples 1 and 2 of the present invention are as follows:

[0064] 1) Instruments and Equipment

[0065] ND-1000 nucleic acid analyzer, ABI 9700PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge, VisualSonics Vevo2100 small animal ultrasound machine

[0066] 2) Reagents and consumables

[0067] GapmeR (purchased from Qiagen), TRIZOL (purchased from Invitrogen), and EdU detection kit (purchased from Guangzhou Ruibo Company);

[0068] Sources of biological materials

[0069] The heart failure patient tissues in Experimental Example 1 of the present invention were from heart transplant patients in Wuhan Union Hospital, and the control samples were from the Red Cross Society, and both signed informed consent forms;

[0070] The HCF (primary human cardiac fibroblasts) cells used in Experimental Example 2 were from ScienCell;

[0071] The C57BL / 6 mice used in Experimental Example 3 were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0072] Group 1 Example: Pharmaceutical Use of RP11-544D21.2

[0073] This group of embodiments provides pharmaceutical uses of RP11-544D21.2.

[0074] The invention proposes for the first time that RP11-544D21.2 can be used for pharmaceutical preparation. Any use of RP11-544D21.2 for pharmaceutical preparation or as a drug component, or any use of synthesizing, expressing, inhibiting, silencing, reducing, knocking down, or knocking out RP11-544D21.2 for treatment falls within the protection scope of the invention.

[0075] The second group of examples, use of RP11-544D21.2 in the preparation of anti-heart failure drugs or heart failure animal models

[0076] This group of embodiments provides the use of RP11-544D21.2 in preparing anti-heart failure drugs or preparing heart failure animal models.

[0077] Any use of RP11-544D21.2 for the preparation of anti-heart failure drugs or as a component of anti-heart failure drugs, or any synthesis, expression, inhibition, silencing, reduction, knockdown, or knockout of RP11-544D21.2 for the treatment of heart failure falls within the scope of protection of the present invention.

[0078] Any use of RP11-544D21.2 to prepare an animal model of heart failure, or any synthesis, expression, inhibition, silencing, reduction, knockdown, or knockout of RP11-544D21.2 for constructing a vector and / or transforming and / or infecting / infecting an animal falls within the scope of protection of the present invention.

[0079] In some embodiments, the heart failure is prevented by inhibiting or silencing or knocking down or knocking out or down-regulating the expression of RP11-544D21.2, and / or binding to or degrading RP11-544D21.2;

[0080] Preferably, the anti-heart failure means: inhibiting the proliferation or differentiation of fibroblasts, and / or increasing the cardiac ejection fraction, and / or improving the cardiac contractile function, and / or improving the cardiac diastolic function.

[0081] The third group of embodiments, the anti-heart failure drug of the present invention

[0082] This group of embodiments provides an anti-heart failure drug. All embodiments in this group have the following common features: the target of the anti-heart failure drug includes: RP11-544D21.2.

[0083] In a specific embodiment, the pharmacologically active ingredients of the anti-heart failure drug include: substances that inhibit or silence or knock down or knock out or down-regulate the expression of RP11-544D21.2, and / or substances that bind to or degrade RP11-544D21.2;

[0084] Preferably, the substance that inhibits or silences or knocks down or knocks out or downregulates the expression of RP11-544D21.2, or the substance that binds to or degrades RP11-544D21.2 comprises: an antisense complementary fragment of RP11-544D21.2;

[0085] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0086] Preferably, the antisense complementary fragment of RP11-544D21.2 is selected from the group consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, and GapmeR-FIRL;

[0087] The target sequence of ASO1 is shown in SEQ ID NO.2;

[0088] The target sequence of ASO2 is shown in SEQ ID NO.3;

[0089] The target sequence of ASO3 is shown in SEQ ID NO.4;

[0090] The target sequence of the siRNA4 is shown in SEQ ID NO.5;

[0091] The target sequence of the siRNA5 is shown in SEQ ID NO.6;

[0092] The target sequence of the siRNA6 is shown in SEQ ID NO.7;

[0093] The target sequence of ASO1 refers to a DNA sequence that is antisense and complementary to the sequence of ASO1 itself. The sequence of ASO1 itself is a single-stranded hybrid structure with 10 DNA bases in the middle and 5 RNA bases modified at both ends.

[0094] The target sequence of ASO2 refers to a DNA sequence that is antisense and complementary to the sequence of ASO2 itself. The sequence of ASO2 itself is a single-stranded hybrid structure with 10 DNA bases in the middle and 5 RNA bases modified at both ends.

[0095] The target sequence of ASO3 refers to a DNA sequence that is antisense and complementary to the sequence of ASO3 itself. The sequence of ASO3 itself is a single-stranded hybrid structure with 10 DNA bases in the middle and 5 RNA bases modified at both ends.

[0096] The target sequence of the siRNA4 refers to a DNA sequence that is antisense and complementary to the sequence of the siRNA4 itself.

[0097] The target sequence of siRNA5 refers to a DNA sequence that is antisense and complementary to the sequence of siRNA5 itself.

[0098] The target sequence of siRNA6 refers to a DNA sequence that is antisense and complementary to the sequence of siRNA6 itself.

[0099] The sequence of the GapmeR-FIRL is shown in SEQ ID NO.8;

[0100] ASO is the abbreviation of antisense oligonucleotides, which means antisense oligonucleotides and has a conventional technical meaning that can be understood by technicians in the field of molecular biology. ASO is a single-stranded DNA that knocks down FIRL through the RNase H pathway after forming a hybrid double strand with the target RNA, and has a better knockdown effect on FIRL in the cell nucleus.

[0101] siRNA is a double-stranded RNA with conventional technical meanings that can be understood by technicians in the field of molecular biology. After siRNA is complementary to the target RNA, it forms a RISC complex to degrade FIRL, mainly knocking down FIRL in the cytoplasm; because the abundance of lncRNA (non-coding RNA) is generally low, it is also difficult to knock down, and it is often distributed in the nucleus. Smartsilencer is one of the means to knock down lncRNA. In theory, the use of one or two of the above-mentioned ASOs or siRNAs alone can achieve a knockdown effect, and the method of mixing multiple nucleotides (i.e., smartsilencer) has a better effect as shown in the following experimental examples of the present invention.

[0102] GapmeR, the full name of which is Antisense LNAGapmeRs, is a type of antisense oligonucleotide that can effectively inhibit the functions of mRNA and lncRNA, and has the conventional technical meaning commonly understood by technicians in the field of molecular biology.

[0103] More preferably, the antisense complementary fragment of RP11-544D21.2 is selected from: a mixture consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, or GapmeR-FIRL.

[0104] Preferably, the medicament further comprises pharmaceutically acceptable excipients, and / or reagents for buffering, synthesizing, and / or purifying the antisense complementary fragment of RP11-544D21.2.

[0105] In a specific embodiment, the active ingredient of the drug for treating heart failure provided uses RP11-544D21.2 as the drug target, and the drug effect of treating heart failure is achieved by knocking down or knocking out RP11-544D21.2. The present invention has found through experiments that the content of RP11-544D21.2 in the heart tissue of patients with heart failure is significantly increased ( Figure 1 ), which suggests that RP11-544D21.2 may play a regulatory role in the pathophysiological process of heart failure, and further experiments have shown that knocking down the expression of RP11-544D21.2 in primary human cardiac fibroblasts can inhibit the proliferation of fibroblasts and inhibit the differentiation of fibroblasts into myofibroblasts.

[0106] Furthermore, the active ingredients of the drug include substances that can reduce RP11-544D21.2. It is well known to those skilled in the art that the degradation of RP11-544D21.2 can be promoted by molecular biological means such as small interfering RNA-siRNA, locked nucleic acid modified RNA-GapmeR, antisense nucleic acid ASO, etc. for long non-coding RNA.

[0107] Preferably, the active ingredient of the drug contains the targeted RP11-544D21.2 sequence fragment. The present invention found that RP11-544D21.2 is located in the cell nucleus and it is difficult to effectively knock down by siRNA alone, so smart silencing is designed for the sequence of RP11-544D21.2, which contains 3 ASOs and 3 si-RNAs, and the specific sequence is shown in SEQ ID NO.2.

[0108] Furthermore, the drug also includes pharmaceutically acceptable excipients, and / or reagents for buffering, culturing, and / or propagating the knockdown of RP11-544D21.2 expression; those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-heart failure drug of the present invention according to objective needs to prepare various dosage forms for easy sales or promotion.

[0109] Group 4 Example: Screening method for anti-heart failure drugs of the present invention

[0110] This group of embodiments provides a method for screening anti-heart failure drugs. All embodiments in this group have the following common features: screening substances that can bind to or degrade RP11-544D21.2 from candidate substances, and / or substances that can inhibit, silence, knock down, knock out or down-regulate the expression of RP11-544D21.2.

[0111] The fifth group of embodiments, the preparation method of the anti-heart failure drug of the present invention

[0112] This group of embodiments provides a method for preparing an anti-heart failure drug. All embodiments in this group have the following common features: the method for preparing an anti-heart failure drug comprises: using a substance that binds to or degrades RP11-544D21.2, and / or a substance that inhibits or silences or knocks down or knocks out or downregulates the expression of RP11-544D21.2 as the pharmacologically active ingredient of the anti-heart failure drug.

[0113] In some embodiments, the substance that binds to or degrades RP11-544D21.2, or, the substance that inhibits or silences or knocks down or knocks out or down-regulates the expression of RP11-544D21.2 comprises: an antisense complementary fragment of RP11-544D21.2;

[0114] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0115] Preferably, the antisense complementary fragment of RP11-544D21.2 is selected from one or more of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, and GapmeR-FIRL;

[0116] The sequence of ASO1 is shown in SEQ ID NO.2;

[0117] The sequence of ASO2 is shown in SEQ ID NO.3;

[0118] The sequence of ASO3 is shown in SEQ ID NO.4;

[0119] The sequence of the siRNA4 is shown in SEQ ID NO.5;

[0120] The sequence of the siRNA5 is shown in SEQ ID NO.6;

[0121] The sequence of the siRNA6 is shown in SEQ ID NO.7;

[0122] The sequence of the GapmeR-FIRL is shown in SEQ ID NO.8;

[0123] More preferably, the antisense complementary fragment of RP11-544D21.2 is selected from: a mixture consisting of ASO1, ASO2, ASO3, siRNA4, siRNA5, siRNA6, or GapmeR-FIRL;

[0124] Preferably, pharmaceutically acceptable excipients are added to the anti-heart failure drug, and / or reagents for buffering, synthesizing, and / or purifying the antisense complementary fragment of RP11-544D21.2.

[0125] Group 6 Example: Method for establishing a heart failure animal model of the present invention

[0126] This group of embodiments provides a method for establishing a heart failure animal model. This group of embodiments has the following common features: overexpressing RP11-544D21.2 in the animal body.

[0127] In a specific embodiment, the overexpression of RP11-544D21.2 refers to: infecting an animal with a recombinant adeno-associated virus expression vector connected with the RP11-544D21.2 sequence;

[0128] Preferably, the sequence of RP11-544D21.2 is shown in SEQ ID NO.1;

[0129] Preferably, the animal is selected from the group consisting of mice, rabbits and monkeys.

[0130] The expression of long non-coding RNA is tissue and cell specific, so it has a higher drugability for RP11-544D21.2. It can not only widely knock down the expression of RP11-544D21.2 in the body, but also use targeted drug delivery to specifically knock down the expression of RP11-544D21.2 in the heart. The specially modified antisense nucleic acid has a long half-life, good stability, high target specificity, few side effects, high patient compliance, convenient production and preparation, low cost and fast cycle, and can achieve long-term inhibition of RP11-544D21.2 after a single injection. In addition, the present invention found that the expression of RP11-544D21.2 was significantly increased in patients with heart failure and animal models of heart failure, such as Figure 1As shown, therefore, any use of detecting RP11-544D21.2 to evaluate the establishment of a heart failure model or to screen drugs for the treatment of heart failure falls within the scope of protection of this patent.

[0131] Experimental Example 1. Expression and subcellular localization of RP11-544D21.2 (FIRL) in cardiac tissues of patients with heart failure and controls (1) qPCR detection of RP11-544D21.2 (FIRL) expression

[0132] Experimental methods: Myocardial tissues were collected from 6 healthy adult males and 10 adult male patients with end-stage heart failure. Total RNA was extracted by TRIZOL method and then reverse transcribed by qPCR to detect the expression level of RP11-544D21.2 (FIRL). The primers used were:

[0133] H-FIRL-F:5'-GCACTCCCAGACCAAGAGAT-3'; (SEQ ID NO.9)

[0134] H-FIRL-R:5'-ACGTAAGTCATGCTTGTGGGT-3'; (SEQ ID NO.10)

[0135] H-GAPDH-F:5'-AGAAGGCTGGGGCTCATTTG-3'; (SEQ ID NO.11)

[0136] H-GAPDH-R: 5'-AGGGGCCATCCACAGTCTTC-3'. (SEQ ID NO. 12).

[0137] Experimental results: Figure 1 -A shows that the content of RP11-544D21.2 in heart tissue of patients with heart failure was significantly upregulated.

[0138] (2) Fluorescence in situ hybridization staining of human heart tissue sections RP11-544D21.2 (FIRL)

[0139] Experimental methods:

[0140] ① Routine dewaxing, rehydration and antigen retrieval of sections;

[0141] ②RNA hybridization:

[0142] a. Add pre-hybridization buffer to cover the tissue and hybridize in a hybridization oven at 55°C for 30 minutes;

[0143] b. Dilute the probe (100uM) with hybridization solution 1:2000, and then hybridize in a hybridization oven at 55°C for 1 hour (note that the hybridization solution should be preheated in advance and the sections should be placed in a humidified box to prevent drying);

[0144] c. Wash with detergent I at 60°C for 3 times, 5 minutes each time;

[0145] d. Wash once with detergent II at 60°C for 5 minutes each time;

[0146] e. Wash once with detergent III at 60°C for 5 minutes each time;

[0147] f. Wash with PBS once, 5 minutes;

[0148] g. 3% H2O2 treatment for 30 minutes to block endogenous peroxidase;

[0149] h. Wash with TN buffer 3 times, 5 minutes each time;

[0150] i. Block with TNB buffer for 30 minutes, then add HRP-labeled digoxigenin primary antibody diluted 1:250 with blocking buffer and incubate at room temperature for 30 minutes;

[0151] j. Wash with TNT buffer 3 times, 5 minutes each time, and wash with PBS once, 5 minutes;

[0152] k. TSA was diluted 1:500 with 0.01% TBST and incubated for 10 minutes in the dark. Note that all subsequent operations in this step should be performed in the dark.

[0153] l. Wash with TNT buffer three times, 5 minutes each time, and wash with PBS once, 5 minutes.

[0154] ③ Detection of protein and cell nucleus staining: After blocking with 5% serum, add primary antibody and incubate overnight at 4°C. After washing with PBST for 3 times, incubate with corresponding secondary antibody at room temperature for 45 minutes. After washing with PBST for 3 times, stain the nucleus for microscopic examination.

[0155] Experimental results: Figure 1 -B shows that the Col1a1-positive staining area is fibroblasts, the cTNT-positive staining area is cardiomyocytes, the CD31-positive staining area is endothelial cells, and the CD45-positive staining area is immune cells. It can be seen from the figure that RP11-544D21.2 is mainly located in the nucleus of cardiac fibroblasts, and a small amount is distributed in other cell types. In heart failure, RP11-544D21.2 in the nucleus of fibroblasts increases significantly, and there is no significant change in other cell types.

[0156] Experimental Example 2: Effects of RP11-544D21.2 (FIRL) on the proliferation and differentiation of cardiac fibroblasts (1) EdU detection of the proliferation ability of cardiac fibroblasts

[0157] Experimental method: Based on the sequence of RP11-544D21.2 (FIRL), an antisense nucleic acid smart silencer (containing 3 siRNAs and 3 ASOs) was designed and transfected into fibroblasts to knock down the expression of RP11-544D21.2 (FIRL). The full-length sequence of RP11-544D21.2 (FIRL) was packaged into an adenovirus expression vector (pADM-CMV-C-FH-mCMV-copGFP) and infected fibroblasts to achieve gene overexpression.

[0158] The infection of fibroblasts is a commonly used technical means in the field of molecular biology. The specific method is: the full-length sequence of RP11-544D21.2 (FIRL) is cloned into an adenovirus expression vector, then packaged into adenovirus in 293 cells, and finally the fibroblasts are infected with adenovirus to achieve overexpression of FIRL.

[0159] The kit was purchased from Guangzhou Ruibo, and the steps are as follows:

[0160] ①Discard the culture medium in the culture plate, pat it dry on a thick stack of toilet paper, and then wash it twice with PBS, 5 minutes each time. Be careful not to repeatedly rinse the cells directly during the operation to avoid washing away the cells. Various buffers should be added slowly along the side wall;

[0161] ② Add 50ul of formaldehyde fixative to each well in a fume hood and treat for 20 minutes;

[0162] ③ Aspirate the formaldehyde fixative, add 50ul 2mg / ml glycine solution to each well to neutralize the formaldehyde, and incubate on a shaker at room temperature for 6 minutes;

[0163] ④ Discard the glycine solution, add 100ul PBS to each well, wash on a horizontal shaker for 5 minutes, repeat the wash once, add 100ul PBST (0.5% TritonX-100 in PBS) to each well for 10 minutes, and then wash with PBS for 5 minutes;

[0164] ⑤ After discarding PBS, add 80ul 1XApollo staining reaction solution to each well, incubate at room temperature for 40 minutes in the dark, then discard the staining reaction solution and wash twice with PBS;

[0165] ⑥ Dilute Hoechst 33342 to 1X with PBS, incubate on a shaker at room temperature for 20 minutes, then wash twice with PBS, 3 minutes each time, and observe under a fluorescence microscope.

[0166] Experimental results: Figure 2As shown in AB, the proliferation ability of HCF was weakened after knocking down the expression of RP11-544D21.2(FIRL) in HCF (si-FIRL); CD, overexpression of RP11-544D21.2(FIRL) (Ad-FIRL) in HCF can promote the proliferation of fibroblasts.

[0167] (2) qPCR detection of cardiac fibroblast differentiation markers

[0168] Experimental methods: HCFs were transfected with si-FIRL or infected with Ad-FIRL and their controls, and then treated with PBS or angiotensin II (1uM) for 24 hours. The expression of differentiation-related markers was detected by qPCR. The primer sequences are as follows:

[0169] H-POSTN-F 5'-TCCCCGTGACTGTCTATAAGC-3'(SEQ ID NO.13)

[0170] H-POSTN-R 5'-ACCTTGGTGACCTCTTCTTGT-3'(SEQ ID NO.14)

[0171] H-ACTA2-F 5'-GTGTTGCCCCTGAAGAGCAT-3'(SEQ ID NO.15)

[0172] H-ACTA2-R 5'-GCTGGGGACATTGAAAGTCTCA-3'(SEQ ID NO.16)

[0173] H-FN1-F 5'-ACAAGCATGTCTCTCTGCCA-3'(SEQ ID NO.17)

[0174] H-FN1-R 5'-TCAGGAAACTCCCAGGGTGA-3' (SEQ ID NO. 18).

[0175] Experimental results: Figure 2As shown in CD, under basal conditions, knocking down the expression of RP11-544D21.2(FIRL) in HCF (si-FIRL) or overexpressing RP11-544D21.2(FIRL) in HCF (Ad-FIRL) had no effect on the expression of fibroblast differentiation-related markers (ACTA2, POSTN, FN1). Under the condition of angiotensin II (AngII) intervention, knocking down the expression of RP11-544D21.2(FIRL) in HCF (si-FIRL) could inhibit the differentiation of fibroblasts, on the contrary, overexpressing RP11-544D21.2(FIRL) (Ad-FIRL) could promote the differentiation of fibroblasts.

[0176] Experimental Example 3: Knockdown of FIRL in mice alleviates TAC-induced cardiac function damage

[0177] Experimental method: Based on the location of RP11-544D21.2 on the chromosome, the applicant found the mouse homologous gene GM26861 and designed GapmeR-FIRL: 5'-ACTTGATGATAGGACA-3' (SEQ ID NO.8) based on its sequence.

[0178] 1. C57BL / 6 mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and kept in the SPF (special pathogen-free) animal room of the Experimental Animal Research Center of Huazhong University of Science and Technology. Newly purchased mice were adaptively raised for one week before intervention.

[0179] 2. Transverse aortic constriction (TAC): 8-week-old male C57BL / 6 mice of uniform weight were selected for modeling. After the mice were successfully anesthetized with 1.5% pentobarbital (50 mg / kg), they were fixed on the operating table in a supine position. Turn on the ventilator and connect the endotracheal tube. Insert the tube into the glottis under direct vision. The respiratory rate is about 80 times / minute. It can be seen that the chest has obvious fluctuations with the ventilator frequency, indicating that the endotracheal intubation is successful. Use a blade to shave the hair on the chest and disinfect the skin with iodine. Cut the skin in the precardiac area, bluntly separate the fascia and muscles to expose the ribs, and shorten the second rib. After fully exposing the field of vision with a spreader, the white thymus covering the aortic arch can be seen. Gently separate the thymus to expose the aortic arch, and place a 27G blunt needle along the direction of the aortic arch. Use 7-0 non-absorbable silk thread to ligate the aortic arch between the brachiocephalic trunk and the left common carotid artery. Gently withdraw the 27G needle. The muscles and skin were sutured layer by layer, the endotracheal tube was removed, and the mice were placed on an electric blanket until they woke up.

[0180] 3. Drug treatment: The mice were randomly divided into the following three groups: sham, TAC+gapmeR-control, and TAC+gapmeR-FIRL; the mice in the sham group only underwent thoracotomy without ligation; the mice in the TAC+gapmeR-control group were not injected with gapmeR, and the mice in the TAC+gapmeR-FIRL group were injected with gapmeR intraperitoneally. GapmeR was designed and synthesized by Qiagen and dissolved in sterile saline. The mice were injected with gapmeR intraperitoneally at a dose of 20 mg / kg. GapmeR was injected 3 days before TAC surgery.

[0181] 4. Ultrasound and hemodynamic detection: VisualSonics Vevo 2100 small animal ultrasound machine was used for mouse cardiac ultrasound, and a 30MHz high-frequency probe was selected as the probe. The specific steps are as follows: After the animal was anesthetized by ether, it was fixed on the operating table, the chest hair was removed, and a coupling agent was applied. Isoflurane was used for maintenance anesthesia to control the heart rate at 500-600 beats per minute. The probe was placed on the left side of the sternum, and the mode was selected as B-mode ultrasound to produce a clear image of the long axis of the ventricle. Switch to M-mode ultrasound and record the ventricular wall motion at the root of the papillary muscle. The probe orientation was changed, and the transverse section of the heart was recorded by B-mode ultrasound, and the ventricular wall motion was recorded and analyzed by M-mode ultrasound. The probe was placed on the right side of the mouse neck, and the aortic ligation site was found in the B-mode ultrasound mode, and the blood flow at the ligation site was analyzed using the Doppler mode. Catheter detection: The mouse was anesthetized with 1% pentobarbital and fixed on the operating table in the supine position. The neck skin was cut along the anterior midline, and the anterior neck muscles were bluntly separated from the middle to both sides to expose the trachea. Separate the surrounding muscle fascia along the carotid artery, and use micro forceps to separate the accompanying vagus nerve. Tie a knot at the distal end of the carotid artery for traction fixation, and use an artery clamp to clamp blood flow at the proximal end of the carotid artery. Under a microscope, use micro scissors to cut a small hole at the distal end of the artery clamp, and carefully insert the heparin-treated Miller catheter along the rupture. After inserting to the artery clamp, loosen the artery clamp and continue to insert the catheter. When the pressure value shows a minimum value of 0mmHg, it has entered the left ventricle, and the intraventricular pressure is monitored at this time.

[0182] Experimental results: Figure 3 -As shown in A, ultrasound results show that knocking down the expression of FIRL (gapmeR-FIRL) can increase the cardiac ejection fraction (Ejection Fraction) and improve cardiac function damage; Figure 3 -B shows that cardiac Miller catheter hemodynamic detection found that knocking down the expression of FIRL (gapmeR-FIRL) can improve the contractile and diastolic functions of the heart.

Claims

1. Use of GapmeR-FIRL shown in SEQ ID NO.8 in the preparation of anti-heart failure drugs.

2. Use of the GapmeR-FIRL shown in SEQ ID NO.8 according to claim 1 in preparing an anti-heart failure drug, characterized in that: The anti-heart failure method refers to: inhibiting the proliferation or differentiation of fibroblasts, and / or increasing the cardiac ejection fraction, and / or improving the cardiac contractile function, and / or improving the cardiac diastolic function.

3. An anti-heart failure drug, characterized in that: The pharmacologically active ingredients include: GapmeR-FIRL as shown in SEQ ID NO.

8.

4. An anti-heart failure drug according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. A method for preparing an anti-heart failure drug, characterized in that: include: GapmeR-FIRL with a sequence as shown in SEQ ID NO.8 is used as the pharmacologically active ingredient of the anti-heart failure drug.

6. The method for preparing an anti-heart failure drug according to claim 5, characterized in that: Pharmaceutically acceptable excipients are added to the anti-heart failure drug.

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