CN116115762A

CN116115762A - SH3 BGR-based method for predicting risk of suffering from Down syndrome and application thereof

Info

Publication number: CN116115762A
Application number: CN202310081723.8A
Authority: CN
Inventors: 时伟丽; 杨帆; 张玉薇; 郝冰涛; 廖世秀
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-01-31
Filing date: 2023-01-31
Publication date: 2023-05-16

Abstract

The invention discloses a SH3 BGR-based method for predicting risk of suffering from Down syndrome and application thereof. The invention utilizes the fetal heart tissue of Down syndrome and AC16 cells of a myocardial cell line to explore a molecular mechanism of SH3BGR abnormal expression in the heart development process of a patient with Down syndrome, and explores the therapeutic effect of inhibiting SH3BGR on heart development of Down syndrome in AC16 cells of a myocardial cell line. In addition, the invention identifies the expression profile of differential genes in ventricular muscle tissue of Down syndrome, revealing a novel mechanism of expression up-regulated SH3BGR in myocardial tissue regulated by METTL3 mediated m6A modification. The invention is helpful to reveal the potential molecular mechanism of DS, and lays a foundation for developing a new treatment strategy.

Description

SH3 BGR-based method for predicting risk of suffering from Down syndrome and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a SH3 BGR-based method for predicting risk of suffering from Down syndrome and application thereof.

Background

Down syndrome (DownSyndrome, DS), also known as the 21-trisomy syndrome (OMIM # 190685), is a chromosomal aneuploidy disease caused by additional replication of the 21 chromosome in whole or in part. Worldwide, the incidence of DS is about 1/1000-1/500 of that of live infants. The incidence rate of the new born infants in China is about 1/800-1/600, and the new born infants are the most common chromosome diseases. The Chinese birth defect prevention report (2012) shows that the total economic burden of the life cycle of the Down syndrome patients born newly in China is over 100 hundred million yuan, and the damage is serious.

One of the most common complications of DS is Congenital Heart Disease (CHD), 40-60% of DS patients have CHD combined, which is the most common cause of death in 2 years after birth of DS patients. DS is also a major cause of CHD onset in the population. There is a literature suggestion that the DSCAM, COL6A2, COL6A1, KCNJ6 and RCAN1 genes are associated with CHD in DS patients. Although studied for many years, the exact mechanism is not completely understood. Focusing on research on the development and function of the heart of DS patients, new candidate genes are found, which is helpful for developing new diagnostic markers and intervention measures.

M6A (N6-methyladenine) modification is commonly present in such genes, and m6A is a modification in which a methyl group is added to the N6 position of adenine (a), and is the most common modification in various RNAs of higher organisms, and has dynamics and reversibility. The m6A modification of mRNA is widely distributed in the transcribed region of genes, affects transcription, splicing, transport, storage and degradation, and participates in regulating various biological processes.

The m6A modification occurs mainly on adenine in the RRACH sequence, consisting mainly of "encoder", "transcoder" (Eraser) and "Reader". The encoder, namely methyltransferase, at present mainly comprises METTL3, METTL14, WTAP, KIAA1429 and the like, wherein the METTL3 and the METTL14 exist in a dimer form, and the two cooperate to carry out methylation modification on adenine loci; the m6A modification is identified by an reader, namely m6A binding protein, and plays a regulatory role; and FTO and ALKBH5 are used as a code eliminator, namely demethyltransferase, so that methylation can be reversed, and the dynamic balance of m6A modification is realized.

Previous studies have found that m6A modification regulates cardiac development related gene expression as an important post-transcriptional regulatory mechanism in human heart disease. m6A modification and methyltransferase METTL3 are necessary during the development of embryonic stem cells into cardiomyocytes during heart development. Previous studies by the inventors found that m6A modification was reduced in expression in DS fetal brain tissue, down-regulated with the expression of methyltransferase METTL3 (see CN112143790a, which is incorporated herein by reference in its entirety). However, it is not clear whether the m6A modification also plays a role in DS fetal heart development. Furthermore, the effect of SH3BGR (SH 3 domain-bindingglutamicad-rich) on cardiac tissue development in DS hearts is not clear and still requires further investigation.

Disclosure of Invention

In order to explore the change of gene expression profile in ventricular muscle tissue of DS patient and the influence of differential expression gene on heart development, the system compares the expression of RNA in DS and normal embryo ventricular muscle tissue, and further explores the molecular mechanism of SH3BGR abnormal expression after identifying differential expression SH3BGR in DS ventricular muscle tissue and explores the influence of abnormal expression on myocardial cell development. Specifically, the present invention includes the following.

In a first aspect of the invention there is provided the use of an agent in the manufacture of a medicament for the prevention, amelioration or treatment of down syndrome, wherein the agent comprises an inhibitor capable of reducing the amount of SH3BGR protein or SH3BGR gene expression, and/or a genetically engineered agent for the overexpression of the METTL3 gene.

In certain embodiments, the use according to the invention, wherein the inhibitor comprises an antibody or a gene knockout agent, wherein the gene knockout agent comprises a CRISPR/Cas9 system, double stranded RNA (dsRNA), antisense oligonucleotide or RNAi molecule; and/or the genetic engineering agent comprises a plasmid, recombinant expression vector or cell line having the METTL3 gene.

In certain embodiments, the use according to the invention, wherein the RNAi molecule comprises a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a small molecule ribonucleic acid (miRNA).

In certain embodiments, the use according to the invention, wherein the preventing, ameliorating or treating comprises at least one of:

(1) Reduce apoptosis of ventricular myocytes;

(2) Improving the cell viability of ventricular myocytes;

(3) Preventing, ameliorating or treating the progression of complications of Down syndrome.

In a second aspect of the invention there is provided the use of a reagent for detecting m6A methylation modification of SH3BGRmRNA in the preparation of a kit for screening for down's syndrome.

In certain embodiments, the use according to the invention, wherein the reagent comprises a primer or probe directed against SH3BGRmRNA, and/or a specific antibody for detecting SH3BGR protein.

In a third aspect of the invention, there is provided a device (or system) for predicting risk of having Down syndrome, which may be used, for example, to assist in diagnosing Down syndrome, wherein the device or system comprises:

a. a data acquisition unit for acquiring data of the amount of SH3BGR protein or SH3BGR gene expression level, and/or the m6A methylation modification level of SH3BGRmRNA in a tissue sample collected from a subject, to obtain a measured value;

b. a judging unit for comparing the measured value with a control value;

c. an output unit for outputting the following result:

outputting that the subject is at high risk of developing down syndrome when the amount of SH3BGR protein or the measured value of SH3BGR gene expression level is above the control value and/or the m6A methylation modification level of SH3BGRmRNA is below the control value;

outputting the subject as having a low risk of down syndrome when the amount of SH3BGR protein or the measured value of SH3BGR gene expression level is equal to or lower than the control value, and/or the m6A methylation modification level of SH3BGRmRNA is equal to or higher than the control value.

In certain embodiments, the device according to the present invention, wherein the control value is a value obtained from a tissue sample of a normal subject of comparable age to the subject.

In certain embodiments, the device according to the present invention, wherein the m6A methylation modification is a METTL3 mediated m6A methylation modification of SH3 BGRmRNA;

the tissue sample is preferably ventricular muscle tissue.

In a fourth aspect of the invention, there is provided a method for identifying a compound useful for down syndrome, wherein the method comprises:

a'. Measuring the amount of SH3BGR protein or the expression level of SH3BGR gene, and/or the m6A methylation modification level of SH3BGRmRNA in the sample to obtain a first measurement value;

a step of measuring the amount of SH3BGR protein or the expression level of SH3BGR gene, or the m6A methylation modification level of SH3BGRmRNA in a sample after the administration of the test compound;

comparing the first measured value with the second measured value; and

(i) identifying the test compound as useful for down syndrome if the amount of SH3BGR protein or the second measurement of SH3BGR gene expression level is lower than the first measurement and/or the m6A methylation modification level of SH3BGRmRNA is higher than the first measurement, i.e., the test compound decreases the amount of SH3BGR protein or SH3BGR gene expression, or increases the m6A methylation modification level of SH3 BGRmRNA;

(ii) If the second measurement of the amount of SH3BGR protein or the level of SH3BGR gene expression is equal to or higher than the first measurement and/or the level of m6A methylation modification of SH3BGRmRNA is equal to or lower than the first measurement, i.e., the test compound has no effect or has an increasing effect on the amount of SH3BGR protein or the SH3BGR gene expression or has no effect or a decreasing effect on the level of m6A methylation modification of SH3BGRmRNA, then the compound is not identified as useful for down syndrome.

In certain embodiments, the method according to the invention for identifying a compound useful for down syndrome, wherein the sample is an AC16 cell line cultured in vitro.

Technical effects of the present invention include, but are not limited to:

the invention utilizes DS fetal heart tissue and myocardial cell line AC16 cells to explore a molecular mechanism of abnormal expression of SH3BGR in the heart development process of DS patients, and explores a therapeutic effect of inhibiting SH3BGR on DS heart development in the myocardial cell line AC16 cells.

The present invention identifies the expression profile of differential genes in DS ventricular musculature. Novel mechanisms of expression upregulation of SH3BGR in myocardial tissue regulated by METTL3 mediated modification of m6A are disclosed. The invention is helpful to reveal the potential molecular mechanism of DS, and lays a foundation for developing a new treatment strategy.

Drawings

FIG. 1 is a graph showing differential expression gene analysis in DS embryo heart ventricular musculature, wherein A: the volcanic plot of RNA-Seq results shows that 3934 expression up-regulated and 3159 expression down-regulated in the DS differential expression genes. B: GO analysis shows that the differential expression genes are mainly enriched in the aspects of striated muscle tissue and cell development, heart process, myocardial contraction and the like. C: KEGG analysis shows that the differential genes are mainly enriched in metabolic signaling pathways, cardiomyopathy, cardiomyocyte contractions and other signaling pathways. D-E: GSEA analysis showed that genes were up-regulated in oxidative phosphorylation and in the myogenic pathway. The myogenic pathway contains the SH3BGR gene whose expression is up-regulated. F: volcanic analysis of the gene on chromosome 21 showed that there were 58 up-regulated and 24 down-regulated expression in the DS differentially expressed genes.

Fig. 2 shows increased SH3BGR expression in DS embryo ventricular musculature, wherein a: RNA-Seq sequencing results showed that SH3BGR expression was up-regulated in DS. B: qPCR results showed that SH3BGR expression was elevated in DS. C-D: westernblotting results show that SH3BGR expression is up-regulated in DS.

FIG. 3 shows that reduced m6A modification in DS embryonic ventricular musculature correlates with reduced METTL3 expression, wherein A: the m6A modification of total RNA in DS cardiac ventricular musculature is reduced. B-C: western blotting results show that METTL3 expression and YTDDF 3 expression in DS are down-regulated, and that METTL3 down-regulation has significant differences. D: merp-qPCR detected a significant decrease in the level of m6A modification of SH3BGRmRNA in DS ventricular musculature RNA.

FIG. 4 shows that SH3BGR is regulated by METTL3 mediated modification of m6A in cardiomyocyte AC16, wherein A-B: knocking down and over-expressing METTL3 in AC16 cells resulted in decreased and increased m6A modification of SH3BGR, respectively. C: after knockdown of METTL3 in AC16 cells, increased SH3BGR mRNA expression was detected. D: westernblotting showed that downregulation of METTL3 expression in AC16 cells resulted in increased expression of SH3 BGR. E: knocking down METTL3 in AC16 cells for RNA-seq detection, total of 89 differential genes whose expression is up-regulated and 138 whose expression is down-regulated, including the SH3BGR gene whose expression is elevated. F: GO analysis shows that the expression up-regulated differential gene is enriched in SH3/SH2 adapter activity and the like.

Fig. 5 shows that METTL3 regulates its expression by m6A modification of SH3BGR3' UTR, wherein a: actinomycin D treatment resulted in slow degradation of SH3BGRmRNA after knocking down METTL3 in AC16 cells. B: the SRAMP website was used to predict the m6Amotif site of SH3BGR against which human SH3BGR3' UTR wild-type and mutant plasmids were constructed. C: after transfection of wild-type or mutant SH3BGR3 'UTRs in AC16 cells transfected with METTL3siNRA, a significant increase in luciferase activity of the wild-type SH3BGR3' UTRs was detected.

Fig. 6 shows that partial restoration of apoptosis caused by METTL3 knockdown by knockdown of SH3BGR in cardiomyocyte AC16, wherein a: knocking down METTL3 in AC16 cells, causing increased apoptosis; simultaneous knockdown of METTL3 and SH3BGR results in partial recovery of apoptotic cells. B: statistical analysis of apoptosis detected by flow cytometry.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

Use of agents for the preparation of a medicament for the prevention, amelioration or treatment of Down syndrome

In a first aspect of the invention there is provided the use of an agent in the manufacture of a medicament for the prevention, amelioration or treatment of down's syndrome, sometimes referred to as a "method of preventing, ameliorating or treating down's syndrome", the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of an agent, wherein the agent comprises an inhibitor capable of reducing the amount of SH3BGR protein or SH3BGR gene expression, and/or a genetically engineered agent for the overexpression of the METTL3 gene. In the present invention, SH3BGR in Down syndrome is used as a target of m6A methylation modification, and the difference of m6A methylation modification level is involved in regulating up-regulation or down-regulation of the expression level.

In the present invention, the SH3BGR gene is located on chromosome 21, and the encoded protein belongs to a small thioredoxin-like family with SH3domain, and is expressed in heart. The DS critical region hypothesis-related critical region of DS-CHD gradually narrows from the earliest 21q22.2-22.3 to the region of D21S3 to the PFKL gene. The SH3BGR gene is located in this region. Precise expression of SH3BGR protein is critical in cardiac development.

The terms "decrease," "inhibit," "decrease," and the like are used interchangeably herein, and the terms "raise," "promote," "activate," "increase," and the like are used interchangeably.

The present invention achieves the above object by reducing the amount of SH3BGR protein or SH3BGR gene expression, and it will be understood by those skilled in the art that the purpose of preventing, ameliorating or treating down syndrome can also be achieved by inhibiting the activity of SH3BGR protein. The reduction in the amount of SH3BGR protein or SH3BGR gene expression may be achieved by, but is not limited to, the following:

(1) Reducing SH3BGR protein expression in the subject; and/or

(2) The METTL3 gene is overexpressed.

In certain embodiments, reducing SH3BGR protein expression in a subject includes, but is not limited to, inhibiting expression of a gene encoding an SH3BGR protein or reducing expression levels of an SH3BGR gene. For example, an inhibitor that inhibits or reduces the expression level of the SH3BGR gene may be administered, such that the inhibitor may be any macromolecular or small molecule inhibitor capable of achieving the above-described objectives, including, but not limited to: an agent that inhibits the transcriptional activity of the SH3BGR gene, an agent that inhibits the transcriptional level of SH3BGRmRNA, an agent that promotes degradation of SH3BGRmRNA, siRNA against the SH3BGR gene, an agent that inhibits translation of SH3BGR mRNA, and an agent that specifically recognizes and cleaves a guide nucleic acid of the SH3BGR gene to reduce the expression level thereof. In certain embodiments, SH3BGR gene expression may be inhibited or its expression level reduced by siRNA administered to the SH3BGR gene. In certain embodiments, suppression or reduction of SH3BGR gene expression may be achieved by knockdown of the SH3BGR gene by administration of a targeting vector.

The specific sequence of the RNAi molecule to the SH3BGR gene is not particularly limited, and one skilled in the art can design and synthesize it from a known gene database. The RNAi molecules of the present invention may be double stranded or single stranded. When the RNAi molecule is double-stranded, one strand is the sense strand and the other strand is the antisense strand; wherein the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence of an SH3BGR protein, or portion thereof, and the sense strand comprises a nucleotide sequence that corresponds to a nucleotide sequence of an SH3BGR protein, or portion thereof. Alternatively, the RNAi molecules may be assembled from individual oligonucleotides, wherein the self-complementary sense and antisense regions of the RNAi molecules may be joined by nucleobase linkers or non-nucleobase linkers. The RNAi molecule can be a polynucleotide having a double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structure. The RNAi molecules can be circular single-stranded polynucleotides having two or more circular structures, as well as stem-like structures comprising self-complementary sense and antisense regions. The circular polynucleotide can be processed into an activated RNAi molecule in vivo or in vitro.

The terms "directed," "targeted," "conjugated," and the like are used interchangeably herein.

In the present invention, examples of RNAi molecules include, but are not limited to: small interfering RNAs (sirnas), small hairpin RNAs (shrnas), or small molecule ribonucleic acids (mirnas). Herein, small interfering RNAs (sirnas) are double-stranded RNA molecules that are capable of inhibiting or reducing expression of their cognate genes. Each strand of the siRNA can be about 8 to about 55 nucleotides in length. The double-stranded siRNA may have about 8 to about 55 base pairs, which is not particularly limited.

The siRNA can be delivered to the cell using a variety of methods known in the art. Such methods include delivery of the synthesized siRNA molecules to cells or vector-based delivery, wherein the vector-based method is the use of vectors to transduce the siRNA in target cells. Certain vector-based siRNA delivery systems can stably and effectively inhibit gene expression. In many vector-based methods, siRNA is produced by preparing small hairpin RNAs or short hairpin RNAs (shrnas). shRNA is a single stranded RNA molecule comprising a stem and hairpin structure. In cells, shRNA is processed into siRNA by action of the Dicer enzyme family. The shRNA may be about 25 to about 85 nucleotides in length. The double-stranded region of ShRNA may have about 8 to 40 base pairs, which is not particularly limited.

In the present invention, small molecule ribonucleic acid (miRNA) molecules, analogs thereof, miRNA precursors (e.g., pre-miRNA and pri-miRNA) may also be used. Such agents may be DNA molecules encoding miRNA or pre-miRNA or pri-miRNA. mirnas are typically single-stranded molecules, whereas precursors of mirnas are typically molecules that are at least partially self-complementary, capable of forming double-stranded parts, such as stem and loop structures.

In the present invention, antisense oligonucleotides include DNA, RNA, or derivatives, analogs, or fragments thereof. Antisense oligonucleotides can inhibit translation of SH3BGR mRNA proteins by binding to specific SH3 bgrmrnas. The length of the antisense oligonucleotide may range from about 5 to about 150 nucleotides, which is not particularly limited.

In the present invention, the term "SH3BGR gene" may be RNA or DNA thereof, and may be single-stranded or double-stranded.

It will be appreciated by those skilled in the art that this may also be achieved by reducing the activity of SH3BGR protein, and that such agents may be, for example, antibodies specific for SH3BGR or small molecule compounds having inhibitory activity. In this context, the activity of the SH3BGR protein is in particular the down syndrome or its complications described herein, preferably the activity mediating a confirmed or suspected down syndrome or its complications, particularly preferably the activity mediating apoptosis of ventricular myocytes with increased ventricular myocytes and decreased cell viability of ventricular myocytes with down syndrome. Herein, a decrease in the activity of a protein includes a decrease in the activity of an SH3BGR protein by at least 30%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even complete inactivity, as compared to its corresponding wild-type protein.

Unless otherwise indicated, the term "level" referred to herein includes the meaning of the amount or activity of the indicated substance.

The inventors found through studies that SH3BGR protein levels are mediated by the METTL3 gene. SH3BGR protein levels decrease when METTL3 gene is overexpressed; in the case of inhibited METTL3 gene expression, SH3BGR protein levels are increased. Furthermore, the inventors have further found that METTL3 regulates SH3BGR expression and stability of SH3BGRmRNA transcripts by mediating methylation of SH3BGRmRNA, thereby affecting apoptosis and cell viability of ventricular myocytes in subjects diagnosed or suspected of having down syndrome. Specifically, when METTL3 gene is overexpressed, the m6A methylation level of SH3BGRmRNA increases and the SH3BGR protein level decreases; whereas in the case of inhibited METTL3 gene expression, the m6A methylation level of SH3BGRmRNA was reduced and the SH3BGR protein level was increased.

Thus, in further embodiments, the METTL3 gene can be overexpressed by genetic engineering means to achieve the objects herein, e.g., using genetic engineering reagents to overexpress the METTL3 gene, examples of which include, but are not limited to, steps using plasmids, recombinant expression vectors, or cell lines with the METTL3 gene to over-express the METTL3 gene and further reduce the amount of SH3BGR protein or SH3BGR gene expression.

It will be appreciated by those skilled in the art that an inhibitor capable of reducing the amount of SH3BGR protein or SH3BGR gene expression may be used alone, or a reagent for genetic engineering that overexpresses the METTL3 gene may be used alone, or an inhibitor capable of reducing the amount of SH3BGR protein (or SH3BGR gene expression) and a reagent for genetic engineering that overexpresses the METTL3 gene may be used in combination.

In the pharmaceutical use of the above-mentioned agent of the present invention, the drug may contain a pharmaceutically acceptable carrier or excipient in addition to the above-mentioned agent in a prophylactically, ameliorated or therapeutically effective amount, and is not particularly limited.

In exemplary embodiments, the objects of the present invention are achieved by transferring an expression vector for over-expression of siRNA and/or METTL3 gene of SH3BGR into an expression cell separately or simultaneously.

In the present invention, the expression vector may be a DNA vector, such as a viral vector or a plasmid, in particular a vector suitable for expressing a nucleic acid in eukaryotic cells, especially mammalian cells. Expression vectors may contain promoters, enhancers or other regulatory elements. Promoters may or may not be cell type specific, and promoters may be constitutive or inducible. Viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, alphaviruses, and the like.

Suitable expression vectors for use in the present invention include, but are not limited to, plasmids derived from pCMV, plasmids derived from pVSVG, and the like, as well as other suitable expression systems for prokaryotic and eukaryotic cells, see Molecular CloningALaboratoryManual,2.sup. Nd. Ed. BySambrook, fritschand Maniatis (Cold spring harbor laboratory publication: 1989).

In another aspect of the invention there is also provided a cell comprising an expression vector as described above, which cell may be any eukaryotic cell including, but not limited to, mammalian cells. The cells may be human, mouse, rat, canine, feline, bovine, ovine, porcine, caprine, equine, primate cells. The cells may be differentiated cells or undifferentiated cells. The invention also provides transgenic organisms, including but not limited to transgenic animals, which may include the expression vectors described above.

As used herein, unless otherwise indicated, the terms "subject" and "patient" are used interchangeably herein to refer to any animal in which the pharmaceutical treatment and/or prevention described herein may be required. Subjects and patients thus include, but are not limited to: primates (including humans), canines, felines, rats and other mammalian subjects. Preferably, the patient is a human.

In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) the progress of an undesired physiological change or disorder, such as Down's syndrome or a complication thereof (congenital heart disease). Beneficial or desired clinical results include, but are not limited to, results that are either detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and palliation (whether partial or total). "treatment" also means an extended lifetime as compared to the lifetime expected when not receiving treatment. The need for treatment includes those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in need of prophylaxis of such a condition or disorder.

The term "preventing" as used herein includes avoiding the occurrence of and/or delaying the onset of a disease, condition or disorder. The avoidance of occurrence, delay of onset, or reduced risk of any statistically significant (p.ltoreq.0.05) as measured by controlled clinical trials can be considered prevention of a disease, condition, or disorder. Subjects suitable for prophylaxis include those subjects at increased risk of a disease, condition or disorder as identified by genetic or biochemical markers.

The term "effective amount" as used herein means the amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or condition, as compared to a corresponding subject that does not receive such an amount. The term also includes within its scope an amount effective to enhance normal physiological function. In general, effective amounts herein will vary depending upon factors such as the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, subject being treated, etc., but can still be routinely determined by one of skill in the art. The effective amount of the medicament of the present invention can be readily determined by one skilled in the art by conventional methods known in the art.

The terms "administering" and "administering" are used interchangeably to refer to contacting an inhibitor of the invention, a drug comprising the same, or the like, with a subject, cell, tissue, organ, or biological fluid when applied to the subject, cell, tissue, organ, or biological fluid, for example. In the case of cells, administration includes contacting the inhibitor of the invention, a drug comprising the same, with the cells (e.g., in vitro or ex vivo), and contacting the inhibitor of the invention, a drug comprising the same, with a fluid, wherein the fluid is in contact with the cells.

Device for predicting or evaluating Down syndrome risk

The invention also provides a device (or system) for predicting or assessing the risk of Down syndrome based on the amount of SH3BGR protein or SH3BGR gene expression, and/or the m6A methylation modification level of SH3 BGRmRNA.

The present invention also provides a computer readable storage medium storing a computer program which when executed by a processor implements the following method to predict or assess risk of having down syndrome: obtaining a measured value by obtaining the amount of SH3BGR protein or the expression level of SH3BGR gene, and/or the m6A methylation modification level of SH3BGRmRNA in a tissue sample collected from a subject; comparing the measured value with a control value; and outputting a prediction result.

The present invention also provides an electronic apparatus, comprising: a processor; and a memory for storing executable instructions of the processor; wherein the processor is configured to perform the following method via execution of the executable instructions: obtaining a measured value by obtaining the amount of SH3BGR protein or the expression level of SH3BGR gene, and/or the m6A methylation modification level of SH3BGRmRNA in a tissue sample collected from a subject; comparing the measured value with a control value; and outputting a prediction result.

The SH3BGR gene or protein can be used as a molecular index and is clinically used for diagnosing or assisting in diagnosing the down syndrome or the development of the complications thereof. Thus, in certain embodiments, the present disclosure also relates to the use of an agent for detecting the amount of SH3BGR protein or SH3BGR gene expression level, and/or m6A methylation modification of SH3BGRmRNA, in the preparation of a kit for screening for down syndrome. Such reagents include, but are not limited to, various primers and probes for detecting the SH3BGR gene, and/or specific antibodies for detecting the SH3BGR protein, etc., and also include other reagents used in preparing SH3BGR gene or protein-containing samples and performing the detection process, such as solvents, etc., including, but not limited to, various reagents necessary for performing PCR, etc. The specific sequences of the primer, probe and antibody are not particularly limited, and those skilled in the art can design and synthesize them by themselves according to known gene or protein databases or can obtain them from commercial products.

Accordingly, also provided herein is a detection or screening kit for Down syndrome comprising reagents for detecting SH3BGR genes and/or proteins as described previously, including, but not limited to, primers and probes required for amplifying and detecting SH3BGR genes, and antibodies specific for SH3BGR proteins. The level of SH3BGR is detected rapidly by the kit, and the level is measured, so that the kit can be used as an alternative index for determining Down syndrome.

Examples

In order to explore the changes in gene expression profile in ventricular muscle tissue of DS patients, RNA is first extracted from ventricular muscle tissue of three DS fetuses and three diploid control fetuses by using RNA-seq technology, and sequencing and analysis are performed after library construction. After verifying the expression of the differential gene SH3BGR in DS, the molecular mechanism of up-regulating SH3BGR expression in DS and the influence of the molecular mechanism on heart development are explored.

1. Experimental method

First, the m6A content of DS ventricular muscle tissue total RNA and the expression of related proteins were examined. Knocking down and over-expressing METTL3 in AC16 cells, detecting m6A modification of SH3BGR and expression of SH3BGR, and determining regulation of the METTL3 on the SH3 BGR; and knocking down METTL3 in AC16 cells to perform RNA-seq detection, and verifying the regulation of SH3BGR genes by METTL 3. Next, by detecting the degradation rate of SH3BGRmRNA after METTL3 knockdown and luciferase experiments, it was determined that METTL 3-mediated m6A methylation modification regulated expression of SH3 BGR. Finally, the flow cytometry detects the apoptosis of the myocardial cells, and clearly inhibits the rescue condition of SH3BGR expression on the apoptosis of the myocardial cells.

The method comprises the following steps:

(1) RNA-Seq: the whole transcriptional activity of ventricular muscle tissue or myocardial cell AC16 is detected at the transcriptional level, and the differential expression genes in two groups are screened out by the following specific methods:

extracting total RNA of the tissue or the cell, and detecting the concentration, the purity and the integrity. mRNA with ployA tail was enriched by Oligo (dT) magnetic beads. Adding a fragmentation buffer solution, randomly breaking mRNA into small fragments of about 300bp, adding a six-base random primer (randomhexamers) under the action of reverse transcriptase, reversely transcribing the mRNA serving as a template to synthesize a first strand of cDNA, and then synthesizing a second strand to form a stable double-stranded structure. The cohesive end double-stranded cDNA was made blunt by addition of EndRepair mix, followed by addition of an "A" base at the 3' end for ligation of the Y-shaped linker. PCR amplification, enrichment of the library followed by IlluminaHiseq sequencing.

(2) QuantitativePCR (qPCR) detection of gene expression: RNA from tissues or cells was extracted by Trisol method, and reverse transcribed into cDNA using a RevertAidFirstStrandcDNA Synthesis kit (Thermo) using 1ug of RNA as a template. Designing a specific primer, taking GAPDH as an internal reference gene, using a SYBRGreenMastermix preparation system, and running a program by an ABIStepOnereal-Time PCR instrument to detect the expression level of mRNA of a target gene.

(3) Expression of the Westernblot detection protein: tissue or cell proteins are extracted and quantified, after SDS-PAGE electrophoresis, membrane transfer is carried out, and the sealing liquid is sealed for 1h at room temperature, and antibodies such as anti-METTL 3, anti-SH 3BGR, anti-GAPDH and the like are used as primary antibodies, and the culture medium is incubated overnight at 4 ℃. After repeated washing of the membrane, the membrane was incubated with horseradish peroxidase (HRP) -labeled secondary antibody, gently shaken at room temperature for 1h, washed, developed and photographed.

(4) RNAm6A content detection: RNA from ventricular muscle tissue of the heart was extracted using Trisol method. The m6A content of total RNA was measured as required by the instructions using the m6ARNAMethylationQuantificationKit (Epigentek) kit. The microplate reader detects the luminosity at 450nm and calculates according to the formula m6A% = [ (sample OD-NCOD)/(S ]/[ (PCOD-NCOD)/(P ]. Times.100%, wherein S represents the amount of RNA in the experimental group and P represents the amount of positive control group.

(5) Merp-qPCR: total RNA in ventricular muscle tissue or cells was extracted and mRNA was purified using Dynabeads mRNA purification kit (Thermo). Then operating according to the NEBEPiMarkm6A enrichment kit, wherein the main steps are as follows: after incubation of the ProteinG beads and m6A antibody for 2h at 4℃1ug of mRNA was added and incubation was continued for 2h at 4 ℃. Washing 3 times with washing buffer, dissolving in RLT buffer (Qiagen), adding washed Dynabeads MyoneS ilane magnetic beads (Life technologies), washing 2 times with 70% ethanol, and dissolving RNA in DEPC water. Nanodrop2000 detects the concentration and purity of the resulting RNA, and qPCR detection is performed after reverse transcription.

(6) Culture and transfection of AC16 cells

Cells with good growth status were seeded in 12-well plates using DMEM: f12+10% fbs+1% diabody culture medium. After 24h of cell inoculation, the Primejet reagent is used for transfection when the cell density is about 50-70%, 50 nM/hole siRNA is added into 100ul of transfection buffer solution during transfection, and the mixture is blown and evenly mixed; then, 2ul of transfection reagent was added, incubated at room temperature for 10-15 minutes, 100ul of the mixed solution was added to each well, and the mixture was placed in an incubator for continuous cultivation.

(7) Expression of METTL3 overexpression in cells: to overexpress METTL3 in cells, the METTL3 overexpressing plasmid pSLenti-EF1-mCherry-P2A-Puro-CMV-METTL3 was constructed and 293T cells were transfected with lentiviral packaging plasmids pVSVG, pCMV-dR8.91 to obtain virus solution. The virus solution and Polybrene (8. Mu.g/ml, sigma) were infected together with AC16 cells. And (3) screening the Puromycin medicament to obtain a stable expression cell strain.

(8) Determination of half-life of mRNA

The AC16 cells with good growth state are inoculated in a 24-well plate, cultured overnight in an incubator, transfected after 24 hours, 1ul of actinomycin D (5 ug/ml) is added into each well after 24 hours of transfection, cells at different time points are collected, RNA is extracted and reverse transcribed, qPCR is used for detecting the expression of the target gene SH3BGR, and the half life of the SH3BGR is calculated.

(9) Double luciferase activity assay

Cells with good growth state are inoculated in a 12-well plate, the cells are transfected after 24 hours in an incubator overnight, and METTL3siRNA is added into 100ul of transfection buffer solution and is blown and evenly mixed. The constructed plasmid psiCHECK2-SH3BGR-3'UTR-WT (3' UTR of wild SH3 BGR) or psiCHECK2-SH3BGR-3'UTR-Mut (3' UTR of mutant SH3BGR, namely T is used for replacing A in m6A die body) is added into transfection buffer solution, and is blown and mixed uniformly. 2ul of transfection reagent is added, incubated at room temperature for 10-15min, 100ul of mixed solution is added to each well, and the mixture is placed in an incubator for culture. After 48h, the cells were lysed, detected using a double luciferase assay kit, and after each well signal was measured by a chemiluminescent instrument, each set of data was normalized.

(10) Detection of apoptosis by flow cytometry

Apoptosis of AC16 cells was detected using an annexin v-FITC apoptosis detection kit (bi yun, C1062S), as follows: cells with good growth status were inoculated in 24-well plates, incubated overnight in an incubator, and transfected with siRNA after 24 h. After 48h, cells were digested with appropriate amount of pancreatin cell digests (EDTA-free). After the digestion was terminated by adding the cell culture solution, the mixture was transferred to a centrifuge tube, centrifuged at 1000g for 5 minutes, and the supernatant was discarded to collect the cells. To the cell resuspension was added 5. Mu.l of Annexin V-FITC and 10. Mu.l of propidium iodide staining solution, and gently mixed. Incubate at room temperature for 10-20 minutes in the dark and then place in ice bath for flow cytometry detection.

2. Experimental results

Through RNA-seq detection, the present invention identified 7093 differentially expressed genes in DS fetal heart ventricular musculature, including 3934 genes whose expression was up-regulated and 3159 genes whose expression was down-regulated, compared to the control group (fig. 1A). GO analysis showed that the differentially expressed genes were associated with striated muscle tissue and cell development, cardiac processes, myocardial contractions, etc. (FIG. 1B). KEGG analysis showed that the differential genes were mainly concentrated in metabolic signaling pathways, cardiomyopathy, cardiomyocyte contractions, etc. (fig. 1C). GSEA analysis showed that the gene was up-regulated in oxidative phosphorylation and in the myogenic pathway (FIGS. 1D-E). Wherein the SH3BGR gene whose expression is up-regulated is located in the myogenic pathway. Analysis was performed around genes on chromosome 21, and a total of 58 genes up-regulated in expression and 24 genes down-regulated in expression were detected for differential expression in DS, including the SH3BGR gene up-regulated in expression (fig. 1F).

The SH3BGR gene is located in a key region on chromosome 21 that is associated with CHD and encodes a protein that belongs to the small thioredoxin-like family with SH3 domains that is expressed in the heart. RNA-Seq sequencing results showed that SH3BGR was up-regulated in DS (FIG. 2A). qPCR and Westernblot assays further confirmed that SH3BGR was up-regulated at mRNA and protein levels, respectively, in DS (fig. 2B-D).

To explore the potential molecular mechanism of up-regulation of SH3BGR expression, in combination with the role of m6A modification in cardiac development, and the regulatory role of m6A modification in DS brain found in earlier studies by the inventors, the level of m6A modification of total RNA in DS ventricular musculature was first examined, and the results showed a significant decrease in DS group (fig. 3A). Further western blotting was performed on methyltransferases, demethylases and binding proteins associated with m6A modification, and the results indicated that METTL3 expression was significantly down-regulated in the DS group (fig. 3B-C), suggesting that it was associated with reduced m6A modification in DS ventricular musculature. Merp-qPCR further detected a decrease in the level of m6A modification of SH3BGRmRNA in DS ventricular musculature RNA (FIG. 3D).

To explore whether SH3BGR expression is up-regulated by METTL3 and its underlying molecular mechanisms, the present invention detects a decrease and increase, respectively, in the m6A modification level of SH3BGR by MeRIP-qPCR after knocking down or over-expressing METTL3 in AC16 cell lines (fig. 4A-B), suggesting that SH3BGR mRNA is a target for METTL3 mediated m6A modification.

Knocking down METTL3 in AC16 cells, increased expression of SH3BGR at both mRNA and protein levels was detected (fig. 4C-D). RNA-seq detection was performed on METTL3 knockdown AC16 cells for a total of 89 differential genes with up-regulated and 138 down-regulated expression, including the expression of the elevated SH3BGR gene (FIG. 4E), confirming the regulation of SH3BGR by METTL 3. GO analysis of the up-regulated differential gene showed that the differential gene was enriched in SH3/SH2 adaptor activity etc. (fig. 4F). The above results suggest that METTL3 regulates expression of SH3BGR by m6A modification of SH3 BGRmRNA.

Methyltransferase METTL3 can affect target mRNA degradation through m6A modification, so to determine if increased expression of SH3BGR is regulated by METTL 3-mediated m6A modification, treatment of cells with actinomycin D after knocking down METTL3 in AC16 cardiomyocytes inhibited transcription, and a significant decrease in the rate of attenuation of SH3BGRmRNA was detected (fig. 5A), suggesting that METTL3 promotes SH3BGRmRNA degradation through m6A modification, affecting its stability.

To further determine whether SH3BGR is m 6A-dependent modified by METTL3, luciferase experiments were performed after knockdown of METTL3 expression in AC16 cell lines. The results show that knocking down METTL3 increases luciferase activity of the wild SH3BGR3' UTR, whereas the activity change of SH3BGR with m6A modification site mutation is not significant (fig. 5B-C). It was further demonstrated that METTL3 regulates SH3BGR expression by m6A methylation modification. The above results indicate that METTL3 modulation of SH3BGR is due to METTL 3-mediated m6A methylation modification of SH3BGRmRNA, affecting the stability of SH3BGRmRNA transcripts.

To explore the effect of SH3BGR on cardiomyocyte development, METTL3 was knockdown in AC16 cells and increased apoptosis was detected by flow cytometry. While simultaneous knockdown of METTL3 and SH3BGR resulted in partial recovery of apoptotic cells (fig. 6A-B). It is shown that METTL3 mediated m6A modification regulates SH3BGR expression, affecting cardiomyocyte apoptosis.

3. Conclusion(s)

While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. Use of an agent in the manufacture of a medicament for the prevention, amelioration or treatment of down syndrome, wherein the agent comprises an inhibitor capable of reducing the amount of SH3BGR protein or SH3BGR gene expression, and/or an agent for genetic engineering for the overexpression of the METTL3 gene.

2. The use of claim 1, wherein the inhibitor comprises a gene knockout agent or antibody, wherein the gene knockout agent comprises a CRISPR/Cas9 system, dsRNA, antisense oligonucleotide, or RNAi molecule; and/or the genetic engineering agent comprises a plasmid, recombinant expression vector or cell line having the METTL3 gene.

3. The use of claim 2, wherein the RNAi molecule comprises a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a small molecule ribonucleic acid (miRNA).

4. The use according to claim 1, wherein the prevention, amelioration or treatment comprises at least one of:

(1) Reduce apoptosis of ventricular myocytes;

(2) Improving the cell viability of ventricular myocytes;

(3) Preventing, improving or treating the progression of down syndrome complications.

5. A device for predicting risk of having down's syndrome, comprising:

a. a data acquisition unit for acquiring data of the amount of SH3BGR protein or SH3BGR gene expression level, and/or the m6A methylation modification level of SH3BGR mRNA in a tissue sample collected from a subject, to obtain a measured value;

b. a judging unit for comparing the measured value with a control value;

c. an output unit for outputting the following result:

outputting the subject as at high risk of down syndrome when the amount of SH3BGR protein or the measured value of SH3BGR gene expression level is above the control value, and/or the m6A methylation modification level of SH3BGR mRNA is below the control value;

outputting the subject as having a low risk of down syndrome when the amount of SH3BGR protein or the measured value of SH3BGR gene expression level is equal to or lower than the control value, and/or the m6A methylation modification level of SH3BGR mRNA is equal to or higher than the control value.

6. The device of claim 5, wherein the control value is a value obtained from a tissue sample of a normal subject of comparable age to the subject.

7. The device of claim 6, wherein the m6A methylation modification is a METTL3 mediated m6A methylation modification of SH3 BGRmRNA;

the tissue sample is preferably ventricular muscle tissue.

8. Use of a reagent for detecting the amount of SH3BGR protein or SH3BGR gene expression, and/or m6A methylation modification of SH3BGR mRNA, in the preparation of a kit for screening down syndrome.

9. The use according to claim 8, wherein the reagent comprises a primer or probe for SH3BGR mRNA and/or a specific antibody for detecting SH3BGR protein.

10. A method for identifying a compound useful for down's syndrome, the method comprising:

a'. Measuring the amount of SH3BGR protein or the expression level of SH3BGR gene, and/or the m6A methylation modification level of SH3BGR mRNA in the sample to obtain a first measurement;

a step of measuring the amount of SH3BGR protein or the expression level of SH3BGR gene, and/or the m6A methylation-modified level of SH3BGR mRNA in the sample after administration of the test compound to obtain a second measurement value;

comparing the first measured value with the second measured value; and

(i) identifying the test compound as useful for down syndrome if the amount of SH3BGR protein or the second measurement of SH3BGR gene expression level is lower than the first measurement, and/or the m6A methylation modification level of SH3BGR mRNA is higher than the first measurement, i.e., the test compound decreases the amount of SH3BGR protein or SH3BGR gene expression, or increases the m6A methylation modification level of SH3BGR mRNA;

(ii) Identifying the compound as not useful in down syndrome if the second measurement of the amount of SH3BGR protein or SH3BGR gene expression level is equal to or higher than the first measurement and/or the m6A methylation modification level of SH3BGR mRNA is equal to or lower than the first measurement, i.e., the test compound has no effect or has an increasing effect on the amount of SH3BGR protein or SH3BGR gene expression, or has no effect or has a decreasing effect on the m6A methylation modification level of SH3BGR mRNA;

the sample is preferably an AC16 cell line cultured in vitro.