CN107604058B - PiRNA-514 nucleotide analogue and application of antisense nucleotide thereof and product using same - Google Patents
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Abstract
The invention provides a piRNA-514 nucleotide analogue, application of antisense nucleotide thereof and a product using the antisense nucleotide, relating to the technical field of medical engineering. The application of the piRNA-514 nucleotide analogue or the bioactive functional fragment or variant thereof in preparing a product for diagnosing and/or prognostically evaluating heart diseases has the potential value of diagnosing and/or prognostically evaluating a plurality of heart diseases by detecting the expression level of the piRNA-514 nucleotide in peripheral blood plasma; moreover, the application of the piRNA-514 antisense nucleotide provided by the invention in the preparation of products for preventing and/or treating heart diseases provides a basis for the prevention and/or treatment of a plurality of heart diseases.
Description
Technical Field
The invention relates to the technical field of medical engineering, in particular to a piRNA-514 nucleotide analogue, application of antisense nucleotide thereof and a product applying the antisense nucleotide.
Background
The global burden of disease report issued by the world health organization indicates that cardiovascular disease has become a leading cause of human death worldwide. According to statistical data in 2014, the number of people suffering from cardiovascular diseases in China is more than 2.7 hundred million, and 300 million people die of the cardiovascular diseases every year. The morbidity and mortality of cardiovascular diseases in middle-aged and elderly people are obviously increased, so that the current situation of the aging population of China is faced, and the important theoretical and practical significance is provided for the pathogenesis and clinical research of the cardiovascular diseases.
Myocardial hypertrophy is the targeting of cardiomyocytes to hemodynamicsAn increased response, usually manifested by thickening of myocardial tissue at the tissue level and an increase in the volume of myocardial cells at the cell level, is a common pathological change in the development of many cardiovascular diseases (hypertension, acute myocardial infarction, congenital heart diseases, etc.). The stimulus factors that induce myocardial hypertrophy include mainly mechanical tension and various neurohumoral factors, such as Angiotensin II (Ang II), isoproterenol (isoproterenol,Iso) Insulin-like growth factor-1 (IGF-1), interleukin-1 (IL-1), and the like.
piRNA (piwi-interacting RNA) is a new non-coding small RNA which is firstly found in male germ cells in 2006 after two types of small RNAs, namely siRNA and miRNA, the length of the piRNA is usually 26-31 nt, the 5' end of the piRNA has strong uracil bias, and the number of the piRNA is more than 50,000.
The past research once thought that piRNA is peculiar to germ cells, therefore, mainly focuses on the functional research of the piRNA in the germ cells, and the existing research results show that the piRNA is mainly involved in the processes of germ cell development, transposon silencing, heterochromatin formation and germ cell DNA integrity maintenance. In 2012, a study published in the Cell journal found that piRNA expressed in the brain of sea rabbits modulated memory function of neural cells by modulating DNA methylation of CREB proteins. Thus, the interest of researchers in functional studies of piRNA in non-germ cells was attracted.
At present, no report is available on the key piRNA in heart diseases such as cardiac hypertrophy and myocardial fibrosis, and the functions exerted by the piRNA are challenges for researchers in the field.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention provides the application of the piRNA-514 nucleotide analogue or the bioactive functional fragment or variant thereof in preparing products for diagnosing and/or prognostically evaluating heart diseases;
the piRNA-514 nucleotide analogue contains a sequence shown in SEQ ID NO. 1.
Further, the product is a medicament or a kit.
Further, each base in the nucleotide sequence of the piRNA-514 nucleotide analogue is chemically modified.
The invention also provides a medicament for the diagnostic and/or prognostic assessment of a cardiac disease, comprising an effective dose of a piRNA-514 nucleotide analogue or a biologically active functional fragment or variant thereof.
The invention also provides a kit for the diagnostic and/or prognostic assessment of heart disease, comprising primers for the specific detection of a piRNA-514 nucleotide analogue.
The invention also provides the application of the piRNA-514 antisense nucleotide in preparing products for preventing and/or treating heart diseases;
the antisense nucleotide of the piRNA-514 contains a sequence shown in SEQ ID NO. 2.
Further, the product is a medicament.
In addition, the invention also provides a medicament for preventing and/or treating heart diseases, which comprises an effective dose of the piRNA-514 antisense nucleotide.
Further, the administration mode of the medicament comprises oral administration or injection administration.
Further, the heart disease includes one or more of myocardial hypertrophy, myocardial fibrosis, coronary heart disease, myocarditis, heart valve disease, hypertension, and heart failure.
Experiments show that the expression of the piRNA-514 nucleotide analogue provided by the invention is obviously reduced in hypertrophic myocardial cells, and the over-expression of the piRNA-514 can obviously inhibit the increase of the surface area of the myocardial cells induced by Iso. As described above, piRNA-514 can inhibit the occurrence of myocardial hypertrophy. This means that piRNA-514 can be used as a biomarker for early diagnosis and early prevention of heart diseases such as myocardial hypertrophy and myocardial fibrosis. Therefore, the expression level of the piRNA-514 nucleotide in the peripheral blood plasma is detected, so that the piRNA-514 nucleotide has potential value in diagnosis and/or prognosis evaluation of a plurality of heart diseases.
Drawings
FIG. 1 is a graph showing the results of changes in the expression level of piRNA-514 in the hearts of a model mouse of a myocardial hypertrophy disease induced by isoproterenol (Iso) and a control mouse provided in example 1 of the present invention;
FIG. 2 is a graph showing the results of changes in the expression level of piRNA-514 during the stimulation of cardiac hypertrophy and isoproterenol (Iso) induced myocardial cell hypertrophy provided in example 2 of the present invention;
FIG. 3 is a graph showing the effect of the transfection of piRNA-514 nucleotide analogues on the surface area of primary cardiomyocytes according to example 3 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides an application of a piRNA-514 nucleotide analogue or a bioactive functional fragment or variant thereof in preparing a product for diagnosing and/or prognostically evaluating heart diseases, wherein the sequence of the piRNA-514 analogue nucleotide is as follows (SEQ ID NO. 1):
5’-AAUCAGAUUUGCACCACUCAUCUUGGUGA-3’。
among them, the biologically active functional fragment or variant of the piRNA-514 nucleotide analog can be, for example, but not limited to, the following (1) to (3):
(1) a recombinant vector containing a gene encoding a piRNA-514 nucleotide analog;
(2) a recombinant virus comprising a gene encoding a piRNA-514 nucleotide analog;
(3) a recombinant viral vector comprising a gene encoding a piRNA-514 nucleotide analog.
In the present invention, the product is a medicament or a kit.
In the present invention, each base in the nucleotide sequence of the piRNA-514 nucleotide analog is chemically modified.
The chemical modification may be, for example, but not limited to, a ribose modification, a base modification or a phosphate backbone modification.
In a preferred embodiment, each base in the nucleotide sequence of the piRNA-514 nucleotide analogue is 2' -methoxy modified.
The invention also provides a medicament for the diagnostic and/or prognostic assessment of heart disease, comprising an effective dose of a piRNA-514 nucleotide analogue or a biologically active functional fragment or variant thereof.
The invention also provides a kit for diagnosing and/or prognostically assessing heart diseases, comprising a probe or primer for specifically detecting the piRNA-514 nucleotide analogue.
Wherein, the primer sequence for specifically detecting the piRNA-514 nucleotide analogue is as follows:
reverse transcription primer:
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACCAAG-3’（SEQ ID NO.3）；
an upstream primer: 5'-CGGGCAATCAGATTTGCACCACTCATCT-3' (SEQ ID NO. 4);
a downstream primer: 5'-TGGTGTCGTGGAGTCG-3' (SEQ ID NO. 5).
In addition, the invention also provides the application of the piRNA-514 antisense nucleotide in preparing products for preventing and/or treating heart diseases, wherein the sequence of the piRNA-514 antisense nucleotide is as follows (SEQ ID NO. 2):
5’-UCACCAAGAUGAGUGGUGCAAAUCUGAUU-3’。
in the present invention, the product is a medicament.
The invention also provides a medicament for preventing and/or treating heart diseases, which comprises an effective dose of the piRNA-514 antisense nucleotide.
In the present invention, the medicament for preventing and/or treating heart diseases further comprises a pharmaceutically acceptable carrier or adjuvant.
The carrier or adjuvant may be, for example, but not limited to, one or more of chitosan, cholesterol, liposome, cyclodextrin, microsphere, and microcapsule.
In the present invention, the administration mode of the drug for preventing and/or treating heart diseases includes oral administration or injection administration.
The injection may be, for example, but not limited to, intravenous injection, intramuscular injection, intracoronary injection, and myocardial injection.
In the present invention, the heart disease includes one or more of cardiac hypertrophy, cardiac fibrosis, coronary heart disease, myocarditis, heart valve disease, hypertension and heart failure.
To facilitate a clearer understanding of the contents of the present invention, reference will now be made in detail to the following specific embodiments. Unless otherwise specified, the experimental procedures referred to in the following examples are molecular biological procedures conventional in the art, all reagents referred to are analytical grade reagents, and all reagents or instruments referred to are commercially available from normal sources.
Unless otherwise indicated, all experimental procedures and procedures described in the following examples, including cell culture, RNA extraction, RCR amplification, fluorescent quantitative PCR, cell staining, etc., can be found in: wang K, et al, miR-361-defined prohibited protein inhibitors in the mitochondrial configuration and apoptosis and protection heart from biochemical in the future, Cell Death Differ. 2015 Jun, 22(6), 1058-68, W. -Q.Tan, et al, Foxo3a inhibition cardiac molecular assay, hyper expression through transport transforming protein J Biol. chem. 2008 October 31, (283 44), 29730-29739, Wang K, et al, miR-9and NFC 3 regulated molecular protein molecular chemistry, J Biol. chem. 2010. 16, (16) miR-03-12, Lin Z, miR-11923, flow of molecular protein kinase 12108, supra, incorporated by reference into the entire text, N.A. No. 11,32, EP 3, see the publication No. 11, et al, EP 63285, et al.
Example 1 detection of changes in the expression level of piRNA-514 in the hearts of model mice and control mice with isoproterenol (Iso) induced myocardial hypertrophy disease
20C 57BL/6J mice are selected and divided into a control group and a model group, and Iso and normal saline are respectively permeated by an implanted capsule osmotic pump to construct a mouse model with myocardial hypertrophy. Total RNA of two groups of mouse hearts is respectively extracted, and the expression level of the piRNA-514 is detected by a real-time fluorescent quantitative PCR technology, and the result is shown in figure 1.
As can be seen from FIG. 1, the expression of piRNA-514 is down-regulated in the heart of mice in the Iso-induced myocardial hypertrophy model group, which indicates that the detection of the change of the expression level of piRNA-514 can be used as the basis for clinical diagnosis of myocardial hypertrophy.
Example 2 detection of altered expression levels of piRNA-514 during isoproterenol (Iso) -induced myocardial cell hypertrophy
For the myocardial cell hypertrophy model, primary myocardial cells of rat suckling mice are cultured by adopting the established method in the laboratory, the myocardial cells are treated by 10 mu M isoproterenol, the total RNA of the cells is extracted at different culture time, and the expression level of the piRNA-514 is detected by the real-time fluorescence quantitative PCR technology. This example uses the expression level of piRNA-514 in heart at different time points after isoproterenol treatment of primary cardiomyocytes for 0 h, 8 h, 16 h and 24 h. Total RNA from the heart was extracted (using TRIZOL kit) and the expression level of piRNA-514 was determined by real-time fluorescent quantitative PCR, as shown in FIG. 2.
As can be seen in FIG. 2, the level of piRNA-514 gradually decreased in a time-dependent manner after 0, 8, 16, 24 hours of Iso treatment, further indicating that piRNA-514 can be a potential marker for clinical diagnosis of myocardial hypertrophy.
Example 3 experiment of overexpression of piRNA-514 to inhibit hypertrophy of Primary cardiomyocytes
In this example, the primary cardiomyocytes provided in example 2 were divided into four groups, blank, Iso-treated, control and experimental, each of which was triplicated (cell mass approximately 1 × 10)6）。
Wherein, the blank group of primary myocardial cells are cultured only by using a normal cell culture solution.
Iso-treated groups primary cardiomyocytes were treated with 10. mu.M isoproterenol.
Experimental group Primary cardiomyocytes were treated with 10. mu.M isoproterenol and transfected with a piRNA-514 nucleotide analog (sequence 5'-AAUCAGAUUUGCACCACUCAUCUUGGUGA-3', SEQ ID NO.1, manufactured by Shanghai Jima pharmaceutical technology Co., Ltd.).
The control group was treated in the same manner as the experimental group except that the piRNA-514 nucleotide analog was replaced with a negative control RNA (agomir-control) having a nonsense sequence.
As can be seen from FIG. 3, overexpression of piRNA-514 agomir-induced piRNA-514 can reduce the increased cell surface area induced by Iso.
In conclusion, the piRNA-514 can inhibit the occurrence of myocardial hypertrophy and can be used as a biomarker for early diagnosis and early prevention of heart diseases such as myocardial hypertrophy, myocardial fibrosis and the like. Therefore, the method has potential value for diagnosing and/or prognostically evaluating a plurality of heart diseases by detecting the expression level of the piRNA-514 in the plasma of peripheral blood; the inhibition of the expression level of the piRNA-514 is detected, so that the piRNA-514 has potential value in prevention and/or treatment of a plurality of heart diseases.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
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Claims (3)
- Use of piRNA-514 nucleotides for the preparation of a product for the diagnostic and/or prognostic assessment of a cardiac disease;the nucleotide of the piRNA-514 is a sequence shown in SEQ ID NO. 1;the heart disease is isoproterenol-induced myocardial hypertrophy.
- 2. Use according to claim 1, wherein the product is a medicament or a kit.
- 3. A kit for the diagnostic and/or prognostic assessment of a cardiac disease, characterized in that the kit comprises a primer for the specific detection of piRNA-514 nucleotides;the primer comprises a reverse transcription primer, an upstream primer and a downstream primer;the reverse transcription primer is a sequence shown as SEQ ID NO. 3;the upstream primer is a sequence shown as SEQ ID NO. 4;the downstream primer is a sequence shown in SEQ ID NO. 5.
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| CN110229879B (en) * | 2019-07-01 | 2022-11-18 | 青岛大学 | PiRNA-500 nucleotide analogue, application of antisense nucleotide thereof and product using same |
| CN111705061B (en) * | 2020-07-22 | 2022-07-05 | 青岛大学 | Antisense nucleotide of piRNA-P1 and piRNA-P1 related to heart disease, application and medicament |
| CN114736956B (en) * | 2022-04-07 | 2024-03-08 | 山东第一医科大学(山东省医学科学院) | Application of piRNA as diagnostic marker in forensic identification of acute myocardial infarction and/or inference of duration of acute myocardial infarction |
| CN114965645B (en) * | 2022-06-10 | 2022-12-09 | 山东大学 | In-vitro piRNA detection method based on photoelectrochemistry and double-strand specific nuclease |
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| CN104645352A (en) * | 2015-01-27 | 2015-05-27 | 青岛大学 | piRNA antisense nucleotide pharmaceutical composition and application thereof |
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