阅读说明：本技术 一种人内含子源性27碱基microRNA及在调节血压中应用 (Human intron-derived 27-base microRNA and application thereof in blood pressure regulation ) 是由 欧和生 杨鹏 罗雪兰 于 2019-12-05 设计创作，主要内容包括：本发明公开了一种人内含子源性27碱基microRNA及在调节血压中应用,该人内含子源性27碱基microRNA是由eNOS第四内含子27碱基重复子重复4次产生,其核苷酸序列如SEQ ID NO.1所示。根据该序列制备其高表达质粒,将其高表达质粒与脂质体按质量比为3：1混合并经生理盐水稀释,经大鼠尾静脉注入SD大鼠体内,证明本发明的人内含子源性27碱基microRNA对大鼠血压具有调节作用,可用于研究高血压疾病,并最终可用于某些降压药物的筛选和制备高血压早期预警芯片,具有潜在的应用前景。(The invention discloses a human intron-derived 27-base microRNA and application thereof in blood pressure regulation, wherein the human intron-derived 27-base microRNA is generated by repeating a 27-base duplicon of a fourth intron of eNOS for 4 times, and the nucleotide sequence of the microRNA is shown in SEQ ID No. 1. Preparing a high expression plasmid according to the sequence, and mixing the high expression plasmid with the liposome according to the mass ratio of 3: 1, diluted by normal saline, injected into SD rat body through tail vein of rat, proving that the human intron-derived 27-base microRNA has regulating effect on rat blood pressure, can be used for researching hypertension disease, and can be finally used for screening certain antihypertensive drugs and preparing hypertension early warning chips, and has potential application prospect.)

1. A human intron-derived 27-base microRNA is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A recombinant expression plasmid, an expression cassette, a transgenic cell line or a recombinant bacterium containing the human intron-derived 27-base microRNA of claim 1.

3. A method for constructing a recombinant expression plasmid containing the human intron-derived 27-base microRNA of claim 1, which comprises the following steps: obtaining a human intron-derived 27-base microRNA nucleotide sequence through a Blast system in NCBI, wherein the nucleotide sequence is shown as SEQ ID NO. 1; amplifying a target gene fragment by PCR, performing double enzyme digestion, performing linear connection, extracting plasmids by using a small-scale extraction kit of Beijing Tiangen Biotechnology Co., Ltd, and taking a positive clone bacterium solution to send to the company for sequencing identification to obtain the human intron-derived 27-base microRNA high-expression plasmid.

4. The construction method according to claim 3, wherein the PCR reaction system is: 10 ng/. mu.L DNA template 1. mu.L, ddH₂O32.5. mu.L, 5 XPS Buffer 10. mu.L, 2.5mM dNTP Mix 4. mu.L, 10. mu.M upstream amplification primer 1. mu.L, 10. mu.M downstream amplification primer 1. mu.L, PrimeSTAR HS DNA polymerase 0.5. mu.L.

5. The construction method according to claim 4, wherein the upstream amplification primer 5'-GGAAGTCTAGACCTGCTGCA-3' is shown as SEQ ID NO. 2;

5'-GTCGGTGTCGTGGAGTCG-3' as shown in SEQ ID NO. 3.

6. The method of claim 3, wherein the PCR reaction conditions are: 5min at 95 ℃ and 1 cycle; 10s at 98 ℃, 10s at 55 ℃, 90s at 72 ℃, 30 cycles, 8min at 72 ℃ and 24h at 4 ℃.

7. The use of the recombinant expression plasmid of the human intron-derived 27-base microRNA of claim 1 and the recombinant expression plasmid of the human intron-derived 27-base microRNA of claims 2 to 6 in the preparation of antihypertensive drugs.

8. The use of claim 7, wherein the drug comprises recombinant expression plasmid and liposome of human intron-derived 27 base microRNA, and the mass ratio of the recombinant expression plasmid to the liposome is 3: 1.

9. the use of the human intron-derived 27-base microRNA of claim 1 and the use of the human intron-derived 27-base microRNA of claims 2 to 6 in the preparation of an early warning chip for hypertension.

Technical Field

The invention belongs to the technical field of rat tail intravenous injection and gene recombination, and particularly relates to a human intron-derived 27-base microRNA and application thereof in blood pressure regulation.

Background

Hypertension is the most common cardiovascular disease, often causes serious heart, brain, kidney complications, affecting 20-50% of the adults worldwide. According to statistics, about 2 hundred million hypertension patients in China currently have hypertension, 1 patient in every 5 adults has hypertension, about 1/5 of the total number of global hypertension, and nearly 760 ten thousand patients die of the hypertension every year in the world. The World Health Organization (WHO) defines hypertension as: under the condition of not using antihypertensive drugs, the systolic pressure is more than or equal to 140mmHg and/or the diastolic pressure is more than or equal to 90 mmHg. Hypertension is again classified into grades 1, 2, 3: 1) high blood pressure level 1, also called mild hypertension, systolic pressure 140-159 mmHg or diastolic pressure 90-99 mmHg; 2) high blood pressure grade 2, also called moderate hypertension, systolic pressure 160-179 mmHg or diastolic pressure 100-109 mmHg; 3) high blood pressure level 3, also called severe hypertension, has systolic pressure not less than 180mmHg or diastolic pressure not less than 110 mmHg. It is worth noting that the American Heart Association modifies the hypertension standard to a systolic pressure of more than or equal to 130mmHg and/or a diastolic pressure of more than or equal to 80mmHg at the end of 2017, which shows that the prevention and treatment of hypertension are more and more highly regarded; according to the modified hypertension standard, the number of global hypertension patients is at least doubled.

miRNA is a type of endogenous non-coding small molecular RNA which is highly conserved in evolution and about 17-25 nucleotides in length. To date, over 1000 mirnas have been identified in the human genome, expressed in a variety of specific tissues, which regulate nearly half of the protein-encoding genes in humans; since the first miRNA (lin-4) was discovered in 1993, 28645 miRNA precursor hairpin structures and 35828 mature miRNAs have been identified in 226 different organisms to date. The miRNAs regulate and control protein expression by degrading target gene mRNA or inhibiting translation of the target gene mRNA, control pathophysiological processes such as cell differentiation, proliferation and apoptosis and participate in related gene expression regulation of major human diseases such as cancer, diabetes, cardiovascular diseases and the like.

Mirnas are currently considered to fall into two broad categories: intergenic mirnas and gene intron-derived mirnas, the latter accounting for approximately 10%, are derived from introns in the mRNA precursor sequences transcribed from certain protein-encoding genes. The discovery and definition of introns dates back to 1977 for the earliest time, and introns in the mRNA precursors of almost all eukaryotic genes were spliced during mRNA editing, and thus have long been recognized as "redundant, useless" garbage RNA sequences. Although some studies have found that its gene regulation effect, for example, the intron site of some nucleoprotein binding sites, plays an important role in p 53-dependent gene activation and transcription, until the discovery of mirnas in recent years, these intron sequences having a strong gene expression regulation function have not been really valued and classified as intron-derived mirnas; to date, 408 intronic miRNA has been identified using bioinformatics methods, accounting for approximately 40% of the total number of human mirnas.

Nitric Oxide (NO) has a very important biological role in cardiovascular regulation, and it can relax vascular smooth muscle, maintain the blood flow of organs relatively stable, control the resting tension of various blood vessels, and is the main blood pressure regulating factor. NO is produced mediated by three different NO Synthase (NOs) isoforms using L-arginine as a substrate: neural (nNOS), Inducible (iNOS) and endothelial (eNOS). eNOS knockout mice exhibit systemic hypertension, which is associated with impaired endothelium-dependent hyperpolarization-mediated relaxation. While a single injection of naked human eNOS plasmid DNA can reduce the blood pressure of SHR rats by 5-6 weeks. The occupation of receptor agonists, or the opening of certain ion channels, leads to an increase in intracellular calcium, activating eNOS to produce NO. The role of NO-cGMP signaling in vascular smooth muscle cell relaxation and blood pressure regulation is well characterized in both humans and animals, and there is increasing evidence that impaired NO signaling is involved in the pathogenesis of hypertension.

miRNAs are associated with hypertension. Relevant researches show that miRNA is involved in a plurality of links of hypertension occurrence and development, and mainly comprises the following three aspects: 1) miRNAs are involved in the functional regulation of the renin-angiotensin-aldosterone system. The renin-angiotensin-aldosterone system is a group of hormone and receptor systems secreted by the kidney and the liver, which interact and mutually regulate, plays a key role in the regulation of hypertension, the over-stimulation reaction of the renin-angiotensin-aldosterone system is a main pathological factor of the hypertension, and angiotensin II is a main biological effector molecule of the RAAS system, which can not only shrink arteriolar vessels, but also activate sympathetic nervous system and promote the production of aldosterone. miR-181a inhibits renal sympathetic nerve activity and reduces renin secretion; angiotensin Converting Enzyme (ACE) is a target gene of miR-143/145, and the increase of miRNA143/145 results in the reduction of ACE gene expression; miR-421 inhibits transcriptional expression of ACE2 protein; miR-124 and miR-135a can inhibit renal salt retention by reducing mineralocorticoid receptor protein; these inhibitory effects of mirnas on RAAS systems will reduce blood pressure. 2) miRNAs affect vascular endothelial cell function. The continuous high pressure in the vascular cavity can cause endothelial cell dysfunction, when the endothelial dysfunction is caused, the secretion of vasodilation factors is reduced, the secretion of vasoconstriction factors is increased, peripheral blood vessels are strongly contracted, the peripheral resistance is obviously increased, and the occurrence and development of hypertension are promoted. miR-125a/b-5p inhibits the expression of vascular endothelial cell endothelin-1, and miR-155 can cause endothelial eNOS transcription to be down-regulated, and the release of vasoactive factors such as NO and the like is reduced, so that the blood pressure regulation is influenced. 3) miRNAs are involved in the regulation of vascular smooth muscle cell proliferation. Stimulation by various factors causes abnormal proliferation and apoptosis of vascular smooth muscle, decrease in vascular compliance, increase in resistance, and increase in blood pressure. miR-122 and miR-222 are involved in the proliferation of vascular smooth muscle, and Ang II regulates the response of smooth muscle cells to Ang II stimulation by regulating transcripts lncRNAs (LongnoonodingRNAs) of the miR-122 and the Ang II, so that the formation of atherosclerosis and hypertension is influenced. miR-145 can directly control proliferation and apoptosis of smooth muscle, miR-143 and miR-145 can regulate expression of various cytoskeletal elements and Serum Response Factors (SRF), play an important role in the conversion process of vascular smooth muscle cell contraction phenotype and synthetic phenotype, and the level of the expression level of the miR-145 and the SRF affects the contractility of blood vessels so as to affect the change of arterial blood pressure.

At present, the cause of hypertension is unknown, and the mechanism of pathological occurrence and development is also unclear, so that a prevention and treatment measure for thoroughly and efficiently curing the disease is lacked. Therefore, the search for new antihypertensive drugs with higher efficacy and no side effects is an important task in the current cardiovascular field.

Disclosure of Invention

In view of the above, the invention provides a human intron-derived 27-base microRNA and application thereof in blood pressure regulation, which can be used for researching hypertension diseases, screening certain antihypertensive drugs and preparing hypertension early warning chips, and has potential application prospects.

In order to solve the technical problems, the invention discloses a human intron-derived 27-base microRNA, and the nucleotide sequence of the microRNA is shown as SEQIDNO.1.

The invention also discloses a recombinant expression plasmid, an expression cassette, a transgenic cell line or a recombinant bacterium containing the human intron-derived 27-base microRNA.

The invention also discloses a construction method of the recombinant expression plasmid containing the human intron-derived 27-base microRNA, which comprises the following steps: obtaining a human intron-derived 27-base microRNA nucleotide sequence through a Blast system in NCBI, wherein the nucleotide sequence is shown as SEQ ID NO. 1; amplifying a target gene fragment by PCR, performing double enzyme digestion, performing linear connection, extracting plasmids by using a small-scale extraction kit of Beijing Tiangen Biotechnology Co., Ltd, and taking a positive clone bacterium solution to send to the company for sequencing identification to obtain the human intron-derived 27-base microRNA high-expression plasmid.

Optionally, the PCR reaction system is: 10 ng/. mu.L DNA template 1. mu.L, ddH₂O32.5. mu.L, 5 XPS Buffer 10. mu.L, 2.5mM dNTP Mix 4. mu.L, 10. mu.M upstream amplification primer 1. mu.L, 10. mu.M downstream amplification primer 1. mu.L, PrimeSTAR HS DNA polymerase 0.5. mu.L.

Optionally, an upstream amplification primer 5'-GGAAGTCTAGACCTGCTGCA-3', shown as SEQ ID NO. 2;

5'-GTCGGTGTCGTGGAGTCG-3' as shown in SEQ ID NO. 3.

Alternatively, the PCR reaction conditions are: 5min at 95 ℃ and 1 cycle; 10s at 98 ℃, 10s at 55 ℃, 90s at 72 ℃, 30 cycles, 8min at 72 ℃ and 24h at 4 ℃.

The invention also discloses the application of the human intron-derived 27-base microRNA and the recombinant expression plasmid of the human intron-derived 27-base microRNA in preparing antihypertensive drugs.

Optionally, the drug comprises recombinant expression plasmid and liposome of human intron-derived 27-base microRNA, and the mass ratio of the recombinant expression plasmid to the liposome is 3: 1.

the invention also discloses the application of the human intron-derived 27-base microRNA and the recombinant expression plasmid of the human intron-derived 27-base microRNA in the preparation of an early hypertension warning chip.

Compared with the prior art, the invention can obtain the following technical effects:

the invention provides a novel molecule with a regulating effect on blood pressure, and the high-expression plasmid of the molecule is obtained by applying a gene recombination technology, can be used for relevant basic research on the hypertension, can be finally used for screening certain antihypertensive drugs and preparing an early warning chip for the hypertension, and has potential application prospect.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a construction diagram of intron-derived 27-base microRNA high-expression plasmid of the invention;

FIG. 2 is the blood pressure change after injecting the human intron-derived 27 base microRNA high expression plasmid;

FIG. 3 is a fluorescent picture of the vascular wall of a rat injected with the human intron-derived 27 base microRNA high expression plasmid; wherein, a represents a 27-base microRNA group, and b represents an empty plasmid group;

FIG. 4 is an immunohistochemical chart of rat vascular eNOS injected with human intron-derived 27 base microRNA high expression plasmid; wherein, a represents a 27-base microRNA group, and b represents an empty plasmid group;

FIG. 5 is an immunohistochemical graph of rat blood vessel SP1 injected with human intron-derived 27 base microRNA high expression plasmid; wherein, a represents a 27-base microRNA group, and b represents an empty plasmid group;

FIG. 6 shows the activity change of rat plasma eNOS enzyme injected with human intron-derived 27 base microRNA high expression plasmid;

FIG. 7 shows the variation of the plasma NO product content of rats injected with the human intron-derived 27-base microRNA high-expression plasmid.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.