阅读说明：本技术 miR-19b-1-5p的应用及提高其表达量的药物组合物 (Application of miR-19b-1-5p and pharmaceutical composition for improving expression level thereof ) 是由 叶文初 黄仕凤 赵贵芳 彭晓飞 于 2021-08-25 设计创作，主要内容包括：本发明公开了miR-19b-1-5p的应用,将miR-19b-1-5p应用在动脉粥样硬化过程中,用于缓解动脉粥样硬化的发生发展。本发明还公开了一种治疗动脉粥样硬化斑块的药物组合物,所述药物包括提高miR-19b-1-5p表达量的试剂或药物,以及至少一种药学上接受的载体或赋形剂。本发明能够应用在动脉粥样硬化治疗过程中,可广泛应用于医疗技术领域。(The invention discloses application of miR-19b-1-5p, and the miR-19b-1-5p is applied to the atherosclerosis process and is used for relieving the occurrence and development of atherosclerosis. The invention also discloses a pharmaceutical composition for treating atherosclerotic plaques, which comprises a reagent or a medicament for improving the expression level of miR-19b-1-5p, and at least one pharmaceutically acceptable carrier or excipient. The invention can be applied to the treatment process of atherosclerosis and can be widely applied to the technical field of medical treatment.)

The application of miR-19b-1-5p is characterized in that miR-19b-1-5p is applied to the atherosclerosis process and is used for relieving the occurrence and development of atherosclerosis.

2. The use of miR-19b-1-5p according to claim 1, wherein the miR-19b-1-5p consists of the following nucleotides: 5'-AGUUUUGCAGGUUUGCAUCCAGC-3' are provided.

3. A pharmaceutical composition for treating atherosclerotic plaques, comprising an agent or medicament for increasing the expression level of miR-19b-1-5p of claim 1 or 2, and at least one pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition of claim 3, wherein the drug is miR-19b-1-5p agomir.

5. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is administered in a unit dose, the route of administration includes enteral, parenteral, and the dosage form is a liquid, solid, or semi-solid dosage form; the pharmaceutical composition can be taken alone or combined with other therapeutic drugs and symptomatic drugs.

Technical Field

The invention relates to the field of medical treatment, in particular to application of miR-19b-1-5p in treatment of atherosclerosis.

Background

Atherosclerosis (As) is the pathological basis of many cardiovascular and cerebrovascular diseases, and seriously harms human health due to its high fatality rate and high disability rate. Disorders of lipid metabolism are one of the most important causes of As formation, in which macrophages take up excess lipids, forming foam cells, which in turn lead to atherosclerotic lesions. The research on the pathogenesis and the targeted therapy of As is always a hot point of attention of researchers at home and abroad. Lipid accumulation of THP-1 macrophage is a core factor of As occurrence and development, and how to inhibit macrophage lipid accumulation is an important entry point for interfering As progression.

With the development of new-generation sequencing technologies and the intensive research of genome, people are gradually aware that non-coding RNA (ncRNA) can play an important role in regulation through a specific regulation mechanism. According to the length of the ncRNA, the ncRNA can be divided into small RNA (microRNA, miRNA) and long non-coding RNA (lncRNA), wherein the miRNAs are single-stranded non-coding RNAs with the length of 19-25 nucleotides, are combined with 3' -UTR of targeted mRNA, inhibit the expression of a target gene at the level after transcription, and are the research hotspots in the fields of life science and medicine at present.

MiR-19b-1 is one of the important members in miR-19b-1-5p family, is often labeled as a tumor promotion factor or a tumor inhibition factor according to the property of the target gene acting on the MiR-19b-1-5p family, and plays an important role in the occurrence and development of malignant tumors. In recent years, abnormal expression of miR-19b-1-5p is found in various diseases, including cardiovascular diseases, Alzheimer's disease and the like, and the expression level of miR-19b-1-5p is closely related to occurrence and development of the cardiovascular diseases.

Therefore, how to treat atherosclerosis by using miR-19b-1-5p is a problem to be solved urgently.

Disclosure of Invention

Aiming at the problems, the invention provides a research on the small RNA gene miR-19b-1-5p in the preparation of atherosclerosis lipid accumulation medicines, and miR-19b-1-5p can effectively relieve the occurrence and development of atherosclerosis.

The technical scheme provided by the invention is as follows:

the application of the miR-19b-1-5p is to apply the miR-19b-1-5p in the atherosclerosis process and is used for relieving the occurrence and development of atherosclerosis.

Further, miR-19b-1-5p is composed of the following nucleotides: 5'-AGUUUUGCAGGUUUGCAUCCAGC-3' are provided.

The invention also provides a technical scheme that:

a pharmaceutical composition for treating atherosclerotic plaques, wherein the medicament comprises an agent or medicament for increasing the expression level of miR-19b-1-5p and at least one pharmaceutically acceptable carrier or excipient.

Further, the medicine is miR-19b-1-5p agomir.

Further, the pharmaceutical composition is administered by unit dose, the administration route comprises intestinal tract and parenteral tract, and the administration dosage form is liquid dosage form, solid dosage form or semisolid dosage form; the medicine composition is separately taken or is used together with other treatment medicines and symptomatic medicines.

Compared with the prior art, the invention has the advantages that:

drawings

FIG. 1. Effect of miR-19b-1-5p on lipid accumulation in THP-1 derived macrophages;

FIG. 2 miR-19b-1-5p at C57 and apoE, respectively^-/-Effect of expression in blood of mice;

FIG. 3 tail vein injection of miR-19b-1-5p agomir vs apoE^-/-Influence of mouse whole blood and expression level of miR-19b-1-5p in aortic vascular tissue;

FIG. 4 reduction of apoE by miR-19b-1-5p agomir^-/-Formation of aortic plaques in mice.

Detailed Description

The present invention is described in further detail below with reference to figures 1-4.

The invention provides a technology for effectively relieving the occurrence and development of atherosclerosis by miR-19b-1-5 p. To prove its effectiveness, the following experiments were performed.

Treating THP-1-derived macrophage by using oxidized low-density lipoprotein (ox-LDL), preparing foam cells, and detecting the expression level of miR-19b-1-5p by using a qRT-PCR technology. The result shows that in the ox-LDL treated THP 1-1-derived macrophage, the expression level of miR-19b-1-5p is obviously lower than that of a normal control group, and the intracellular lipid accumulation is promoted; in THP 1-1-derived macrophages, miR-19b-1-5p mimics are transfected, the expression level of miR-19b-1-5p is detected by using a qRT-PCR technology, and the lipid accumulation condition in cells is detected by using oil red O staining. The result shows that miR-19b-1-5p mimics are transfected to remarkably up-regulate miR-19b-1-5p expression and reduce intracellular lipid accumulation. The result shows that miR-19b-1-5p inhibits lipid accumulation in cells, and has important reference significance for guiding treatment of atherosclerosis.

The invention selects 6 male apoE with 6 weeks of age^-/-Mice and 6C 57 mice were used as controls, and were raised on high-fat diet for 8 weeks, blood was collected and miR-19b-1-5p expression level was detected by qRT-PCR technique. The result shows that the expression level of miR-19b-1-5p in a mouse disease model is obviously lower than that of a normal control group. The method has important reference significance for clinical diagnosis and guidance treatment of atherosclerosis.

To further explore the role of miR-19b-1-5p in the formation of atherosclerotic plaques, the invention uses tail vein injection of miR-19b-1-5p agomir (agonist) to ensure that the gene is highly expressed in mouse arterial vascular tissues. The experimental results show that apoE is obtained after molding^-/-The mice are injected with miR-19b-1-5p agomir in tail vein, which reduces the formation of aortic arch root plaque, and further proves that miR-19b-1-5p agomir plays a protective role in the formation process of atherosclerotic plaque.

The present invention will be further described below with reference to specific embodiments.

The miR-19b-1-5p provided by the invention consists of the following nucleotides: 5'-AGUUUUGCAGGUUUGCAUCCAGC-3' are provided. The medicine is a reagent or a medicine for improving the expression quantity of miR-19b-1-5p, in particular to miR-19b-1-5p agomir.

Then, the invention discloses a pharmaceutical composition for treating atherosclerotic plaques, and the pharmaceutical composition comprises an agent or a medicament for increasing the expression amount of miR-19b-1-5p and one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition can be administered in unit dosage form, and the administration route can be intestinal or parenteral.

In order to encapsulate the administration units, the pharmaceutically active ingredient can be mixed with diluents and glidants and the mixture can be placed directly into hard or soft capsules. Or mixing the effective components with diluent, binder, and disintegrating agent, making into granule or pellet, and placing into hard capsule or soft capsule. The diluent, the adhesive, the wetting agent, the disintegrating agent and the glidant used for preparing the tablets can also be used for preparing capsules.

The pharmaceutical composition of the present invention can be taken alone or in combination with other therapeutic agents or symptomatic drugs. When the pharmaceutical composition of the present invention is used in combination with other therapeutic agents, its dosage should be adjusted according to the actual situation.

In addition, the invention also provides application of the miR-19b-1-5p in treatment of atherosclerosis. The miR-19b-1-5p provided by the invention consists of the following nucleotides: 5'-AGUUUUGCAGGUUUGCAUCCAGC-3' are provided.

The medicine is a reagent or a medicine for improving the expression quantity of miR-19b-1-5p, and the specific medicine is miR-19b-1-5p agomir.

The miR-19b-1-5p provided by the invention is low expressed in ox-LDL induced THP-1-derived macrophages and the aortic blood vessels of an atherosclerotic disease model mouse, and miR-19b-1-5p imic [ mimics ] is injected into tail veins of the atherosclerotic disease model mouse to ensure that after the miR-19b-1-5p mimic [ mimics ] is highly expressed in the aortic blood vessels of the atherosclerotic disease model mouse, the aortic arch plaques of the disease model mouse are remarkably reduced, which indicates that miR-19b-1-5p can effectively reduce the area of atherosclerotic plaques, and an effective way is provided for treating cardiovascular diseases.

Atherosclerosis (As) is a chronic inflammatory pathophysiological process with disturbances in lipid metabolism, in which excessive accumulation of macrophage lipids is a key factor in its pathogenesis. Macrophages cause the macrophages to foam by ingesting excess lipids such as ox-LDL, causing the accumulation of foam cells in the arterial vessel wall and the formation of fatty streaks. The macrophage foaming process can be used As an important target in the As prevention and treatment process, so that the research on influencing factors and related molecular mechanisms of foam cell formation has very important significance for the prevention and treatment of As diseases.

The miRNA mimics is synthesized by a chemical synthesis method, and simulates miRNAs endogenous to organisms to regulate and control the biological function of a target gene. The miRNA agomir is double-stranded small RNA which is specially marked and chemically modified, and regulates and controls the biological function of a target gene by simulating endogenous miRNA. In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, cell and animal experiments are described in detail below as examples with reference to the accompanying drawings.

Example 1

And (3) carrying out oil red O staining to detect the lipid accumulation condition of the THP-1-derived macrophage.

THP-1 cells are plated in a 12-well plate, 50ng/mL Phorbol (phosphor 12-myristate 13-acetate, PMA) is added for treatment for 48h to induce the THP-1 cells to differentiate into macrophages, 100 mu g/mL oxidized low density lipoprotein (ox-LDL) is added for treatment for 24h, and the result of oil red O staining microscopy shows that the ox-LDL can remarkably promote the lipid accumulation of the THP-1-derived macrophages (figure 1).

In FIG. 1, THP-1 cells were recovered and cultured, the THP-1 cells were plated in 12-well plates, 50ng/mL Phorbol (Phorbol 12-myrristate 13-acetate, PMA) was added to induce the differentiation of THP-1 cells into macrophages, and 100. mu.g/mL oxidized low density lipoprotein (ox-LDL) was added for 24 hours. Wherein, A is the expression condition of ox-LDL to miR-19B-1-5p detected by RT-PCR, and B is oil red O staining (oil red O) microscopic examination. Inducing THP-1 cells to be differentiated into macrophages under PMA, and transfecting miR-19b-1-5p mimics (mimics); c is the expression condition of miR-19b-1-5p detected by RT-PCR to miR-19b-1-5 p. D is oil red O (oil red O) staining microscopy. NC mics: and in the control group, miR-19b-1-5p mimics are miR-19b-1-5p mimics. P <0.001, compared to Control group.

Total cellular RNA was extracted using Trizol reagent from SIAMA. The specific extraction steps are as follows:

(1) directly adding 1mL of Trizol lysis cells into a 6-hole culture plate, and blowing and uniformly mixing by using a pipette;

(2) placing the lysed sample or homogenate at room temperature for 5-10min to completely separate the nucleoprotein and the nucleic acid;

(3) adding 0.2ml chloroform, shaking vigorously for 15sec, standing at room temperature for 5min, and centrifuging at 12000rpm at 4 deg.C for 10 min;

(4) absorbing the upper aqueous phase, transferring to a clean 1.5mL EP tube without RNase, adding isopropanol with the same volume, mixing uniformly, standing at room temperature for 10min, centrifuging at 12000rpm at 4 ℃ for 10min, and removing the supernatant;

(5) the precipitate was washed by adding 1ml of 75% ethanol. Centrifuging at 12000rpm at 4 deg.C for 3min, removing supernatant, and drying at room temperature for 5-10 min;

(6) 30-50. mu.l of RNase-free ddH2O was added to dissolve the RNA sufficiently, and the resulting RNA solution was stored at-70 ℃ or used for subsequent experiments.

Reverse transcription PCR was performed using the One Step PrimeScript mirNacDNA Synthesis kit from Takara. Because miRNA is different from mRNA, miRNA has no Poly (A) structure, but miRNA in the transcription kit and small non-coding RNA thereof can be subjected to Poly (A) tailing reaction simultaneously, then tailed RNA and cDNA obtained by reverse transcription reaction of mRNA are subjected to reverse transcription reaction by using a Universal Adaptor Primer, and a binding site of a Uni-miR qPCR Primer is introduced, and any cDNA in a sample can be subjected to quantitative PCR reaction by using the position.

Example 2

And RT-PCR is used for detecting the expression condition of miR-19b-1-5p in THP-1-derived macrophages.

THP-1 cells were plated in 6-well plates, PMA (50ng/mL) was added to induce differentiation of THP-1 cells into macrophages, ox-LDL (100. mu.g/mL) was added for 24h, total RNA was extracted and reverse transcribed into cDNA. RT-PCR detection results show that ox-LDL down-regulates miR-19b-1-5p expression (figure 2), and that miR-19b-1-5p is possibly involved in inhibiting lipid accumulation in THP-1-derived macrophages.

In FIG. 2, 12 mice of 6 weeks old C57 and apoE-/-males were purchased, divided into 2 groups, and after 1 week of general diet acclimation, high fat diet (21% fat, 0.3% cholesterol) was kept for 8 weeks, sacrificed after anesthesia, and blood was taken. And detecting the expression condition of the miR-19b-1-5P in the mice by RT-PCR (reverse transcription-polymerase chain reaction). P <0.001, and comparing with the Control group.

The specific steps of transfection specifically include the following steps:

(1) siRNA synthesis: selecting a target sequence of miR-19b-1-5p from NCBI, and designing a mimics sequence;

(2) cell transfection: transfecting an miR-19b-1-5p mimics overexpression vector; the specific steps of transfection of the NC mimics and the miR-19b-1-5p mimics overexpression vector are as follows:

(2.1) plating THP-1 cells two days before transfection, adding PMA (50ng/mL) for treatment for 48h, and inducing the differentiation of the THP-1 cells into macrophages;

(2.2) preparation of transfection complexes: taking a 6-well plate as an example, adding 200pmoL of miR-19B-1-5p mimics into each well, diluting to 500 mu l of Opti MEM culture medium as solution A, dissolving 4 mu l of Lipofectamine TM 2000 in the Opti MEM culture medium as solution B, mixing the solution B for 5min, mixing the solution A and the solution B, standing for 15min, and adding into a cell culture plate;

(2.3) 5% CO at 37 ℃₂After the incubator is incubated for 4-6 hours, the incubator is replaced by a primary cell growth culture medium;

(2.4) one day after transfection, cells were treated with ox-LDL (100. mu.g/mL) for 24 h.

Oil Red O staining microscopy for post-transfection results

(1) Preparing an oil red O dye solution storage liquid: weighing 0.5g of oil red O powder, dissolving the powder in isopropanol, quantifying the volume of the liquid to 100mL, uniformly mixing, and storing at 4 ℃ in a brown bottle;

(2) preparing an oil red O dye solution working solution: mixing oil red O storage liquid and double distilled water according to the weight ratio of 3: 2, uniformly mixing, filtering for 2 times by using filter paper, and standing at room temperature;

(3) cell staining preparation: after the cells are prepared, removing the culture medium, washing with PBS for 2 times, and adding 4% paraformaldehyde for fixation;

preparation of frozen sections: fresh tissue (mouse heart) was fixed in 4% paraformaldehyde;

(4) dyeing: removing the fixing solution, washing with PBS for 2-3 times, dripping oil red O working solution onto cells or heart tissue, and staining for 30 min;

(5) color matching: rinsing with 60% isopropanol to remove excess oil red O dye;

(6) counterdyeing: dripping hematoxylin into heart tissue or cells, dyeing for 2min, removing redundant dye solution, and re-dyeing with running water;

(7) mounting and taking pictures by a microscope.

Example 3

And RT-PCR is used for detecting the expression condition of miR-19b-1-5p mimics in THP-1-derived macrophages.

Total RNA of THP-1 derived macrophages induced by ox-LDL was extracted and reverse transcribed into cDNA. RT-PCR detection results show that miR-19b-1-5p mimics are transfected, and miR-19b-1-5p expression is up-regulated (figure 3).

In FIG. 3, purchase of 6 week old apoE^-/-The male mice were divided into 2 groups of 12 mice, and after 1 week of adaptation to a common diet, the mice were kept on a high-fat diet (21% fat, 0.3% cholesterol) for 8 weeks, and were given to the tail vein of the mice, respectively, NC agomir and miR-19b-1-5p agomir were injected 1 time per week and 8 times in total. After anesthesia, the patient is sacrificed and blood, aortic vessels, heart, etc. are collected. RT-PCR detects the expression of mouse blood (A) and aortic vascular tissue (B) miR-19B-1-5 p. NC agomir: a control group, miR-19b-1-5p agomir, miR-19b-1-5p agonist. P<0.001, compared to Control group.

Example 4

And (4) observing the influence of miR-19b-1-5p on intracellular lipid accumulation by oil red O (O-staining microscopy).

miR-19b-1-5p mimics are transfected, and the result of treating THP-1-derived macrophages with ox-LDL for 24h shows that miR-19b-1-5p inhibits intracellular lipid accumulation (figure 4).

In fig. 4, a is the whole aorta from the aortic arch to the iliac artery branch, and the aortic arch and three branches (brachiocephalic trunk, left common carotid artery, left subclavian artery) thereof are observed with a stereomicroscope to form As plaque, and photographed. B is the middle section of the aortic arch, oil red O staining microscopic examination. C is frozen section, oil red O staining detects the lipid accumulation condition of aortic arch root.

Example 5

And RT-PCR is used for detecting the expression conditions of miR-146a-5p in blood and blood vessels of the mice.

Male C57 mice and male apoE-/-mice were purchased from south beijing violet technologies ltd, respectively, were raised on high fat diet for 8 weeks, mice were sacrificed under anesthesia, and blood was drawn into an anticoagulation tube. In this example, total RNA in blood was extracted from blood collection solution or plasma RNA extraction Kit (Beeby Biotechnology Co., Ltd.), and after reverse transcription of Mir-X mirNafirst-Strand Synthesis Kit into cDNA solution, Mir-X miRNA qRT-PCR TBAnd carrying out qRT-PCR detection on the miR-19b-1-5p by Kit. PCR using20 mu L of system, the reaction liquid system is prepared according to the proportion of the specification, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10sec, 40 cycles (denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 20sec), and 3 duplicate wells of the sample using U6 as an internal control, and relative expression analysis was performed using 2-. DELTA.CT as the average CT value. The qRT-PCR result shows that the expression level of miR-19b-1-5p in whole blood of high-fat fed apoE-/-mice is reduced compared with that of a normal control group (C57 mice).

Example 6

Establishing a mouse atherosclerosis model and interfering miR-19b-1-5p agomir.

12 apoE-/-male mice at 6 weeks of age, after 1 week of normal diet acclimation, the mice were fed a high fat diet (21% fat, 0.3% cholesterol), and were given a tail vein injection of NC agomir and miR-19b-1-5p agomir, respectively, 1 time per week for 8 weeks. Groups of apoE-/-mice were sacrificed and whole blood was taken, aortic vessels and hearts for the following experiments.

The experimental groups were as follows: (apoE)^-/-Male mice each group 6)

1) Control group: NC ago

2) miR-19b-1-5p group: miR-19b-1-5p agomir

Example 7

And (3) detecting the expression levels of whole blood and blood vessel tissue miR-19b-1-5p of mice injected with miR-19b-1-5p agomir in tail vein by qRT-PCR.

This example used a whole blood and animal tissue total RNA extraction kit (Beebel Biotech, Inc.) to extract whole blood and aortic vascular tissue total RNA, the specific procedures being performed according to the instructions. Reverse transcription and qRT-PCR detection were performed as in example 1. Results show that the content of miR-19b-1-5p in an animal disease model of atherosclerosis is reduced, and the miR-19b-1-5p agomir tail vein injection treatment can obviously improve the expression level of miR-19b-1-5p in blood and aortic tissues.

Example 7

The aortic arch and three branches (brachiocephalic trunk, left common carotid artery and left subclavian artery) thereof are observed by a stereomicroscope to form atheromatous plaques.

The result shows that by using miR-19b-1-5p agomir to inject apoE-/-mice into tail vein, the formation of atheromatous plaque can be obviously reduced.

Example 8

Oil red O detects aortic vascular plaque status in mice.

Adding oil red O dye solution into artery blood vessel of separated aortic arch and three branches (brachiocephalic trunk, left common carotid artery, left subclavian artery), incubating at room temperature for 20min, and washing with running water. Oil red O staining results show that by using miR-19b-1-5p agomir to inject apoE-/-mice into tail vein, the formation of atheromatous plaques can be remarkably reduced.

Example 9

And detecting the area of the aortic root plaque by oil red O staining detection.

The results of oil-red-O staining microscopic examination of the heart and the aortic root of a mouse by using a frozen section show that the formation of the atherosclerotic plaque can be obviously reduced by using miR-19b-1-5p agomir for tail vein injection of apoE-/-mice.

The present invention and its embodiments have been described above, and the description is not intended to be limiting, and what is shown in the drawings is only one embodiment of the present invention, and the actual inventive content is not limited thereto. In summary, those skilled in the art should appreciate that embodiments and examples similar to those of the present invention can be devised without departing from the spirit and scope of the invention.