CN108524534A - MiR-214 antagonists are preparing the application in treating hypertension product - Google Patents

Abstract

The invention discloses the application of miR-214 antagonist in the preparation of products for treating hypertension. This application is based on the fact that miR-214 antagonists and their analogs can strongly competitively bind to mature miR-214 in vivo, prevent the complementary pairing of miR-214 and its target gene mRNA, inhibit miR-214 from functioning, and then increase monoxide Nitrogen synthase expression, so as to achieve the effect of lowering blood pressure. Compared with the prior art, the miR-214 antagonist and its analogues have obvious antihypertensive effect without obvious side effects.

Description

Application of miR-214 antagonist in the preparation of products for the treatment of hypertension

technical field

The invention relates to the field of biotechnology, in particular to the application of a miR-214 antagonist in the preparation of products for treating hypertension.

Background technique

Hypertension is the most common chronic disease and the main risk factor for cardiovascular and cerebrovascular diseases. Previous survey data show that the prevalence and absolute number of hypertension in China are increasing rapidly, and only 28.8% of people's blood pressure can be effectively controlled. Therefore, how to effectively treat and prevent hypertension is one of the current hot issues. In addition to non-drug interventions for hypertensive patients, the production of various antihypertensive drugs can undoubtedly effectively control blood pressure in patients. There are six common antihypertensive drugs, including diuretics, β-blockers, calcium antagonists, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists and α-blockers. However, the side effects of antihypertensive drugs in the course of treatment limit their wide application. For example, calcium antagonists (dihydropyridine dipines) have a rapid and powerful effect, and the individual differences in curative effect are small, but they will cause tachycardia in patients. Ankle edema, headache, dizziness, etc. Individual patients may experience nausea, stomach discomfort, and allergic reactions. Therefore, how to avoid toxic and side effects and to find new hypertension drugs are the key issues to be solved urgently.

Studies have found that the occurrence of hypertension is closely related to the dysfunction of vascular endothelial cells. Vascular endothelial cells are a layer of mononuclear cells between the blood flow and the vascular wall tissue. They can secrete a series of vasoactive substances such as NO, PGI2, and ET-1 through autocrine, endocrine, and paracrine pathways to regulate the blood vessels. Tonicity, anti-thrombosis, inhibition of smooth muscle cell proliferation and vascular wall inflammatory response and other functions. NO is the most important vasodilation factor produced by endothelial cells. It is produced by the NO synthase (eNOs) of endothelial cells acting on L-arginine. NO can diffuse to the smooth muscle cells of the vessel wall to activate ornithine cyclase and mediate cGMP Regulated vasodilation. Not only that, NO also has the functions of inhibiting platelet aggregation, inhibiting the adhesion of monocytes to endothelial cells, and inhibiting the proliferation of smooth muscle cells. However, when the vascular endothelium is affected by a series of harmful factors, the vasodilator factors released by the endothelial cells decrease and the vasoconstrictor factors increase, which breaks the homeostasis of blood vessels. Almost all patients with essential hypertension have vascular endothelial injury. Although the cause and effect of endothelial injury and hypertension are not yet clear, the current research results tend to believe that endothelial injury in hypertensive patients is secondary to hypertension. A sharp increase in blood pressure damages endothelial cells, which reduces the release of NO from endothelial cells and reduces the bioavailability of NO.

MicroRNA (miRNA, miRNA) is secreted in the body. Because it is widely expressed in multiple tissues and organs, participates in blood circulation, and can effectively regulate multiple targets at the same time, it has become a research hotspot in various fields. The regulatory role of miRNAs in the kidney and cardiovascular system has received extensive attention. It has been confirmed that miRNAs are involved in the regulation of cardiac hypertrophy, atherosclerosis, heart failure and congenital heart disease. Current research has found that there are more than 2,000 mature miRNAs in the human body, which regulate the expression and translation of about 60% of mRNAs.

In recent years, studies have found that miR-214 is mostly associated with malignant tumors, and miR-199a/miR-214 aggregates promote high glucose-induced peritoneal fibrosis by targeting E-Cadherin and Claudin-2. In addition, the regulation of miR-214 The abnormality also leads to hypoxic pulmonary hypertension, but its mechanism in systemic hypertension remains unclear.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide an application of miR-214 antagonist in the preparation of products for treating hypertension.

Application of miR-214 antagonist in the preparation of products for treating hypertension.

As an improvement, the hypertension is induced by angiotensin II.

As an improvement, the product is a medicine.

As an improvement, the sequence of the miR-214 antagonist is 5' ACUGCCUGUCUGUGCCUGCUGU 3'.

A drug for treating hypertension, the active ingredient is miR-214 antagonist and its analogues.

The analogs of the above-mentioned miR-214 antagonists can inhibit the function of miR-214 by strongly competitively combining with mature miR-214 in vivo, preventing the complementary pairing of miR-214 and its target gene mRNA.

Beneficial effect:

Our results showed that intraperitoneal injection of miR-214 antagonists in C57BL/6 mice could significantly reduce the systolic blood pressure (7-8 mmHg) of the mice, which had little effect on the body functions of the mice under normal physiological conditions. However, injection of miR-214 antagonist into C57BL/6 mice treated with angiotensin II can significantly reduce the systolic blood pressure (20-30 mmHg) of the mice, and the degree of blood pressure reduction is significantly higher than that of the physiological state. In addition, conditional miR-214 knockout mice treated with angiotensin II (AngII) in vascular endothelial cells found that the blood pressure of miR-214 knockout mice (miR-214 ^fl/fl ; Tek-Cre) was significantly lower than that of controls. mouse (miR-214 ^fl/fl ). This is consistent with the effect of intraperitoneal injection of miR-214 antagonists in C57BL/6 mice. At the same time, the levels of eNOS in the aorta of miR-214 knockout mice were significantly increased regardless of administration of miR-214 antagonist or endothelial cell conditional miR-214 knockout mice. In addition, the level of miR-214 in serum of hypertensive patients was also significantly higher than that of normal controls (as shown in Figure 11). Therefore, miR-214 may lower blood pressure in mice by targeting nitric oxide synthase (eNOS), which is an effective means to treat hypertension, thus providing the possibility for the later development of related hypertension drugs. Compared with the prior art, miR-214 antagonists and their analogs can lower blood pressure, indicating that miR-214 antagonists have the effect of lowering blood pressure; conditional knockout of miR-214 in vascular endothelium can significantly reduce the blood pressure induced by angiotensin Ⅱ. Hypertension, and the reduction of blood pressure may be mediated through the regulation of the increased expression of eNOS.

Description of drawings

Figure 1 shows the changes in blood pressure of C57BL/6 mice after intraperitoneal injection of mir-214 antagonists;

Figure 2 shows the changes in blood pressure of C57BL/6 mice after intraperitoneal injection of mir-214 antagonists to C57BL/6 mice treated with angiotensin II;

Figure 3 shows the changes in systolic blood pressure of vascular endothelial conditional miR-214 knockout mice;

Figure 4 shows the changes in the relative expression of miR-214 in the aorta of C57BL/6 mice after treatment with miR-214 antagonists;

Figure 5 shows the changes in the mRNA expression of total eNOS in the aorta of C57BL/6 mice after treatment with miR-214 antagonists;

Figure 6 shows the changes in aortic phosphorylation and total eNOS protein expression in C57BL/6 mice after treatment with miR-214 antagonists, wherein, 1-control group, 2-miR-214 antagonists;

Figure 7 shows the changes in aortic phosphorylation and total eNOS protein expression of C57BL/6 mice treated with angiotensin II after intraperitoneal injection of mir-214 antagonists. Angiotensin II, 4-administration of angiotensin II and miR-214 antagonists;

Figure 8 shows the changes in the expression level of miR-214 in the mouse aorta after conditional knockout of miR-214 in vascular endothelial cells;

Figure 9 shows the changes in the mRNA expression of total eNOS in mouse aorta after conditional knockout of miR-214 in vascular endothelial cells;

Figure 10 shows the changes in aortic phosphorylation and total eNOS protein expression in mice after conditional knockout of miR-214 in vascular endothelial cells, wherein, 5-control group, 6-miR-214 knockout mice;

Figure 11 shows that the expression level of miR-214 in serum of hypertensive patients was significantly higher than that of normal control group.

Detailed ways

The fermentation method of the present invention is described and illustrated in detail below in conjunction with specific examples. Its content is an explanation of the present invention rather than limiting the protection scope of the present invention.

Main instruments and reagents:

Small animal non-invasive blood pressure analysis system (BP-2000-M6-R4, Visitech Systems, USA); ABI Prism7500 fluorescence quantitative PCR instrument (Applied Biosystems, USA); angiotensin II (sigma, USA), miR-214 antagonist (Gimma, Shanghai); Trizol (Invitrogen); SYBR Prime Script RT-PCR kit (TaKaRa). Total eNOS antibody was purchased from Abcam (Cat. #ab73980); p -eNOS (Ser1179 Cat. # 9574) was purchased from CST (Danvers, MA).

Experimental animals:

Eight 8-week-old C57BL/6 mice (provided by Nanjing Institute of Model Animals) were divided into two groups. miR-214 ^fl/fl mice were imported from Nanjing Institute of Model Animals, and Tek-Cre transgenic mice were imported from Jackson Corporation of the United States. A conditional miR-214 knockout mouse model of vascular endothelial cells (miR-214 ^fl/fl ; Tek-Cre) has been successfully prepared. Eight weeks-old control group (miR-214 ^fl/fl ) and miR-214 knockout mice (miR-214 ^fl/fl ; Tek-Cre) were divided into two groups.

Statistical methods: Experimental data are expressed as mean ± standard error. Statistical software SPSS11.5 was used to analyze the differences among the groups by t test, and P <0.05 was considered significant.

Example 1

Take 8-week-old C57BL/6 mice and divide them into two groups. After blood pressure monitoring training, record the systolic blood pressure of the tail of the mice for about 3 days, inject miR-214 antagonist (10mg/kg) intraperitoneally, and measure blood pressure Determination.

The results are shown in Figure 1. The blood pressure of the mice given miR-214 antagonists was significantly lower than that of the control group, especially on the first day. After a delay, the blood pressure of the mice in the experimental group gradually increased. The control group was close, indicating that the effect of the miR-214 antagonist can last for about four days. In the figure, days (-1)-(-3) represent the basal blood pressure values of mice without injection of miR-214 antagonists.

Example 2 Effect of miR-214 antagonist on AngII-induced hypertension model

Eight-week-old C57BL/6 mice were divided into two groups. After blood pressure monitoring training, the systolic blood pressure of the tail of the mice was recorded for about 4 days to prepare an AngII-induced hypertension model. After the mice were anesthetized with 3% pentobarbital, a 14-day AngII slow-release osmotic micro-vacuum pump (1.4mg/kg/d) was implanted in the subcutaneous area in the middle of the scapula on both sides, and sutured, and the blood pressure was monitored on the next day . Three days after pumping, miR-214 antagonist (10 mg/kg) was injected intraperitoneally, and blood pressure was measured the next day. The miR-214 antagonist was injected intraperitoneally once every 4 days.

The results are shown in Figure 2. The blood pressure of mice given miR-214 antagonists was significantly lower than that of the control group (that is, only angiotensin II was given), and the results were particularly obvious on the first day. The blood pressure of the mice gradually increased, and was close to that of the control group after four days, indicating that the effect of the miR-214 antagonist can last for about four days. In the figure, days (-1)-(-4) represent the basal blood pressure values of mice without administration of miR-214 antagonists and AngII.

Example 3 The effect of vascular endothelial conditional knockout of miR-214 on AngII-induced hypertension model

The 8-week-old vascular endothelial cells conditional miR-214 knockout mice (miR-214 ^fl/fl ; Tek-Cre) that had undergone blood pressure monitoring training were used to prepare the AngII-induced hypertension model, and miR-214 ^fl/ The hypertensive model prepared from ^fl mice was used as the control group. After the mice were anesthetized with 3% pentobarbital, a 14-day AngII slow-release osmotic micro-vacuum pump (1.4mg/kg/d) was implanted in the subcutaneous area in the middle of the scapula on both sides, and sutured, and the blood pressure was monitored on the next day .

The BP2000 blood pressure analysis system was used to monitor the blood pressure of the mice. All the mice had to adapt to the blood pressure analyzer for at least one week before blood pressure measurement. During the measurement, the instrument parameters were set to three cycles, each cycle was repeated 15 times. Each mouse can only be measured once a day, and at least three days can be used as valid data.

The results are shown in Figure 3. The blood pressure of the two groups of mice increased significantly the next day, but compared with the control group, the increase in blood pressure of the miR-214-knockout mice was lower than that of the control group (the mice in the control group increased 30mmHg higher, and the blood pressure of the miR-214 knockout mice increased by about 10mmHg), and over time, the blood pressure of the mice in the control group continued to rise, while the blood pressure of the mice in the miR-214 knockout group was basically Maintain at the same level (around 130mmHg). Thirteen days after the final modeling, the blood pressure of the two groups of mice showed a downward trend due to the decrease in the content of AngII. In the figure, days (-1)-(-4) represent the basal blood pressure values of mice without AngII administration.

Example 4

The mice were anesthetized with 3% pentobarbital and killed immediately, and the thoracoabdominal aorta was separated and placed on ice. The connective tissue around the blood vessels was stripped under a microscope, and the transparent blood vessels were taken and extracted by adding Trizol (TaKaRa) Tissue total RNA. ABIPrism 7500 fluorescent quantitative PCR instrument was used for amplification.

The primer kit Bulge-loop ^TM miRNA qRT-PCR Primer Sets for miR-214 quantitative PCR was designed and synthesized by Guangzhou Ruibo (Ribo, Guangzhou).

eNOS upstream primer: 5'-CGTCCTGCAAACCGTGCAGA-3',

Downstream primer: 5'-TCCTGG GTGCGCAATGTGAG-3';

GAPDH upstream primer: 5'-GTCTTCACTACCATGGAGAAGG-3',

Downstream primer: 5'-TCATGGATGACCTTGGCCAG-3'.

PCR reaction conditions: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, extension at 60°C for 1 minute, 40 cycles in total; 3 parallel wells were set for each sample to take the average value. According to the fluorescence (SYBR Green) reaction curve, the ΔCt method was used to analyze the difference between the expression of the sample target gene and the internal reference gene (U6 or GAPDH), where the Ct value was the number of cycles experienced when the fluorescence signal in each reaction tube reached the set threshold, and ΔCt was The difference between the target gene Ct value and the reference gene (U6 or GAPDH) Ct value. According to the principle of linear amplification of PCR in an ideal state, 2-ΔCt was used to calculate the fold change of the expression of the target gene relative to the internal reference gene.

After the mouse blood pressure was measured, the thoracoabdominal aorta of the mouse was taken to extract the total mRNA, which was reverse transcribed into cDNA and amplified by ABI Prism 7500 fluorescent quantitative PCR instrument. It can be seen from Figure 4 and Figure 5 that miR-214 antagonists can significantly increase the expression level of total eNOS while reducing the expression level of miR-214. The expression level of total eNOS in the aorta of mice except miR-214 was significantly lower than that of the control group.

Example 5

The mice were anesthetized with 3% pentobarbital and killed immediately, and the thoracoabdominal aorta was separated and placed on ice. The connective tissue around the blood vessel was stripped under a microscope, and the transparent blood vessel was added to RIPA lysate to separate the protein. Reserve after denaturation. Samples were loaded on 8% polyacrylamide gel for SDS-PAGE electrophoresis with a constant flow of 300mA for 1.5 hours. Blocking solution (0.01% n-butanol, 0.02% Tween, 5% skimmed milk powder, 0.02% sodium azide w/v) to block PVDF membrane for 2 hours, TBST for 1 hour, combined with I antibody (GAPDH monoclonal antibody, the concentration is 1: 2000; eNOS monoclonal antibody at a concentration of 1:500; p -eNOS antibody at a concentration of 1:500), overnight at 4°C, rinsed three times with TBST, and added horseradish-enzyme-labeled goat anti-rabbit IgII antibody (1: 2000) were incubated at room temperature for 1 h, washed 3 times with TBST, and developed.

Take transparent blood vessels and add RIPA lysate to separate proteins, and run SD-PAGE gel after denaturation. It can be seen from Figure 6 and Figure 7 that miR-214 antagonists can increase the phosphorylation of C57BL/6 mouse aorta and the protein expression level of total eNOS. In addition, Figure 10 shows that the conditional knockout of miR-214 in the vascular endothelium The levels of phosphorylation and total eNOS in mouse aortic blood vessels also showed an increasing trend, indicating that miR-214 increased the expression level of total eNOS, and then increased the phosphorylation level of eNOS.

sequence listing

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Claims (5)

1.miR-214 antagonists are preparing the application in treating hypertension product.

2. application of the miR-214 antagonists according to claim 1 in preparing treatment hypertension product, feature exist In the hypertension is caused by hypertensinⅡinduction.

3. application of the miR-214 antagonists according to claim 1 in preparing treatment hypertension product, feature exist In the product is drug.

4. application of the miR-214 antagonists according to claim 1 in preparing treatment hypertension product, feature exist In the sequence of the miR-214 antagonists is 5 ' ACUGCCUGUCUGUGCCUGCUGU 3 '.

5. a kind of drug for treating hypertension, which is characterized in that active constituent is miR-214 antagonists and the like.