Application of gene inhibitor in preparation of medicine for treating ischemic heart disease
阅读说明：本技术 一种基因抑制剂在制备缺血性心脏病治疗药物中的应用 (Application of gene inhibitor in preparation of medicine for treating ischemic heart disease ) 是由 刘婧星 王颖翠 于 2021-01-15 设计创作，主要内容包括：本发明提供了一种基因抑制剂在制备缺血性心脏病治疗药物中的应用,属于心脏医学技术领域。本发明所提供的基因抑制剂为下调基因RP11-71G12.1的基因抑制剂,该基因抑制剂能够有效的促进冠状动脉内皮细胞增殖和血管生成,因此可将该基因抑制剂用于制备治疗缺血性心脏病治疗药物,为治疗缺血性心脏病提供新的治疗方向。(The invention provides an application of a gene inhibitor in preparing a medicine for treating ischemic heart disease, belonging to the technical field of cardiac medicine. The gene inhibitor provided by the invention is a gene inhibitor for down-regulating the gene RP11-71G12.1, and the gene inhibitor can effectively promote the proliferation and angiogenesis of endothelial cells of coronary arteries, so that the gene inhibitor can be used for preparing a therapeutic drug for treating ischemic heart diseases, and provides a new therapeutic direction for treating the ischemic heart diseases.)
1. The application of the inhibitor of the gene RP11-71G12.1 in preparing the medicine for treating ischemic heart disease is characterized in that the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 3.
2. The application of an inhibitor of a gene RP11-71G12.1 in preparing a human coronary artery endothelial cell angiogenesis promoter, wherein the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sense strand sequence of the siRNA is shown as SEQ ID NO.2, and the antisense strand of the siRNA is shown as SEQ ID NO. 3.
3. The application of an inhibitor of a gene RP11-71G12.1 in preparing a protein expression promoter of a vascular endothelial growth factor A of a human coronary endothelial cell, wherein the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sequence of a sense strand of the siRNA is shown as SEQ ID NO.2, and the sequence of an antisense strand of the siRNA is shown as SEQ ID NO. 3.
Technical Field
The invention belongs to the technical field of cardiac medicine, and particularly relates to application of a gene inhibitor in preparation of a medicine for treating ischemic heart disease.
Background
Ischemic heart disease is also called coronary heart disease, and refers to heart disease caused by myocardial ischemia, hypoxia or necrosis due to stenosis or obstruction of an organ caused by coronary atherosclerosis, and includes asymptomatic myocardial ischemia, angina pectoris, myocardial infarction, ischemic heart failure and cardiac arrest. With the increasing living standard, ischemic heart disease has become the leading cause of death in the world. Patients with ischemic heart disease are mainly supplemented and compensated by the establishment of collateral circulation in the heart. In the case of acute vascular occlusion in a patient, treatment may be provided by in situ enlargement or prolonged growth of the vessel and dilation of the lumen. Whereas, when a patient is chronically occluded in the coronary arteries, treatment may be effected in a manner that promotes the production of capillaries by angiogenic factors.
Although. At present, a plurality of medicines for clinically treating the ischemic heart disease exist, but the pathological mechanism and the physiological mechanism of the ischemic heart disease are very complex, so the effect of single-target-point medicine treatment is not ideal. In this case, angiogenesis is a new and potentially promising research direction. The therapeutic angiogenesis can effectively promote the growth of blood vessels around ischemic cardiac muscle, establish collateral circulation and improve the symptoms of ischemia and anoxia of patients. Therefore, studying the angiogenesis of coronary arteries helps to further treat ischemic heart disease.
Disclosure of Invention
The invention aims to provide a coronary artery endothelial cell angiogenesis promoter.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides application of an RP11-71G12.1 gene as a target molecule in promoting angiogenesis, wherein the External Transcript ID of the RP11-71G12.1 gene is ENST00000457239.1, and the sequence of ENST00000457239.1 is shown in SEQ ID NO. 1.
In addition, the invention provides siRNA for treating ischemic heart disease, wherein the siRNA is siRNA of gene RP11-71G12.1, the sense strand sequence of the siRNA is shown as SEQ ID NO.2, and the antisense strand sequence of the siRNA is shown as SEQ ID NO. 3.
In addition, the invention provides application of an inhibitor of the gene RP11-71G12.1 in preparing a medicament for treating ischemic heart disease.
Preferably, the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 3.
In addition, the invention provides application of an inhibitor of the gene RP11-71G12.1 in preparing a human coronary artery endothelial cell proliferation promoter.
Preferably, the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 3.
In addition, the application of the promoter of the gene RP11-71G12.1 in preparing the human coronary artery endothelial cell angiogenesis promoter.
Preferably, the inhibitor of the gene RP11-71G12.1 is siRNA of RP11-71G12.1, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 3.
The invention has the beneficial effects that:
the invention provides siRNA capable of effectively inhibiting gene RP11-71G 12.1; secondly, the invention discovers that the down-regulation of RP11-71G12.1 can effectively promote the proliferation and angiogenesis of endothelial cells of coronary arteries for the first time, so that the siRNA of RP11-71G12.1 can be used for preparing the medicine for treating the ischemic heart disease, and a new treatment direction is provided for treating the ischemic heart disease.
Drawings
FIG. 1 is a knock-down effect assay of si-RP11-71G 12.1;
FIG. 2 downregulation of the effect of si-RP11-71G12.1 on cell proliferation of HCAEC cells;
FIG. 3 downregulation of the effect of si-RP11-71G12.1 on HCAEC cell vascularization;
FIG. 4 Down-regulates the effect of si-RP11-71G12.1 on vascular endothelial growth factor A expression.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
Design and verification of siRNA of RP11-71G12.1
(A) Design of RP11-71G12.1 siRNA
The siRNA designed according to the RP11-71G12.1 gene sequence is as follows:
sense strand: 5' -UUCUUCUUCUUCUUCUUCCCA-3, SEQ ID NO. 2;
antisense strand: 5'-GGAAGAAGAAGAAGAAGAAAG-3', SEQ ID NO. 3;
(B) verification of the inhibitory Effect of si-RP11-71G12.1
1. Extraction of RNA from human coronary endothelial cells (HCAEC cells) transfected with si-NC and si-RP11-71G12.1
(1) HCAEC cells were seeded in cell culture plates and si-NC and si-RP11-71G12.1 were transfected according to lip2000 instructions, with 3 replicates per set;
(2) after transfection for 48h, removing the culture medium, washing the cells for 3 times by using PBS, adding 1ml of Trizol into each hole, blowing and uniformly mixing, and standing for 5min at room temperature;
(3) transferring the cells into an EP tube, adding 200 mu l of chloroform, uniformly mixing, and standing at room temperature for 5 min;
(4) adjusting the parameters of the centrifuge to 4 ℃, 12000rpm/min, and centrifuging for 15 min;
(5) after centrifugation, carefully suck the upper aqueous phase into a new EP tube;
(6) adding isovoluminal precooled isopropanol, uniformly mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ at 12000rpm/min for 10 min;
(7) discarding the supernatant, and adding 75% ethanol prepared from DEPC water to wash and precipitate;
(8) adjusting the parameters of the centrifuge to 4 ℃, 8000rpm/min, and centrifuging for 5 min;
(9) the supernatant was discarded, dried in a clean bench until the ethanol evaporated completely, and 30. mu.l of DEPC water was added.
2. Reverse transcription to obtain cDNA
(1) Removal of genomic DNA
The reaction system is as follows:
reagent
Adding amount of
5×gDNA Eraser Buffer
2.0 μl
gDNA Eraser
1μl
Total RNA
1μg
RNase Free dH2O
up to 10 μl
The reaction conditions were as follows: stored at 42 ℃ for 2 minutes and 4 ℃.
(2) Reverse transcription reaction
The reaction system is as follows:
reagent
Adding amount of
Reaction solution of step (1)
10μl
PrimeScript RT Enzyme Mix I
1.0μl
RT Primer Mix
1.0 μl
5×PrimeScript Buffer 2
4.0 μl
RNase Free dH2O
4.0 μl
Total
20 μl
The reaction conditions were as follows: 15min at 37 ℃; 5s at 85 ℃; storing at 4 ℃.
3. Detecting the inhibitory effect by RT-PCR
(1) An RP11-71G12.1 primer is designed, and the primer sequence is as follows:
an upstream primer: 5'-AGCCTGTGTCAAGGTTCATGT-3', SEQ ID NO. 4;
a downstream primer: 5'-TCATACCATGCATTGCTGTTCA-3', SEQ ID NO. 5;
(2) and performing PCR reaction by taking GAPDH as an internal reference, wherein the PCR reaction system is as follows:
reagent
Adding amount of
cDNA
1μl
TaKaRa Ex Taq
0.5μl
10×Ex Taq buffer
5μl
dNTP mixture
4μl
Primer F
2μl
Primer R
2μl
ddH2O
35.5μl
PCR reaction procedure:
Step 1 94℃ 3min
Step 2 94℃ 1min， 60℃ 1min， 72℃ 1min， 35 Cycles
Step 3 72℃ 5 min
Step 4 4℃ forever；
(3) after the PCR is finished, the inhibition effect is detected by electrophoresis, and the result is shown in FIG. 1, and it can be seen from the figure that the expression of RP11-71G12.1 can be effectively inhibited by down-regulating RP11-71G 12.1.
Example 2
Down-regulation of RP11-71G12.1 promotes proliferation of endothelial cells of human coronary artery
(1) HCAEC cells were seeded in 96-well plates and transfected individually with si-NC and si-RP11-71G12.1, with 3 replicates per group;
(2) after 48h of transfection, the proliferation of the cells was measured and counted using CCK-8, and the statistics are shown in FIG. 2.
The results show that the down-regulation of RP11-71G12.1 can effectively promote the proliferation of human coronary endothelial cells (the relative proliferation rate of the si-RP11-71G12.1 group cells is 1.381 +/-0.046, which is obviously higher than that of the si-NC group, and the difference has statistical significance).
Example 3
Down-regulation of RP11-71G12.1 promotes angiogenesis of human coronary endothelial cells
(1) Adding 50 mul of Matrigel glue into a 96-well plate, slightly shaking the 96-well plate to enable the Matrigel glue to be paved in the well, and placing for 2 hours at 37 ℃;
(2) inoculating HCAEC cells into a 6-well plate, transfecting si-NC and si-RP11-71G12.1 respectively, and collecting the cells after transfecting for 48 hours;
(3) the cells were prepared as single cell suspensions and added to a 96-well plate supplemented with Matrigel gel at 1X 10 per well4(ii) individual cells;
(4) the 96-well plate was placed in a cell incubator, and after 24 hours of incubation, the vessel lumen formation was observed under an inverted microscope, and the results of the pictures are shown in FIG. 3.
The results show that the down-regulation of RP11-71G12.1 can obviously increase the number of HCARC cell lumen formation lumens, and the down-regulation of RP11-71G12.1 can effectively promote the angiogenesis of human coronary artery endothelial cells.
Example 4
Down-regulation of protein expression of RP11-71G12.1 promotion Vascular Endothelial Growth Factor A (VEGFA)
(1) HCAEC cells were seeded in 6-well plates, transfected with si-NC and si-RP11-71G12.1, triplicates per set;
(2) after transfection for 48h, the cells were washed with PBS, 100. mu.l of RIPA cell lysate was added to each well, and the cells were scraped off using a cell scraper and transferred to an EP tube;
(3) after cell lysis for 30min, placing in a centrifuge at 1200rpm/min, centrifuging for 10min, and taking the supernatant to a new EP tube;
(4) quantifying protein by using a BCA method, adding 5 Xloading buffer solution, and boiling for 5min with boiling water to obtain a protein sample;
(5) preparing 5% of upper layer glue and 12% of lower layer glue, assembling an electrophoresis tank, adding 20 mug of protein sample and a Marker indicator, and adding electrophoresis liquid for electrophoresis;
(6) after electrophoresis is finished, assembling an electric rotating clamp, and rotating the membrane for 1.5 hours at 200 mA;
(7) after the electrotransformation is finished, transferring the PVDF membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;
(8) after washing the membrane, incubating VEGFA and GAPDH primary antibody overnight at 4 ℃;
(9) after washing the membrane, incubating corresponding secondary antibody, and incubating for 1h in a shaking table at room temperature;
(10) in the dark, development exposure was performed, and the experimental results are shown in fig. 4.
The results show that the down regulation of RP11-71G12.1 promotes the protein expression of vascular endothelial growth factor A, and further prove that the down regulation of RP11-71G12.1 can effectively promote the angiogenesis of human coronary artery endothelial cells.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
Sequence listing
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