CN108796070A - Purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit - Google Patents

Purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit Download PDF

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CN108796070A
CN108796070A CN201810775428.1A CN201810775428A CN108796070A CN 108796070 A CN108796070 A CN 108796070A CN 201810775428 A CN201810775428 A CN 201810775428A CN 108796070 A CN108796070 A CN 108796070A
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陈文娜
郭隽馥
成泽东
王丹
李曦明
孙宏伟
王继伟
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Liaoning University of Traditional Chinese Medicine
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Abstract

The invention belongs to biomedical professional domains, and in particular to purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit.The diagnostic kit is that the expression quantity using miR-125a-3p albumen as marker to it in blood plasma is detected, and is early diagnosed to angiocardiopathy.Meanwhile to the function controlling significant effect of mononuclear macrophage, implemented effectively and speedily promote its phagocytosis to vascular lipids composition, and then control the level of inflammation of hyperlipidemia and artery sclerosis, there is extraordinary application prospect.

Description

Purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit
Technical field
The invention belongs to biomedical professional domains, and in particular to miR-125a-3p is preparing cardiovascular disease diagnosis examination Purposes in agent box.
Background technology
Angiocardiopathy is to endanger the serious disease of human health, is murderous one of the main reasons.Because of its morbidity Rate height, disability rate height, death rate height, high recurrence rate, complication are more, seriously threaten the life and health of the mankind.Currently, in China In the death notation of resident, angiocardiopathy has become No. second killer for being only second to malignant tumour, and it is annual separately have number with The people of ten thousand meters leads to deformity because suffering from the disease.Atherosclerosis (atherosclerosis, AS) is the common disease, more in the whole world Morbidity, is the Etiological for leading to a variety of diseases such as coronary heart disease, cerebral apoplexy, severely compromises human health and existence matter Amount, it has also become the focus of attention of cardiovascular field.Its Etiological is since many reasons cause excess fat in the blood vessels Wall deposits, and leads to vessel wall thickening, influences blood flow, while lipid excessive in blood can activate inflammatory reaction, gradually invade Vascular wall is lost, atherosclerosis is caused.With the exacerbation of the artery sclerosis state of an illness, vascular wall can be hardened further, mistake in blood More lipids can cause the obstruction of blood vessel, prevent blood from normal through and form thrombus, to cause apoplexy, myocardial infarction. Angiocardiopathy develops to the later stage, since to pump blood for a long time bad for heart, systemic organs all may extravasated blood anoxic and by different journeys The damage of degree, such as there are pulmonary infection, hepatic sclerosis, hypertension, renal failure multiple complications, these symptoms can aggravate again in turn The state of an illness of angiocardiopathy causes body even more serious damage.
MicroRNAs（It is abbreviated as miRNAs, miRs）It is that a kind of length is non-for the endogenous of 18~25 nucleotide single-chains Encoding tiny RNA is generated by precursor under the processing of enzyme, and the sequence of precursor is generally 70~120 nucleotide.MiRNA is acted on Mechanism is mainly shown as degradation or inhibits target gene.MiRNA5 ' holds 2-8 base to be referred to as miRNA seed sequences.Pass through seed The areas 5'-UTR and 3'-UTR ˊ of sequence and said target mrna interact, and gene or protein expression are adjusted in transcription or translation skill, Various biological process is participated in, evolutionary process is conservative.Each miRNA can be positioned at a variety of mRNA, it is inhibited to transcribe or make It is degraded, therefore a variety of transcriptons may be fine-tuned by miRNA, so that transcription-translation system is effectively operated, to make Cell is able to rapidly and efficiently control various physiology or pathologic process.Generally believe that the function of miRNA is to participate in life at present Some basic processes such as growth and development, orga- nogenesis, hematopoiesis, Cell apoptosis and proliferation, stress reaction and tumour generate etc..Cause MiRNA is stable in the presence of in the body fluid and hematological system of people, has repeatability, can from peripheral blood stable detection, can be with Biological marker as disease.
Having found that it is likely that for angiocardiopathy correlation miRNA generates great influence to the related gene treatment of the disease.It is logical The miRNA Differential expression analysis to cardiovascular patient and normal population is crossed, can identify to obtain relevant sensitivity miRNA, And then inhibited or by viral vectors or plasmid vector overexpression by introducing the GEM 132 of the miRNA complementations of specificity Method, regulate and control the expression quantity of correlation miRNA or its precursor, to achieve the purpose that treatment angiocardiopathy.Thus, it is found that special The miRNA of opposite sex expression has very important significance for the diagnosing and treating of angiocardiopathy.Studies have shown that a variety of serum MicroRNAs and artery sclerosis occurrence and development process are closely related, and its expression shows organ specificity and tissue Specificity.The variation of specific microRNA express spectras can prompt specific pathology, physiology course in vivo.Research confirmation, MiR-21-3p can promote vascular smooth muscle cell proliferation, to promote atherosclerosis；MiR-155 and oxidized low density fat egg It is white to wait vasculitics reaction closely related；The close phase of expression of miR-126-3p and vascular stability, integrality and adhesion molecule It closes；MiR-210 can promote vascular proliferation under anaerobic condition, be anoxic specificity microRNA；MiR-221-3p and miR- 222-3p has anti-angiogenic proliferation function, inhibits endothelial cell migration proliferation and angiogenesis function.But miR-125a-3p is in the heart The effect being especially in hyperlipidemia and atherosclerosis in angiosis has no document report.
Hyperlipidemia, atherosclerosis patient due to lipids contents it is higher, the inflammatory cell such as monokaryon in blood is huge Phagocyte is acted on by chemotactic factor (CF) focuses on impaired vascular wall, swallows lipide component, forms foam cells.When monokaryon macrophage Cell by such environmental effects immune function weaken, cannot timely and effective removing endovascular lipide component when, will result in fat Matter is gathered and artery sclerosis accelerated development, eventually leads to the serious consequences such as apoplexy, myocardial infarction.Seminar's early period where inventor Studies have shown that the expression quantity specificity of hyperlipidemia, miR-125a-3p in Atheromatosis human peripheral blood mononuclear cell Ground reduces, can be as the marker of hyperlipidemia and atherosclerosis using this characteristic performance；Due to MiR-125a-3p has apparent adjustment effect for mononuclear macrophage function, therefore viral vectors or plasmid can be used to carry The method that body is overexpressed, makes the miR-125a-3p expression quantity in patient's mononuclear macrophage increase, can normally swallow With handle endovascular lipide component, and then efficiently control the development of hyperlipidemia and the artery sclerosis state of an illness.miR-125a-3p The discovery of this function suffers from the diagnosing and treating of the angiocardiopathies such as hyperlipidemia, atherosclerosis of crucial importance Meaning.
People source miR-125a-3p provided by the present invention（has-miR-125a-3p,MIMAT0004602）With mouse source miR- 125a-3p（mmu-miR-125a-3p, MIMAT0004528）Ripe body includes following sequence：
5’- ACAGGUGAGGUUCUUGGGAGCC -3’(SEQ ID1)。
mir-125a（MI0000151）Loop-stem structure sequence is：
CUGGGUCCCUGAGACCCUUUAACCUGUGAGGACGUCCAGGGUCACAGGUGAGGUUCUUGGGAGCCUGG(SEQ ID2)。
Invention content
The purpose of the present invention is to provide a kind of new applications of miR-125a-3p.The present inventor's premenstruum (premenstrua) is studied, and finds such as Lower SEQ ID1, SEQ ID2 can be used in hyperlipidemia and the diseases related marker of atherosclerosis, especially miR- 125a-3p mimic achieve good technique effect for improving hyperlipidemia and atherosclerosis microenvironment, from And complete the present invention.
Present invention offer adopts the following technical scheme that：
Purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit, the diagnostic kit are with miR-125a-3p Albumen is detected its expression quantity in blood plasma as marker, is early diagnosed to angiocardiopathy.
The diagnostic kit contains following component：For miR-125a-3p 10 × miRNA reverse transcriptase primers, 20 × MiRNA sense primers, 20 × miRNA universal primers, 4 × One step miRNA RT Solution, 5 × SYBR Green qPCR Mix、50×ROX Reference Dye。
The miR-125a-3p can inhibit the expression of mononuclear macrophage inflammatory factor, cardiovascular as treatment is prepared Purposes in disease medicament, the angiocardiopathy are hyperlipidemia or atherosclerosis.
Specifically, the present invention is with human peripheral blood mononuclear cell, C57BL/6 wild-type mices（WT mouse）, ApoE genes Knock-out mice（ApoE-/-Mouse）Heart, the aorta vessel of hyperlipemia model are that material carries out the real-time glimmering of miRNA molecule Fluorescent Quantitative PCR is analyzed.Using conventional molecular biological technology, total serum IgE is extracted from cell and tissue, by the primer of specificity Carry out reverse transcription and PCR amplification.Using fluorescence real-time quantitative（qRT-PCR）Technology testing result shows miR-125a-3p in height Its expression quantity is substantially reduced under smectic state；Inhibit the expression of miR-125a-3p, monokaryon with the GEM 132 of complementary specificity Cellular inflammation factor level up-regulated expression.And the miR-125a-3p precursor RNAs of synthesis is used to increase intracellular miR-125a-3p Expression, can significantly reduce the expression of inflammatory factor.
Hsa-miR-125a-3p target genes are predicted by websites such as miRBase, TargetScan, miRPathDB Analysis, the results showed that miR-125a-3p regulation and control target gene include mainly：CCL4,NCR2,IL10,TLR9,IL-33,IL- 1R1, CCR3, IL-4R, CCL25 etc. and the relevant cell factor of inflammation.
Therefore the present invention provides miR-125a-3p in hyperlipidemia and the diseases related diagnostic kit of atherosclerosis In application, when sequence SEQ ID1, the SEQ ID2 of miRNA are respectively applied to subject, to the function of mononuclear macrophage Regulating effect is notable, implemented effectively and speedily promotes its phagocytosis to vascular lipids composition, and then controls hyperlipidemia and move The level of inflammation of arteries and veins hardening.
Description of the drawings
Fig. 1 is hyperlipidemia, in atherosclerotic's peripheral blood mononuclear cells miR-125a-3p expression.
Fig. 2 is ApoE-/-The expression of mouse model blood vessel and heart tissue miR-125a-3p.
The expression that Fig. 3 is miR-125a-3p in mononuclear macrophage under environment high in fat.
Fig. 4 is influences of the miR-125a-3p mimic to inflammatory factor level in mononuclear macrophage under environment high in fat.
Specific implementation mode
It prepares in a kind of cardiovascular disease diagnosis kit, which contains following component：For miR-125a-3p's 10 × miRNA reverse transcriptase primers, 20 × miRNA sense primers, 20 × miRNA universal primers, 4 × One step miRNA RT Solution、 5×SYBR Green qPCR Mix、50×ROX Reference Dye。
The present invention is further elaborated by the following examples.
RNA sequence disclosed in this invention can be obtained with artificial synthesized method or other biological methods.
Example 1：Healthy People and miR-125a-3p expression quantity in hyperlipidemia disease human peripheral blood mononuclear cell Compare.
1. human peripheral blood mononuclear cell detaches
Using Ficoll density-gradient centrifugation method separating periphery blood monocytic cells, concrete operations mode is as follows：
（1）10ml anticoagulant heparin venous wholes are transferred in 50ml centrifuge tubes, 10ml phosphate buffers are added（PBS）Solution is dilute It releases, gently mixing；
（2）Two 15ml centrifuge tubes are taken, 5ml Ficoll solution is first added.Then diluted blood is gently added to two centrifugations The Ficoll liquid upper layer of pipe has to softly, avoid two kinds of solution from mixing, every centrifuge tube adds 10ml diluted bloods Liquid；
（3）800g is centrifuged 30 minutes, pays attention to having to be arranged to no break in reduction of speed setting, centrifugation finishes, and three are divided into pipe Layer, upper layer are blood plasma and PBS, and lower layer is mainly red blood cell and granulocyte.Have at upper and lower bed boundary and one is with mononuclearcell Main white cloud and mist layer narrow band is drawn the confluent monolayer cells in another clean 15ml centrifuge tubes with suction pipe.
（4）5 times of PBS with upper volume are added, room temperature centrifuges 800g × 5 minute, and washing cell is twice.
（5）Erythrocyte cracked liquid is added after cell is resuspended, 800g × 5 minute are centrifuged after being placed at room temperature for 3 minutes.
（6）Supernatant is abandoned after centrifugation, the RPMI1640 culture solutions without serum is added, cell is resuspended.Room temperature centrifuges 800g × 5 Minute, wash cell twice with the RPMI1640 without serum.It is eventually adding the RPMI1640 cultures containing 10% calf serum Cell is resuspended in liquid.
（7）It takes a drop cell suspension to drip 0.2% with one and expects that blue dye liquor mixes, on blood counting chamber, it is total to count cell It counts and detects cell viability.Living cells percentage can continue subsequent experimental 95% or more.
2. cell total rna extracts (TRIzol methods)
（1）Harvest cell 1-5 × 107Cell moves into 1.5ml centrifuge tubes, and 1ml Trizol are added, and mixing is stored at room temperature 5min。
（2）0.2ml chloroforms are added, vibrate 15s, stand 2min.
（3）4 DEG C of centrifugations, 12000g × 15min take supernatant.
（4）0.5ml isopropanols are added, by the gently mixing of liquid in pipe, are stored at room temperature 10min.
（5）4 DEG C of centrifugations, 12000g × 10min abandon supernatant.
（6）1ml75% ethyl alcohol is added, gently washing precipitation.4 DEG C, 7500g × 5min abandons supernatant.
（7）Sample is dried, suitable DEPC water dissolutions (65 DEG C of dissolution 10-15min) are added.Measure OD260 and OD280 Value, tentatively concludes RNA mass.
（8）Total RNAs extraction is using the DNA enzymatic I processing of no RNA enzyme, QIAGEN RNeasy kits total serum IgEs, in detail Operating principle and method are shown in kit specification.
3.qRT-PCR detects the expression quantity of intracellular miR-125a-3p.
Using person monocytic cell's total serum IgE as template, with the primer amplification miR-125a-3p genes in this kit and carry out The expression quantity of intracellular miR-125a-3p is quantitatively detected, detailed operating principle and method are shown in kit specification.
4. statistical procedures
All data are indicated with mean ± standard deviation, are compared between multigroup sample using variance analysis, are compared use between two groups Kruskal-Wallis is examined, p<0.05 thinks with statistical significance.
The results show that in hyperlipidemia, atherosclerotic's peripheral blood mononuclear cells miR-125a-3p expression quantity It is substantially reduced compared with normal healthy people（p<0.01）, there is statistical significance.The result prompts, in peripheral blood in monocyte MiR-125a-3p expressions can be shown in Fig. 1 as the marker of diagnosis hyperlipidemia, atherosclerosis, concrete outcome.
Example 2：WT mouse and ApoE-/-The difference of different tissues of mice miR-125a-3p expression quantity.
1. conventional method detaches WT mouse and ApoE-/-The heart and aorta of mouse.
2. total tissue RNA extracts (TRIzol methods)
It takes 50-100mg tissues to set in 1.5ml centrifuge tubes, 1ml Trizol is added and are fully homogenized, are stored at room temperature 5min.Follow-up step The rapid b-h with reference in step (2) in embodiment 1.
3.qRT-PCR detects the expression quantity of miR-125a-3p in different tissues
Using mouse heart and aorta vessel total serum IgE as template, qRT-PCR detections are carried out（Method and step in embodiment 1 the same as walking Suddenly（3）.
4. statistical procedures
Using Prism Graphpad softwares it is for statistical analysis to result and mapping, all data are with mean ± standard deviation table Show, compares between two samples and examined with t, with p<0.05 has statistical significance.
The results show that as hyperlipidemia and the ApoE of artery sclerosis research model-/-Mouse, aorta vessel MiR-125a-3p expression quantity is significantly lower than normal wild type WT mouse（p<0.01）, there is statistical significance；And heart tissue MiR-125a-3p expression quantity does not have notable difference（p>0.05）, should the result shows that miR-125a-3p expression and regulation and control there are Certain tissue specificity, i.e., it is more notable compared with heart tissue in the reduction degree of hyperlipidemia and atherosclerotic blood vessel tissue, Concrete outcome is shown in Fig. 2.
Embodiment 3：Detect the expression quantity and cellular inflammation factor expression water of the intracellular miR-125a-3p of RAW264.7 It is flat.
1. detection state culture WT and ApoE high in fat-/-The expression quantity of the intracellular miR-125a-3p of mouse RAW264.7
（1）Cell inoculation is in 24 well culture plates, per hole cell number 4 × 104。
（2）Ox-LDL is added in the DMEM culture solutions without lipid makes its concentration reach 50ug/ml.
（3）It puts it into the cell incubator of standard and is incubated 72 hours.
（4）It collects cell total rna and carries out the expression quantity that qRT-PCR detects intracellular miR-125a-3p.Specific method reference Embodiment 2.
2. adjusting the expression of intracellular miR-125a-3p with miR-125a-3p mimic
（1）Cell inoculation is in 24 well culture plates, per hole cell number 4 × 104。
（2）By the OPTI-MEM I culture solutions of the transfection reagent siPORT NeoFX (AM4510, Ambion) of 1ul and 25ul (Invitrogen) mixing is positioned over incubation at room temperature 10 minutes.
（3）MiR-125a-3pmimic is diluted with OPTI-MEM I culture solutions, and incubation at room temperature 10 is positioned over after the two mixing Minute.As a control group with non-functional miRNA sequence processing cell (Scrambled miRNA, unrelated sequences miRNA).
（4）The miR-125a-3p mimic and transfection reagent that mixing has diluted, being positioned over incubation at room temperature makes its shape in 10 minutes At transfection composite.
（5）Tissue culture plate is added in culture solution containing transfection composite, is incubated jointly with cell 72 hours.
（6）It collects cell total rna and carries out the expression quantity that qRT-PCR detects intracellular miR-125a-3p, specific method reference Embodiment 2.
（7）It collects cell total rna progress qRT-PCR and detects intracellular inflammatory factor TNF-a, IL-10, IL-6, iNOS Expression quantity, specific method is with reference to embodiment 2.
3. statistical procedures
All data are indicated with mean ± standard deviation, are compared between multigroup sample using variance analysis, are compared use between two groups Kruskal-Wallis is examined,p<0.05 thinks with statistical significance.
The results show that state high in fat（50ug/ml ox-LDL）Cultivate mononuclear macrophage（RAW264.7）Interior miR- The expression quantity of 125a-3p reduces, *p<0.01, it is statistically significant compared with negative control group；MiR-125a-3p mimic can To increase the expressions of miR-125a-3p in the cell, *p<0.01, statistically significant compared with negative control group, #p< 0.01, it is statistically significant compared with ox-LDL control groups（Fig. 3）.MiR-125a-3p mimic can reduce state high in fat （50ug/ml ox-LDL）Cultivate mononuclear macrophage（RAW264.7）Interior level of inflammation, *p<0.01, with Normal group （control）Compared to statistically significant, #p<0.01, with model control group（ox-LDL）Compared to statistically significant, Fig. 4 is seen.
SEQUENCE LISTING
<110>Liaoning University of TCM
<120>Purposes of the miR-125a-3p in preparing cardiovascular disease diagnosis kit
<130> 2
<160> 2
<170> PatentIn version 3.3
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<212> DNA
<213>It is unknown
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acaggugagg uucuugggag cc 22
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<213>It is unknown
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Claims (5)

1.miR-125a-3p the purposes in preparing cardiovascular disease diagnosis kit.
2. purposes according to claim 1, which is characterized in that the diagnostic kit is made with miR-125a-3p albumen Its expression quantity in blood plasma is detected for marker, angiocardiopathy is early diagnosed.
3. purposes according to claim 1, which is characterized in that the diagnostic kit contains following component：For miR- 10 × miRNA reverse transcriptase primers, 20 × miRNA sense primers, 20 × miRNA universal primers, the 4 × One of 125a-3p step miRNA RT Solution、 5×SYBR Green qPCR Mix、50×ROX Reference Dye。
4.miR-125a-3p as the purposes treated in cardiovascular disease medicine is prepared, which is characterized in that the miR-125a- 3p can inhibit the expression of mononuclear macrophage inflammatory factor.
5. purposes according to claim 4, which is characterized in that the angiocardiopathy is hyperlipidemia or Atherosclerosis Change.
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