CN104357555B - Hprt minigene acid hsa-miR574-3p application in hypertension diagnosis - Google Patents

Hprt minigene acid hsa-miR574-3p application in hypertension diagnosis Download PDF

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CN104357555B
CN104357555B CN201410543912.3A CN201410543912A CN104357555B CN 104357555 B CN104357555 B CN 104357555B CN 201410543912 A CN201410543912 A CN 201410543912A CN 104357555 B CN104357555 B CN 104357555B
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hypertension
prehypertensive
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mir574
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CN104357555A (en
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张腾
陈瑜
贾成林
杨琴波
王培伟
李黎
崔金刚
杜宵烨
宁冰冰
刘丽
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Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
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Abstract

The present invention relates to hprt minigene acid hsa-miR574-3p application in hypertension diagnosis. The invention has the advantages that: the present invention collects the blood plasma of prehypertensive, hyperpietic and normal person and full-length genome miR express spectra is studied. It is relevant to prehypertensive and hypertension that result of study discloses a series of miRs, and the miRs of these differential expressions contributes to the molecular mechanism of explaination hypertension incidence; Progress assessment for prehypertensive and hypertension provides molecular marked compound; For effectively controlling blood pressure, treatment prehypertensive and hypertension provide brand-new therapy target, adopt single miR or in conjunction with the antagonist of multiple miRs, simulant, for the means that the treatment offer of prehypertensive and hypertension is brand-new simultaneously.

Description

Hprt minigene acid hsa-miR574-3p application in hypertension diagnosis
The application is November 26 2013 applying date, application number 201310605650.4, and invention and created name is the divisional application of " hprt minigene acid is in hypertension diagnosis and the application preparing in Altace Ramipril ".
Technical field
The present invention relates to hprt minigene acid new application, specifically, be hprt minigene acid (microRNA) hsa-miR574-3p application in hypertension diagnosis.
Background technology
Hypertension (hypertension) is a kind of common to continue to raise the chronic disease into main manifestations with arteriotony, refers to systolic arterial pressure >=140mmHg and/or diastolic pressure >=90mmHg under quiescent condition. And contraction is pressed in 120-139mmHg and/or diastolic pressure and is called prehypertensive or normal high level between 80-89mmHg.
Hypertension is the topmost risk factor of cardiovascular and cerebrovascular disease, the major complications such as its apoplexy, myocardial infarction, heart failure and chronic kidney disease, not only disable, fatality rate high, and seriously consume medical treatment and social resources, cause heavy burden to family and country. Clinical observation long-term in a large number shows that prehypertensive is also the independent hazard factor of cardiovascular and cerebrovascular disease and nephropathy. Population of China is the obvious ascendant trend of Prevalence of Hypertension over 50 years. According to survey data in 2002, more than 18 years old adult hypertension prevalence of China was 18.8%, estimates that current China there are about 200,000,000 hyperpietics, just has 2 people to suffer from hypertension in every 10 adults, accounted for the 1/5 of whole world hypertension total number of persons.Prehypertensive is about 44%-60% at the sickness rate of China, based on the young and the middle aged; Wherein the prehypertensive of 45% developed into hypertension in 10 years, was China hyperpietic " reserves ". Both domestic and external it was verified that hypertension is the disease can prevented and control, reduce the blood pressure level of hyperpietic, can obviously reduce apoplexy and the events of heart attack, significantly improve the life quality of patient, effectively reduce Disease Spectrum.
Hprt minigene acid (microRNA, miR) is generally made up of 15-22 nucleotide, is short-chain, conservative non-coding RNAs. Gene expression is had important adjusting function by miR, mainly through degraded target mRNA or suppress its translation and reach after suppressor gene is transcribed express effect. The gene expression regulation of miR mediation is characterized as being a miR generally multiple target gene, relates to multiple signal path; And gene can be subject to the regulation and control of multiple miR simultaneously, therefore relevant for miR gene expression regulation has network-like and relative conversations feature. The biological effect of miR relates to biological process widely, including cell proliferation, differentiation, growth, apoptosis etc. MiRNAs abnormal expression relates to the mechanism of multiple pathological and physiological condition, including allelotaxis, aging, tumor, heart disease, nerve retrograde affection etc. Therefore, miRs is important molecule link biology understanding disease development, is also the important target spot for the treatment of correlative diseases simultaneously. The approach of two aspects is mainly included: miR antagonist (miRantagonists) or inhibitor (microRNAinhibitors) and miR simulant (microRNAmimics) based on the Therapeutic mode of miRs. MiR antagonist is mainly used in suppressing the rise of endogenous miRs expression or function under morbid state, and miR simulant is primarily used to recover the downward of endogenous miRs expression or function in lysis. By the adjustment to miR, the expression of a series of target genes of its regulation and control can be carried out collaborative effective adjustment, thus playing the intervention effect to disease.
Summary of the invention
It is an object of the invention to for deficiency of the prior art, it is provided that hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments preparing office hypertension.
Second purpose of the present invention is to provide hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments of the progression of disease of preparation assessment hypertension.
3rd purpose of the present invention be to provide hprt minigene acid hsa-miR574-3p as target spot screening blood pressure lowering and target-organ protection medicine in application.
For realizing above-mentioned first purpose, the present invention adopts the technical scheme that: hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments preparing office hypertension, and the medical apparatus and instruments of described office hypertension is using hprt minigene acid as diagnosing molecular marked compound.
For realizing above-mentioned second purpose, the present invention adopts the technical scheme that: hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments of the progression of disease of preparation assessment hypertension, described medical apparatus and instruments is using hprt minigene acid as diagnosing molecular marked compound.
For realizing above-mentioned 3rd purpose, the present invention adopts the technical scheme that: hprt minigene acid hsa-miR574-3p as target spot screening blood pressure lowering and target-organ protection medicine in application.
The invention has the advantages that:
The present invention collects the blood plasma of prehypertensive, hyperpietic and normal person and full-length genome miR express spectra is studied.It is relevant to prehypertensive and hypertension that result of study discloses a series of miRs, and the miRs of these differential expressions contributes to the molecular mechanism of explaination hypertension incidence; Progress assessment for prehypertensive and hypertension provides molecular marked compound; For effectively controlling blood pressure, treatment prehypertensive and hypertension provide brand-new therapy target, adopt single miR or in conjunction with the antagonist of multiple miRs, simulant, for the means that the treatment offer of prehypertensive and hypertension is brand-new simultaneously.
Accompanying drawing explanation
Fig. 1. at normal vs prehypertensive, normal vs hypertension and there is in prehypertensive vs hypertension comparative analysis between two the miRs of significant difference (p < 0.05) occur simultaneously.
Fig. 2. at normal vs prehypertensive, normal vs hypertension and in prehypertensive vs hypertension comparative analysis between two the differential expression multiple miRs more than 2 occur simultaneously.
Detailed description of the invention
Below in conjunction with accompanying drawing, detailed description of the invention provided by the invention is elaborated.
Embodiment 1
In the present embodiment, the present invention, mainly through the research means of miR chip of expression spectrum, inquires into and specify that the miRs of differential expression in prehypertensive and hypertension forming process. This research discloses the miRs expressing change that prehypertensive and hypertension are relevant first; not only facilitate the molecular mechanism illustrating hypertension incidence more fully hereinafter; assessment for high blood pressure disease progress simultaneously provides a series of brand-new molecular marker; and provide a series of brand-new therapy target based on miR, for based on miR reduce blood pressure and the research and development for the treatment of means of hypertension target-organ protection are had laid a good foundation.
One, method
1. clinical research sample collection
Sample for the present invention all is from the court, and all object of study are all conscientiously read and sign Informed Consent Form, meet ethical standard. Object of study is male, and the age is between 36-50 year. When not using medicine, use mercurial sphygmomanometer that object of study arteriotony is measured, and in triplicate. Shrink pressure��120mmHg and diastolic pressure��90mmHg and include Normal group in; The object of study shrinking pressure >=140mmHg and/or diastolic pressure >=90mmHg includes hypertension group in; And use 24 h ABP meter monitoring whole day average shrinkage to be pressed in 120-139mmHg and/or diastolic pressure and include prehypertensive group in the object of study of 80-89mmHg. All object of study have a heart disease by carrying out electrocardiogram and the detection eliminating of other biochemical indicators, and nephropathy and diabetes use EDTA anticoagulant blood-collecting pipe to collect 2ml blood simultaneously. The blood sample collected centrifugal (3000 revs/min) 10 minutes, takes upper plasma for this research.
2. blood plasma miR chip of expression spectrum
Adopt mirVanaTMMiRNAIsolationKit (Cat#AM1561, Ambion, Austin, TX, US) Standard Operating Procedure and according to production firm provided carries out the totalRNA extracting of plasma sample, and extracting gained totalRNA is through AgilentBioanalyzer2100 (Agilenttechnologies, SantaClara, CA, US) electrophoresis quality inspection qualified after standby. Laboratory sample RNA adopts the test kit that AgilentmiRNA chip is supporting, miRNACompleteLabelingandHybKit (Cat#5190-0456, Agilenttechnologies, SantaClara, CA, US), according to the mark part of Standard Operating Procedure, the miRNA molecule in sample is carried out fluorescent labeling, carry out full-length genome miR expression pattern analysis for AgilenthumanmiRNAmicroarray.Array analysis is according to the Standard Operating Procedure of the supporting offer of AgilentmiRNA chip and matched reagent box, miRNACompleteLabelingandHybKit (Cat#5190-0456, Agilenttechnologies, SantaClara, CA, US) hybridization portion, carries out the hybrid experiment of sample. In rolling hybrid heater, HybridizationOven (Cat#G2545A, Agilenttechnologies, SantaClara, CA, US), 55 DEG C, 20rpm, roll hybridization 20 hours. Cylinder stainingdishes (Cat#121 is being washed after having hybridized, ThermoShandon, Waltham, MA, US) in develop a film, reagent used of developing a film is GeneExpressionWashBufferKit (Cat#5188-5327, Agilenttechnologies, SantaClara, CA, US). Chip results adopts AgilentMicroarrayScanner (Cat#G2565BA, Agilenttechnologies, SantaClara, CA, US) it is scanned, with FeatureExtractionsoftware10.7 (Agilenttechnologies, SantaClara, CA, US) data are read, software design patterns Scanresolution=5 ��m, PMT100%, 5% finally adopts GeneSpringSoftware11.0 (Agilenttechnologies, SantaClara, CA, US) it is normalized, algorithm used is Quantile.
3. statistical analysis
T inspection and fold difference (FoldChange, FC) calculating sifting is adopted to carry out statistical analysis the chip results of two groups of samples.
Two, result
1. each group sample clinical data
To normal control, prehypertensive and three groups of sample clinical informations of hypertension are analyzed, statistical result is as shown in table 1, it was shown that hypertension group shrinks pressure and diastolic pressure apparently higher than Normal group and prehypertensive group, and prehypertensive group is apparently higher than Normal group. Hypertension group creatinine level is apparently higher than prehypertensive and Normal group, and compares with prehypertensive group and have significant difference. Age, BMI index, carbamide, uric acid, triglyceride, T-CHOL, HDL and LDL between three groups all without significant difference.
Table 1. institute sample clinical information is added up
* compared with normal control, p < 0.05; # compared with prehypertensive, p < 0.05.
2. according to the T-test result (p < 0.05) compared between two, the differential expression miRs relevant to hypertension incidence
Such as table 2, compared with Normal group, having 4 miRs to express in prehypertensive group and have significant difference, 3 miRs express in hypertension group has significant difference. And compare with prehypertensive group, there are 12 miRs differential expressions in hypertension group. By will result of the comparison occur simultaneously between two, it has been found that miR939 and miR574-3p is at normal ratio prehypertensive, and prehypertensive all has significant difference than in hypertension, such as Fig. 1.
2. 3 groups of table compares the miRs with significant difference between two
3. normal group compares with prehypertensive group, the differential expression multiple miRs more than 2
As shown in table 3,9 miRs express rising in prehypertensive, have 18 miRs to express decline in prehypertensive.
Table 3. and the prehypertensive correlated expression fold differences miRs more than 2
4. normal group compares with hypertension group, the differential expression multiple miRs more than 2
As shown in table 4,17 miRs raise at hypertensive patient's plasma levels, have 33 miRs level in hypertension human blood sample to decline.
Table 4. and the hypertension correlated expression fold differences miRs more than 2
5. prehypertensive group compares with hypertension group, the differential expression multiple miRs more than 2
As shown in table 5, higher blood pressure early stage, 25 miRs raise at hypertensive patient's plasma levels, and 26 miRs decline at hypertensive patient's plasma levels.
Table 5. compares with prehypertensive, the differential expression multiple miRs more than 2 in hypertension
6. in pair comparing between two, the differential expression multiple miRs more than 2 occurs simultaneously
As in figure 2 it is shown, 10 miRs are overlapping in normal group vs prehypertensive group and normal group vs hypertension group; 17 miRs are overlapping in normal group vs prehypertensive group and prehypertensive group vs hypertension group; 22 miRs are overlapping in normal group vs hypertension group and prehypertensive group vs hypertension group. Wherein 5 miRs both are greater than 2 at three groups of relatively middle differential expression multiples, and they are hsa-miR-3620 respectively, hsv1-miR-H18, hsa-miR-1226*, hsa-miR-1237, ebv-miR-BART13.
The present invention collects the blood plasma of prehypertensive, hyperpietic and normal person and full-length genome miR express spectra is studied. It is relevant to prehypertensive and hypertension that result of study discloses a series of miRs, and the miRs of these differential expressions contributes to the molecular mechanism of explaination hypertension incidence; Progress assessment for prehypertensive and hypertension provides molecular marked compound; For effectively controlling blood pressure, treatment prehypertensive and hypertension provide brand-new therapy target, adopt single miR or in conjunction with the antagonist of multiple miRs, simulant, for the means that the treatment offer of prehypertensive and hypertension is brand-new simultaneously.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the inventive method; can also making some improvement and supplement, these improve and supplement and also should be regarded as protection scope of the present invention.

Claims (2)

1. hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments preparing office hypertension, the medical apparatus and instruments of described office hypertension is using hprt minigene acid as diagnosing molecular marked compound.
2. hprt minigene acid hsa-miR574-3p application in the medical apparatus and instruments of the progression of disease of preparation assessment hypertension, described medical apparatus and instruments is using hprt minigene acid as diagnosing molecular marked compound.
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CN102115787A (en) * 2010-11-22 2011-07-06 中国科学院动物研究所 MicroRNA (Ribose Nucleic Acid) and application of antisensenucleic acid of microRNA to diagnosis, prevention, treatment and/or prognostic evaluation of heart disease

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CN102115787A (en) * 2010-11-22 2011-07-06 中国科学院动物研究所 MicroRNA (Ribose Nucleic Acid) and application of antisensenucleic acid of microRNA to diagnosis, prevention, treatment and/or prognostic evaluation of heart disease

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