CN104357554B - Hprt minigene acid hsa-miR939 application in hypertension diagnosis - Google Patents

Hprt minigene acid hsa-miR939 application in hypertension diagnosis Download PDF

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CN104357554B
CN104357554B CN201410543613.XA CN201410543613A CN104357554B CN 104357554 B CN104357554 B CN 104357554B CN 201410543613 A CN201410543613 A CN 201410543613A CN 104357554 B CN104357554 B CN 104357554B
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hypertension
prehypertensive
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mir939
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CN104357554A (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 miR939 application in hypertension diagnosis.The invention has the advantages that: the present invention collects the blood plasma of prehypertensive, hyperpietic and normal person and studies full-length genome miR express spectra.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 explaining the molecular mechanism of 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, use single miR simultaneously or combine the antagonist of multiple miRs, simulant, for the means that the treatment offer of prehypertensive and hypertension is brand-new.

Description

Hprt minigene acid hsa-miR939 application in hypertension diagnosis
The application is November 26 2013 applying date, application number 201310605650.4, innovation and creation The divisional application of entitled " hprt minigene acid is in hypertension diagnosis and the application prepared in Altace Ramipril ".
Technical field
The present invention relates to hprt minigene acid new application, specifically, be hprt minigene acid (microRNA) Hsa-miR939 application in hypertension diagnosis.
Background technology
Hypertension (hypertension) is a kind of common persistently to rise the slow of a height of main performance with arteriotony Property disease, 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 referred to as prehypertensive or just between 80-89mmHg Often high level.
Hypertension is the topmost risk factor of cardiovascular and cerebrovascular disease, its apoplexy, myocardial infarction, heart failure And the major complications such as chronic kidney disease, not only disable, fatality rate high, and seriously consume medical and social Resource, causes heavy burden to family and country.The most long-term clinical observation shows that prehypertensive is also Cardiovascular and cerebrovascular disease and the independent hazard factor of nephropathy.Population of China Prevalence of Hypertension obvious over 50 years Ascendant trend.According to survey data in 2002, more than 18 years old adult hypertension prevalence of China was 18.8%, Estimate that current China there are about 200,000,000 hyperpietics, every 10 adults just have 2 people to suffer from hypertension, Account for the 1/5 of whole world hypertension total number of persons.Prehypertensive is about 44%-60% at the sickness rate of China, with The young and the middle aged is main；Wherein the prehypertensive of 45% developed into hypertension in 10 years, was that China's hypertension is suffered from " reserves " of person.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 improves the life of patient Deposit quality, effectively reduce Disease Spectrum.
Hprt minigene acid (microRNA, miR) is generally made up of 15-22 nucleotide, is short-chain, Conservative non-coding RNAs.MiR has important adjusting function to gene expression, mainly by degraded mesh Mark mRNA or suppress its translation and reach the effect expressed after suppressor gene is transcribed.The gene of miR mediation Expression regulation feature is that a miR generally has multiple target gene, relates to multiple signal path；And a gene Can be regulated and controled by multiple miR, therefore relevant for miR gene expression regulation has network-like and phase simultaneously Close the feature of dialogue.The biological effect of miR relates to biological process widely, including cell proliferation, divides Change, growth, apoptosis etc..MiRNAs abnormal expression relates to the mechanism of multiple pathological and physiological condition, bag Include allelotaxis, aging, tumor, heart disease, nerve retrograde affection etc..Therefore, miRs is reason Solve important molecule link biology of disease development, be also the important target spot for the treatment of correlative diseases simultaneously. Therapeutic mode based on miRs mainly include of both approach: miR antagonist (miR antagonists) Or inhibitor (microRNA inhibitors) and miR simulant (microRNA mimics).MiR antagonist It is mainly used in suppression endogenous miRs under morbid state to express or the rise of function, and miR simulant master If being used for recovering endogenous miRs in lysis to express or the downward of function.By the regulation to miR, The expression of a series of target genes that it regulates and controls can be carried out collaborative effective regulation, thus play and disease is done Preliminary effect.
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-miR939 exists Prepare the application in the medical apparatus and instruments of office hypertension.
Second object of the present invention is to provide hprt minigene acid hsa-miR939 in preparation assessment hypertension Progression of disease medical apparatus and instruments in application.
Third object of the present invention is to provide hprt minigene acid hsa-miR939 as target spot in screening fall Application in the medicine of blood pressure and target-organ protection.
For realizing above-mentioned first purpose, the present invention adopts the technical scheme that: hprt minigene acid Hsa-miR939 application in the medical apparatus and instruments preparing office hypertension, the medical treatment of described office hypertension Apparatus is using hprt minigene acid as diagnosis molecular marked compound.
For realizing above-mentioned second purpose, the present invention adopts the technical scheme that: hprt minigene acid Hsa-miR939 application in the medical apparatus and instruments of the progression of disease of preparation assessment hypertension, described Medical treatment device Tool is using hprt minigene acid as diagnosis molecular marked compound.
For realizing above-mentioned 3rd purpose, the present invention adopts the technical scheme that: hprt minigene acid Hsa-miR939 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 expresses full-length genome miR Spectrum is studied.It is relevant to prehypertensive and hypertension that result of study discloses a series of miRs, these The miRs of differential expression contributes to explaining the molecular mechanism of hypertension incidence；For prehypertensive and hypertension Progress assessment provide molecular marked compound；For effectively controlling blood pressure, treatment prehypertensive and hypertension provide Brand-new therapy target, uses single miR simultaneously or combines the antagonist of multiple miRs, simulant, for The treatment of prehypertensive and hypertension provides brand-new means.
Accompanying drawing explanation
Fig. 1. in normal vs prehypertensive, normal vs hypertension and prehypertensive vs hypertension two-by-two The miRs in comparative analysis with significant difference (p < 0.05) occurs simultaneously.
Fig. 2. in normal vs prehypertensive, normal vs hypertension and prehypertensive vs hypertension two-by-two In comparative analysis, the differential expression multiple miRs more than 2 occurs simultaneously.
Detailed description of the invention
The detailed description of the invention provided the present invention below in conjunction with the accompanying drawings elaborates.
Embodiment 1
In the present embodiment, the present invention, mainly by the research means of miR chip of expression spectrum, inquires into and bright The really miRs of differential expression in prehypertensive and hypertension forming process.This research discloses height first The miRs expressing change that blood pressure early stage and hypertension are correlated with, not only facilitates and illustrates high blood more fully hereinafter The molecular mechanism that Hair Fixer is sick, the assessment for high blood pressure disease progress simultaneously provides a series of brand-new molecule mark Will thing, and provide a series of brand-new therapy target based on miR, reduce blood pressure for based on miR And the research and development of the treatment means of hypertension target-organ protection have 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 the most conscientiously read and signed and know Feelings letter of consent, meets ethical standard.Object of study is male, and the age is between 36-50 year.Not In the case of using medicine, use mercurial sphygmomanometer that object of study arteriotony is measured, and repeat three Secondary.Shrink pressure≤120mmHg and diastolic pressure≤90mmHg and include Normal group in；Shrink pressure >=140mmHg And/or the object of study of diastolic pressure >=90mmHg includes hypertension group in；And use 24 h ABP meters Monitoring whole day average shrinkage is pressed in 120-139mmHg and/or the diastolic pressure object of study at 80-89mmHg Include prehypertensive group in.All object of study get rid of trouble by carrying out electrocardiogram and the detection of other biochemical indicators There are heart disease, nephropathy and diabetes, use EDTA anticoagulant blood-collecting pipe to collect 2ml blood simultaneously.Collect Blood sample centrifugal (3000 revs/min) 10 minutes, takes upper plasma for this research.
2. blood plasma miR chip of expression spectrum
Use mirVanaTMmiRNA Isolation Kit(Cat#AM1561,Ambion,Austin,TX,US) And the Standard Operating Procedure provided according to production firm carries out the total RNA extracting of plasma sample, extracting Gained total RNA through Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US) electrophoresis quality inspection qualified after the most standby.Laboratory sample RNA uses Agilent miRNA chip supporting Test kit, miRNA Complete Labeling and Hyb Kit (Cat#5190-0456, Agilent Technologies, Santa Clara, CA, US), according to the mark part of Standard Operating Procedure in sample MiRNA molecule carries out fluorescent labeling, carries out full genome for Agilent human miRNA microarray Group miR expression pattern analysis.Array analysis is according to the standard operation stream of the Agilent supporting offer of miRNA chip Journey and matched reagent box, miRNA Complete Labeling and Hyb Kit (Cat#5190-0456, Agilent Technologies, Santa Clara, CA, US) hybridization portion, carry out the hybrid experiment of sample.Miscellaneous rolling Hand in stove, Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), 55 DEG C, 20rpm, roll hybridization 20 hours.After having hybridized wash cylinder staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) in develop a film, reagent used of developing a film is Gene Expression Wash Buffer Kit(Cat#5188-5327,Agilent technologies,Santa Clara, CA,US).Chip results uses Agilent Microarray Scanner (Cat#G2565BA, Agilent Technologies, Santa Clara, CA, US) it is scanned, with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) reads data, software design patterns Scan Resolution=5 μm, PMT 100%, 5% finally uses Gene Spring Software 11.0 (Agilent Technologies, Santa Clara, CA, US) it is normalized, algorithm used is Quantile.
3. statistical analysis
T inspection and fold difference (Fold Change, FC) is used to calculate sieve the chip results of two groups of samples Choosing carries out statistical analysis.
Two, result
1. each group sample clinical data
Normal control, prehypertensive and three groups of sample clinical informations of hypertension are analyzed, statistical result As shown in table 1, show that hypertension group shrinks pressure and diastolic pressure apparently higher than Normal group and prehypertensive Group, prehypertensive group is apparently higher than Normal group.Hypertension group creatinine level is apparently higher than before hypertension Phase and Normal group, and compare with prehypertensive group there is significant difference.Age, BMI index, urine Element, uric acid, triglyceride, T-CHOL, HDL and LDL is not significantly different between three groups.
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 two-by-two, the differential expression relevant to hypertension incidence miRs
Such as table 2, compared with Normal group, have 4 miRs express in prehypertensive group have aobvious Writing 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 two-by-two, find MiR939 and miR574-3p is at normal ratio prehypertensive, and prehypertensive is more aobvious than all having in hypertension Write difference, such as Fig. 1.
2. 3 groups of table compares the miRs with significant difference two-by-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 at height Blood pressure early stage is expressed and declines.
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 to exist In hypertension human blood sample, level declines.
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, 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 two-by-two, the differential expression multiple miRs more than 2 occurs simultaneously
As in figure 2 it is shown, 10 miRs are in normal group vs prehypertensive group and normal group vs hypertension group Middle overlap；17 miRs are in normal group vs prehypertensive group and prehypertensive group vs hypertension group Overlapping；22 miRs are overlapping in normal group vs hypertension group and prehypertensive group vs hypertension group. Wherein 5 miRs are both greater than 2 at three groups of relatively middle differential expression multiples, and they are respectively hsa-miR-3620, 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 expresses full-length genome miR Spectrum is studied.It is relevant to prehypertensive and hypertension that result of study discloses a series of miRs, these The miRs of differential expression contributes to explaining the molecular mechanism of hypertension incidence；For prehypertensive and hypertension Progress assessment provide molecular marked compound；For effectively controlling blood pressure, treatment prehypertensive and hypertension provide Brand-new therapy target, uses single miR simultaneously or combines the antagonist of multiple miRs, simulant, for The treatment of prehypertensive and hypertension provides brand-new means.
The above is only the preferred embodiment of the present invention, it is noted that common for the art Technical staff, on the premise of without departing from the inventive method, it is also possible to makes some improvement and supplements, these Improve and supplement and also should be regarded as protection scope of the present invention.

Claims (2)

1. hprt minigene acid hsa-miR939 answering in the medical apparatus and instruments preparing office hypertension With, the medical apparatus and instruments of described office hypertension is using hprt minigene acid as diagnosis molecular marker Thing.
2. hprt minigene acid hsa-miR939 is in the medical treatment of the progression of disease of preparation assessment hypertension Application in apparatus, described medical apparatus and instruments is using hprt minigene acid as diagnosis molecular marked compound.
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