CN105483230B - MiR-3197 molecular marked compounds and its amplimer for detecting diabetes B retinopathy and application - Google Patents

MiR-3197 molecular marked compounds and its amplimer for detecting diabetes B retinopathy and application Download PDF

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CN105483230B
CN105483230B CN201510980953.3A CN201510980953A CN105483230B CN 105483230 B CN105483230 B CN 105483230B CN 201510980953 A CN201510980953 A CN 201510980953A CN 105483230 B CN105483230 B CN 105483230B
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retinopathy
diabetes
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韩丽媛
陆南佳
刘艳芬
段东辉
汪凯悦
顾凯波
张璐
段世伟
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Ningbo University
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Abstract

The invention discloses for detecting diabetes B retinopathy 3197 molecular marked compounds of miR and its amplimer and application, feature is that molecular marked compound is 3197 molecular markers of miR, its nucleotides sequence is classified as 5 ' GGAGGCGCAGGCUCGGAAAGGCG 3 ', the nucleotides sequence of its positive amplimer is classified as 5 ' GGA GGC GCA GGC TCG GAA 3 ', it can be applied in the kit of detection diabetes B retinopathy, advantage is detection efficiency height, it is with strong points, in auxiliary diagnosis, detection or screening drug that group's diabetes B retinopathy can be used.

Description

MiR-3197 molecular marked compounds for detecting diabetes B retinopathy and its Amplimer and application
Technical field
The present invention relates to a kind of miRNA molecule markers, more particularly, to one kind for detecting diabetes B retinopathy The miR-3197 molecular marked compounds and its amplimer of change and application.
Background technology
Diabetes B（Type 2 diabetes mellitus, T2DM）Also known as adult-onset diabetes, it is sugar The most important type of urine disease, accounts for about 90%（23499527）.It is that one group of multifactor metabolic disorder characterized by chronic hyperglycemia is comprehensive Simulator sickness, it be it is global it is main it is lethal, one of disable and expend huge disease.Diabetic retinopathy (Diabetic Retinopathy, DR) one of microvascular complication as diabetes most serious, it has also become the first place of diabetic's blinding Reason, current whole world DR patient about 93,000,000（22301125）.China diabetic 92,400,000 occupies the whole world first Position, wherein DR illness rates are 37%（20335585）.And the extension of the increase and the average life span with diabetes morbidity, The main reason for illness rate of DR is just continuously increased, and prompt future DR will be visual loss and visual function damage.Diabetes view The goldstandard of film pathological changes diagnosis is fluorescence fundus angiography, but it is invasive detection, has traumatic, and price is high It is expensive, it is time-consuming.Therefore, the prediction of some biomarkers or the generation of warning DR are found, has important meaning to reducing its blind rate Justice.
Currently, some researches show that cycle miRNAs is a kind of new bio mark with potential diagnostic value Object.But currently, disclosing not yet both at home and abroad any about pre- as detection diabetes B retinopathy using miRNA molecule It surveys or the molecular marked compound of diagnosis is reported to detect the correlative study of diabetes B retinopathy.
Invention content
Technical problem to be solved by the invention is to provide a kind of miR- for detecting diabetes B retinopathy 3197 molecular marked compounds and its amplimer and application, wherein cycle miR-3197 expressions and diabetes B retina Lesion illness rate is proportionate, and detection efficiency is high, with strong points.
Technical solution is used by the present invention solves above-mentioned technical problem：One kind is for detecting diabetes B retina The miR-3197 molecular marked compounds of lesion, the miRNA molecule marker are miR-3197 molecular markers, molecular marked compound The nucleotides sequence of miR-3197 genetic fragments is classified as 5 ' GGAGGCGCAGGCUCGGAAAGGCG-3 '.
The nucleotides sequence of the positive amplimer of above-mentioned miR-3197 molecular markers is classified as 5 '-GGA GGC GCA GGC TCG GAA -3 ', reversed amplimer are universal primer.
A kind of kit for detecting diabetes B retinopathy, the kit include expanding above-mentioned miR-3197 The positive amplimer of genetic fragment：5 '-GGA GGC GCA GGC TCG GAA -3 ' and cel-miR-39 reference genes The nucleotides sequence of positive amplimer is classified as：5’-CGTGAGTCGATCCATATTGTA-3’.
A kind of detection method using above-mentioned miR-3197 molecular labelings analyte detection diabetes B retinopathy, step It is as follows：
A, the extraction of total serum IgE；
B, reverse transcription is carried out to miRNA using miScript II RT Kit kits；
C, PCR amplification：It is carried out using 7500real-time PCR system instruments, cel-miR-39 is added as internal reference Gene, 25ul amplification systems:12.5ulSYBR Green mixed liquors, 2.5ul universal primers, 2.5ul specific primers, 2ulcDNA solution and 5.5ul are without RNA enzyme water；Amplification reaction condition：Initial activation is at 95 DEG C, 15 minutes；94 DEG C of 15 s, 55 DEG C 30 s, 70 DEG C of 30 s, totally 40 cycles；Melting curve analysis is carried out after the completion of reaction；Wherein primer solution includes amplification The positive amplimer of miR-3197 genetic fragments：In 5 '-GGA GGC GCA GGC TCG GAA -3 ' and cel-miR-39 Join the positive amplimer of gene, nucleotides sequence is classified as：5 '-CGTGAGTCGATCCATATTGTA-3 ', reverse primer are logical Use primer；Each primer concentration is 10pmol/ul；
D, it identifies：After the completion of amplification, 2 are utilized-△△CTMethod analyzes gene expression amount, if miR-3197 genetic fragments Gao Shui Flat expression, and express multiple and be considered at pathogenesis of diabetic retinopathy early stage higher than 1.7.
Compared with the prior art, the advantages of the present invention are as follows：Present invention firstly discloses for detecting diabetes B view The miR-3197 molecular marked compounds and its amplimer of film lesion and application, wherein molecular marked compound are miR-3197 molecular markers Object, reference gene are cel-miR-39 and expand the forward primer of miR-3197, cel-miR-39；Detection method is total serum IgE Extraction, cDNA synthesis, PCR amplification and identification, identification is quick, easy, special and effective.MiR-3197 expressions are recycled to increase Diabetic retinopathy can be led to by adding, and be proportionate with pathogenesis of diabetic retinopathy rate.The excavation of the marker The detection to diabetes B retinopathy can be conveniently and efficiently realized on a molecular scale with application, and detection efficiency is high, needle It is strong to property, meanwhile, it is expected to assist as diabetes B retinopathy as the drug of target spot to recycle miR-3197 expressions A kind of new tool of diagnosis, detection and screening.
Description of the drawings
Fig. 1 is the expression otherness figure for recycling miR-3197 in DR and T2DM；
Fig. 2 is the ROC curve figure for the miR-3197 genetic fragments that PCR amplification obtains.
Specific implementation mode
Below in conjunction with attached drawing embodiment, present invention is further described in detail.
Specific embodiment
1, the collection of research object
All samples of this research are all in the approval of the unit medical research ethics committee and patient's informed consent situation Lower collection, wherein health group serum sample 144, prediabetes group serum sample 70, T2DM serum samples 117 and DR Serum sample 75.It collects and requires：1. diagnose confirms that DR is verified microaneurysm through fluorescence fundus angiography inspection, Bleeding, hard exudate, the symptoms such as soft exudation of flocculence；Healthy group, the verified eyes mydriasis inspection of prediabetes group, T2DM groups It is normal to look into eyeground.Exclude 1. acute and chronic infectious diseases；2. severe cardiac, brain, hepatic disorder and influence glycometabolism it is other Endocrine system disease；3. simple eye or eyes refracting media is obviously muddy, the person that influences fundus observation.
2, with genetic chip in 5 prediabetes, 5 Healthy Peoples, 5 T2DM and 5 DR（According to the age, gender, BMI and medical history（>10 years）Stringent pairing）Filtered out in serum sample it is variant it is apparent, closely related follow may be developed with DR Ring miRNAs.
According to chip detection technique, cycle miR-3197, miR-2116-5P, miR-3939 and miR-1910- are as a result shown 3p differential expressions in DR and T2DM groups are notable.The nucleotide sequence of each molecular marked compound genetic fragment is as follows：Molecular marked compound The nucleotides sequence of miR-3197 is classified as 5 '-GGAGGCGCAGGCUCGGAAAGGCG-3 '；
The nucleotides sequence of miR-2116-5P is classified as：5’-GGUUCUUAGCAUAGGAGGUCU-3’；
The nucleotides sequence of miR-3939 is classified as：5’-UACGCGCAGACCACAGGAUGUC-3’；
The nucleotides sequence of miR-1910-3p is classified as：5’-GAGGCAGAAGCAGGAUGACA-3’；
3, by according to age, gender, BMI and medical history（>10 years）It is matched, finally matches out 45 DR serum and 45 Example T2DM serum samples, and further enlarged sample verification.
4, the serum miRNA of sample is extracted using miRNeasy Serum/Plasma Kit (Qiagen) kit, then is led to Cross spectrophotometer detection gained miRNA concentration, for cycle miR-3197, miR-2116-5P, miR-3939 and MiR-1910-3p fluorescence quantitative PCR detections.
5, RT-PCR is detected
This research uses quantitative fluorescent PCR（RT-PCR）To cycle miR-3197, miR-2116-5P, miR-3939 and MiR-1910-3p is detected in DR and T2D expressions.The basic principle of this technology：In PCR reaction systems, it was added SYBR fluorescent dyes are measured, after SYBR fluorescent dyes non-specifically mix DNA double chain, emit fluorescence signal, without mixing in chain SYBR dye molecules will not emit any fluorescence signal, to ensure fluorescence signal increase and PCR product increase it is complete It is synchronous.SYBR is only combined with double-stranded DNA, therefore can determine whether PCR reactions are special by solubility curve.For reality The miR-3197 PCR amplification forward primer sequences tested are as follows：5’-GGA GGC GCA GGC TCG GAA -3’、miR-2116- 5PPCR amplification forward primer sequences：5 '-GGG TTC TTA GCA TAG GAG GTC -3 ', miR-3939 PCR amplifications are just To primer sequence：5 '-TTA CGC GCA GAC CAC AGG AT-3 ', miR-1910-3pPCR amplification forward primer sequences： 5’- TGG AGG CAG AAG CAG GAT GA-3’.Reverse primer is universal primer（Nucleotide sequence such as SEQ ID Shown in NO.9：GAATCGAGCACCAGTTACGCATG）.The nucleotides sequence of cel-miR-39 reference genes is classified as：5’- CCUGAGUCGAUCGAUAUAGGA-3’.The nucleotides sequence for expanding the forward primer of cel-miR-39 reference genes is classified as：5’- CGTGAGTCGATCCATATTGTA-3 ' is reversely universal primer to primer（Nucleotide sequence is as shown in SEQ ID NO.9： GAATCGAGCACCAGTTACGCATG）, volume 2.5ul.
Above-mentioned amplification the specific steps are：
（1）MiRNeasy serum extracts kit (Qiagen) extracts serum miRNAs, in the serum miRNA extracted 3.5 μ l miRNA-39 are as internal control for middle addition；
（2）MiRNA is inverted using miScript II reverse transcription reagent box (Qiagen, Germany) kit Record；
（3）Step（2）Middle reverse transcription at cDNA in quantitative polyase chain reaction（PCR）Cel- is added in 11 times of dilution MiR-39 is normalized outer ginseng control, 25ul amplification systems：12.5ul SYBR PCR mixed liquors, 2.5ul universal primers, 2.5ul specific primers, 2ulcDNA solution and 5.5ul are without RNA enzyme water.Amplification step includes initial activation at 95 DEG C, 15 points Clock；94 DEG C of 15 s, 55 DEG C of 30 s, 70 DEG C of 30 s carry out 40 cycles altogether.And in 7500real-time PCR System (Applied Biosystems) instrument is operated.（Note：Tm is determined according to race PCR gradient temperatures in an experiment）Respectively Primer concentration is 10pmol/ul.
6, data analysis
This research uses 2-△△CTMethod carries out data in the case figure that finishing analysis obtains（As shown in Figure 1）, as a result send out It is existing：Detected cycle miR-3197 is notable in DR and differential expression in T2DM, P=0.0033, is less than 0.01, there is statistics meaning Justice.
The ROC curve analyzed by 18.0 softwares of SPSS（Receiver operating curve）Figure（As shown in Figure 2） In, area under the curve（AUC）It is 0.967, is more than 0.5, this illustrates that the expression degree for recycling miR-3197 is used for assisting examining Disconnected diabetes B retinopathy has very strong diagnostic value.The expression journey of cycle miR-3197 is detected by kit Degree carrys out auxiliary diagnosis diabetic retinopathy and has the certain significance.Note：Reference line, that is, chance diagonal line, area under reference line （AUC be) 0.5, under this line then the diagnostic method completely without diagnostic value.
Identification：After the completion of amplification, 2 are utilized-△△CTMethod analyzes gene expression amount, if miR-3197 genetic fragment high levels Expression, and express multiple and be considered at pathogenesis of diabetic retinopathy early stage higher than 1.7.
The measurement for recycling miRNAs expressions is analyzed by kit, it has been found that：MiR-3197 is 2 in total group There is difference between patients with type Ⅰ DM patient with retinopathy and diabetes B control group, i.e., in diabetes B patient with retinopathy Expression to be apparently higher than control group.
The present invention can be used for detecting and the relevant cycle miR-3197 expression degree of diabetes B retinopathy Kit has the advantages that accurate and reliable, flexible quick and economy.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, and should also belong to the protection of the present invention Range, protection scope of the present invention are subject to claims.

Claims (1)

1. a kind of primer pair for detecting miR-3197 molecular marked compounds is preparing auxiliary diagnosis diabetes B retinopathy Drug in application, it is characterised in that：The nucleotides sequence of the miR-3197 molecular marked compounds is classified as 5 ' GGAGGCGCAGGCUCGGAAAGGCG-3 ', the nucleotide sequence of the positive amplimer of the miR-3197 molecular markers For 5 '-GGA GGC GCA GGC TCG GAA -3 ', reversed amplimer is 5 '-GAATCGAGCACCAGTTACGCATG - 3’。
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CN106119385B (en) * 2016-08-11 2019-03-05 河南大学 A kind of miRNA molecule marker hsa-miR-149-3p of diagnosed type 2 diabetic and its application
CN106434872A (en) * 2016-08-11 2017-02-22 河南大学 MiRNA molecule marker hsa-miR-152-3p for diagnosing type 2 diabetes, and application thereof
CN107385035B (en) * 2017-07-18 2021-01-08 深圳大学 Serum/plasma miRNA marker related to type 2 diabetes retinopathy and application thereof
CN107557467B (en) * 2017-08-10 2021-05-11 李永东 Clinical marker related to cerebral aneurysm and application thereof
CN108796063A (en) * 2018-06-13 2018-11-13 云南省第二人民医院 Application of Micro-15b in preparation of reagent for diagnosing diabetic retinopathy
CN109680058A (en) * 2019-01-10 2019-04-26 华中科技大学同济医学院附属协和医院 Application and kit of the miR-101 level detection in diabetic microvascular complication diagnostic reagent

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