CN105483231B - MiR-2116-5p molecular marked compounds and its amplimer for detecting diabetes B retinopathy and application - Google Patents

MiR-2116-5p molecular marked compounds and its amplimer for detecting diabetes B retinopathy and application Download PDF

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CN105483231B
CN105483231B CN201510980980.0A CN201510980980A CN105483231B CN 105483231 B CN105483231 B CN 105483231B CN 201510980980 A CN201510980980 A CN 201510980980A CN 105483231 B CN105483231 B CN 105483231B
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mir
retinopathy
diabetes
amplimer
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CN105483231A (en
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刘艳芬
韩丽媛
段东辉
陆南佳
汪凯悦
顾凯波
张璐
段世伟
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Ningbo University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses for detecting diabetes B retinopathy 2116 5p molecular marked compounds of miR and its amplimer and application, feature is that molecular marked compound is 2116 5p molecular markers of miR, its nucleotides sequence is classified as 5 ' GGUUCUUAGCAUAGGAGGUCU 3 ', the nucleotides sequence of its positive amplimer is classified as 5 ' GGG TTC TTA GCA TAG GAG GTC 3 ', it can be applied in the kit of detection diabetes B retinopathy, advantage is detection efficiency height, it is with strong points, the auxiliary diagnosis of group's diabetes B retinopathy can be used, in detection or screening drug.

Description

MiR-2116-5p 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-2116-5p 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 2116-5p molecular marked compounds and its amplimer and application, wherein cycle miR-2116-5p expressions and diabetes B Retinopathy 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-2116-5p molecular marked compounds of lesion, the molecular marked compound are miR-2116-5p molecular markers, molecular labeling The nucleotides sequence of object miR-2116-5p genetic fragments is classified as.
The nucleotides sequence of the positive amplimer of above-mentioned miR-2116-5p molecular markers is classified as 5 '-GGG TTC TTA GCA TAG GAG GTC -3 ', reversed amplimer are universal primer.
A kind of kit for detecting diabetes B retinopathy, the kit include expanding above-mentioned miR-2116- The positive amplimer of 5p genetic fragments：In 5 '-GGG TTC TTA GCA TAG GAG GTC -3 ' and cel-miR-39 The nucleotides sequence for joining the positive amplimer of gene is classified as：5’-CGTGAGTCGATCCATATTGTA-3’.
A kind of detection method using above-mentioned miR-2116-5p molecular labelings analyte detection diabetes B retinopathy, step It is rapid as follows：
A, the extraction of total serum IgE；
B, reverse transcription is carried out to miRNA using miScript II reverse transcription reagent box；
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-2116-5p genetic fragments：5 '-GGG TTC TTA GCA TAG GAG GTC -3 ' and cel- The nucleotides sequence of the positive amplimer of miR-39 reference genes is classified as：5-CGTGAGTCGATCCATATTGTA-3 ' reversely draws Object is universal 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-2116-5p genetic fragments are high Horizontal expression, and express multiple and be considered at pathogenesis of diabetic retinopathy early stage higher than 1.6.
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-2116-5p molecular marked compounds and its amplimer of film lesion and application, wherein molecular marked compound are miR-2116-5p points Sub- marker, reference gene are cel-miR-39 and expand the forward primer of miR-2116-5p, cel-miR-39；Detection side Method is that the extraction of total serum IgE, cDNA synthesis, PCR amplification and identification, identification are quick, easy, special and effective.Recycle miR-2116- The increase of 5p expressions can lead to diabetic retinopathy, be proportionate with pathogenesis of diabetic retinopathy rate.It should The excavation and application of marker can conveniently and efficiently realize the detection to diabetes B retinopathy on a molecular scale, Detection efficiency is high, with strong points, meanwhile, it is expected to as 2 type glycosurias as the drug of target spot using recycling miR-2116-5p expressions Sick retinopathy auxiliary diagnosis, detection and a kind of new tool of screening.
Description of the drawings
Fig. 1 is the expression otherness figure for recycling miR-2116-5p in DR and T2DM；
Fig. 2 is the ROC curve figure for the miR-2116-5p 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- are as a result shown 1910-3p differential expressions in DR and T2DM groups are notable.The nucleotide sequence of each molecular marked compound genetic fragment is as follows：Molecule mark The nucleotides sequence of note object miR-3197 is classified as 5 ' GGAGGCGCAGGCUCGGAAAGGCG-3 '；
The nucleotides sequence of miR-2116-5P is classified as：GGUUCUUAGCAUAGGAGGUCU
The nucleotides sequence of miR-3939 is classified as：UACGCGCAGACCACAGGAUGUC
The nucleotides sequence of miR-1910-3p is classified as：GAGGCAGAAGCAGGAUGACA
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 extracts kit (Qiagen) kit, then is passed through The concentration of spectrophotometer detection gained miRNA, for recycling 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-3939PCR are expanded just To primer sequence：5 '-TTA CGC GCA GAC CAC AGG AT-3 ', miR-1910-3p amplification forward primer sequences：5’- TGG AGG CAG AAG CAG GAT GA-3’.Reverse primer is universal primer（Nucleotide sequence such as SEQ ID NO.9 institutes Show：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）.
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 added as internal control；
（2）Reverse transcription is carried out to miRNA using miScript II reverse transcriptions (Qiagen, Germany) kit；
（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-2116-5p is notable in DR and differential expression in T2DM, P<0.001, it is statistically significant.
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 0.760, is more than 0.5, this illustrates that the expression degree for recycling miR-2116-5p is used for assisting Diagnosed type 2 diabetic retinopathy has very strong diagnostic value.The expression water of cycle miR-2116-5p is detected by kit Flat 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-2116-5p genetic fragments Gao Shui Flat expression, and express multiple and be considered at pathogenesis of diabetic retinopathy early stage higher than 1.6.
The measurement for recycling miRNAs expressions is analyzed by kit, it has been found that：MiR-2116-5p in total group There is difference between diabetes B patient with retinopathy and diabetes B control group, i.e., in diabetes B retinopathy The expression of patient will be apparently higher than control group.
The present invention can be used for detecting and the relevant cycle miR-2116-5p expression journeys of diabetes B retinopathy The kit of degree 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-2116-5p molecular marked compounds is preparing auxiliary diagnosis diabetes B retina Application in the drug of lesion, it is characterised in that：The nucleotides sequence of the miR-2116-5p molecular marked compounds is classified as 5 ' GGUUCUUAGCAUAGGAGGUCU-3 ', the nucleotides sequence of the positive amplimer of the miR-2116-5p molecular markers 5 '-GGG TTC TTA GCA TAG GAG GTC -3 ' are classified as, reversed amplimer is 5 ' - GAATCGAGCACCAGTTACGCATG -3’。
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