CN106434872A - MiRNA molecule marker hsa-miR-152-3p for diagnosing type 2 diabetes, and application thereof - Google Patents
MiRNA molecule marker hsa-miR-152-3p for diagnosing type 2 diabetes, and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and relates to a miRNA molecule marker hsa-miR-152-3p for diagnosing type 2 diabetes, and the application thereof. The method comprises the steps of extracting blood total RNA and preparing cDNA; detecting the level of hsa-miR-152-3p through the fluorescent quantitative PCR; and evaluating the miRNA molecule marker. According to the technical scheme of the invention, the result of the large sample detection process shows that, the level of a novel peripheral blood miRNA marker hsa-miR-152-3p in type 2 diabetes patients is significantly increased. Meanwhile, the molecular level of the miRNA marker hsa-miR-152-3p is significantly in positive correlation with the blood glucose level of subjects. The detection method is simple and convenient in operation, and the trauma degree of subjects is small. Meanwhile, the method is high in sensitivity and strong in specificity, so that the method can be used for diagnosing type 2 diabetes at the early stage or at different stages. The invention also provides a primer for detecting the blood hsa-miRNA-152-3p, a kit and a detection method, which have obvious clinical application value.
Description
Technical field
The invention belongs to biomedicine field, relate to the miRNA molecule mark of a kind of diagnosed type 2 diabetic and answer
With.
Background technology
Diabetes are a kind of chronic metabolic disease with hyperglycaemia as principal character, and the hyperglycaemia of continuation causes simultaneously
Send out disease and can seriously damage the life and health of patient.IDF's latest data shows, whole world diabetes adult in 2015
Number of patients nearly 400,000,000 people, and the ill ratio of China occupies about 1/4, brings white elephant day by day to society and family.
Diabetes are broadly divided into 1 type and diabetes B according to pathogenesis.Diabetes B is also referred to as Adult Onset's patients with type Ⅰ DM, accounts for sugar
About the 94% of the sick ratio of urine.Diabetes B still can not be cured completely, and in early days without obvious clinical symptoms, patient often exists
Health check-up or occur that typical symptom is just diagnosed.If prediabetes can find in time and by changing lifestyles or
Pharmaceutical intervention can prevent the appearance of complication.The clinical diagnosis index of current diabetes B mainly includes：Fasting blood-glucose
(FBG), dextrose tolerance test (OGTT), glycosylated hemoglobin (GHb) etc., but these indexs are used for examining of having diabetes
Break and parting, it is impossible to carry out the early diagnosis of diabetes.
MicroRNA (miRNA) is the non-coding tiny RNA of a class high conservative, and length is about 18-25 nucleotides.
MiRNA is widely present in protokaryon and eucaryote body, to the propagation of cell, differentiation, apoptosis, the growth of embryo, the shape of organ
Becoming, endocrine regulation and control, generation, the development of disease all play important regulation effect.MiRNA is by the one or more base of targeting
3 ' non-coding regions of cause, degrade its mRNA or suppress its carrying out translated, thus reaches the regulating and controlling effect to gene expression.
MiRNA is closely related with the generation of many diseases such as tumour, cardiovascular and cerebrovascular disease, endocrine system disease.Have substantial amounts of grinding at present
Study carefully and show miRNA from each rank such as pancreas islet growth, the apoptosis of islet cells, the generation of insulin, secretion and insulin actions
Section participates in the morbidity of diabetes, but research means is confined to animal model or illing tissue more, and needs by traumatic side
Formula obtains.
Blood also exists thousands of kinds of miRNA, these microRNAs not only rich content, stable in properties, it is simple to detection,
And there is significant relevance with specific diseases.MiRNA in blood is obtained by tissue secretion, analyzes in blood
MiRNA can reflect the miRNA level in tissue indirectly.Therefore by Non-Invasive means, quick, sensitive, specific fixed
Quantity measuring method, in screening diabetes B human blood, the circulation miRNA of specific expressed or unconventionality expression is as molecular marker
Thing, develops the auxiliary diagnostic box of diabetes early stage, is used for monitoring diabetes B Susceptible population, assessment risk and adopts
Taking effective intervening measure, for containment, current fulminant onset diabetes trend has significant social value.
Content of the invention
The invention discloses a kind of miRNA molecule mark hsa-miR-152-3p for diagnosed type 2 diabetic, provide
Application in preparing diabetes B auxiliary diagnostic box.
For solving the problems referred to above, the present invention uses techniques below method：
The application of the miRNA molecule mark hsa-miR-152-3p of a kind of diagnosed type 2 diabetic, step is as follows：
(1) preparation of the extracting of blood total serum IgE and cDNA；
(2) fluorescence quantitative PCR detection hsa-miR-152-3p level；
(3) assessment of miRNA molecule mark.
Described miRNA molecule mark hsa-miR-152-3p, its miRNA sequence such as SEQ ID NO：Shown in 1, DNA sequence
Row are such as SEQ ID NO：Shown in 2.
The reverse transcription primer sequence such as SEQ ID NO that in described step (1), the preparation of cDNA is used：Shown in 3.
The detection of described step (2) real-time fluorescence quantitative PCR is according to dye method detection, sonde method detection.
The forward primer such as SEQ ID NO of quantitative fluorescent PCR in described step (2)：4 shown, reverse primer such as SEQ ID
NO：Shown in 5.
The forward primer of the miRNA molecule mark hsa-miR-152-3p of diagnosed type 2 diabetic and reverse primer, in system
Application in standby diabetes B auxiliary diagnostic box.
The beneficial effects of the present invention is：
First：The present invention by introducing exogenous miRNA as reference gene, by miRNA fluorescence quantitative PCR detection outside
Hsa-miR-152-3p level in all blood；Detect as reference gene relative to traditional use endogenous serum miRNA
Method, it is to avoid the data deviation that between individual, internal reference miRNA difference causes, significantly improve the stability of testing result.
Second：The present invention is detected by large sample, finds new peripheral blood miRNA marker hsa-miR-152-3p 2
In diabetes mellitus type significantly raised, and its molecular level and person under inspection's blood sugar level be notable positive correlation；This detection method is grasped
Make simplicity, traumatic little and highly sensitive to person under inspection, high specificity, can be used in early days and different phase diabetes B
Diagnosis.On this basis, the present invention develops the primer of detection blood hsa-miR-152-3p, kit and detection method, tool
There is obvious clinical value.
Brief description
Fig. 1 is the expression schematic diagram of hsa-miRNA-152-3p in diabetes B case group and normal healthy controls group.
Fig. 2 is the correlation analysis schematic diagram of hsa-miRNA-152-3p level in detection sample and fasting blood glucose level.
Detailed description of the invention
Further describe the present invention by the following examples, including use material and specifically originate.It is to be understood that
, these are exemplary, and the unrestricted present invention.With following reagent, the type of instrument and model, or character or function
Similar or identical material may be incorporated for the enforcement of the present invention.Unless otherwise stated, the reagent used in the present invention can be
Any suitable commercial reagent.
Method in following example is commonsense method if no special instructions.
Main material：
1. the collection of clinical sample
Inventor in 2015 start so far from attached Huaihe River hospital of He'nan University have collected substantial amounts of diabetes B people and
The peripheral blood sample of normal Check-up crowd.Whole collection and subsequent experimental process meet medical ethical moral requirement and follow strictly
The principle of secrecy of case-data.The sampling of study sample, packing, preservation condition are consistent.By the arrangement to medical history information, invention
People have selected 25 cases and meets the laboratory sample that the sample of following standard detects as real-time fluorescence quantitative PCR.
Healthy population between 3.9-6.1mmol/L for the fasting blood blood sugar is defined as normal healthy controls group；
Fasting blood blood sugar is more than or equal to 7.0mmol/L, and double replication result is close, by endocrine doctor
It is diagnosed as diabetes B, and be defined as case group without the crowd of any drug therapy.
2. the extracting of total serum IgE in blood
The fresh blood sample of every 200 μ L, adds 1 μ L, the outer ginseng (Tiangen) of miRNA detection of 1 μM, adds after mixing again
Entering 600 μ L Tri reagent, whirlpool concussion fully cracking haemocyte, left at room temperature, after 5 minutes, adds 1/10 times of Tri
Reagen volume BCP solution, vortex mixes that 15 seconds rear chamber are gentle and quiet puts 10 minutes.4 DEG C, 13400g room temperature centrifuges 15 minutes；
Supernatant is transferred to new 1.5mL centrifuge tube, adds the isopropanol of equimultiple supernatant volume, overturn after mixing for several times gently ,-80
Stand 1 hour under the conditions of DEG C, 4 DEG C, after 13400g centrifuges 1 hour, absorb supernatant, add 500 μ L freshly prepared without RNase water
75% ethanol solution, featheriness suspends and cleans RNA, 4 DEG C, and 13400g centrifuges 5 minutes precipitation RNA.It after absorbing supernatant, is placed in room temperature and leads to
Dry at wind, be dried about 5 minutes.Add appropriate nuclease free water to be placed in 55 DEG C of water-baths 10 minutes, survey after fully dissolving
Determine OD260 and OD280 absorption value.It is generally acknowledged that A260/A280 between 1.8 and 2.1 can be preferable with preliminary judgement total serum IgE quality.
3. fluorescence quantitative PCR detection hsa-miR-152-3p level
Taking 2 μ g total serum IgE as template, it is right for the cDNA the first chain synthetic agent box (BioTeke) of miRNA to use
MiRNA carries out adding Poly (A) end reaction, and reaction prepares reverse transcription system after terminating：
37 DEG C are reacted 60 minutes, and reverse transcription becomes cDNA.CDNA is diluted to the mould that 4ng/ μ L reacts as quantitative fluorescent PCR
Plate.Use forward primer, general reverse primer and the 2 × SYBR Green qPCR for hsa-miR-152-3p, outer ginseng gene
Mixture, expands on ABI 7500 quantitative real time PCR Instrument.
The reverse transcription primer of miRNA such as SEQ ID NO：3 shown, forward primer such as SEQ ID NO：Shown in 4 and general instead
To primer such as SEQ ID NO：Shown in 5.
The primer combination of the detection blood miRNA that the present invention provides designs based on poly (A) polymerase tailing method and obtains.?
In some specific embodiments, the primer of described real-time fluorescence quantitative PCR detection hsa-miRNA-152-3p also can root
Design according to stem ring method and obtain, it is not limited to its design principle.Reaction system is as follows：
Outer ginseng gene system is：
Hsa-miR-152-3p system is：
PCR condition is：50 DEG C 20 seconds；95 DEG C 10 minutes；95 DEG C 1 minute；60 DEG C 1 minute, repeat 40 circulations；Record sample
The CT value of product miR-152-3p amplification, the CT value joining gene magnification in addition is standardized correction.
4. data process＆analysis
The ratio of two groups of blood sample miRNA expressions uses 2-ΔΔCTMethod calculates, wherein Δ Δ CT=[CT1
(miRNA)-CT1 (ginseng outward)]-[CT2 (miRNA)-CT2 (ginseng outward)], CT miRNA is the CT of sample miR-152-3p amplification
Value, outside CT, ginseng is the CT value joining gene magnification outside sample, and CT1 is case group or the CT value of normal healthy controls group sample amplification, and CT2 is
The CT value of one case normal healthy controls group sample amplification.Concrete outcome is as shown in the table：
Table 1. fluorescence quantitative PCR detection is good for the expression of hsa-miR-152-3p in control group and case group blood
| Sample number into spectrum | Diabetes B | Hsa-miR-152-3p relative expression levels |
| 1 | 0 | 1.324 |
| 2 | 0 | 0.814 |
| 3 | 0 | 1.389 |
| 4 | 0 | 1.104 |
| 5 | 0 | 0.595 |
| 6 | 0 | 0.463 |
| 7 | 0 | 1.520 |
| 8 | 0 | 0.818 |
| 9 | 0 | 0.973 |
| 10 | 1 | 2.469 |
| 11 | 1 | 2.265 |
| 12 | 1 | 1.027 |
| 13 | 1 | 1.737 |
| 14 | 1 | 1.812 |
| 15 | 1 | 1.533 |
| 16 | 1 | 1.557 |
| 17 | 1 | 1.416 |
| 18 | 1 | 2.574 |
| 19 | 1 | 1.178 |
| 20 | 1 | 1.224 |
| 21 | 1 | 2.915 |
| 22 | 1 | 1.480 |
| 23 | 1 | 1.750 |
| 24 | 1 | 1.292 |
In table 1, " 0 " represents healthy population, and " 1 " represents diabetes B patient.
Carrying out statistical analysis through SPSS software, hsa-miR-152-3p is in diabetes B case group and normal healthy controls group
Expression there is significant difference (P=0.002), and hsa-miR-152-3p level is obvious positive with fasting blood glucose level
Closing (R=0.62, P=0.001), result is as shown in Figure 1.P<0.05 thinks have statistical significance.
Thus it was determined that hsa-miR-152-3p is significantly raised in the blood of diabetes B patient, can be as 2
The detection molecules mark of patients with type Ⅰ DM.
Claims (6)
1. the application of the miRNA molecule mark of a diagnosed type 2 diabetic, it is characterised in that：Step is as follows：
（1）The extracting of blood total serum IgE and the preparation of cDNA；
（2）Fluorescence quantitative PCR detection hsa-miR-152-3p level；
（3）The assessment of miRNA molecule mark.
2. the miRNA molecule mark of diagnosed type 2 diabetic as claimed in claim 1, it is characterised in that：Described miRNA divides
Sub-mark is hsa-miR-152-3p, its miRNA sequence such as SEQ ID NO：Shown in 1, DNA sequence dna such as SEQ ID NO：2 institutes
Show.
3. the application of the miRNA molecule mark of diagnosed type 2 diabetic as claimed in claim 1, it is characterised in that：Described step
Suddenly（1）The reverse transcription primer sequence such as SEQ ID NO used by preparation of middle cDNA：Shown in 3.
4. the application of the miRNA molecule mark of diagnosed type 2 diabetic as claimed in claim 1, it is characterised in that：Described step
Suddenly（2）Real-time fluorescence quantitative PCR detection is according to dye method detection, sonde method detection.
5. the application of the miRNA molecule mark of diagnosed type 2 diabetic as claimed in claim 1, it is characterised in that：Described step
Suddenly（2）The forward primer of middle quantitative fluorescent PCR such as SEQ ID NO：4 shown, reverse primer such as SEQ ID NO：Shown in 5.
6. the forward primer of the miRNA molecule mark of diagnosed type 2 diabetic as claimed in claim 5 and reverse primer,
Prepare the application in diabetes B auxiliary diagnostic box.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109852688A (en) * | 2019-04-02 | 2019-06-07 | 中国药科大学 | A kind of application of the diagnostic primers and kit and non-coding RNA molecular marker of diabetes B |
| CN110396542A (en) * | 2019-05-20 | 2019-11-01 | 徐州市疾病预防控制中心(徐州市健康教育所) | A kind of LncRNA marker and its application in diabetes |
| CN113462769A (en) * | 2021-08-05 | 2021-10-01 | 中国医学科学院医药生物技术研究所 | inhibitor/CaMKII system and application thereof as biomarker |
| WO2023032452A1 (en) * | 2021-08-31 | 2023-03-09 | 国立大学法人大阪大学 | Test method for type-2 diabetes and colon cancer |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852688A (en) * | 2019-04-02 | 2019-06-07 | 中国药科大学 | A kind of application of the diagnostic primers and kit and non-coding RNA molecular marker of diabetes B |
| CN109852688B (en) * | 2019-04-02 | 2021-03-16 | 中国药科大学 | Diagnostic primer and kit for type 2 diabetes mellitus and application of non-coding RNA molecular marker |
| CN110396542A (en) * | 2019-05-20 | 2019-11-01 | 徐州市疾病预防控制中心(徐州市健康教育所) | A kind of LncRNA marker and its application in diabetes |
| CN113462769A (en) * | 2021-08-05 | 2021-10-01 | 中国医学科学院医药生物技术研究所 | inhibitor/CaMKII system and application thereof as biomarker |
| WO2023032452A1 (en) * | 2021-08-31 | 2023-03-09 | 国立大学法人大阪大学 | Test method for type-2 diabetes and colon cancer |
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