CN111073973A - MicroRNA sequence for early diagnosis of type 2 diabetes and application thereof - Google Patents
MicroRNA sequence for early diagnosis of type 2 diabetes and application thereof Download PDFInfo
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Abstract
The invention discloses a microRNA sequence for early diagnosis of type 2 diabetes and application thereof, belonging to the technical field of medicines, wherein the sequence is microRNA-X, and the sequence of the microRNA is shown as SEQ: ID: NO: 1, the sequence of the designed primer is shown as SEQ: ID: NO: 2-SEQ: ID: NO: 3, respectively. The invention collects 562 serum of Uygur individual, and utilizes Illumina Infinium Global Screening Array-24 v1.0(GSA) Bead Chip technology to analyze, and finds a section of microRNA sequence which is obviously and positively correlated with fasting blood glucose.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a microRNA sequence for early diagnosis of type 2 diabetes and application thereof.
Background
The incidence of Type 2 diabetes (T2 DM) has increased worldwide year by year in recent years, becoming the 5 th leading cause of death in the world. According to the prediction of the World Health Organization (WHO), the number of diabetic patients reaches 3 hundred million worldwide by 2025, 2.3 hundred million of the diabetic patients are in developing countries, and the newly increased number of diabetes in China accounts for the first of the newly increased number of diabetes in the world. The results of the investigation by Ning G et al show that the prevalence of T2DM in adults in China is currently 11.6%, and 50.1% of the adult population is in the pre-diabetic state. Epidemiological data show that 80% of patients with T2DM are associated with obesity, which is an important risk factor for IR and T2 DM. Recent studies have shown that in the adult population in china, the male obesity rate increases from 0.33% in 1980 to 5.02% in 2015, and the female obesity rate increases from 0.9% to 5.51%. Recent literature indicates that obesity causes T2DM, but the specific mechanism is not well defined.
microRNA is a non-coding single-stranded RNA molecule with the length of about 22 nucleotides, which is coded by endogenous genes and is involved in the regulation and control of the expression of genes after transcription in animals and plants. Recent literature indicates that most of in vivo circulating exosome microRNAs are derived from adipose tissues and reach target tissue organs through blood circulation, influence the expression of key genes of carbohydrate and lipid metabolism, and cause diseases such as fatty liver, atherosclerosis, cancer, diabetes and the like. In recent years, the role of microRNAs in the field of glycolipid metabolism is more and more emphasized, and a large number of reports show that microRNAs are important factors for regulating and controlling Insulin sensitivity, are abnormally expressed in an obese body, and induce Insulin Resistance (IR). For treating obesity and related chronic diseases, microRNAs have great potential and can be used as diagnosis and treatment targets.
Disclosure of Invention
The invention aims to provide a microRNA sequence for early diagnosis of type 2 diabetes and application thereof.
The specific technical scheme is as follows:
a microRNA sequence for early diagnosis of type 2 diabetes mellitus is microRNA-X, and the sequence of the microRNA is shown as SEQ: ID: NO: 1, the sequence of the designed primer is shown as SEQ: ID: NO: 2-SEQ: ID: NO: 3, respectively.
The microRNA sequence for early diagnosis of type 2 diabetes mellitus is applied to the preparation process of the medicament for treating type 2 diabetes mellitus.
Compared with the prior art, the invention has the beneficial effects that:
the invention collects 562 serum of Uygur individual, and utilizes Illumina Infinium GlobalScaring Array-24 v1.0(GSA) Bead Chip technology to analyze, and finds a section of microRNA sequence which is obviously and positively correlated with fasting blood glucose. Then, the verification is carried out on normal, obese and diabetic serum samples of three different nationalities, namely Han, Ha and Wei, and the remarkable high expression of the microRNA in the obese and diabetic serum samples is found, and the expression of the microRNA is remarkably and positively correlated with the related indexes of blood sugar and blood fat; in vitro culture of human liver cancer cell line HepG2 cells, the results show that a group of triglyceride transfected by the microRNA mimic has increased synthesis and reduced glucose utilization rate and insulin sensitivity, and the microRNA can influence sugar and lipid metabolism in liver cells. The expression of the microRNA shows a gradually increasing trend in three groups of normal, obese and diabetic, and compared with the prior art, the microRNA is more sensitive to early diabetes and more accurate and more advantageous to diagnosis of the early diabetes.
Drawings
FIG. 1 the expression level of miR-X in the serum of normal, obese and diabetic individuals was significantly increased by the expression level of miR-X in 24 cases of A. Uygur family normal serum samples, 46 cases of obese serum samples, 23 cases of diabetic serum samples; B. 21 Kazakh normal serum samples, 48 obesity serum samples, 10 diabetes serum samples and miR-X expression level; C. 21 Han family normal serum samples, 22 obese serum samples, 20 diabetic serum samples and miR-X expression level. Rank-sum test, # P <0.05, # P <0.01, the difference being statistically significant;
FIG. 2A shows the results of 400-fold microscopic oil red O staining 24h after FFA stimulation and 50nM miR-X-Mimic transfection of HepG2 cells; figure 2b.570nm absorbance of oil red, t-test,. P <0.001, the difference was statistically significant; B.0.1mmol/L FFA stimulates and 50nM miR-X-Mimic transfects HepG2 cell 0h and 24h, the content of triglyceride in cell, t test, P <0.01, compared with NC group, the difference has statistical significance;
figure 3 a. HepG2 glucose utilization after 24h of miR-NC, miR-X-mimic transfection, compared to the NC group, rank sum test,. P <0.01, differences were statistically significant;
FIG. 3B, miR-NC and miR-X-imic are transfected for 22h, then 100 mug/ml insulin is added to stimulate HepG2, and the insulin sensitivity of HepG2 cells is measured;
FIG. 4 is a technical roadmap of the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.
Example 1
1. Subject groups collect 562 serum of Uygur individual, and analyze the serum by using Illumina Infinium Globalscreening Array-24 v1.0(GSA) Bead Chip technology to find a section of microRNA sequence which is significantly and positively correlated with fasting blood glucose, and determine that the sequence is microRNA-X through database comparison, and the sequence of the microRNA is AAAGGUAAUUGUGGUUUCUGC. The subject group was designed with the following primers:
F:5’-AAAGGUAAUUGUGGUUUCUGC-3’
R:5’-AGAAACCACAAUUACCUUUUU-3’
2. collecting a human body sample:
(1) sample grouping and source: 24 serum samples of normal individuals of Uygur family, 48 serum samples of obese individuals and 24 serum samples of diabetic individuals are collected in Kaishi area, Ili area and Shihe sub-area of Uygur autonomous region of Xinjiang in 2016-2018 for 12 months respectively; 24 serum samples of Kazak normal individuals, 48 serum samples of obese individuals and 10 serum samples of diabetic individuals; serum samples of Han normal individuals are 21, obese individuals are 22 and diabetic individuals are 20.
(2) Inclusion and exclusion criteria for samples: normal group: age 40 < age < 50; body Mass Index (BMI) <24kg/m 2; fasting blood glucose is less than 6.1 mmol/l; triglyceride (TG) <1.7 mmol/l; total Cholesterol (TC) <5.17 mmol/l; sex: 1: 1, pairing; the obesity group: age 40 < age < 50; BMI is more than or equal to 28kg/m 2; fasting blood glucose is less than 6.1 mmol/l; sex: 1: 1, pairing; t2DM group: age 38 < age < 50; the fasting blood sugar is more than or equal to 7.0 mmol/l; sex: 1: 1 pairing.
(3) Collection and preservation of blood samples: after the sample is collected, the sample is centrifuged at 4000rpm for 5min at room temperature, and the supernatant serum is sucked into a 1.5ml RNase-Free EP tube and stored in a refrigerator at-80 ℃.
(4) The extraction method of microRNA in the blood sample comprises the following steps:
1) sample treatment: adding 900 mul of lysate MZA into every 200 mul of serum or plasma, oscillating and mixing evenly for 30sec by an oscillator until the serum is completely homogenized, and reversing and mixing evenly;
2) standing at room temperature for 5min to completely separate nucleic acid protein complex;
3) adding 200 μ L chloroform, covering the tube cover, shaking vigorously for 15sec, and standing at room temperature for 5 min;
4) after centrifugation at 12000rpm for 15min at 4 ℃, the sample will be divided into 3 layers: a yellow organic phase, a white intermediate layer and a colorless aqueous phase, wherein RNA is mainly in the aqueous phase, and the aqueous phase is transferred to a new tube for the next operation;
5) measuring the volume of the transfer solution, slowly adding 2 times of anhydrous ethanol, mixing, transferring the obtained solution and precipitate into an adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm for 30sec, centrifuging, removing effluent, and keeping the adsorption column miRelute;
6) adding 700 μ L deproteinized solution MRD into adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm at room temperature for 30sec, and discarding waste liquid;
7) adding 500 μ L of rinsing solution RW into adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm at room temperature for 30sec, and discarding the waste solution;
8) repeating the step 7 once;
9) centrifuging at 12000rpm for 2min at room temperature, and discarding the collection tube;
10) transferring the adsorption column miRelute into a new RNase-Free 1.5ml centrifuge tube, and adding 15-30 μ L RNase-Free ddH to the center of the adsorption membrane2O, standing at room temperature for 2min, and centrifuging at room temperature 12000 for 2 min.
(5) Reaction system and conditions for in vitro amplification:
1) the qRT-PCR reaction system is shown in table 1:
TABLE 1 qRT-PCR reaction System
2) The real-time quantitative PCR amplification procedure is shown in table 2:
TABLE 2 real-time quantitative PCR amplification procedure
3. On the basis of in vitro culture of a human liver cancer cell line HepG2, a microRNA-X in vitro mimic sequence (AAAGGUAAUUGUGGUUUCUGC) is used for observing the influence on the carbohydrate and lipid metabolism capability of HepG2 cells after the microRNA-X is up-regulated:
(1) human hepatoma cell line HepG2 in vitro culture: the cells were purchased from Shanghai cell Bank of Chinese academy of sciences, DMED medium, 10% FBS (fetal bovine serum), penicillin (100 units/mL) and streptomycin (100ug/mL), and were routinely cultured at 37 ℃ in the presence of 5% CO2In the incubator.
(2) Synthesis of mimic mimetic sequences: the mimetic synthesis sequences were purchased from the germa gene,
F:5’-AAAGGUAAUUGUGGUUUCUGC-3’
R:5’-AGAAACCACAAUUACCUUUUU-3’
(3) the method for detecting the carbohydrate metabolism capability of the cells comprises the following steps:
cell sugar consumption experiments:
1) after the normal culture medium is cultured for 12 hours, replacing a sugar-free culture medium to deprive glucose for 12 hours;
2) mock was transfected into cells using sugarless DMEM and lipo 2000;
3) after 4h, the serum-containing culture medium of high-glucose DMEM is replaced, and the glucose content in the culture medium is detected for 0h and 24 h.
Cellular insulin sensitivity assay:
1) replacing serum-free culture medium for starvation treatment for 12h after the normal culture medium is cultured for 12 h;
2) mock was transfected into cells using normal DMEM and lipo 2000;
3) and after 4h, replacing a normal DMEM serum-containing culture medium for 22h, adding a high-sugar culture medium containing high-concentration insulin and serum, and detecting the glucose content in the culture medium for 0min, 30min and 120 min.
(4) The method for detecting the lipid metabolism capability of the cells comprises the following steps:
intracellular triglyceride assay:
1) replacing serum-free culture medium for starvation treatment for 12h after the normal culture medium is cultured for 12 h;
2) mock was transfected into cells using normal DMEM and lipo 2000;
3) after 4h, the normal culture medium containing 0.1mmol/L free fatty acid and serum is replaced, and the content of triglyceride in the cells is detected within 0h and 24 h.
Oil red O staining to identify lipid droplet generation:
1) replacing serum-free culture medium for starvation treatment for 12h after the normal culture medium is cultured for 12 h;
2) mock was transfected into cells using normal DMEM and lipo 2000;
3) replacing a normal culture medium containing 0.1mmol/L free fatty acid and serum after 4h, and culturing for 24 h;
4) after 24h, 10% formaldehyde phosphate buffer was added to cover all cells and fixed for one hour at room temperature.
5) The formaldehyde phosphate buffer was removed by suction and washed 2-3 times with 1 XPBS.
6) Adding oil red O working solution, and dyeing at room temperature or 37 deg.C for about 30 min.
7) And (3) absorbing the oil red O working solution, flushing each hole of a six-hole plate for 2-3 times by using 1-2mL of 1 XPBS (phosphate buffer solution), observing no precipitate or impurity by naked eyes, and observing the red dyeing effect of the lipid droplets under a microscope.
4. The statistical method comprises the following steps: the data of the detection results are expressed by mean value plus or minus standard deviation (x plus or minus s), and the statistical analysis of the data is carried out by using SPSS 25.0 software. The data analysis adopts t test and rank sum test, the multi-group comparison adopts one-factor variance analysis, the data correlation analysis adopts Pearson and Spearman correlation analysis, and P <0.05 is considered to have statistical significance.
Example 2miR-X is closely related to obesity and type 2 diabetes
miR-X is remarkably and highly expressed in bodies of obese and type 2 diabetes mellitus individuals
The copy number of microRNA-X (AAAGGUAAUUGGGUUCUGC) in the serum of 24 normal individuals, 48 obese individuals, 24 individuals of T2DM Uygur families, 48 obese individuals and 10 individuals of T2DM Kazak families is verified, and the result shows that the copy number of the sequence in the obese group and the T2DM group is remarkably increased (P <0.05) compared with the normal group, and the microRNA-X (AAAGGUAAUUGUGUGUGUGUGUGUGUGUGUGUGUGUUCUGC) is probably closely related to the occurrence and development of type 2 diabetes.
Significant positive correlation between miR-X expression and glycolipid metabolism
SPSS 25.0 analyzes the correlation between miR-X and glycolipid metabolism related indexes, and the Spearman correlation analysis result shows that miR-X is significantly related to BMI, fasting blood glucose, TG, TC, LDL and HDL.
TABLE 3 correlation analysis of miR-X and glycolipid metabolism-related indices
Spearman correlation analysis, P <0.05, P <0.01, correlation was statistically significant
miR-X promotes fat synthesis capability of liver cells
After 50nm of miR-X-micic is transfected into liver cells (HepG2) for 24 hours and simultaneously stimulated by adding 0.1mM of Free Fatty Acid (FFA), oil red O staining identifies lipid droplets in the cells, and the content of Triglyceride (TG) in the cells is detected by an enzyme method, so that the results suggest that: intracellular lipid droplet synthesis of the transfected miR-X is increased, and the content of triglyceride is obviously increased.
MiR-X reduces cellular glucose consumption and insulin sensitivity
50nm miR-X-mici is transfected into a liver cell (HepG2) for 24 hours to detect the glucose content in the cell for 0h and 24h, 50nm miR-X-mici is transfected into the liver cell (HepG2) for 22 hours and then large-concentration insulin is added to stimulate simultaneously to detect the glucose content for 0h, 30min and 2h, and the results show that: the glucose consumption capability of cells transfected with miR-X is remarkably reduced, and the insulin sensitivity is remarkably reduced.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> river university
<120> microRNA sequence for early diagnosis of type 2 diabetes and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>21
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aaagguaauu gugguuucug c 21
<210>2
<211>21
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aaagguaauu gugguuucug c 21
<210>3
<211>21
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
agaaaccaca auuaccuuuu u 21
Claims (2)
1. A microRNA sequence for early diagnosis of type 2 diabetes is characterized in that the sequence is microRNA-X, and the sequence of the microRNA is shown as SEQ: ID: NO: 1, the sequence of the designed primer is shown as SEQ: ID: NO: 2-SEQ: ID: NO: 3, respectively.
2. The application of the microRNA sequence for early diagnosis of type 2 diabetes mellitus of claim 1 in the preparation process of a medicament for treating type 2 diabetes mellitus.
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