阅读说明：本技术 一种糖尿病合并冠心病的生物标志物 (Biomarker for diabetes complicated with coronary heart disease ) 是由 王兆平 王革 王颖翠 于 2020-12-29 设计创作，主要内容包括：本发明提供了一种糖尿病合并冠心病的生物标志物,属于生物医药技术领域。本发明通过提取RNA发现,相较于健康人体血清,糖尿病合并冠心病患者血清中基因AC021188.4的表达量显著降低,且AC021188.4的表达量在单纯冠心病患者和单纯糖尿病患者血清中没有显著的变化,因此,可将AC021188.4作为诊断糖尿病合并冠心病的基因标志物。(The invention provides a biomarker for diabetes complicated with coronary heart disease, and belongs to the technical field of biological medicines. According to the invention, compared with the serum of a healthy human body, the expression level of the gene AC021188.4 in the serum of a patient with diabetes and coronary heart disease is obviously reduced by extracting RNA, and the expression level of AC021188.4 has no obvious change in the serum of the patient with simple coronary heart disease and the serum of the patient with simple diabetes, so that AC021188.4 can be used as a gene marker for diagnosing the patient with diabetes and coronary heart disease.)

1. A marker for diagnosing diabetes combined with coronary heart disease is characterized in that the marker is a gene AC021188.4, and the transcript sequence of the gene AC021188.4 is ENST 00000421534.1.

2. Application of a reagent for detecting the expression level of the gene AC021188.4 in preparing a product for pre-diagnosing diabetes complicated with coronary heart disease.

3. The use according to claim 2, wherein the product is a gene chip, a kit or a biological agent.

4. The use according to claim 2, wherein a primer specific to AC021188.4 is included in the reagent, and the sequence of the primer is shown in SEQ ID No.2 and SEQ ID No. 3.

5. The pre-diagnosis detection kit for diabetes complicated with coronary heart disease is characterized by comprising a specific primer for detecting the expression quantity of AC021188.4, wherein the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

6. The application of the reagent for detecting the expression quantity of the gene AC021188.4 in preparing a product for prognosis evaluation of diabetes complicated with coronary heart disease.

7. The use of claim 6, wherein the product is a gene chip, a kit or a biological agent.

8. The use according to claim 6, wherein the reagent comprises a primer specific to AC021188.4, and the sequence of the primer is shown as SEQ ID No.2 and SEQ ID No. 3.

9. The kit for the prognosis evaluation detection of diabetes complicated with coronary heart disease is characterized by comprising a specific primer for detecting the expression quantity of AC021188.4, wherein the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a biomarker for diabetes complicated with coronary heart disease.

Background

With the aging population becoming more severe, the incidence of coronary heart disease and diabetes is increasing year by year. Relevant research data show that the incidence of coronary heart disease of diabetic patients is 2-4 times of that of normal people. Compared with the patients with simple coronary heart disease, the patients with diabetes complicated with coronary heart disease have more diffuse and serious coronary artery pathological changes. It can be seen that diabetes combined with coronary heart disease has become a major disease that seriously threatens human life and health. Therefore, the early diagnosis of the diabetes and coronary heart disease is beneficial to the early treatment of the diabetes and coronary heart disease, and the cure rate of patients is improved.

lncRNA is a non-coding RNA with more than 200 nucleotides, and has wide regulation and control functions in various life activities of a human body. Research in recent years shows that the differential expression of lncRNA is closely related to the occurrence and development of human diseases. At present, no report related to lncRNA and diabetes-associated coronary heart disease exists, so that research on lncRNA differential expression in blood of patients with diabetes-associated coronary heart disease provides a new direction for diagnosing and treating diabetes-associated coronary heart disease.

Disclosure of Invention

The invention aims to provide a new direction for diagnosing and treating a diabetic coronary heart disease patient by using differential lncRNA in blood of the diabetic coronary heart disease patient.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a marker for diagnosing diabetes combined with coronary heart disease, wherein the marker is gene AC021188.4, the transcript sequence of the gene AC021188.4 is ENST00000421534.1, and ENST00000421534.1 is shown as SEQ ID No. 1.

Secondly, the invention provides application of a reagent for detecting the expression level of the gene AC021188.4 in preparing a product for pre-diagnosing diabetes complicated with coronary heart disease.

Preferably, the product is a gene chip, a kit or a biological agent.

Preferably, a primer specific to AC021188.4 is included in the reagent, and the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention also provides a diabetes combined coronary heart disease pre-diagnosis detection kit, which comprises a specific primer for detecting the expression quantity of AC021188.4, wherein the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

Fourthly, the invention provides application of a reagent for detecting the expression level of the gene AC021188.4 in preparation of a product for prognosis evaluation of diabetes mellitus with coronary heart disease.

Preferably, the product is a gene chip, a kit or a biological agent.

Preferably, a primer specific to AC021188.4 is included in the reagent, and the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention also provides a diabetes-associated coronary heart disease prognosis evaluation detection kit, which comprises a specific primer for detecting the expression quantity of AC021188.4, wherein the sequences of the primer are shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention has the beneficial effects that:

the gene AC021188.4 is found to be low expressed in patients with diabetes complicated with coronary heart disease for the first time, the expression of the gene AC021188.4 has no obvious difference between the patients with simple coronary heart disease and the patients with simple diabetes, and the gene AC021188.4 has excellent specificity and sensitivity as a marker of the diabetes complicated with coronary heart disease, so the gene AC021188.4 can be used for preparing a pre-diagnosis kit of the diabetes complicated with coronary heart disease and a prognosis diagnosis kit of the diabetes complicated with coronary heart disease.

Drawings

FIG. 1 expression differences of AC021188.4 in healthy human serum (group A), diabetic combined coronary serum (group B), simple coronary serum (group C) and simple diabetic serum (group D).

FIG. 2 ROC curves of AC021188.4 between healthy human serum (group A) and serum from diabetic patients combined with coronary disease (group B).

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.

Example 1

Extraction of serum RNA

(1) Collecting 30 healthy human blood (set as group a), 22 diabetic combined coronary blood (set as group B) and 25 simple coronary blood (set as group C) and 20 simple diabetic blood (set as group D);

(2) placing the blood plasma of each group in a centrifuge, centrifuging for 15min at 3000rpm/min, and taking the supernatant to obtain serum;

(3) respectively taking 250ul of serum from each group, placing the serum into a centrifuge tube, adding 750ul of Invitrogen TRIzol reagent, blowing, uniformly mixing, and standing at room temperature for 5 min;

(4) adding 200ul chloroform, shaking and mixing uniformly, and standing at room temperature for 15 min;

(5) placing the centrifuge tube in a low-temperature centrifuge, and centrifuging at 4 ℃ and 12000rpm/min for 15 min;

(6) sucking the supernatant into a new centrifugal tube, adding isopropanol with the same volume, uniformly mixing, and standing on ice for 10 min;

(7) placing the centrifuge tube in a low-temperature centrifuge, and centrifuging at 4 ℃ and 12000rpm/min for 15 min;

(8) discarding the supernatant, adding 1ml of 75% ethanol prepared by DEPC water into the precipitate, and shaking the centrifugal tube to suspend the precipitate in the 75% ethanol;

(9) placing the centrifuge tube in a low temperature centrifuge, centrifuging at 4 deg.C 7000rpm/min for 5min, discarding supernatant, placing in a superclean bench, drying for 8min, and adding 30ul DEPC water for dissolving;

(10) the purity and concentration of the RNA were determined for subsequent experiments.

Example 2

Preparation of cDNA for each group of sera

(1) Removal of genomic DNA

The reaction system is as follows:

reagent	Amount of the composition used
		5×gDNA Eraser Buffer	2.0 μl
gDNA Eraser	1μl
		Total RNA	1μg
RNase Free dH2O	up to 10 μl

The reaction conditions were as follows: 42 ℃ for 2 minutes, 4 ℃.

(2) Reverse transcription reaction

The reaction system is as follows:

reagent	Amount of the composition used
		Reaction solution of step (1)	10μl
PrimeScript RT Enzyme Mix I	1.0μl
		RT Primer Mix	1.0 μl
5×PrimeScript Buffer 2	4.0 μl
		RNase Free dH2O	4.0 μl
Total	20 μl

The reaction conditions were as follows: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ is prepared.

Example 3

Detection of differential expression of AC021188.4 in each group

(1) Specific primers of AC021188.4 were designed, and the primer sequences were as follows:

AC021188.4 upstream primer: 5'-CACATCGAGGCTCTAGTGACC-3'

AC021188.4 downstream primer: 5'-GCTTGCCAGCACGATTTACAA-3'

GAPDH upstream primer: 5'-CCCATCACCATCTTCCAGGAG-3'

GAPDH downstream primer: 5'-TTCTCCATGGTGGTGAAGACG-3'

(2) Performing fluorescent quantitative PCR reaction:

reagent	Amount of the composition used
		SYBR Green Premix Ex Taq（2×）	10μl
Upstream primer	0.4μl
		Downstream primer	0.4μl
cDNA template	2μl
		Sterile water	7.2μl
Total	20μl

Reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃, 35s at 60 ℃ and 40 cycles; 45s at 70 ℃.

(3) Use 2^-△△CtThe method calculates the differential expression level of AC021188.4 in each group.

As shown in FIG. 1, it can be seen from the results that the relative expression level of AC021188.4 in group B (diabetic patients with coronary heart disease serum) was 0.439. + -. 0.048, the relative expression level of AC021188.4 in group C (simple coronary heart disease serum) was 0.914. + -. 0.030, and the relative expression level of AC021188.4 in group D (simple diabetic patients serum) was 0.983. + -. 0.041.

From the results, the relative expression level of AC021188.4 in the serum of the patients with diabetes and coronary heart disease is obviously lower than that of the serum of healthy people, and the difference has statistical significance; the relative expression level of AC021188.4 in the serum of the coronary heart disease patient and the diabetes patient is not obviously different from the expression level of AC021188.4 in the serum of a healthy human body.

The ROC curve was plotted based on the data of groups a and B, and as a result, as shown in fig. 2, it can be seen that the AUC value of the ROC curve is 0.9621 (std. error is 0.02232,95% confidence interval 0.9184-1.006, P value < 0.0001), indicating that AC021188.4 has excellent specificity and sensitivity as a marker for diabetes combined with coronary heart disease.

In conclusion, the AC021188.4 is low in expression in patients with diabetes complicated with coronary heart disease, but has no significant difference in expression in patients with simple coronary heart disease and patients with simple diabetes, and the AC021188.4 has excellent specificity and sensitivity as a marker of the diabetes complicated with coronary heart disease, so that the AC021188.4 can be used for preparing a pre-diagnosis kit and a prognosis kit for the diabetes complicated with coronary heart disease.

The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.

Sequence listing

<110> Shandong university's Qilu hospital (Qingdao)

<120> biomarker for diabetes complicated with coronary heart disease

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 740

<212> DNA

<213> Human source (Human)

<400> 1

ggcacatcga ggctctagtg accgcattat gaattagtcc tttccttttc tcctcatgtg 60

attgcagcga gcgctcgtct tgtttgtaaa tcgtgctggc aagccccgtt ttaaccaact 120

acgaatttca tttcggaatg aaaaaggaga cgcactttcg gacagggcag cggttgccgc 180

cggtccttcc tctgagctac cggtccctag aaggaagacc tgccttcgac cgatctggaa 240

atgagtccca gctgcaagcc cagggcggcg gaagccccac gggccgagcc gaagaaagac 300

gaggcggggg acggggagcg tagtgccgcg tgccgggacc cacaacccct ttcccctggg 360

cgctcttccc cgagcggaca ggaaaaagct ctcctttgcc gctgcccatc ccccagggcc 420

caagcgatgc ttcctaaccg cgtggacgca gcttgatggg ggagaaagag ctctgggagc 480

agggcttaga gcctcgtctc gcctcctgcc aactcggtcc ggcaaggttc cgaacctccc 540

tgagcctcag cttcctaatc tggcaaacat gacgacaaaa gtccttctct tttaaggttt 600

gcccacagcc cagctgcatc tgtgaacatg aatgctactt ctacagaaac agagtggaat 660

ctccgaaata cctttgagac cctttcgtgt caggtgggtg ggccacagcc tgatcatgga 720

ataaaagtca gccttaagga 740

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cacatcgagg ctctagtgac c 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcttgccagc acgatttaca a 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cccatcacca tcttccagga g 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttctccatgg tggtgaagac g 21