CN114457150B - Annular RNA marker for diagnosing diabetic retinopathy and application thereof - Google Patents
Annular RNA marker for diagnosing diabetic retinopathy and application thereof Download PDFInfo
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Abstract
The invention relates to a circular RNA hsa _ circ _0095008 and a specific amplification primer thereof, wherein the cDNA nucleotide sequence corresponding to the circular RNA is shown as SEQ ID NO. 1, the specific amplification primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3. Compared with normal persons and diabetic patients, the expression of hsa _ circ _0095008 is abnormally reduced in peripheral blood mononuclear cells of diabetic retinopathy patients, so that hsa _ circ _0095008 can be used for screening persons at risk of diabetic retinopathy.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a circular RNA for diagnosing diabetic retinopathy.
Background
Diabetic Retinopathy (DR) is the most prominent factor in the visual impairment of diabetic patients. It is now known that approximately 50% of patients with diabetic retinopathy develop Diabetic Macular Edema (DME) complications. The most prominent pathological features of DME include a breakdown of the blood-retinal barrier (abnormal vascular architecture) and a chronic inflammatory response, the latter mediated multiple inflammatory factors, chemokines, are often important factors in retinal and choroidal leakage.
The development of diabetic retinopathy is a long-term pathological process, usually as a result of multifactorial effects. Diabetic retinopathy, which is found early in clinical work, can be delayed in its course of disease progression or even restored to normal visual function by rational treatment, and therefore an appropriate diagnostic method should be selected. The common relevant ocular fundus examination method for ophthalmology is a golden standard for diabetic retinopathy diagnosis, detection of relevant diabetic retinopathy risk factors can provide strong evidence for early diagnosis and disease condition evaluation of diabetic retinopathy, combination of the two methods is beneficial to early discovery of diabetic retinopathy, the blindness incidence rate of diabetic retinopathy patients can be reduced, and the method is worthy of popularization and use in clinic.
Circular RNA (circular RNA) is a special non-coding RNA, is in a closed circular structure, is not influenced by RNA exonuclease, is more stable in expression and is not easy to degrade. circRNA is ubiquitous in the body, and is widely involved in regulation of life activities (e.g., affecting RNA polymerase elongation), regulation of gene expression after transcription and transcription, as a miRNA inhibitor, acting as competitive endogenous RNA (cerana) to inhibit expression of target genes, regulation of translation processes by interaction with RNA-binding proteins, and the like. There is increasing evidence that circRNA is closely associated with a variety of human diseases, such as tumors, diabetes, and the like.
The circRNA has strong specificity of tissues or development stages, higher stability and tolerance to temperature change, and is an ideal molecular marker and therapeutic target for disease diagnosis. The circRNA can play a role in disease susceptibility detection, disease diagnosis, treatment, prognosis and the like, and is a research object with high application value. Therefore, the finding and screening of the circRNA markers of diabetes and complications thereof have important significance and value for early prevention and treatment of diabetic retinopathy.
Disclosure of Invention
In order to solve the above technical problems, the present invention includes the following aspects:
the first aspect of the present invention provides a specific amplification primer for amplifying circular RNAhsa _ circ _0095008, the specific amplification primer comprising an upstream primer and a downstream primer, wherein,
the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the cDNA nucleotide sequence corresponding to the circular RNA is shown as SEQ ID NO. 1.
Specifically, the cDNA nucleotide sequence corresponding to the circular RNAhsa _ circ _0095008 is as follows: AGAAGCTCATGTTCAAGAATGCGCCAACCCCACAGGAGTTCCGGGAGGGGGAAGATGCCGTGATTGTGTGTGATGTGGTCAGCTCCCTCCCACCAACCATCATCTGGAAACACAAAGGCCGAGATGTCATCCTGAAAAAAGATGTCCGATTCATAGTCCTGTCCAACAACTACCTGCAGATCCGGGGCATCAAGAAAACAGATGAGGGCACTTATCGCTGTGAGGGCAGAATCCTGGCACGGGGGGAGATCAACTTCAAGGACATTCAGGTCATTGTGAATGTGCCACCTACCATCCAGGCCAGGCAGAATATTGTGAATGCCACCGCCAACCTCGGCCAGTCCGTCACCCTGGTGTGCGATGCCGAAGGCTTCCCAGAGCCCACCATGAGCTGGACAAAGGATGGGGAACAGATAGAGCAAGAGGAAGACGATGAGAAGTACATCTTCAGCGACGATAGTTCCCAGCTGACCATCAAAAAGGTGGATAAGAACGACGAGGCTGAGTACATCTGCATTGCTGAGAACAAGGCTGGCGAGCAGGATGCGACCATCCACCTCAAAGTCTTTG are provided.
Specifically, the nucleotide sequence of the upstream primer is as follows: 5'-ATGCGACCATCCACCTCAAAG-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-ACATCACACACAATCACGGCA-3' are provided.
The second aspect of the invention provides a kit for screening diabetic retinopathy, which comprises the specific amplification primer, a reference gene GAPDH primer and an amplification system.
Preferably, the nucleotide sequence of the upstream primer of the reference gene GAPDH is shown as SEQ ID NO. 4; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5.
Specifically, the nucleotide sequence of the upstream primer is as follows: 5'-ATGGAAATCCCATCACCATCTT-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-CGCCCCACTTGATTTTGG-3' are provided.
Preferably, the amplification system is a real-time fluorescent quantitative PCR amplification system and comprises Ex Taq enzyme, dNTP mix and Mg 2+ Tli RNaseH and TB Green.
In a third aspect, the invention provides the use of the kit as described above in the preparation of a reagent for screening for diabetic retinopathy.
In a fourth aspect, the invention provides the use of hsa _ circ _0095008 or a substance that increases the expression level of hsa _ circ _0095008 in the manufacture of a medicament for the treatment of diabetic retinopathy.
The invention has the following beneficial effects:
1. the inventor finds that the circular hsa _ circ _0095008 is obviously related to the occurrence, development, treatment and prognosis of diabetic retinopathy, and provides a novel detection marker and a prognosis evaluation method for the diabetic retinopathy. Research shows that hsa _ circ _0095008 expression is abnormally reduced in peripheral blood mononuclear cells of diabetic retinopathy patients compared with normal persons and diabetic patients, and the finding has important diagnostic significance for distinguishing healthy persons, diabetic patients and diabetic retinopathy patients, so that the annular hsa _ circ _0095008 can be used for screening subjects at risk of having diabetic retinopathy.
2. The circular hsa _ circ _0095008 is amplified by using the specific amplification primer provided by the invention, and the amplification result is analyzed, so that the area under the ROC curve of healthy people and diabetic retinopathy patients is 0.804, the sensitivity is 71.43%, and the specificity is 85%, which indicates that the primer has high sensitivity and strong specificity.
Drawings
FIG. 1 shows a graph of the difference in expression levels of hsa _ circ _0095008 in healthy persons, diabetic patients, diabetic retinopathy patients;
FIG. 2 shows a ROC plot of hsa _ circ _0095008 for diagnosis of diabetic retinopathy.
Detailed Description
To further illustrate the objects and embodiments of the present invention, the following description will be given with reference to examples. It is to be understood that this description is primarily intended to illustrate the invention in detail, and not to limit its application.
The reagents and equipment used in this embodiment are, unless otherwise noted, conventional commercial reagents and equipment used in the practice of the present invention in accordance with manufacturer's instructions.
The room temperature in the embodiment of the invention refers to natural room temperature conditions, and is generally controlled within the range of 15-25 ℃, and the sample is not subjected to separate heating or cooling treatment.
Peripheral blood mononuclear cells contain a large number of immune cells, including lymphocytes, monocytes, phagocytes, and the like. The detection of peripheral blood mononuclear cells is widely applied to the diagnosis and research of various diseases.
Peripheral blood mononuclear cells can be obtained by Ficoll-hypaque (polysucrose-diatrizoate) density gradient centrifugation.
Test example 1, hsa _ circ _0095008 expression level differences among healthy persons, diabetic patients, and diabetic retinopathy patients
First, test method
Blood sample and treatment: peripheral blood mononuclear cells were collected from 42 diabetic retinopathy patients, 60 diabetic patients, and 20 normal persons. Total RNA was extracted using TRIzol (Invitrogen) reagent and stored at-80 ℃ until use.
The specific experimental procedures are as follows:
1. collecting a blood sample of a subject
Blood samples of healthy people, diabetic patients and diabetic retinopathy patients are collected by using an anticoagulation tube, and about 5ml of blood is collected by each person to be tested.
2. Isolation of peripheral blood mononuclear cells
(1) The 15ml centrifuge tubes were removed in 3 replicates and labeled.
(2) To the first centrifuge tube, 3ml of fresh blood was added, followed by 3ml of PBS buffer (blood sample: PBS buffer 1: 1).
(3) 2ml of the separation solution is added into the second centrifugal tube, and then the mixed solution in the first centrifugal tube is gently and slowly added, so that the mixed solution is positioned above the separation solution.
(4) And placing the second centrifugal tube into a centrifuge for trim centrifugation at 1500r/min for 40 min.
(5) And slowly taking out the centrifuge tube after the centrifugation is finished, sucking the white floccule, namely the mononuclear cells in the middle layer into a third centrifuge tube by using a pipette gun, adding PBS buffer solution into the third centrifuge tube to 6ml, shaking up the third centrifuge tube at 1700r/min for 10 min.
(6) Centrifuging, removing supernatant to obtain precipitate as mononuclear cell, and storing in refrigerator at-80 deg.C.
3. Advancing Total RNA in samples
(1) 1ml of Trizol was added to the sample, the lysed cells were blown with a pipette to complete lysis, and transferred to a 1.5ml centrifuge tube.
(2) 200 mul of chloroform was added, followed by vigorous shaking for 30s and standing on ice for 5 min.
(3) Centrifuge at 12000rpm for 15min at 4 ℃. Automatic layering after centrifugation: the upper layer is water phase, the middle layer is protein layer, and the lower layer is phenol phase. The upper aqueous phase was carefully transferred to a new 1.5ml centrifuge tube using a rnase-free wall head.
(4) Adding equal amount of isopropanol into the upper water phase, slightly turning upside down, mixing, and standing at room temperature for 30-60 min.
(5) Centrifugation was carried out at 10000rpm for 5min at 4 ℃ and a feathery white precipitate was visible at the bottom of the EP tube after centrifugation, and the supernatant was discarded.
(6) Adding 1ml anhydrous ethanol for rinsing, slightly inverting by hand, centrifuging at 4 deg.C and 10000rpm for 5min, and discarding the supernatant.
(7) Drying at room temperature, and adding appropriate amount of DEPC water to dissolve the precipitate.
(8) The purity and concentration of the RNA sample is measured by an electron spectrophotometer.
4. Reverse transcription assay
The reagent in the reverse transcription kit of TaKaRa company is thawed and shaken and mixed evenly. Reagents of interest and RNA were added to the PCR tube according to the instructions of the TaKaRa reverse transcription kit.
The PCR procedure was as follows: 15min at 37 ℃, 85 ℃ and 5 sec.
5. Real-time fluorescent quantitative PCR detection of hsa _ circ _0095008 expression level
A real-time fluorescent quantitative PCR system (20. mu.L) was prepared using a SYBR Premix Mater mix kit from Takara corporation with reference to the instructions: 10. mu.L of SYBR premix, 1. mu.L of forward primer, and 2. mu.L of sample of reverse primer 1. mu. L, cDNA were made up with ultrapure water for the remainder.
Setting real-time fluorescent quantitative PCR reaction conditions:
5min at 92 deg.C, (92 deg.C, 30 sec; 55 deg.C, 30 sec; 72 deg.C, 30sec, 40 cycles), 5min at 72 deg.C.
The sequence of the upstream primer that specifically recognizes hsa _ circ _0095008 is as follows: 5'-ATGCGACCATCCACCTCAAAG-3' (SEQ ID NO:2), the sequence of the downstream primer is as follows: 5'-ACATCACACACAATCACGGCA-3' (SEQ ID NO: 3); the sequence of the upstream primer specifically recognizing GAPDH is as follows: 5'-ATGGAAATCCCATCACCATCTT-3' (SEQ ID NO:4), the sequence of the downstream primer is as follows: 5'-CGCCCCACTTGATTTTGG-3' (SEQ ID NO: 5).
6. Analysis of expression level
The same sample was tested in triplicate and the average calculated. Normalization was performed using internal reference GAPDH, using
The method analyzes the relative expression level of the circRNA in the experimental sample and the control group sample, and analyzes the possibility of the experimental sample suffering from the diabetic retinopathy.
Second, test results
The expression levels of hsa _ circ _0095008 in diabetic retinopathy patients (DR, n-42), diabetic patients (DM, n-60) and healthy controls (Control, n-20) are shown in fig. 1. The clinical value of hsa _ circ _0095008 was obtained using ROC statistics using Graphpad prism 9.0 software, as shown in FIG. 2, where healthy persons and diabetic retinopathy patients had an area under the ROC curve (DR vs Control) of 0.804 and diabetic patients and diabetic retinopathy patients had an area under the ROC curve (DR vs DM) of 0.671.
The cutoff value for distinguishing healthy persons from diabetic retinopathy patients was 0.294, the hsa _ circ _0095008 sensitivity was 71.43%, the specificity was 85%, the positive predictive value was 90.9%, and the negative predictive value was 58.6%.
The cutoff value for distinguishing between diabetic and diabetic retinopathy patients was 0.447, the hsa _ circ _0095008 sensitivity was 52.38%, the specificity was 81.67%, the positive predictive value was 66.7%, and the negative predictive value was 71%.
Test example 2 application of expression level of hsa _ circ _0095008 in clinical diagnosis of diabetic retinopathy patients
First, test method
Referring to the method of example 1, peripheral blood of suspected diabetic retinopathy patients was collected and tested for hsa _ circ _0095008 expression levels in PBMCs.
Second, test results
The relative expression level of hsa _ circ _0095008 in the suspected patient was 0.0448911, less than 0.294. The clinical diagnosis is diabetic retinopathy patients.
Sequence listing
<110> Ningbo university subsidiary people hospital
<120> a circular RNA marker for diagnosing diabetic retinopathy and use thereof
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agaagctcat gttcaagaat gcgccaaccc cacaggagtt ccgggagggg gaagatgccg 60
tgattgtgtg tgatgtggtc agctccctcc caccaaccat catctggaaa cacaaaggcc 120
gagatgtcat cctgaaaaaa gatgtccgat tcatagtcct gtccaacaac tacctgcaga 180
tccggggcat caagaaaaca gatgagggca cttatcgctg tgagggcaga atcctggcac 240
ggggggagat caacttcaag gacattcagg tcattgtgaa tgtgccacct accatccagg 300
ccaggcagaa tattgtgaat gccaccgcca acctcggcca gtccgtcacc ctggtgtgcg 360
atgccgaagg cttcccagag cccaccatga gctggacaaa ggatggggaa cagatagagc 420
aagaggaaga cgatgagaag tacatcttca gcgacgatag ttcccagctg accatcaaaa 480
aggtggataa gaacgacgag gctgagtaca tctgcattgc tgagaacaag gctggcgagc 540
aggatgcgac catccacctc aaagtctttg 570
<210> 2
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acatcacaca caatcacggc a 21
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atggaaatcc catcaccatc tt 22
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Claims (3)
1. The application of a kit in preparing a reagent for screening diabetic retinopathy is characterized in that the kit comprises a specific amplification primer for amplifying circular RNA hsa _ circ _0095008, an internal reference gene GAPDH primer and an amplification system, wherein the specific amplification primer comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3; the cDNA nucleotide sequence corresponding to the circular RNA is shown as SEQ ID NO. 1.
2. The use of claim 1, wherein the nucleotide sequence of the upstream primer of the reference gene GAPDH is shown as SEQ ID NO. 4; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5.
3. Root of herbaceous plantThe use of claim 1, wherein the amplification system is a real-time fluorescent quantitative PCR amplification system comprising Ex Taq enzyme, dNTP mix, Mg 2+ Tli RNaseH and TB Green.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355373A (en) * | 2018-11-29 | 2019-02-19 | 复旦大学附属眼耳鼻喉科医院 | CircRNA is preparing the application in proliferative vitreoretinopathy diagnostic reagent |
| CN110106248A (en) * | 2019-04-12 | 2019-08-09 | 江苏省人民医院（南京医科大学第一附属医院） | Application of circular RNA hsa _ circ _0001543 in preparation of retinal degeneration disease diagnostic reagent |
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| CN109355373A (en) * | 2018-11-29 | 2019-02-19 | 复旦大学附属眼耳鼻喉科医院 | CircRNA is preparing the application in proliferative vitreoretinopathy diagnostic reagent |
| CN110106248A (en) * | 2019-04-12 | 2019-08-09 | 江苏省人民医院（南京医科大学第一附属医院） | Application of circular RNA hsa _ circ _0001543 in preparation of retinal degeneration disease diagnostic reagent |
Non-Patent Citations (2)
| Title |
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| CircRNA expression profiles in human visceral preadipocytes and adipocytes.;WENXING SUN等;《MOLECULAR MEDICINE REPORTS》;20201231;第21卷;第817页左栏第1段，表S1 * |
| Circular Rims2 Deficiency Causes Retinal Degeneration;Lan-Fang Sun等;《Adv. Biology》;20211105;第5卷;第2100906篇第1-11页 * |
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