CN109355373B - Application of circRNA in preparation of proliferative vitreoretinopathy diagnostic reagent - Google Patents
Application of circRNA in preparation of proliferative vitreoretinopathy diagnostic reagent Download PDFInfo
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Abstract
The invention discloses an application of circRNA in preparing a Proliferative Vitreoretinopathy (PVR) diagnostic reagent. The invention also discloses a circRNA detection kit for PVR diagnosis. The kit mainly comprises an RNA extraction system, a reverse transcription reaction system and a PCR reaction system. The relative content of circRNA in the vitreous body and the serum is detected by a real-time fluorescent quantitative PCR technology, and the method is used for the early diagnosis of PVR. The method has the advantages of small wound, high sensitivity and simple operation.
Description
Technical Field
The invention belongs to the technical field of medical biological detection, and particularly relates to application of circRNA in preparation of a Proliferative Vitreoretinopathy (PVR) diagnostic reagent, a circRNA kit for assisting in early diagnosis of PVR diseases, application of the circRNA kit in assisting in early diagnosis of PVR and a detection method.
Background
Proliferative Vitreoretinopathy (PVR) is a complex pathological response composed of a mixture of cellular components, vitreous, extracellular matrix, and a number of autocrine or paracrine cytokines. The extensive fibroproliferative membrane on the surface of the retina and behind the vitreous body contracts and stretches, thereby causing the retinal detachment. PVR has become a major cause of retinal reattachment surgery failure and retinal re-detachment, which can lead to severe impairment of visual function and even blindness. PVR is commonly seen in diseases such as coagulation, trauma, huge retinal tears, multiple retinal tears, long-term porogenic retinal detachment, multiple intraocular surgeries, eye trauma and the like, and the risk factors of PVR are not completely clear. PVR is a pathological process which is simultaneously involved in a plurality of cells, cytokines and growth factors, and the pathogenesis of the PVR is not clear. For the above reasons, there is currently no effective therapeutic PVR strategy, which is mainly based on vitrectomy and assisted by drug therapy (including antimetabolites, glucocorticoids, etc.). However, many patients have PVR proliferation membranes that cannot be removed completely by surgery, or have intraocular cells that are prone to proliferation recurrence after surgery, resulting in surgical failure, even with drug-assisted therapies. Therefore, there is a need to discover biomarkers for PVR to make early diagnosis and prognosis assessment of the occurrence of PVR.
Circular RNA (circular RNA) is a covalently closed circular RNA molecule and has the characteristics of universality, conservation, tissue specificity and high-stability expression. The circRNA can play a regulating role in the aspects of transcription, posttranscription, translation, epigenetics and the like. The circRNA has multiple physiological functions, can be involved in regulating and controlling processes such as cell proliferation, cell migration, cell differentiation and apoptosis and is abnormally expressed in the occurrence and development processes of a plurality of human diseases (including tumors, diabetes, cardiovascular diseases, nervous system diseases, blood system diseases and the like). In view of the characteristics and disease correlation of the circRNA, the circRNA is expected to become a novel clinical disease diagnosis marker.
There has been no study of circRNA as an early diagnostic marker for proliferative vitreoretinopathy to date.
Disclosure of Invention
The invention aims to provide a novel application of circRNA, which can be used as a diagnostic reagent for diagnosis of proliferative vitreoretinopathy, in particular to early diagnosis of proliferative vitreoretinopathy.
The specific technical scheme of the invention is as follows:
the application of circular RNA circ _0043144 in preparing a proliferative vitreoretinopathy diagnostic reagent, wherein the circBase ID of the circRNA is circ _0043144, and the cDNA nucleotide sequence of the circRNA is shown as SEQ ID NO: 1. The nucleotide sequence of the circRNA is complementary with the nucleotide sequence shown in SEQ ID NO. 1, and the head and the tail of the nucleotide sequence are connected into a circular structure.
Another object of the present invention is to provide a circRNA detection kit for proliferative vitreoretinopathy diagnosis, comprising an RNA extraction system, an RNA reverse transcription reaction system and a PCR reaction system, wherein the PCR reaction system contains a primer sequence for specifically amplifying the nucleotide sequence of SEQ ID NO. 1 according to claim 1. Further, the primer sequences are upstream and downstream primers of the circRNA specific qRT-PCR.
In the detection kit, the RNA extraction system comprises an RNA extraction reagent; the RNA reverse transcription reaction system comprises reverse transcriptase, a reverse transcription system buffer solution and an RNase inhibitor; the PCR reaction system comprises an amplification system and a primer system, wherein the amplification system is SYBR Premix Ex TaqTMA reagent; the primer system comprises RNA reverse transcription random primers and primers of circRNA specific qRT-PCR, wherein,
the random primer for RNA reverse transcription is a GAPDH quantitative PCR primer sequence, the upstream primer sequence is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3;
the upstream primer of the circRNA specific qRT-PCR is shown as SEQ ID NO. 4, and the downstream primer is shown as SEQ ID NO. 5.
The detection kit comprises:
(a) an extraction system:
1) trizol reagent, 1 tube, 5000. mu.L/tube;
2) chloroform, 1 tube, 1000 μ L/tube;
3) absolute ethyl alcohol, 1 tube, 10000 uL/tube;
4)DEPC ddH2o, 1 tube, 10000 uL/tube;
5)ddH2o, 1 tube, 10000 uL/tube;
6) isopropanol, 10000 μ L/tube;
(b) reverse transcription system:
1) total RNA reverse transcription primers (Random 6 mers), 1 tube, concentration: 50 μ M, 100 μ L/tube;
2) reverse transcriptase (200U/. mu.L) 100. mu.L;
3）dNTP Mixture (10 mM each) 100 μL；
4) reverse transcription buffer 100 μ L;
(c) and (3) PCR system:
1) SYBR Premix Ex Taq enzyme;
2）buffer 100 μL；
3) circ _0043144 specific qRT-PCR upstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
the circ _0043144 specific qRT-PCR downstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
18S rRNA quantitative PCR upstream primer, 1 tube, 10 MuM, 100 MuL/tube;
18S rRNA quantitative PCR downstream primer, 1 tube, 10 μ M, 100 μ L/tube;
4）dNTP Mixture (10 mM each) 100 μL。
the differential expression profiles of the retinal proliferation membranes of PVR diseases and other ophthalmic diseases are screened out by using the microarray of Agilent company, and the chip analysis results are verified by jointly applying a quantitative PCR method. Finally, circular RNA circ _0043144 is selected as a biomarker for assisting in early diagnosis of PVR diseases.
The method for determining the circ _0043144 as the biomarker for early diagnosis of the PVR disease comprises the following steps:
the first step is as follows: sample preparation: pre-retinal membrane specimens (experimental group, n = 30) and cataract proliferation membrane specimens (control group, n = 30) after ophthalmic vitrectomy, RNA was extracted using trizol (invitrogen) reagent and stored at-80 ℃ for use.
The second step is that: differential expression screening: analyzing circRNA related to PVR disease occurrence by adopting an expression profile chip of American Aglient company; the analysis comprises the following specific steps: amplifying and marking sample total RNA by using an Agilent expression profiling chip matched Kit, a Low Input Quick Amp WT labelling Kit (Cat. # 5190-; according to the Hybridization standard flow and the Kit provided by the Agilent Expression profiling chip, Gene Expression Hybridization Kit (Cat. # 5188-; the hybridized chip is scanned by an Agilent Microarray Scanner, data is read by a software Feature Extraction software 10.7 (Agilent technologies), and finally normalization processing is carried out by a limma packet in R software, wherein the algorithm is Quantum. The original data are subjected to line normalization processing, and the overall characteristics of sample data distribution are observed by using a Box plot (Box plot) on the normalized data. The original data is normalized by limma package in software R software, and then the differential genes are screened by using Fold-change (expression difference multiple) and T test (Student's T-test) statistical methods, and then a target point is determined for subsequent determination by combining GO and KEGG signal channel analysis.
The third step: and (3) verifying the experimental result of chip analysis by adopting quantitative PCR.
The fourth step: verifying the expression difference of the target point circ _0043144 in the serum of PVR patients, normal persons and operation treatment PVR patients.
The fifth step: based on in vitro cell experiments, the basic principle aspect reveals that circ _0043144 defines the regulation function of RPE cells in the pathological process of PVR by regulating the proliferation and migration of the RPE cells.
The invention also aims to provide application of circ _0043144 siRNA in preparing a medicament for treating proliferative vitreoretinopathy.
The invention proves that the detection of the expression of circ _0043144 in the serum of patients to realize the diagnosis of early PVR is completely feasible through a quantitative PCR technology.
The kit of the invention is also suitable for: patients who are initially suspected of PVR based on their age, medical history, and local signs.
The kit of the invention is used for detecting the content of circ _0043144 in the serum of a plurality of known PVR patients and a plurality of normal patients respectively, and the content is used as a standard curve. The content of circ _0043144 in the serum of unknown patients is determined by the same method, and the patients can be preliminarily judged whether to have PVR or not by comparing the standard data so as to further confirm the diagnosis.
The kit disclosed by the invention utilizes a quantitative PCR method for the first time, assists the diagnosis of PVR by detecting the content of circ _0043144 in serum, has the characteristics of small wound and simple operability, and ensures that the circ _0043144 is expected to be a biomarker for scientifically judging the generation of PVR and the prognosis judgment of patients.
Drawings
FIG. 1 is a graph showing the results of chip analysis for PVR disease screening and verification (graph A is the quality of analysis of a boxplot chip analysis in which 10 samples are mixed to form a biological duplication to eliminate individual differences; graph B is a graph of volcano plot screening for CRICRNA associated with PVR; and graph C is a graph of cluster analysis of the upstream 10 circRNA and downstream expression of PVR).
FIG. 2 is a graph demonstrating the expression of circ _0043144 in human PVR proliferation membranes. (FIG. A shows the expression of circ-0043144 in the PVR growth membrane by quantitative PCR analysis; FIG. B shows the expression change of PVR-associated genes (as a positive control) by quantitative PCR analysis).
FIG. 3 is a graph showing the analysis of the expression of circ _0043144 in PVR vitreous body and serum (graph A shows the quantitative PCR analysis of the difference between the expression of circ _0043144 in the vitreous body of normal person and PVR patient, graph B shows the quantitative PCR analysis of the difference between the expression of circ _0043144 in the serum of normal person and PVR patient, and graph C shows the quantitative PCR analysis of the difference between the expression of circ _0043144 in the serum of normal person and PVR post-operation patient).
FIG. 4 is a graph of the effect of circ _0043144 intervention on PVR-related pathological processes (graph A is a quantitative PCR analysis of the effect of circ _0043144 siRNA transfection on circ _0043144 expression; graph B is a graph of the effect of circ _0043144 intervention on RPE proliferation analyzed using Ki67 immunofluorescence, wherein the left panel is a fluorescence staining graph and the right panel is a statistical analysis graph; graph C is a graph of the effect of circ _0043144 intervention on RPE migration analyzed using the Transwell method, wherein the left panel is a fluorescence staining graph and the right panel is a statistical analysis graph; and graphs D, E, F and G analyze the effect of the circ _0043144 intervention on RPE analysis of CCL-2, CCL-8, IL-6 and VEGF-A.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1 circ _0043144 correlation with proliferative vitreoretinopathy
The first step is as follows: sample preparation: pre-retinal membrane specimens (experimental group, n = 30) and cataract proliferation membrane specimens (control group, n = 30) after ophthalmic vitrectomy, total RNA was extracted using trizol (invitrogen) reagent and stored at-80 ℃ for use.
The second step is that: differential expression screening:
analyzing circRNA related to PVR disease occurrence by adopting an expression profile chip of American Aglient company; the analysis comprises the following specific steps: amplifying and marking sample total RNA by adopting an Agilent expression profile chip matched Kit, a Low Input Quick Amp WT labelling Kit (Cat. # 5190-; according to the Hybridization standard flow and the Kit provided by the Agilent Expression profiling chip, Gene Expression Hybridization Kit (Cat. # 5188-; the hybridized chip is scanned by an Agilent Microarray Scanner, data is read by a software Feature Extraction software 10.7 (Agilent technologies), and finally normalization processing is carried out by a limma packet in R software, wherein the algorithm is Quantum. The original data are subjected to line normalization processing, and the overall characteristics of sample data distribution are observed by using a Box plot (Box plot) on the normalized data. After normalization of the original data by limma package in software R software, different genes were screened lines using Fold-change (Fold differential expression) and T-test (Student's T-test) statistical methods. The distribution of each sample is shown in FIG. 1A; screening circRNA molecules related to PVR occurrence by using volcano charts, as shown in figure 1B; the 10 circrnas with the most pronounced up-and down-regulation were analyzed by clustering analysis, as shown in figure 1C.
The third step: the expression of circ _0043144 in human PVR proliferation membranes was verified (panel A is quantitative PCR analysis of the expression of circ _0043144 in PVR proliferation membranes; panel B is quantitative PCR analysis of the expression change of PVR-associated genes (as positive control)).
The fourth step: verifying the expression difference of the target point circ _0043144 in the PVR proliferation membrane (experimental group) and the pre-cataract membrane (control group), as shown in FIG. 3A; verifying the expression difference of the target point circ _0043144 in the serum of PVR patients, normal persons and patients treated by operation, as shown in FIG. 3B; verifying the expression difference of target circ _0043144 in serum before and after PVR patients, as shown in FIG. 3C.
The fifth step: based on in vitro cell experiments, it is revealed from the basic principle aspect that circ _0043144 is involved in the regulation of PVR pathological process by regulating RPE cell function, as shown in FIG. 4.
Example 2: preparation of the kit of the invention
The sequence of circ _0043144 is shown in SEQ ID NO 1. The specific quantitative PCR upstream and downstream primers are synthesized by Shanghai' S chemical company, the purity is PAGE grade, the synthesized primers are dissolved by DEPC H2O, the total concentration is 10 mu M, and the internal reference primer is 18S rRNA.
Preparing a kit comprising the following components:
(a) an extraction system:
1) trizol reagent, 1 tube, 5000. mu.L/tube;
2) chloroform, 1 tube, 1000 μ L/tube;
3) absolute ethyl alcohol, 1 tube, 10000 uL/tube;
4)DEPC ddH2o, 1 tube, 10000 uL/tube;
5)ddH2o, 1 tube, 10000 uL/tube;
6) isopropanol, 10000 μ L/tube;
(b) reverse transcription system:
1) total RNA reverse transcription primers (Random 6 mers), 1 tube, concentration: 50 μ M, 100 μ L/tube;
2) reverse transcriptase (200U/. mu.L) 100. mu.L;
3）dNTP Mixture (10 mM each) 100 μL；
4) reverse transcription buffer 100 μ L;
(c) and (3) PCR system:
1) SYBR Premix Ex Taq enzyme;
2）buffer 100 μL；
3) circ _0043144 specific qRT-PCR upstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
the circ _0043144 specific qRT-PCR downstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
18S rRNA quantitative PCR upstream primer, 1 tube, 10 MuM, 100 MuL/tube;
18S rRNA quantitative PCR downstream primer, 1 tube, 10 μ M, 100 μ L/tube;
4）dNTP Mixture (10 mM each) 100 μL。
example 3: kit detection of the invention
First, separation of serum
Blood samples of the tested individual and the healthy individual as a reference are collected, and serum and blood cells are separated from the blood samples by centrifugation using a heparin anticoagulation tube for detection. Centrifugation conditions were 4 ℃, 12,000 rpm, 10 min.
Second, RNA extraction of serum and vitreous samples
By adopting the total RNA extraction system, TRIzol is added and then the mixture is placed at room temperature for 10 min, so that the sample is fully cracked (if the next operation is not carried out, the sample can be stored for a long time at minus 80 ℃). Adding 200 μ l chloroform into 1 ml TRIzol, vigorously shaking, mixing, standing at room temperature for 3-5 min, and naturally separating phases. Centrifuge at 12,000 rpm for 15 min at 4 ℃. The sample will be divided into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the aqueous phase is transferred to a new tube. An equal volume of ice-cold isopropanol was added to the supernatant and left at room temperature for 15 min. Centrifugation was carried out at 12,000 rpm at 4 ℃ for 10 min, the supernatant was discarded, and RNA was precipitated at the bottom of the tube. 1 ml of 75% ethanol (prepared with RNase-free water) was added to the RNA precipitate, and the pellet was suspended by gently shaking the centrifuge tube. 1 ml of 75% ethanol was added per 1 ml of TRIzol. Centrifuge at 8,000 rpm for 5min at 4 ℃ and discard the supernatant. After air-drying at room temperature, 50. mu.l of RNase-free water was added to the pellet to dissolve RNA sufficiently, and the pellet was stored at-80 ℃.
Third, RNA quality detection
Measuring the concentration of RNA at the absorbance positions of 260 nm and 280 nm by adopting an ultraviolet spectrophotometer; the ratio of A260/A280 of the RNA solution is the RNA purity, and the ratio ranges from 1.8 to 2.1.
IV, reverse transcription reaction
The reverse transcription reaction adopts an RNA reverse transcription reversing system of the kit of the invention: PrimeScriptTMThe RT reagent Kit, 25. mu.l of the system was added:
5×PrimeScriptTM Buffer 5 μl
PrimeScriptTM RT Enzyme Mix I 1.25 μl
Random 6mers（100μM） 6.25 μl
Total RNA 2.5 μl
RNase free ddH2O 16.5 μl
the above system was placed in an Rnase-free EP tube and inverted to cDNA according to the following procedure: the cDNA was stored at-20 ℃ for 15 min at 37 ℃ and 10 sec at 80 ℃.
Four, real-time fluorescent quantitative PCR
A PCR reaction system (50 mu l) of the kit is prepared as follows: SYBR Green Mix 25 mul, circ _0043144 specific qRT-PCR upstream primer, circ _0043144 specific qRT-PCR downstream primer, 18S rRNA quantitative PCR upstream primer, 18S rRNA quantitative PCR downstream primer 1 mul each, dNTP 2 mul, to-be-detected sample cDNA 3 mul, ddH2O 18 μl。
Quantitative PCR reaction conditions
5min at 92 deg.C, (92 deg.C, 30 s, 54 deg.C, 30 s, 72 deg.C, 30 s 45 cycles), 72 deg.C, 10 min.
Fifth, data analysis
And respectively detecting target RNA and internal reference RNA of the same sample, normalizing the target RNA by taking the expression quantity of the internal reference as a reference, and then analyzing the relative expression quantity of the target RNA by adopting a delta delta delta Ct method commonly used in the field. Finally, the disease susceptibility of the patient to be detected is judged by comparing the expression difference of the circ _0043144 of the sample to be detected and the target sample.
Sequence listing
<110> eye, ear, nose and throat department hospital affiliated to the university of Compound Dan
Application of circRNA in preparing proliferative vitreoretinopathy diagnostic reagent
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<213> Artificial Sequence (Artificial Sequence)
<400> 1
cttcctaacc aagcgaagcc ggcaggtctg tgctgacccc agtgaggagt gggtccagaa 60
atatgtcagc gacctggagc tgagtgcctg aggggtccag aagcttcgag gcccagcgac 120
ctcggtgggc ccagtgggga ggagcaggag cctgagcctt gggaacatgc gtgtgacctc 180
cacagctacc tcttctatgg actggttgtt gccaaacagc cacactgtgg gactcttctt 240
aacttaaatt ttaatttatt tatactattt agtttttgta atttattttc gatttcacag 300
tgtgtttgtg attgtttgct ctgagagttc ccctgtcccc tcccccttcc ctcacaccgc 360
gtctggtgac aaccgagtgg ctgtcatcag cctgtgtagg cagtcatggc accaaagcca 420
ccagactgac aaatgtgtat cggatgcttt tgttcagggc tgtgatcggc ctggggaaat 480
aataaagatg ctcttttaaa aggtaa 506
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<213> Artificial Sequence (Artificial Sequence)
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ggtaacttcc taaccaagcg a 21
Claims (6)
1. The application of a reagent for detecting circular RNA circ _0043144 in preparing a proliferative vitreoretinopathy diagnostic reagent, wherein the cDNA nucleotide sequence of the circular RNA is shown as SEQ ID NO: 1.
2. A circRNA detection kit for proliferative vitreoretinopathy diagnosis is characterized in that the kit comprises an RNA extraction system, an RNA reverse transcription system and a PCR reaction system; wherein the PCR reaction system contains a primer sequence for specifically amplifying the nucleotide sequence of SEQ ID NO. 1 as set forth in claim 1.
3. The test kit according to claim 2, wherein the primer sequence is an upstream primer and a downstream primer of a circRNA-specific fluorescent real-time quantitative PCR.
4. The test kit of claim 2, wherein the RNA extraction system comprises an RNA extraction reagent; the RNA reverse transcription reaction system comprises reverse transcriptase, a reverse transcription system buffer solution and an RNase inhibitor; the PCR reaction system comprises an amplification system and a primer system, wherein the amplification system is SYBR Premix Ex TaqTMA reagent; the primer system comprises reverse transcription random primers and circRNA specific qRT-PCR primers, wherein the RNA reverse transcription random primers are quantitative PCR primer sequences of 18S rRNA, an upstream primer sequence is shown as SEQ ID NO. 2, and a downstream primer sequence is shown as SEQ ID NO. 3; the upstream primer of the circRNA specific qRT-PCR is shown as SEQ ID NO. 4, and the downstream primer is shown as SEQ ID NO. 5.
5. The test kit of claim 4, wherein the kit comprises:
(a) an extraction system:
1) trizol reagent, 1 tube, 5000. mu.L/tube;
2) chloroform, 1 tube, 1000 μ L/tube;
3) absolute ethyl alcohol, 1 tube, 10000 uL/tube;
4) DEPC ddH2o, 1 tube, 10000 uL/tube;
5) ddH2o, 1 tube, 10000 uL/tube;
6) isopropanol, 10000 μ L/tube;
(b) reverse transcription system:
1) total RNA reverse transcription primers, Random 6 mers, 1 tube, concentration: 50 μ M, 100 μ L/tube;
2) reverse transcriptase, 200U/muL, 100 muL;
3）dNTP Mixture ，10 mM each，100 μL；
4) reverse transcription buffer 100 μ L;
(c) and (3) PCR system:
1) SYBR Premix Ex Taq enzyme;
2）buffer 100 μL；
3) circ _0043144 specific qRT-PCR upstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
the circ _0043144 specific qRT-PCR downstream primer, 1 tube, 10. mu.M, 100. mu.L/tube;
18S rRNA quantitative PCR upstream primer, 1 tube, 10 MuM, 100 MuL/tube;
18S rRNA quantitative PCR downstream primer, 1 tube, 10 μ M, 100 μ L/tube;
4）dNTP Mixture ，10 mM each，100 μL。
6. the use of siRNA interfering with circRNA expression according to claim 1 in the preparation of a medicament for the treatment of proliferative vitreoretinopathy, the nucleotide sequence of which is shown in SEQ ID NO. 6.
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| CN104962654A (en) * | 2014-11-18 | 2015-10-07 | 南京医科大学眼科医院 | Application of lncRNA-MALAT1 in preparing proliferative vitroretinopathy (PVR) diagnosis reagent |
| CN108064176A (en) * | 2015-04-22 | 2018-05-22 | 库瑞瓦格股份公司 | For treating the composition containing RNA of tumor disease |
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