CN110106248A - Application of circular RNA hsa _ circ _0001543 in preparation of retinal degeneration disease diagnostic reagent - Google Patents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses an application of circular RNA hsa _ circ _0001543 in preparing a retinal degeneration disease diagnostic reagent. Application of a reagent for detecting expression quantity of hsa _ circ _0001543(SEQ ID NO.4) in preparation of a kit for diagnosing retinal degeneration diseases. Research shows that the expression quantity of hsa _ circ _0001543 is reduced to be used as an auxiliary diagnosis basis of the retinal degeneration diseases, and the early diagnosis of the retinal degeneration diseases is facilitated. The protective effect of hsa _ circ _0001543 on the retina suggests that it can be used in the preparation and screening of drugs for the treatment of retinal degenerative diseases. The invention provides a novel detection marker and a novel therapeutic target for retinal degenerative diseases, and provides brand-new molecular information and biological basis for diagnosis and treatment of the diseases.
Description
Technical field
The invention belongs to fields of biomedicine, are related to circular rna hsa_circ_0001543 preparation diagnosis retinosis
Application in the kit of disease.
Background technique
Blinding eye disease is the third-largest influence human health disease of World Health Organization, global about 2.5 hundred million eyesights
Disabled person and China accounts for nearly 20%.Retinal degenerative disease there is no effective treatment method at present, is important blinding eye disease, is
The sciences problems and great public health problem urgently captured.The pathogenesis multiplicity of retinal degenerative disease, can according to the cause of disease
To be divided into the hereditary retinal dystrophy disease as caused by single-gene defect and the complexity retina as caused by multifactor damage
Degenerative disease.
Hereditary retinal dystrophy disease be with inherent cause it is closely related, with primary degeneration of retina be main pathology
Change, be badly damaged with visual functions such as eyesight and the visuals field as main clinical phenotypes.Hereditary retinal dystrophy disease is world's model
The primary blinding eye disease of disease incidence in interior work age groups is enclosed, disease incidence is about 1/3000 in developed country, at me
State is then higher, and only the disease incidence of retinal pigment degeneration is as high as 1/1000 or so, estimates the Inherited retinal in China accordingly
Degenerative disease patient is more than 1,300,000.Clinically it there is no effective therapeutic modality for hereditary retinal dystrophy disease at present, sternly
The health of compatriots is compromised again.
Age-related macular degeneration (age-related macular degeneration, AMD) is most commonly seen
A kind of complexity retinal degenerative disease, also referred to as senile macular degeneration are that the aging of macula lutea plot structure sexually revises, the head of patient
Sending out clinical symptoms is metamorphopsia and visual impairment.AMD mostly occurs in 45 years old or more crowd, illness rate with the growth at age and by
Step increases, and is the irreversible blind first cause of elderly population in world wide.In China, with the acceleration of population aging, AMD
Illness rate increase year by year, show AMD in each age group crowd of Shanghai City according to the cri dernier cri disease statistical result in multiple cities
Recall rate respectively be up to 6.23% (60~69 years old), 14.98% (70~79 years old) and 29.91% (80 years old or more), Wuxi City
The recall rate of AMD then be respectively 3.43% (50~59 years old), 7.73% (60~69 years old), 14.69% (70~79 years old) and
16.51% (80 years old or more), more some researches show that, Hangzhou cause 70 years old and the cause of disease of above the elderly's inpairment of vision in,
AMD accounts for 45.64%, these epidemic datas all show AMD gradually as the irreversible vision impairment of China's elderly population
Main cause.Can be divided AMD according to the course of disease of disease in clinical manifestation then can further divide for early and late AMD, advanced stage AMD
For stemness and moist amphitypy.Clinical effective therapeutic modality still without being directed to stemness AMD at present.It is clinical at present for moist AMD
Upper master is to be used to be treated for anti-vegf, and anti-VEGF antibody can effectively inhibit the expression of VEGF, inhibit the generation of CNV and reduction
Vascular leakage.However, there is also a series of defects for anti vegf agents treatment, multiple intraocular injection is generally required, is increased
The pain of patient has also aggravated financial burden, and the therapeutic effect of anti vegf agents differs greatly with different patients, part
Patient's unsatisfactory curative effect after the treatment of multiple anti-vegf, eyesight still can not improve or improve unobvious.Retinal degenerative disease is not only
The quality of life of our people is drastically influenced, also brings harm to the development of national economy.Therefore, to retinosis disease
The early diagnosis of disease is intervened, and finds novel, sensitive and applies convenient disease detection marker, view can effectively be controlled by finding
The treatment and prevention means of nethike embrane degenerative disease disease, are of great practical significance.
Circular rna (circular RNA, circRNA) is a kind of special non-coding RNA, and circRNA molecule is in closing
Cyclic structure, expression is stablized, not degradable.CircRNA does not encode albumen, but it is rich in Microrna (microRNA, miRNA)
Binding site, can in the cell by competitive endogenous RNA (competing endogenous RNA, ceRNA) mechanism,
As miRNA sponge, miRNA is released to the inhibiting effect of target gene, to regulate and control expression of target gene.CircRNA participates in cell
The regulation of interior various procedures, plays a significant role in numerous vital movements, at the same also with the generation of many diseases, development there is
Very closely connection.Nearest research finds circHIPK3 (Shan K, Liu C, Liu BH et al.Circular
Noncoding RNA HIPK3Mediates Retinal Vascular Dysfunction in Diabetes
Mellitus.Circulation 2017;136 (17): 1629-1642) and circ_0005015 (Zhang SJ, Chen X, Li
CP et al.Identification and Characterization of Circular RNAs as a New Class
of Putative Biomarkers in Diabetes Retinopathy.Invest Ophthalmol Vis
Sci.2017;58 (14): 6500-6509) expression change and take part in the occurrence and development of diabetic retinopathy;
The expression of circ_0043144 changes occurrence and development (Yao J, the Hu LL, Li for also affecting proliferative retinopathy
XM et al.Comprehensive circular RNA profiling of proliferative
vitreoretinopathy and its clinical significance.Biomed Pharmacother.2018;111:
548-554).These results of study prompt to deposit between circRNA expression and the variation of specificity and the occurrence and development of disease
In inevitable connection.Inventor discloses LncRNA ZNF503-AS1 expression quantity in the CN107099589A that early period applies
Decline can be used as the auxiliary diagnosis foundation of retinal degenerative disease, help the early diagnosis for realizing retinal degenerative disease.
LncRNA ZNF503-AS1 prompts it to can be used for preparing and screening for treating retinal degeneration the protective effect of retina
The drug of property disease.But the diagnosing and treating of retinal degenerative disease also needs more markers and candidate circRNA.
Summary of the invention
The object of the present invention is to provide the new opplications of hsa_circ_0001543.
The purpose of the present invention can be achieved through the following technical solutions:
It is a kind of detect hsa_circ_0001543 (SEQ IDNO.4) expression quantity reagent preparation diagnose retinosis
Application in the kit of disease.
The reagent of the detection hsa_circ_0001543 expression quantity preferably has inspection to hsa_circ_0001543
Survey probe, genetic chip or real-time quantitative PCR (the quantitative real-time polymerase chain of specificity
Reaction, Q-PCR) primer.
Described has the probe sequence of detection specificity preferably as follows hsa_circ_0001543:
GCTATTCAAGTTGATATTCACTGATGGACTCCAAAGAATCATTAACTCCTGGTAGAGAAG(SEQID
NO.1)。
Described has the PCR primer of detection specificity preferably as follows hsa_circ_0001543:
Upstream primer: ACAACTTGACTTCTCTGGGGAC (SEQ ID NO.2);
Downstream primer: GTCCATCACATCTCCCCTCTC (SEQ ID NO.3).
A kind of diagnostic kit of retinal degenerative disease, it is characterised in that include detection hsa_circ_0001543 expression
The reagent of amount.
The kit is preferably comprised to the hsa_circ_0001543 probe with detection specificity or to hsa_
Circ_0001543 has the real-time quantitative PCR primer of detection specificity.
Hsa_circ_0001543 or the substance for increasing hsa_circ_0001543 expression quantity treat retinal degeneration in preparation
Application in the drug of property disease.
Hsa_circ_0001543 answering in the drug of screening treatment retinal degenerative disease as drug target
With.
The utility model has the advantages that
1. applicant filters out hsa_circ_0001543 work on the basis of being engaged in clinical treatment and basic research for many years
For the detection marker of retinal degenerative disease, and hsa_circ_0001543 and retinal degeneration are specified by the method for the invention
The relationship of property disease.
2. inventor is it has been investigated that the decline of hsa_circ_0001543 expression quantity can be used as the auxiliary of retinal degenerative disease
Diagnosis basis is helped, the early diagnosis for realizing retinal degenerative disease is helped.Protectiveness of the hsa_circ_0001543 to retina
Effect prompts it to can be used for preparing and screening the drug for treating retinal degenerative disease.The present invention provides new views
The detection marker and therapy target of film degenerative disease provide completely new molecular information and life for the diagnosing and treating of the disease
Object basis.
Detailed description of the invention
Fig. 1 hsa_circ_0001543 is in the retinal tissue of 6 normal persons and 9 retinal degenerative disease patients
Expression compare
U6,18S and hsa_circ_0001543 that Fig. 2 in situ hybridization is shown are in adult human retinal pigment epithelial
Positioning in (adult retinal pigment epithelial, ARPE19) cell
The expression of Fig. 3 U6, GAPDH and hsa_circ_0001543 in ARPE19 cytoplasm and nucleus compares
Hsa_circ_0001543 strikes the (hsa_circ_ that comes down to a lower group in hsa_circ_0001543 in Fig. 4 ARPE19 cell
0001543siRNA) compared with the expression in control group (scramble siRNA)
Retinal specific marker gene strikes in hsa_circ_0001543 and comes down to a lower group and control group in Fig. 5 ARPE19 cell
In expression compare
Retinal specific labelled protein BEST1 is in hsa_circ_ in the ARPE19 cell that Fig. 6 immunofluorescence dyeing is shown
0001543 strikes and comes down to a lower group compared with the expression in control group
Cell connects specific marker proteins ZO-1 in hsa_ in the ARPE19 cell that Fig. 7 immunofluorescence dyeing is shown
Circ_0001543, which strikes, to come down to a lower group compared with the expression in control group
Cell connection specific marker proteins β-catenin exists in the ARPE19 cell that Fig. 8 immunofluorescence dyeing is shown
Hsa_circ_0001543, which strikes, to come down to a lower group compared with the expression in control group
The microsphere particle swallowed in the ARPE19 cell that Fig. 9 immunofluorescence dyeing is shown strikes drop in hsa_circ_0001543
Group is compared with the content situation in control group
The microsphere particle swallowed in Figure 10 ARPE19 cell strikes to come down to a lower group in hsa_circ_0001543 to be contained with control group
The quantization of amount is compared
ARPE19 cell Mitochondria active oxygen (the reactive oxygen that Figure 11 immunofluorescence dyeing is shown
Species, ROS) it strikes and is come down to a lower group compared with content in control group in hsa_circ_0001543
Figure 12 ARPE19 cell Mitochondria ROS strikes the amount come down to a lower group with content in control group in hsa_circ_0001543
Change is compared
The cell of the EdU positive strikes drop in hsa_circ_0001543 in the ARPE19 cell that Figure 13 immunofluorescence dyeing is shown
Group is compared in control group
In Figure 14 ARPE19 cell the cell proportion of the EdU positive hsa_circ_0001543 strike come down to a lower group in control group
Comparison
PTEN, p-AKT, AKT, p-mTOR, mTOR, p-p70S6K in the ARPE19 cell of Figure 15 immunoblotting dyeing display
And p70S6K strikes in hsa_circ_0001543 and comes down to a lower group compared in control group
PTEN, p-AKT/AKT, p-mTOR/mTOR and p-p70S6K/p70S6K are in hsa_ in Figure 16 ARPE19 cell
Circ_0001543, which strikes, to come down to a lower group compared with the quantization in control group
Mouse Retina pigment epithelium that Figure 17 immunofluorescence dyeing is shown (retinal pigment epithelium,
RPE) in tissue the expression quantity of ZO-1 albumen struck in mmu_circ_0007330 come down to a lower group (mmu_circ_0007330siRNA) with it is right
Compare according to the expression in group
The expression quantity of β-catenin albumen is in mmu_circ_ in the mouse RPE tissue that Figure 18 immunofluorescence dyeing is shown
0007330 strikes and comes down to a lower group compared with the expression in control group
Specific embodiment
Embodiment 1:
Our induce multi-potent stem cell-retinal color to each different divergaence time points by circRNA chip early periods
Plain epithelial cell (induced pluripotent stem cells-retinal pigment epithelium, iPSC-
RPE the detection for) having carried out circRNA expression quantity finds the expression quantity of hsa_circ_0001543 with RPE cell differentiation
Increase and increase, height prompts potential important function of the hsa_circ_0001543 in RPE cell differentiation procedure.For into one
Pathology sense of the clear hsa_circ_0001543 in retinal degenerative disease is walked, we are first in normal control and retina
The expression quantity of hsa_circ_0001543 is detected in the retinal tissue of degenerative disease patient.
Experimental method:
1. the acquisition of normal control and the blood preparation of retinal degenerative disease patient:
Collect the blood sample of patient and 6 normal controls that 9 clinical definites are retinal degenerative disease voluntarily contributed
This, carries out RNA extraction.With 5~8mL of peripheric venous blood of EDTA anticoagulant blood-collecting pipe collection research object, total serum IgE is extracted.
2.Q-PCR detection:
Further by reverse transcription PCR (reverse transcription-PCR, RT-PCR) and Q-PCR to blood rna
The expression quantity of hsa_circ_0001543 is detected in sample.
RT-PCR:
Reverse transcription reaction is carried out by following system using Takara Reverse Transcriptase kit, mRNA is transcribed into cDNA, is reacted
System (40 μ l) is as follows:
Reaction condition: room temperature 10min, 42 DEG C of 45min, 95 DEG C of 5min, 4 DEG C of 5min.
Q-PCR:
Q-PCR reaction is carried out using the cDNA template generated by RT-PCR, reaction system is as follows:
Involved primer sequence is referring to following table:
Q-PCR reaction condition:
According to formula R (relative expression quantity)=2-ΔΔct=2[(ct sample-ct internal reference)-(ct blank-ct internal reference blank)]Calculate relative expression quantity.
Experimental result:
Chip test result discovery hsa_circ_0001543 is deposited in retinal degenerative disease patient and Normal group
In differential expression, the expression in the retina group of retinal degenerative disease patient significantly reduces (figure compared with normal control
1).Disease associated, the hsa_circ_0001543 of hsa_circ_0001543 and retinal degenerative disease is further prompted
It may play the role of to the course of disease of retinal degenerative disease protective.
The analysis of embodiment 2hsa_circ_0001543 intracellular targeting
By the fluorescent staining of in situ hybridization combined immunization and caryoplasm separation RNA quantitative detection experiment to hsa_circ_
0001543 expresses in ARPE19 cell and is positioned, and U6 is set as nucleus specific localization reference, 18S is that cytoplasm is special
Opposite sex positioning reference.It is Ribo used in situ hybridization combined immunization fluorescent stainingTMLncRNA FISH Probe Mix examination
Agent box (Rui Bo biotech firm), it is PARIS that caryoplasm, which separates used in RNA extraction,TMKit (Invitrogen company), RNA
Quantitative detecting method is referring to embodiment 1.It is operated in strict accordance with kit specification.
Experimental result:
The fluorescent staining of in situ hybridization combined immunization and caryoplasm separation RNA are extracted and are found the main table of hsa_circ_0001543
Up to (Fig. 2,3) in the cytoplasm in ARPE19 cell.
Influence of the embodiment 3hsa_circ_0001543 to RPE cell function related genes and protein expression
In retinal degenerative disease, the expression of RPE cell degeneration, RPE cell function related genes and albumen is lowered,
RPE cell is caused dysfunction occur.For pathological effect of the clear hsa_circ_0001543 in retinal degenerative disease, I
Have detected its influence to RPE cell function related genes and protein expression.It is set at two groups of different ARPE19 cells altogether
Reason group, respectively hsa_circ_0001543 strike come down to a lower group (hsa_circ_0001543siRNA) and its control group (scramble
siRNA).Wherein, scramble siRNA and hsa_circ_0001543siRNA is purchased from Rui Bo biotech firm.
1. the RNA in cell is extracted first, in accordance with the method in embodiment 1, and further by RT-PCR and Q-PCR, it is right
In different disposal group ARPE19 cell hsa_circ_0001543 and retinal specific marker gene (including MITF,
MERTK, BEST1, KRT18, CTNNB1 and TJP1) expression detected.
Involved primer sequence is referring to the sequence and following table in embodiment 1:
2. by immunofluorescence dyeing, to retinal specific labelled protein BEST1 in different disposal group ARPE19 cell,
The expression of the connection of retina R PE cell specific marker proteins β-catenin and ZO-1 detect, and specific experiment step is such as
Under:
(1) cell climbing sheet: cell is laid in 8 hole Chamber slide tissue culture plates (Millipore company), reference
Cell culture part;
(2) go out from incubator and take out cell, after PBS washing, 4% paraformaldehyde (paraformaldehyde, PFA) is fixed
30~60min;
(3) after PBS rinses 10min, 0.5%Triton X-100 perforation 20min;
(4) PBS is rinsed 2 times, and after each 10min, 1%BSA closes 30min;
(5) the diluted primary antibody of 1%BSA is added, in 4 DEG C of hybridized overnights;
(6) PBS is rinsed 2 times, and after each 10min, the diluted fluorescence secondary antibody of 1%BSA, 37 DEG C of hybridization 2h are added;
(7) PBS is rinsed 2 times, and after each 10min, DAPI (Sigma company) the dye core containing anti-quencher simultaneously prevents fluorescence
It is quenched, mounting;
(8) it Olympus IX70 confocal laser scanning microscope and takes pictures.
Related antibody information is referring to following table:
Experimental result:
The result of Q-PCR prompts, hsa_circ_0001543 and retinal specific marker gene (packet in ARPE19 cell
Include MITF, MERTK, BEST1, KRT18, CTNNB1 and TJP1) expression in hsa_circ_0001543 strikes and comes down to a lower group compared with
Control group significantly reduces (Fig. 4,5).The result of immunofluorescence dyeing prompts, and retinal specific marks egg in ARPE19 cell
White BEST1, cell connection specific marker proteins ZO-1 and β-catenin are relatively compareed in hsa_circ_0001543 strikes and comes down to a lower group
The fluorescence intensity of group obviously weakens (Fig. 6,7,8), and the expression quantity of these albumen is further prompted to strike in hsa_circ_0001543
It is significantly reduced in coming down to a lower group compared with control group.
To sum up, ours as a result, it has been found that hsa_circ_0001543 expression quantity deficiency can inhibit RPE cell function correlation
The expression of albumen causes RPE cell dysfunction, further prompts hsa_circ_0001543 to RPE cell and in retina
Potential important protective effect in the degenerative disease course of disease.
Influence of the embodiment 4hsa_circ_0001543 to RPE cytophagy
Two groups of different ARPE19 cell processing groups are set altogether, and respectively hsa_circ_0001543, which strikes, to be come down to a lower group and its compare
Group, with embodiment 3.Polystyrene latex microballoon (Sigma company) the specific detection different disposal group modified by carboxylate
The phagocytic function of APRE19 cell.The microsphere particle has green fluorescence, and can be swallowed by RPE cell, by detecting and comparing
Compared with the intensity of the intracellular green fluorescence of different disposal group APRE19, to help to assess the phagocytic activity of RPE cell.It is intracellular green
Color fluorescence intensity is higher, shows that the microsphere particle of cell phagocytosis is more, the phagocytic activity of RPE cell is stronger.Experimental implementation is stringent
It is carried out according to kit specification.Experimental result:
The intracellular green fluorescence intensity of ARPE19 obviously weakens (figure in hsa_circ_0001543 strikes and comes down to a lower group compared with control group
9,10) microsphere particle, swallowed in prompt RPE substantially reduces in hsa_circ_0001543 strikes and comes down to a lower group compared with control group.We
The result shows that hsa_circ_0001543 can promote the phagocytosis of RPE cell, further prompted it to RPE cell and regarded
Potential important protective effect in the nethike embrane degenerative disease course of disease.
Embodiment 5hsa_circ_0001543 to RPE cellular oxidation stress influence
Two groups of different ARPE19 cell processing groups are set altogether, and respectively hsa_circ_0001543, which strikes, to be come down to a lower group and its compare
Group;With embodiment 3.By MitoSOX red mitochondria superoxides fluorescence probe, (Thermo Fisher Scientific is public
Department) content of mitochondria activity oxygen (ROS) in each processing group living cells of specific detection, to help to assess the oxygen of RPE cell
Change stress situation.Intracellular red fluorescence intensity is higher, shows that the ROS content generated into the cell is higher, and RPE cell is in oxidation
Stress situation.Experimental implementation is carried out in strict accordance with kit specification.
Experimental result:
By Figure 11 and Figure 12 as it can be seen that the fluorescence intensity that hsa_circ_0001543 strikes ARPE19 cell of coming down to a lower group is significantly stronger than pair
According to group, show that the intracellular ROS content of ARPE19 significantly increases in hsa_circ_0001543 strikes and comes down to a lower group compared with control group.We
The result shows that hsa_circ_0001543 is able to suppress the oxidative stress of RPE cell, further prompted it to RPE cell and
Potential important protective effect in the retinal degenerative disease course of disease.
Influence of the embodiment 6hsa_circ_0001543 to RPE ability of cell proliferation
Two groups of different ARPE19 cell processing groups are set altogether, and respectively hsa_circ_0001543, which strikes, to be come down to a lower group and its compare
Group;With embodiment 3.ByEdU Alexa488Imaging Kit (Invitrogen company) statistics
The EdU positive cell ratio of different disposal group ARPE19 cell, to help to assess the proliferative capacity of cell.Experimental implementation is stringent
It is carried out according to kit specification.
Experimental result:
Figure 13 and Figure 14 are shown: the cell proportion that hsa_circ_0001543 strikes the EdU positive in ARPE19 cell of coming down to a lower group is aobvious
It writes and is higher than control group.As it can be seen that the proliferative capacity of ARPE19 cell is significant compared with control group in hsa_circ_0001543 strikes and comes down to a lower group
It improves.Our result indicate that hsa_circ_0001543 is able to suppress the proliferation of RPE cell, prompt it in RPE cell differentiation
Potential facilitation in the process has further prompted it potential heavy to RPE cell and in the retinal degenerative disease course of disease
Want protective effect.
Influence of the embodiment 7hsa_circ_0001543 in RPE cell to PTEN/AKT/mTOR signal path
Multiple researchs find that the abnormal activation of mTOR signal path can induce RPE cell de-differentiation, cause RPE thin
The dysfunction of born of the same parents.Accordingly, we have detected hsa_circ_0001543 and make to the regulation of PTEN/AKT/mTOR signal path
With.Set two groups of different ARPE19 cell processing groups altogether, respectively hsa_circ_0001543, which strikes, to come down to a lower group and its control group;With real
Apply example 3.It is dyed by immunoblotting, to PTEN/AKT/mTOR signal path correlation egg in different disposal group ARPE19 cell
The expression of white (PTEN, p-AKT, AKT, p-mTOR, mTOR, p-p70S6K and p70S6K) is detected.
Experimental result:
It is prompted according to the result that immunoblotting in Figure 15 and Figure 16 dyes, the expression quantity of PTEN is in hsa_ in ARPE19 cell
Circ_0001543 strikes come down to a lower group in significantly reduced compared with control group, and p-AKT/AKT, p-mTOR/mTOR and p-p70S6K/
The ratio of p70S6K significantly increases in hsa_circ_0001543 strikes and comes down to a lower group, and prompts mTOR signal path in hsa_circ_
0001543 strike come down to a lower group it is middle by abnormal activation.Our result indicate that hsa_circ_0001543 is able to suppress PTEN/AKT/mTOR
The activation of signal path has further prompted its potential important protection to RPE cell and in the retinal degenerative disease course of disease
Property effect.
Influence of the embodiment 8mmu_circ_0007330 in mouse to its RPE form and functional dependency protein expression
Two eyes of same mouse are set to mmu_circ_0007330 by the C57BL/6 mouse for selecting 5-6 week old
Strike (the mmu_circ_0007330siRNA that comes down to a lower group;Mmu_circ_0007330 is that hsa_circ_0001543 is corresponding in mouse
Homologous circRNA, coding gene sequence is as shown in SEQ ID NO.5) and control group (scramble siRNA), concrete operations
Method is as follows:
(1) 4.3% chloraldurate 0.01ml/g intraperitoneal anesthesia mouse;
(2) Tropicamide and Phenylephrine eyedrop eyes mydriasis, preoperative double ocellus table anaesthetic keep eye with methylcellulose in art
Table is wet;
(3) the 1 μ L (mmu_circ_ of injection drug of 1nmol/ μ L is drawn with 33G syringe (Hamilton company)
0007330siRNA or scramble siRNA is purchased from Rui Bo biotech firm), 1mm avoids cutting at blood vessel after corneoscleral junction
Mouthful, needle point is vertically into towards vitreous chamber center.It is slowly injected after syringe needle enters vitreous chamber, let the acupuncture needle remain at a certain point after pushing pin 0.5-
1min, rapidly needle out.
Mouse is spaced after injecting for the first time goes identical injection for 2 weeks again, and second of injection took out eyeball of mouse after 2 weeks, removes
Prosthomere is simultaneously fixed on 4%PFA, isolated RPE tela chorioidea, by immunofluorescence dyeing to retinal specific albumen
Expression is observed and is compared, and concrete operation method is referring to embodiment 3.
Experimental result:
By Figure 17 with Figure 18 as it can be seen that cell connects specific marker in immunofluorescence dyeing result prompt mouse RPE tissue
The expression quantity of albumen ZO-1 and β-catenin significantly reduce in mmu_circ_0007330 strikes and comes down to a lower group compared with control group.We
The result shows that mmu_circ_0007330 expression quantity deficiency can inhibit the expression of RPE cell function related proteins, cause RPE thin
Born of the same parents' dysfunction has further prompted mmu_circ_0007330 latent to RPE cell and in the retinal degenerative disease course of disease
In important protective effect.
Sequence table
<110>Jiangsu Prov. People's Hospital (No.1 Attached Hospital, Nanjing Medical Univ)
<120>circular rna hsa_circ_0001543 is preparing the application in retinal degenerative disease diagnostic reagent
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gctattcaag ttgatattca ctgatggact ccaaagaatc attaactcct ggtagagaag 60
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acaacttgac ttctctgggg ac 22
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtccatcaca tctcccctct c 21
<210> 4
<211> 1197
<212> DNA
<213>mankind (Homo sapiens)
<400> 4
cttgaatagc cattagaaaa aactgttcga ccagggaagt tcagagtccc cagagaagtc 60
aagttgtcat ctccagatcc ttggcaccta ttccaatttt cggaaccaac gggaattggt 120
ggaatgacat taaaaatagg cttctgatcc tgctgttgag aaagggatgc tgtattcatg 180
tcatagtggt acatctgtcc tccagaggta ctcacaccat gaacagaaat ggcagacatt 240
ttattaccaa ttatatttgc tccaggaaag cttgcctgac agtaaactgt gcccagtttc 300
tcttgcttaa ttaccccagg ggtgcagagt tcgatgaaat cttctttttc tgttttcact 360
tggggcagtg ttacattact ggggcttgac aaaaccagat ctccattatc cttaattttg 420
ggtttagtgt ccggtaaaat gagaggcttg cagtcctcat tcgagtttcc ttccaaaagg 480
aatgaatcgt cttctcccgc cagaggagaa agcaaacagt tttcatctat caacaggtct 540
gatctccaag gactctcatt cgtctcttta cctggggacc cagaagaaaa ctccaaatcc 600
tgcaaaatgt caaaggtgct ttggtctgtg gtatacaatt tcacattgcc accgttggtg 660
ccagtctggc ccttcaaatg ttgctgttct gaagatacat cagagtgagt ttttggaaac 720
tccttctctg tgggggcagc agacacagca gtggatgctg aactcttggg gttctctgga 780
acactggtcg acctattgag gtttgcaatg ctttcttcca aaagctttaa gtctgtttcc 840
cccgaggaaa ggctgatttg gccctgctgt gggaatccca ggtcatttcc catcactttt 900
gtttctgtct ctcccatata cagtcccatt gagagtgaaa ctgctttgga cagatctggc 960
tgctgcgcat tgcttactga gccttttgga aaatcaacca aaagtcttcg ctgcttggag 1020
tctgattgag aagcgacagc cagtgagggt gaagacgcag aaaccttcac agtagctcct 1080
cctcttaggg ttttatagaa gtccatcaca tctcccctct cctgagcaag cacactgctg 1140
gggttttctt ctctaccagg agttaatgat tctttggagt ccatcagtga atatcaa 1197
<210> 5
<211> 1248
<212> DNA
<213>mankind (Homo sapiens)
<400> 5
cttgaatatc cattagaaaa cactgagcgg cctgcgaagt tcatagcccc caaggaagtc 60
aggttgtcct ctccagaccc ttggcaccta ttccagtttt cagaaccaac aggaattggt 120
ggaatgacat taaaaacagg cttctgatcc tgctgctgag aaagggatgc tgtattcatg 180
tcatagtggt acatctgtcc tccagaggta ctcacgccat gaacagaaat ggcagacatt 240
ttattcccaa ttatatttgt cccagaaaag cttgcctggc aataaaccgg gcccagtttc 300
tcttgcttaa ttaccccagg ggtgcaaagc tcaatgaaat catctttctc tgttttcact 360
tggggcagtg ccacactgct ggggcttgat aagattgtat ctccagtatc ctgaatttta 420
ggtttagtgt ccggtaaaat aagaggcttg caatcctcat tcacgtcccc ttccagaagg 480
aatggatcat cttctcccgc caaaggagaa agcaagtttt catctatcaa caggtctgac 540
ctccaaggac tctcgtttgt ctctttacct ggggacccgg cagaaaactc caaatcctgc 600
aagatgtcaa aggtgctttg gtctgtggta tacaatttca cactgccacc gttggtgcca 660
ggctggcttt ttctattttg ctgttctgaa gatggatcag agtgagtctg gggaaactcc 720
ttctctgtcg gggtagcaca cccagctgca ggtgttgaac tcttggggtt ctctggacgg 780
ctggtcgacc tattgaggtt tgcaatgctt tcttccagaa gccgaaagtc tgtttcccca 840
gaggagaggc caagctggcc ctgctgtggg tagcccaagt cattccccat cacttttgtt 900
tcggtctctc ccatatacag tcccatggac agtgaaacgg ctttggataa atctggctgc 960
ggctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgcgcattg 1020
cttgctgagc cttttgaaaa atcaaggaga atcctctgct gcttggaatc tgcctgagaa 1080
gcagcagcca ctgagggtga agacgcagaa accttgactg tagctccacc cctcagggtt 1140
ttatacaagt ccatcacgct tcccctcccc cggccaagca aactgctggg gacttcgtct 1200
ctaccagggg gagctaagga ttctttggag tccattggca aatattaa 1248
Claims (10)
1.hsa_circ_0001543 as application of the biomarker in the kit of preparation diagnosis retinal degenerative disease.
2. a kind of reagent for detecting hsa_circ_0001543 expression quantity is in the kit of preparation diagnosis retinal degenerative disease
Application.
3. application according to claim 2, it is characterised in that the examination of the detection hsa_circ_0001543 expression quantity
Agent is probe, genetic chip or the real-time quantitative PCR primer for having detection specificity to hsa_circ_0001543.
4. application according to claim 3, it is characterised in that described to have detection special hsa_circ_0001543
The probe sequence of property is as shown in SEQ ID NO.1.
5. application according to claim 3, it is characterised in that described to have detection special hsa_circ_0001543
The real-time quantitative PCR primer of property is as shown in SEQ ID NO.2 and SEQ ID NO.3.
6. a kind of diagnostic kit of retinal degenerative disease, it is characterised in that include detection hsa_circ_0001543 expression quantity
Reagent.
7. diagnostic kit according to claim 6, it is characterised in that the kit includes to hsa_circ_
0001543 with the specific probe of detection or to hsa_circ_0001543 there is the real-time quantitative PCR of detection specificity to draw
Object.
8. diagnostic kit according to claim 6, it is characterised in that described that there is inspection to hsa_circ_0001543
The probe sequence of specificity is surveyed as shown in SEQ ID NO.1;Described has detection specificity to hsa_circ_0001543
Real-time quantitative PCR primer is as shown in SEQ ID NO.2 and SEQ ID NO.3.
9.hsa_circ_0001543 or increasing the substance of hsa_circ_0001543 expression quantity in preparation treatment retinosis
Application in the drug of disease.
10.hsa_circ_0001543 as drug target answering in the drug of screening treatment retinal degenerative disease
With.
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