WO2018223848A1 - 两种用于青光眼诊断的分子标记物、试剂盒及应用 - Google Patents

两种用于青光眼诊断的分子标记物、试剂盒及应用 Download PDF

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WO2018223848A1
WO2018223848A1 PCT/CN2018/088324 CN2018088324W WO2018223848A1 WO 2018223848 A1 WO2018223848 A1 WO 2018223848A1 CN 2018088324 W CN2018088324 W CN 2018088324W WO 2018223848 A1 WO2018223848 A1 WO 2018223848A1
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lncrnas
glaucoma
expression level
real
detecting
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江冰
谢犁犁
张璐丝
黄伟
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Second Xiangya Hospital of Central South University
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Second Xiangya Hospital of Central South University
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Priority claimed from CN201710423197.3A external-priority patent/CN107142316B/zh
Priority claimed from CN201710424168.9A external-priority patent/CN107034303B/zh
Application filed by Second Xiangya Hospital of Central South University filed Critical Second Xiangya Hospital of Central South University
Priority to EP18813073.6A priority Critical patent/EP3643794A4/en
Priority to US16/619,945 priority patent/US20200181705A1/en
Publication of WO2018223848A1 publication Critical patent/WO2018223848A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the field of molecular diagnostic techniques, and in particular to long-chain non-coding RNA markers, kits and applications that contribute to the diagnosis of glaucoma.
  • the eye is a very important sensory organ of the human body. It can receive external light stimulation and transmit light impulses to the brain center to cause vision. Da Vinci once said: "The eyes are the windows of the soul. Through the eyes, people can embrace and appreciate the infinite beauty of the world, and the soul can live in the body.” In the information age, about 90% of the information that people get from the outside world through sensory organs is done by the eyes. According to the World Health Organization, ophthalmology has become the third hazard after cancer and cardiovascular disease and a disease that affects people's quality of life. Among all eye diseases, glaucoma is the first irreversible blinding eye disease. The reason for its impact on visual quality is to cause blindness by threatening and damaging the optic nerve and its pathways, thus seriously threatening human visual health, giving individuals, families and Society causes incalculable losses.
  • the main hazard caused by glaucoma is affecting visual function. Even in developed countries, only about 50% of glaucoma patients can get timely diagnosis and treatment, and the pathogenesis and genetic laws of glaucoma are unknown. It is necessary to scientifically grasp the law of its occurrence and development, so as to carry out early diagnosis and early treatment to avoid blindness in patients with glaucoma.
  • diagnosis of glaucoma mainly depends on medical history, morphology and functional examinations, such as intraocular pressure measurement, ultrasound biomicroscopy, fundus photography, optical coherence tomography and visual field examination. Although these tests can diagnose glaucoma, studies have shown that glaucoma diagnosed by morphological and functional means has a visual impairment of more than 50%.
  • the biochemical examination, serological screening and detection criteria related to glaucoma are still in a relatively blank state. Therefore, it is especially important to find a marker with high sensitivity and high specificity for the diagnosis and monitoring of glaucoma.
  • ncRNA transcripts with a sequence length greater than 200 bases are long noncoding RNAs (lncRNAs), which have attracted much attention due to their functional richness and diversity of mechanisms of action.
  • lncRNAs regulate the expression of protein-coding genes at various levels of transcription, post-transcription and translation, and thus participate extensively in important life processes including cell differentiation and organism development. Their abnormal expression is also closely related to the occurrence of many major human diseases. Related.
  • lncRNA is highly conserved and tissue-specific and is abundant in the brain. lncRNA not only participates in the growth and function of the nervous system, but also makes the nervous system grow and develop in a certain time sequence and in a certain space, and participate in the function of the nervous system. lncRNA participates in the development and function of the nervous system through various mechanisms, including cis-acting elements and trans-acting factors involved in gene imprinting, chromatin remodeling, cell cycle regulation, splicing regulation, mRNA degradation and translational regulation. . Therefore, it is a feasible means to detect the physiological and pathological state of the nervous system by detecting changes in the composition and expression level of lncRNAs.
  • the aqueous humor belongs to the contents of the eye and is produced by the ciliary body. It enters the blood through the pupil, trabecular meshwork, etc., and is in the dynamic cycle. Its composition is closely related to the local physiological and pathological environment of the eyeball.
  • Exosomes are important mediators of intercellular communication between the exosomes, including lncRNAs, mRNAs, proteins and lipids. The substances contained in the exosomes can remain stable for a long time due to special protection mechanisms. Therefore, molecules such as lncRNAs in aqueous humor can exchange information between cells in the form of exosomes, and can enter the blood with the dynamic circulation of aqueous humor. Therefore, glaucoma-related lncRNAs research has the potential to provide a theoretical basis for the detection of glaucoma-related biomarkers.
  • the technical problem to be solved by the present invention is to provide two molecular markers lncRNAs T267384, lncRNAsENST00000564363, kits and applications for glaucoma diagnosis.
  • One of the objects of the present invention is to provide a long-chain non-coding RNA for early diagnosis of glaucoma, which is of great significance for the detection of glaucoma.
  • the molecular marker lncRNAsT267384 for early diagnosis of glaucoma whose sequence is SEQ ID NO: 1 is shown.
  • Another object of the present invention is to provide the use of the above molecular marker lncRNAs T267384.
  • the product for detecting the expression level of lncRNAs T267384 comprises: a preparation for detecting the expression level of lncRNAs T267384 by real-time fluorescent quantitative PCR.
  • the preparation for detecting the expression level of lncRNAs T267384 by real-time fluorescent quantitative PCR includes primers for specifically amplifying lncRNAs T267384.
  • the primers for specific amplification of lncRNAs T267384 by real-time PCR were used to detect the expression level of lncRNAsT267384 as:
  • a third object of the present invention is to provide a kit for early diagnosis of glaucoma comprising an agent for detecting the expression level of lncRNAs T267384. Specifically, it comprises an agent for detecting the expression level of lncRNAs T267384 by real-time fluorescent quantitative PCR.
  • the reagent for detecting the expression level of lncRNAs T267384 by real-time fluorescent quantitative PCR comprises a primer for specifically amplifying lncRNAs T267384 by real-time fluorescent quantitative PCR.
  • the reagent for detecting the expression level of lncRNAs T267384 by real-time fluorescent quantitative PCR comprises a pair of primers for specifically amplifying lncRNAs T267384 by real-time fluorescent quantitative PCR, and the primer sequence is:
  • aqueous humor has the highest diagnostic significance, and the sensitivity/specificity of three lncRNAs for the diagnosis of glaucoma in aqueous humor is greater than 90%.
  • the possible cause of this phenomenon is due to the presence of the blood-aqueous barrier, which significantly limits the entry of blood-derived immune cells and signaling molecules from other parts of the body into the aqueous humor. Therefore, compared with serum, aqueous humor can more specifically represent the physiological and pathological state of the eye. Therefore, in future studies, aqueous humor may have a greater diagnostic value for glaucoma than serum.
  • Another object of the present invention is to provide another long-chain non-coding RNA for early diagnosis of glaucoma, which is of great significance for the detection of glaucoma.
  • a molecular marker for the early diagnosis of glaucoma lncRNAsENST00000564363, whose sequence is SEQ. ID NO: 4 is shown.
  • a fifth object of the present invention is to provide the use of the above molecular marker lncRNAsENST00000564363.
  • the product for detecting the expression level of lncRNAs ENST00000564363 comprises: a preparation for detecting the expression level of lncRNAs ENST00000564363 by real-time fluorescent quantitative PCR.
  • Detection of lncRNAs by real-time fluorescent quantitative PCR ENST00000564363 expression level preparations include specific amplification of lncRNAs Primer for ENST00000564363.
  • ENST00000564363 expression level specific amplification of lncRNAs The primer sequence of ENST00000564363 is:
  • R 5'AGAGCACCCCCAAGCCCT3'.
  • a sixth object of the present invention is to provide another kit for early diagnosis of glaucoma comprising an agent for detecting the expression level of lncRNAs ENST00000564363. Specifically, it comprises detecting lncRNAs by real-time fluorescent quantitative PCR. ENST00000564363 Expression level of reagent.
  • the ENST00000564363 expression level of the reagent comprises a primer that specifically amplifies lncRNAs ENST00000564363 by real-time fluorescent quantitative PCR.
  • the ENST00000564363 expression level reagent comprises a pair of primers that specifically amplify lncRNAs ENST00000564363 by real-time fluorescent quantitative PCR, the primer sequences of which are:
  • R 5'AGAGCACCCCCAAGCCCT3'.
  • T267384 was up-regulated in both aqueous humor and serum of glaucoma patients relative to the control population.
  • T267384 is a high-expression lncRNAs in glaucoma and a biomarker for glaucoma diagnosis.
  • the invention provides a strong molecular biological basis for the diagnosis of glaucoma, and has far-reaching clinical significance and popularization.
  • lncRNAs:ENST00000564363 was up-regulated in the aqueous humor of glaucoma patients relative to the control group. It is suggested that ENST00000564363 is a highly expressed lncRNAs in glaucoma and is a biomarker for glaucoma diagnosis.
  • the invention provides a strong molecular biological basis for the diagnosis of glaucoma, and has far-reaching clinical significance and popularization.
  • Figure 1 shows the expression profiles of lncRNAs and mRNAs in aqueous humor of patients with glaucoma
  • A Clustering map of lncRNAs expression profiles in aqueous humor of patients with glaucoma
  • B Clustering analysis of expression profiles of mRNAs in aqueous humor of patients with glaucoma.
  • Figure 2 shows differential expression of lncRNAs and mRNAs in aqueous humor of patients with glaucoma
  • A Compared with age-related cataracts, glaucoma patients have 2-fold differentially expressed lncRNAs in aqueous humor; B: 2 times differentially expressed mRNAs in aqueous humor of glaucoma patients compared with age-related cataracts.
  • Figure 3 is a CNC analysis of the expression of lncRNAs and mRNAs in aqueous humor of patients with glaucoma;
  • Red dots represent lncRNAs
  • blue dots represent mRNAs
  • solid lines represent positive correlations
  • dashed lines represent negative correlations.
  • Figure 4 shows the expression levels of lncRNAs in aqueous humor of patients with glaucoma and age-related cataract
  • A-C scatter plots of T267384, ENST00000607393, and T342877 in aqueous humor of glaucoma and age-related cataract patients
  • D-F T267384, ENST00000607393, and T342877 expressed in the aqueous humor of patients with glaucoma and age-related cataracts.
  • Figure 5 shows the expression levels of lncRNAs in iris tissues of control subjects in glaucoma patients
  • A-C scatter plots of T267384, ENST00000607393, and T342877 expression in iris tissue of glaucoma patients and control subjects;
  • D-F box plots of T267384, ENST00000607393, and T342877 expressed in iris tissue of glaucoma patients.
  • Figure 6 shows the expression levels of lncRNAs in serum of glaucoma patients and control subjects
  • A-C scatter plots of T267384, ENST00000607393, and T342877 expression in serum of glaucoma patients and controls;
  • D-F box plots of T267384, ENST00000607393, and T342877 expressed in serum of glaucoma patients.
  • Figure 7 is a plot of ROC for the diagnosis of glaucoma in different tissues using lncRNAs
  • A The ROC curve of glaucoma was diagnosed in aqueous humor by T267384. The area under the curve was 0.998.
  • B The ROC curve of glaucoma was diagnosed in aqueous humor by ENST00000607393. The area under the curve was 0.998.
  • C The ROC curve of glaucoma was diagnosed in aqueous humor by T342877.
  • the area under the curve is 0.983; D: the ROC curve of glaucoma is diagnosed in the iris tissue by ENST00000607393, the area under the curve is 0.793; E: the ROC curve of glaucoma is diagnosed in serum by T267384, the area under the curve is 0.620; F: using ENST00000607393 The ROC curve of glaucoma was diagnosed in serum with an area under the curve of 0.638.
  • Figure 8 shows the expression levels of another group of lncRNAs in aqueous humor of patients with glaucoma and age-related cataract
  • A-D is a scatter plot of lncRNA:ENST00000564363, NR_026887, TCONS_00025577, and ENST00000508241 expressed in the aqueous humor of glaucoma and age-related cataract patients.
  • Figure 9 is a plot of ROC for the diagnosis of glaucoma in aqueous humor using another set of lncRNAs
  • A-D is: lncRNA ENST00000564363, NR_026887, TCONS_00025577 and ENST00000508241
  • the area under the ROC curve for the diagnosis of glaucoma in aqueous samples is 1.000, 0.920, 0.940 and 0.880, respectively.
  • Example 1 Expression profiles of lncRNAs and mRNAs in aqueous humor of patients with glaucoma:
  • centrifuges and cryogenic centrifuges were purchased from Eppendorf; high-speed centrifuges were purchased from Beckman; Real-Time PCR instruments were purchased from ABI; pipettes and electric pipetting guns were purchased from Eppendorf; Q5000 was purchased from Quawell Technology; The ice machine was purchased from Sanyan.
  • centrifuge tubes and PCR tubes were purchased from Axygen; DEPC water was purchased from Dingguo Changsheng Company; Trizol was purchased from Invitrogen; TargetAmp TM 1-Round aRNA Amplification kit was purchased from Epicentre; Transcriptor First Strand cDNA Synthesis Kit Purchased from Roche; Quick Amp Labeling Kit, One-Color kit purchased from Agilent Technologies; human lncRNAs microarray chip purchased from Arraystar.
  • the reagents used in various immunoblots were configured according to the methods provided in Molecular Cloning (Third Edition).
  • Typical glaucomatous visual field defect D Anterior chamber angle is open. Have the above four items or have A, D, B or C.
  • Normal tension glaucoma It has optic disc change similar to POAG, RNFL and visual field damage, 24h intraocular pressure measurement is ⁇ 21mmHg, and the angle of the anterior chamber is open.
  • Primary angle-closure glaucoma optic disc changes with glaucoma, RNFL and visual field damage, intraocular pressure >21 mmHg, anterior chamber angle narrowed or closed 4.
  • Exclusion criteria 1. Associated with systemic diseases such as hypertension and diabetes.
  • Secondary glaucoma An ophthalmic or neurological disease that may affect vision, optic nerve, or color vision. 4. Reliability criteria for visual field detection: fixation loss rate, false positive rate and/or false negative rate >25%. 5. Age-related cataracts: exclude complicated, traumatic, congenital cataracts and exclude those with other intraocular diseases, and exclude over-mature cases.
  • RNA concentration of each tube was measured by a Q5000 instrument.
  • Use TargetAmp TM 1-Round aRNA press manufacturers the step shown Amplification Kit 103 (epicentre) instructions for RNA amplification.
  • the general procedure is as follows: First, a single-stranded cDNA: RNA hybridization product is synthesized from an RNA sample, and then the hybridization product is digested into small fragment sequences using RNase H enzyme, and these small fragment sequences can assist in the synthesis of double-stranded cDNA. Finally, antisense RNA is synthesized by double-stranded cDNA transcription.
  • the fluorescence intensity of the chip was scanned using a GenePix 4000B chip scanner and the results were converted to digital data storage. P values ⁇ 0.05 were considered statistically significant.
  • glaucoma patients up-regulated 2,783 mRNAs in the aqueous humor and 2,716 down-regulated mRNAs.
  • aqueous humor is a valuable research object for the study of biomarkers of ocular diseases.
  • aqueous humor has the potential to be applied to geological studies related to glaucoma in the future.
  • the human aqueous sample is small in size due to sampling limitations.
  • lncRNAs microarray analysis method 11728 lncRNAs and 6686 mRNAs were detected in 10 glaucoma aqueous samples, which were significantly different from the expression profiles of lncRNAs and mRNAs in aqueous humor of age-related cataract patients. Therefore, lncRNAs in aqueous humor And mRNAs expression profiles exhibit individual specificity and disease specificity. To the best of our knowledge, this is the first study of the expression of lncRNAs and mRNAs in glaucoma aqueous humor.
  • Example 2 Defining the correlation between specific lncRNAs and mRNAs in aqueous humor by CNC analysis
  • CNC coding-noncoding gene co-expression
  • the mRNAs that were differentially expressed in aqueous humor and correlated with the development of glaucoma were selected, and the expression data of these mRNAs in the aqueous humor of different glaucoma patients were averaged; the data after standardization of selected mRNAs and the differentially expressed lncRNAs in the aqueous humor of glaucoma patients were selected. Pearson correlation coefficient between related data (Pearson Correlation Coefficient, PCC) and False Discovery (False Discovery) Rate, FDR); records with PCC ⁇ 0.90 and FDR ⁇ 0.05; using the relevant records, drawing with the Cytoscape 2.8.3 tool.
  • PCC Pearson Correlation Coefficient
  • FDR False Discovery
  • mRNAs that are differentially expressed in the aqueous humor of glaucoma patients and are associated with the development of glaucoma are as follows: bone morphogenetic protein 2 (bone) Morphogenetic protein 2, BMP2), ependymin related 1, EPDR1, transforming growth factor beta 1 (TGFB1), forkhead protein E3 (forkhead box E3, FOXE3), growth hormone secretagogue receptor (growth hormone secretagogue) Receptor, GHSR), forkhead protein C1 (forkhead Box C1, FOXC1), transmembrane and coiled-coil domain 1 (transmembrane And coiled-coil domains 1, TMCO1), PH domain A7 (pleckstrin Homology domain containing A7, PLEKHA7), optic nerve protein (optineurin, OPTN) and integrin subunit ⁇ 5 (integrin Subunit beta 5, ITGB5).
  • bone morphogenetic protein 2 bone Morphogenetic protein 2, BMP2
  • centrifuges and cryogenic centrifuges were purchased from Eppendorf; high-speed centrifuges were purchased from Beckman; Real-Time PCR instruments were purchased from ABI; pipettes and electric pipetting guns were purchased from Eppendorf; Q5000 was purchased from Quawell Technology; The ice machine was purchased from Sanyan.
  • centrifuge tubes and PCR tubes were purchased from Axygen; DEPC water was purchased from Dingguo Changsheng Company; Trizol was purchased from Invitrogen; reverse transcription kits and fluorescent quantitative kits were purchased from Roche.
  • the reagents used in various immunoblots were configured according to the methods provided in Molecular Cloning (Third Edition).
  • the aqueous sample is prepared as described in 1.2.1.
  • Serum samples were taken from glaucoma patients and healthy controls.
  • the inclusion and exclusion criteria for glaucoma patients were the same as those described in 1.2.1.
  • the serum from the control group was obtained from healthy individuals matched with age and gender in the glaucoma group.
  • the iris samples were taken from the glaucoma patient group and the control group.
  • the inclusion and exclusion criteria for glaucoma patients were the same as those described in 1.2.1.
  • the iris samples of the control group were obtained from the iris of the cornea donors of the Xiangya Second Hospital, which matched the age and gender of the glaucoma patients. organization.
  • RNA concentration of each well was measured by a Q5000 instrument. Peripheral venous blood was allowed to stand for 30 minutes, placed in a centrifuge, centrifuged at 3200 rpm for 10 minutes, and the supernatant serum was taken using a sterile pipette, and RNA was extracted by the same procedure as above.
  • the reverse transcription enzyme is first used to reverse transcribe the RNA into cDNA, and then the cDNA is amplified by PCR, and the amount of the quantitatively amplified product is detected in real time by measuring the fluorescence intensity signal.
  • aqueous sample size To further expand the aqueous sample size to verify the expression levels of related lncRNAs and to explore the expression levels of specific lncRNAs in serum and iris of glaucoma patients, we used qRT-PCR to detect 60 aqueous samples (30 glaucoma patients with aqueous samples, 30 Age- and sex-matched age-related cataract patients with aqueous humor), 50 iris tissues (40 glaucoma patients with iris tissue samples, 10 age- and sex-matched corneal donors with iris tissue) and 158 serum samples (103 Serum samples from glaucoma patients, serum samples from 55 age- and sex-matched healthy populations), T267384, ENST00000607393, and T342877 expression levels.
  • ENST00000607393 The area under the ROC curve for the diagnosis of glaucoma in iris tissue was 0.793; the area under the ROC curve for the diagnosis of glaucoma in serum samples by T267384 and ENST00000607393 was 0.620 and 0.638, respectively.
  • the diagnostic values of T267384, ENST00000607393, and T342877 in the aqueous humor were set to 1.5437, 1.1485, and 2.1052, the diagnostic sensitivity/specificity was 0.967/0.967, 0.967/0.967, and 0.967/0.900, respectively; when ENST00000607393 was in the iris tissue.
  • the sensitivity/specificity of diagnosis is 0.700/0.700; when the diagnostic values of T267384 and ENST00000607393 in serum are set to 0.7191 and 0.8376, the sensitivity/specificity of diagnosis is 0.612/0.600. And 0.680/0.600.
  • centrifuges and cryogenic centrifuges were purchased from Eppendorf; high-speed centrifuges were purchased from Beckman; Real-Time PCR instruments were purchased from ABI; pipettes and electric pipetting guns were purchased from Eppendorf; Q5000 was purchased from Quawell Technology; The ice machine was purchased from Sanyan.
  • centrifuge tubes and PCR tubes were purchased from Axygen; DEPC water was purchased from Dingguo Changsheng Company; Trizol was purchased from Invitrogen; reverse transcription kits and fluorescent quantitative kits were purchased from Roche.
  • the reagents used in various immunoblots were configured according to the methods provided in Molecular Cloning (Third Edition).
  • the aqueous sample is prepared as described in 1.2.1.
  • RNA concentration of each well was measured by a Q5000 instrument.
  • the reverse transcription enzyme is first used to reverse transcribe the RNA into cDNA, and then the cDNA is amplified by PCR, and the amount of the quantitatively amplified product is detected in real time by measuring the fluorescence intensity signal.
  • the areas under the ROC curve for the diagnosis of glaucoma in the aqueous samples of ENST00000564363, NR_026887, TCONS_00025577 and ENST00000508241 were found to be 1.000, 0.920, 0.940 and 0.880, respectively.
  • the diagnostic values of ENST00000564363, NR_026887, TCONS_00025577, and ENST00000508241 in the aqueous humor are set to 0.0068, 0.0056, 0.0074, and 0.0048, the diagnostic sensitivity/specificity is 1.000/1.000, 0.900/0.900, 0.900/0.900, and 0.800/0.800, respectively. (Table 6).
  • Tccgtaatta Tctactggat ccatttggtc tattgtgcag attaagtctg atgtttcttt 540
  • Attgatttttt Ctgcctggaa gatccatcca gtgctgaaag tggggtgtcg aagtctccag 600
  • Gagccacata Aagccggcgg tggagtgaca caagcatccc tgtggccacc accactatga 1800
  • Gccttctgtc Acgcccgcat aagggccgct tgagggctcc ttggtcaagc ggtaatgcca 3960
  • Atcctaacaa Cagtctcata agtataagag ttatgagtag atgaactgta ttcataaaga 4800
  • Atgtttttac Atacatgcac aatgcttaca caaattcagc ttcgtgataa tgcactttca 5100
  • Gcctggccaa Catagtgaaa ccctgtctgt agtaaaata caaaaattaa cacttgagcc 5340
  • Tattgtctaa Aatggtctat gaagtgctct gttgtgtttt tatgtttctc aaatctcctt 5520
  • Gtgagatttt Catcagggca aaatgatgct gacggatcaa atgggacaag aaatgtgaaa 6480
  • Tcttgctttg Tcagcattaa tactaacaga taacatgaga agaggcaaaa gtgttggcta 6780
  • Aagttcttag Atgaaagaga gctgggttac tgtgaaccaa aatctcctgc t 6891

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Abstract

本发明公开了两种用于青光眼诊断的分子标记物、试剂盒及应用。相对于对照组人群，lncRNAs:T267384在青光眼患者的房水和血清中均表达上调，lncRNAs:ENST00000564363在青光眼患者的房水中表达上调。提示T267384、ENST00000564363均是青光眼中的高表达lncRNAs，是有助于青光眼诊断的生物标志物。

Description

两种用于青光眼诊断的分子标记物、试剂盒及应用 技术领域
本发明涉及分子诊断技术领域，具体涉及有助于青光眼诊断的长链非编码RNA标志物、试剂盒及应用。
 背景技术
眼睛是人体十分重要的感觉器官，能够接受外部的光刺激，并将光冲动传送到大脑中枢而引起视觉。达芬奇曾说：“眼睛是心灵的窗户，通过眼睛，人们得以拥抱和欣赏世界的无限美妙，灵魂才得以安居于体内”。信息时代，人通过感觉器官从外界获得的信息中，大约90%是由眼睛来完成的。世界卫生组织的资料显示，眼科疾病已成为继肿瘤、心血管疾病之后的第三位危害及影响人们生存质量的疾病。在所有眼科疾病中，青光眼则是首位不可逆转的致盲性眼病，其影响视觉质量的原因是通过威胁和损害视神经及其通路而导致失明，从而严重威胁人类的视觉健康，给个人、家庭和社会造成难以估量的损失。
青光眼造成的主要危害是影响视觉功能，即便是在发达国家中，也只有50%左右的青光眼患者能够得到及时的诊断和治疗，而且，青光眼的发病因素、遗传学规律等均不明了，因此我们要科学地掌握其发生发展规律，从而进行早期诊断和早期治疗，避免青光眼患者的失明。目前，青光眼的诊断主要是依靠病史、形态学和功能学方面的检查，如眼压测量、超声生物显微镜、眼底照相、光学相干断层扫描和视野检查等。虽然这些检查可以诊断青光眼，但研究表明，通过形态学和功能学手段诊断的青光眼，患者视觉功能的损害已经超过50%。而青光眼相关的生化检查、血清学筛查及检测标准仍处于相对空白状态，因此寻找一种高灵敏度和高特异性的标志物对青光眼诊断和监控尤为重要。
基因组计划研究表明，在组成人类基因组的30亿个碱基对中，仅有1.5%的核苷酸序列用于蛋白质编码，其余98.5%的基因组为非蛋白编码序列。这些序列曾被认为是在进化过程中累积的“垃圾序列”而未予以关注，在随后启动的人类基因组DNA元件百科全书（encyclopedia of DNA elements, ENCODE）计划中，研究表明75%的基因序列能够被转录成RNA，其中近74%的转录产物为非编码RNA（non-coding RNAs，ncRNAs）。在ncRNA中，序列长度大于200个碱基的转录本为长链非编码RNAs (long noncoding RNA, lncRNAs), 它们由于功能的丰富性及作用机制的多样性而备受关注。lncRNAs能够在转录、转录后和翻译多个层面上调节蛋白编码基因的表达，从而广泛地参与包括细胞分化和机体发育在内的重要生命过程，其异常表达还与人类多种重大疾病的发生密切相关。
lncRNA具有高度保守性及组织特异性，在脑部含量非常丰富。lncRNA不仅参与了神经系统的生长发育和功能完善，使神经系统按照一定的时间顺序和在一定的空间内进行生长和发育，并且参与执行神经系统的功能。lncRNA通过多种机制参与到神经系统的发育及功能执行中，包括作为顺式作用元件及反式作用因子参与基因印记、染色质重塑、细胞周期调控、剪接调控、mRNA降解和翻译调控等过程。因此，利用检测lncRNAs的组成及表达水平的变化来反映神经系统的生理及病理状态是可行的手段。
房水属于眼内容物，由睫状体产生，经过瞳孔、小梁网等最终进入血液，且处于动态循环之中，其成分构成与眼球局部生理及病理环境密切相关。外泌体作为机体至关重要的细胞间相互交流的中介物质，包含了lncRNAs、mRNAs、蛋白质及脂质等。外泌体内包含的物质由于特殊保护机制能长期保持稳定状态。因此，房水中的lncRNAs等分子可以外泌体的形式传递细胞间的相互交流信息，并可随房水的动态循环进入血液。因此青光眼相关的lncRNAs研究具有为青光眼相关生物标志物检测提供理论基础的潜能。
 技术问题
本发明要解决的技术问题是提供两种用于青光眼诊断的分子标记物lncRNAsT267384，lncRNAsENST00000564363、试剂盒及应用。
 技术解决方案
发明内容
本发明的目的之一是提供一种用于青光眼早期诊断的长链非编码RNA，对青光眼的发病检测有重要意义。
用于青光眼早期诊断的分子标记物lncRNAsT267384，其序列如SEQ ID NO:1所示。
本发明的目的之二是提供上述分子标记物lncRNAsT267384的应用。检测lncRNAsT267384表达水平的产品在制备青光眼早期诊断工具中的应用。
所述的检测lncRNAsT267384表达水平的产品包括：通过实时荧光定量PCR检测lncRNAsT267384表达水平的制剂。
用实时荧光定量PCR 检测lncRNAsT267384表达水平的制剂包括特异性扩增lncRNAsT267384的引物。
用实时荧光定量PCR 检测lncRNAsT267384表达水平的特异性扩增lncRNAsT267384的引物序列为：
F:5'-TCTGGGCATTGAAGAGTGAAG-3’，
 R:5'-AGAGGGATGGGTACAAATAAGC-3’。
本发明的目的之三是提供一种用于青光眼早期诊断的试剂盒，包含检测lncRNAsT267384表达水平的试剂。具体是包含通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂。
所述的通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂包含通过实时荧光定量PCR特异性扩增lncRNAsT267384的引物。
所述的通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂包含一对通过实时荧光定量PCR特异性扩增lncRNAsT267384的引物，其引物序列为：
F:5'-TCTGGGCATTGAAGAGTGAAG-3’，
 R:5'-AGAGGGATGGGTACAAATAAGC-3’。
我们运用qRT-PCR检测方法验证lncRNAs：T267384、ENST00000607393和T342877在房水、虹膜组织和血清中的表达水平并分析其在两类房水中表达量的差异。在三种组织中，房水具有最高的诊断意义，三种lncRNAs在房水中诊断青光眼的敏感性/特异性均大于90%。这种现象的可能原因是由于血-房水屏障的存在，显著限制了血源性免疫细胞和来自全身其他部位的信号分子进入房水。因此较血清而言，房水更能特异性的代表眼部的生理及病理状态。因此，在今后的研究中房水可能较血清具有更大的青光眼诊断价值。
本发明的目的之四是提供另一种用于青光眼早期诊断的长链非编码RNA，对青光眼的发病检测有重要意义。
用于青光眼早期诊断的分子标记物lncRNAsENST00000564363，其序列如SEQ ID NO:4所示。
本发明的目的之五是提供上述分子标记物lncRNAsENST00000564363的应用。检测lncRNAsENST00000564363表达水平的产品在制备青光眼早期诊断工具中的应用。
所述的检测lncRNAs ENST00000564363表达水平的产品包括：通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的制剂。
所述的通过实时荧光定量PCR 检测lncRNAs ENST00000564363表达水平的制剂包括特异性扩增lncRNAs ENST00000564363的引物。
用实时荧光定量PCR 检测lncRNAs ENST00000564363表达水平的特异性扩增lncRNAs ENST00000564363的引物序列为：
F:5' ACAAAAAACATAAAGGCCGGG3’，
R:5’AGAGCACCCCCAAGCCCT3’。
本发明的目的之六是提供另一种用于青光眼早期诊断的试剂盒，包含检测lncRNAs ENST00000564363表达水平的试剂。具体是包含通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂。
所述的通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂包含通过实时荧光定量PCR特异性扩增lncRNAs ENST00000564363的引物。
所述的通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂包含一对通过实时荧光定量PCR特异性扩增lncRNAs ENST00000564363的引物，其引物序列为：
F:5' ACAAAAAACATAAAGGCCGGG3’，
R:5’AGAGCACCCCCAAGCCCT3’。
 有益效果
对于本申请而言，申请人发现相对于对照组人群，lncRNAs:T267384在青光眼患者的房水和血清中均表达上调。提示T267384是青光眼中的高表达lncRNAs，是有助于青光眼诊断的生物标志物。本发明为青光眼诊断提供了强有力的分子生物学基础，具有深远的临床意义和推广性。
申请人发现相对于对照组人群，lncRNAs:ENST00000564363在青光眼患者的房水中表达上调。提示ENST00000564363是青光眼中的高表达lncRNAs，是有助于青光眼诊断的生物标志物。本发明为青光眼诊断提供了强有力的分子生物学基础，具有深远的临床意义和推广性。
 附图说明
图1为青光眼患者房水中lncRNAs和mRNAs表达谱；
A：青光眼患者房水中lncRNAs表达谱分析聚类图；B：青光眼患者房水中mRNAs表达谱分析聚类图。
图2为青光眼患者房水中差异表达的lncRNAs和mRNAs；
A：较年龄相关性白内障而言，青光眼患者房水中2倍差异表达的lncRNAs；B：较年龄相关性白内障而言，青光眼患者房水中2倍差异表达的mRNAs。
图3为青光眼患者房水中lncRNAs和mRNAs表达的CNC分析图；
红色点代表lncRNAs，蓝色点代表mRNAs，实线代表正相关，虚线代表负相关。
图4为lncRNAs在青光眼及年龄相关性白内障患者房水中的表达量；
A-C： T267384、ENST00000607393和T342877在青光眼及年龄相关性白内障患者房水中表达的散点图；D-F：T267384、ENST00000607393和T342877在青光眼及年龄相关性白内障患者房水中表达的箱图。
图5为lncRNAs在青光眼患者对照人群虹膜组织中的表达量；
A-C：T267384、ENST00000607393和T342877在青光眼患者及对照人群虹膜组织中表达的散点图；D-F：T267384、ENST00000607393和T342877在青光眼患者对照人群虹膜组织中表达的箱图。
图6为lncRNAs在青光眼患者及对照人群血清中的表达量；
A-C：T267384、ENST00000607393和T342877在青光眼患者及对照人群血清中表达的散点图；D-F：T267384、ENST00000607393和T342877在青光眼患者对照人群血清中表达的箱图。
图7为利用lncRNAs在不同组织中诊断青光眼的ROC曲线；
A：利用T267384在房水中诊断青光眼的ROC曲线，曲线下面积为0.998；B：利用ENST00000607393在房水中诊断青光眼的ROC曲线，曲线下面积为0.998；C：利用T342877在房水中诊断青光眼的ROC曲线，曲线下面积为0.983；D：利用ENST00000607393在虹膜组织中诊断青光眼的ROC曲线，曲线下面积为0.793；E：利用T267384在血清中诊断青光眼的ROC曲线，曲线下面积为0.620；F：利用ENST00000607393在血清中诊断青光眼的ROC曲线，曲线下面积为0.638。
图8为另一组lncRNAs在青光眼及年龄相关性白内障患者房水中的表达量；
A-D为：lncRNA:ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241在青光眼及年龄相关性白内障患者房水中表达的散点图。
图9为利用另一组lncRNAs在房水中诊断青光眼的ROC曲线；
A-D为：lncRNA ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241在房水样本中诊断青光眼的ROC曲线下面积分别为1.000、0.920、0.940和0.880。
 本发明的实施方式
以下结合实施例旨在进一步说明本发明，而非限制本发明。
实施例1：青光眼患者房水中lncRNAs及mRNAs的表达谱：
1.1 材料与试剂
1.1.1 主要仪器
常用离心机及低温离心机购自Eppendorf公司；高速离心机购自Beckman公司；Real-Time PCR仪器购自ABI公司；移液器和电动移液枪购自Eppendorf公司；Q5000购自Quawell Technology公司；制冰机购自Sanyan公司。
1.1.2 材料与试剂
    不同型号的离心管及PCR管均购自Axygen公司；DEPC水购自鼎国昌盛公司；Trizol购自Invitrogen公司；TargetAmp TM1-Round aRNA Amplification试剂盒购自epicentre公司；Transcriptor First Strand cDNA Synthesis试剂盒购自Roche公司；Quick Amp Labeling Kit, One-Color试剂盒购自Agilent Technologies公司；人lncRNAs microarray芯片购自Arraystar公司。
1.1.3 试剂配置
各种免疫印迹所用试剂根据《分子克隆（第三版）》中提供的方法配置。
1.2 方法
1.2.1 房水样本准备
该研究已通过中南大学湘雅二医院伦理委员会审批。术前与所有研究对象签署知情同意书，在正式手术步骤开始前利用1ml的一次性无菌注射器于角膜缘抽取未稀释的房水标本0.1ml，立即注入高压灭菌的0.5mlEP管中，置于-80℃低温冰箱避光保存备用。所有入选患者为在我院住院需要进行手术治疗的青光眼患者和年龄相关性白内障患者。纳入标准：1. 原发性开角型青光眼：A. 眼压>21mmHg B. 青光眼性视盘损害和/RNFL缺损 C.典型的青光眼性视野缺损 D. 前房角开放。具有以上四项或具有其中的A, D, B或C者。2. 正常眼压性青光眼：具有类似于POAG的视盘改变，RNFL及视野损害，24h眼压测量均≤21mmHg, 房角开放。3. 原发性闭角型青光眼：具有青光眼的视盘改变，RNFL及视野损害，眼压>21mmHg, 前房角变窄或关闭4. 年龄相关性白内障（对照组）：晶状体呈皮质性和/或核型浑浊和/或后囊下性混浊。排除标准：1. 伴发全身性疾病如高血压和糖尿病等。2. 继发性青光眼3. 可能影响视野、视神经或色觉的眼科或神经科疾病。4. 视野检测可靠性标准：固视丢失率、假阳性率和/或假阴性率>25%。5. 年龄相关性白内障：排除并发性、外伤性、先天性白内障并排除伴有其他眼内疾病者、排除过熟期病例。
1.2.2 RNA提取
取房水0.1ml加入0.5mlTrizol试剂，剧烈震荡后加入0.1毫升氯仿。而后经离心、沉淀及75%乙醇清洗后溶于10微升DPEC水，经Q5000仪器测量各管RNA浓度。
1.2.3RNA扩增
使用TargetAmp TM1-Round aRNA Amplification Kit 103（epicentre）按生厂商说明书所示步骤对RNA进行扩增。大致步骤如下：首先根据RNA样本合成单成单链cDNA: RNA杂交产物，然后使用RNase H酶将上述杂交产物消化为小片段序列，这些小片段序列可协助双链cDNA的合成。最后由双链cDNA转录合成反义RNA。
1.2.4 cDNA合成和标记
    使用Transcriptor First Strand cDNA Synthesis Kit（Roche）按生厂商说明书所示步骤合成cDNA。使用Quick Amp Labeling Kit, One-Color (Agilent Technologies)对合成的cDNA进行标记。  
1.2.5标记效率质量检测
    取1.5微升已标记的cDNA样本，使用NanoDrop ND-1000检测荧光标记效率。
1.2.6芯片杂交
    在标准条件下将标记好的探针和高密度芯片（Human lncRNA microarray V4.0, Arraystar公司）进行杂交。该芯片共检测40173种lncRNAs及20730种mRNAs表达水平。
1.2.7图像采集和数据分析
    使用GenePix 4000B芯片扫描仪扫描芯片的荧光强度，并将实验结果转换成数字型数据保存。 P值<0.05为差异有统计学意义。
1.3 结果
1.3.1 青光眼患者房水中lncRNAs及mRNAs的表达谱
    在10份青光眼患者房水样本中平均检测到20653±569.9种lncRNAs和11265±268.3种mRNAs。其中，10份青光眼患者房水样本中均检测到的lncRNAs为11728种，均检测到的mRNAs为6686种。
1.3.2 青光眼患者房水中差异表达的lncRNAs及mRNAs
较年龄相关性白内障而言，青光眼患者房水中2倍上调表达的lncRNAs为4372种，2倍下调表达的lncRNAs为2602种。较年龄相关性白内障而言，青光眼患者房水中2倍上调表达的mRNAs为2783种，2倍下调表达的mRNAs为1617种。
1.4 结果
由于其易取性、个体特异性及相对受机体其他器官影响较小的特点，房水是一种眼部疾病生物标志物相关研究的价值较大的研究对象。并且，尽管目前已有多种研究报道青光眼相关的流行病学及遗传学危险因素，但以房水为研究对象的相关研究相对较少。因此，我们认为房水具有应用于今后青光眼相关遗传学研究的潜能。由于取样的限制，人源性房水样本体积较小。为克服该问题，我们在进行芯片杂交前增加RNA扩增步骤，如此以保证后续步骤的顺利进行。通过使用lncRNAs芯片微阵列分析方法，11728种lncRNAs及6686种mRNAs在10份青光眼房水样本中均检测到，这与年龄相关性白内障患者房水中lncRNAs和mRNAs表达谱具有显著差异，因此房水中lncRNAs和mRNAs表达谱表现出个体特异性及疾病特异性。就我们所知，这是第一个青光眼房水相关lncRNAs和mRNAs表达谱研究。
实施例2：通过CNC分析明确房水中特定lncRNAs与mRNAs的相关性
为进一步探讨青光眼患者房水中表达的lncRNAs的可能功能，我们运用CNC（coding-noncoding gene co-expression）分析明确与青光眼相关基因的mRNAs表达具有显著相关性的lncRNAs。CNS分析是一种通过lncRNA和mRNA共表达数据，将lncRNA与mRNA联系起来的分析方法。通过CNC分析可以发现与某个lncRNA具有相同表达模式的mRNA，通过这些mRNA的功能，可以将lncRNA与特定信号通路或疾病状况联系起来，从而便于预测lncRNA的功能，并揭示其作用机制。
2.1 方法
    挑选房水中差异表达并且与青光眼发生发展具有相关性的mRNAs，将这些mRNAs在不同青光眼患者房水中的表达数据求平均值；求挑选出的mRNAs标准化之后的数据与青光眼患者房水中差异表达的lncRNAs相关数据之间的皮尔逊相关系数（Pearson Correlation Coefficient, PCC）与错误发现率（False Discovery Rate, FDR）；挑选出PCC≥0.90并且FDR≤0.05的记录；使用相关记录，利用Cytoscape2.8.3工具绘图。
2.2 结果
较年龄相关性白内障而言，在青光眼患者房水中差异表达且与青光眼发生发展具有相关性的mRNAs为如下十种：骨形态生成蛋白2（bone morphogenetic protein 2, BMP2）、室管膜相关基因1（ependymin related 1, EPDR1）、转化生长因子β1（transforming growth factor beta 1, TGFB1）、叉头蛋白E3（forkhead box E3, FOXE3）、生长激素促分泌素受体（growth hormone secretagogue receptor, GHSR）、叉头蛋白C1（forkhead box C1, FOXC1）、跨膜及卷曲螺旋结构域1（transmembrane and coiled-coil domains 1, TMCO1）、PH结构域A7（pleckstrin homology domain containing A7, PLEKHA7）、视神经蛋白（optineurin, OPTN）和整合素亚基β5（integrin subunit beta 5, ITGB5）。在青光眼患者房水中，与这些mRNAs表达的皮尔逊相关系数大于0.9并且错误发现率小于0.05的lncRNAs有10种。
实施例3
我们利用qRT-PCR在青光眼及年龄相关白内障患者房水中验证实施例2中的10种lncRNAs中，7种lncRNAs在两类房水中的差异表达量具有统计学差异，3种lncRNAs在两类房水中的差异表达量无统计学差异。然后选取差异表达量最显著的3种T267384、ENST00000607393和T342877，进行进一步验证。
实施例4
我们运用qRT-PCR检测方法验证实施例3中差异最显著的3种lncRNAs分子在房水、虹膜组织和血清中的表达水平，并分析其在青光眼患者和正常人中表达量的差异。
4.1 材料与试剂
4.1.1 主要仪器
常用离心机及低温离心机购自Eppendorf公司；高速离心机购自Beckman公司；Real-Time PCR仪器购自ABI公司；移液器和电动移液枪购自Eppendorf公司；Q5000购自Quawell Technology公司；制冰机购自Sanyan公司。
4.1.2 材料与试剂
不同型号的离心管及PCR管均购自Axygen公司；DEPC水购自鼎国昌盛公司；Trizol购自Invitrogen公司；逆转录试剂盒和荧光定量试剂盒均购自Roche公司。
4.1.3 试剂配置
各种免疫印迹所用试剂根据《分子克隆（第三版）》中提供的方法配置。
4.2方法
4.2.1 房水、血清及虹膜样本准备
    房水样本按1.2.1所述步骤准备。血清样本取自青光眼患者组及健康对照组人群，其中青光眼患者的纳入及排除标准同1.2.1所述，对照组血清取自与青光眼组患者年龄与性别匹配的健康人群。虹膜样本取自青光眼患者组及对照组，其中青光眼患者的纳入及排除标准同1.2.1所述，对照组虹膜样本取自与青光眼患者年龄与性别匹配的湘雅二医院角膜捐赠自愿者的虹膜组织。
4.2.2 RNA提取
    0.1毫升房水或虹膜组织加入0.5毫升Trizol试剂，剧烈震荡后加入0.1毫升氯仿。而后经离心、沉淀及75%乙醇清洗后溶于20微升DPEC水，经Q5000仪器测量各孔RNA浓度。外周静脉血静置30分钟后放入离心机中，在3200转/分钟的条件下离心10分钟, 使用灭菌的移液管取上层血清，然经由上述同样步骤提取RNA。
4.2.3 qRT-PCR
根据生厂商说明书首先依赖反转录酶将RNA反转录成cDNA，然后用PCR方法扩增 cDNA，通过测定荧光强度信号实时检测定量扩增的产物量。
4.2.2 数据分析
采用SPSS19.0版统计学软件进行数据处理。计量资料以中位数±四分位数间距表示，青光眼/白内障代表两组中位数比值，比较进行Mann-whitney U检验。 P值<0.05为差异有统计学意义。
4.3 结果
为进一步扩大房水样本量验证相关lncRNAs的表达水平并且探究特定lncRNAs在青光眼患者血清及虹膜中的表达量，我们利用qRT-PCR检测60个房水样本（30个青光眼患者房水样本，30个年龄与性别匹配的年龄相关性白内障患者房水标本）、50个虹膜组织（40个青光眼患者虹膜组织样本，10个年龄与性别匹配的角膜捐赠自愿者的虹膜组织）和158个血清样本（103个青光眼患者血清样本，55个年龄与性别匹配的健康人群血清样本）中T267384、ENST00000607393和T342877表达水平。结果表明，在两类房水样本中，三种lncRNAs的表达量均有统计学差异，其在青光眼患者房水中表达量依次为在年龄相关性白内障患者房水中表达量的4.4036、2.1467和2.9692倍；在两类虹膜组织中ENST00000607393的表达量具有统计学差异，其在青光眼患者虹膜组织中表达量为在对照组虹膜组织中表达量的3.3436倍，而T267384和T342877的表达量无统计学差异；在两类血清样本中，T267384及ENST00000607393的表达量具有统计学差异，其在青光眼患者血清中表达量依次为在对照组血清中表达量的1.2878和1.5301倍，而T342877的表达量无统计学差异（表1,2,3）（图4,5,6）。为进一步了解这三种lncRNAs在不同种组织中诊断青光眼的有效性，我们利用所得数据绘制受试者工作特征（receiver operating characteristic, ROC）曲线（图7）。发现T267384、ENST00000607393和T342877在房水样本中诊断青光眼的ROC曲线下面积分别为0.998、0.998和0.983。ENST00000607393在虹膜组织中诊断青光眼的ROC曲线下面积为0.793；T267384和ENST00000607393在血清样本中诊断青光眼的ROC曲线下面积分别为0.620和0.638。当T267384、ENST00000607393和T342877在房水中的诊断值设为1.5437、1.1485和2.1052时，其诊断的敏感性/特异性依次为0.967/0.967、0.967/0.967和0.967/0.900；当ENST00000607393在虹膜组织中的诊断值设为0.2470时，其诊断的敏感性/特异性为0.700/0.700;当T267384和ENST00000607393在血清中的诊断值设为0.7191和0.8376时，其诊断的敏感性/特异性依次为0.612/0.600和0.680/0.600。
                表1            3种lncRNAs在房水中的表达量
Figure 769909dest_path_image001
                  表2            3种lncRNAs在虹膜组织中的表达量
Figure 749366dest_path_image002
                       表3             3种lncRNAs在血清中的表达量
Figure 840688dest_path_image003
            表4         3种lncRNAs在不同组织中诊断的敏感性和特异性
Figure 235897dest_path_image004
实施例5
我们利用qRT-PCR在青光眼及年龄相关白内障患者房水中验证实施例2中的10种lncRNAs中，7种lncRNAs在两类房水中的差异表达量具有统计学差异，3种lncRNAs在两类房水中的差异表达量无统计学差异。然后选取另一组具有统计学差异的lncRNA:ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241进行进一步验证。
实施例6
我们运用qRT-PCR检测方法验证lncRNA:ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241分子在房水中的表达水平，并分析其在青光眼患者和对照组人群中表达量的差异。
6.1 材料与试剂
6.1.1主要仪器
常用离心机及低温离心机购自Eppendorf公司；高速离心机购自Beckman公司；Real-Time PCR仪器购自ABI公司；移液器和电动移液枪购自Eppendorf公司；Q5000购自Quawell Technology公司；制冰机购自Sanyan公司。
6.1.2材料与试剂
不同型号的离心管及PCR管均购自Axygen公司；DEPC水购自鼎国昌盛公司；Trizol购自Invitrogen公司；逆转录试剂盒和荧光定量试剂盒均购自Roche公司。
6.1.3试剂配置
各种免疫印迹所用试剂根据《分子克隆（第三版）》中提供的方法配置。
6.2方法
6.2.1房水样本准备
    房水样本按1.2.1所述步骤准备。
6.2.2 RNA提取
    0.1毫升房水加入0.5毫升Trizol试剂，剧烈震荡后加入0.1毫升氯仿。而后经离心、沉淀及75%乙醇清洗后溶于20微升DPEC水，经Q5000仪器测量各孔RNA浓度。
6.2.3 qRT-PCR
根据生厂商说明书首先依赖反转录酶将RNA反转录成cDNA，然后用PCR方法扩增 cDNA，通过测定荧光强度信号实时检测定量扩增的产物量。
6.2.4数据分析
采用SPSS19.0版统计学软件进行数据处理。计量资料以中位数±四分位数间距表示，青光眼/白内障代表两组中位数比值，比较进行Mann-whitney U检验。 P值<0.05为差异有统计学意义。
6.3 结果
为进一步扩大房水样本量验证相关lncRNAs的表达水平并且探究特定lncRNAs在青光眼患者房水中的表达量，我们利用qRT-PCR检测20个房水样本（10个青光眼患者房水样本，10个年龄与性别匹配的年龄相关性白内障患者房水标本）中ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241表达水平。结果表明，在两类房水样本中，ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241的表达量均有统计学差异，其在青光眼患者房水中表达量依次为在年龄相关性白内障患者房水中表达量的1.6200、1.9500、1.9302和1.8378倍（表5）（图8）。为进一步了解ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241在房水中诊断青光眼的有效性，我们利用所得数据绘制受试者工作特征（receiver operating characteristic, ROC）曲线（图9）。发现ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241在房水样本中诊断青光眼的ROC曲线下面积分别为1.000、0.920、0.940和0.880。当ENST00000564363、NR_026887、TCONS_00025577和ENST00000508241在房水中的诊断值设为0.0068、0.0056、0.0074和0.0048时，其诊断的敏感性/特异性依次为1.000/1.000、0.900/0.900、0.900/0.900和0.800/0.800（表6）。
                            表5  lncRNAs在房水中的表达量
 
Figure 50270dest_path_image005
                   表6  lncRNAs在房水中诊断青光眼的敏感性和特异性
Figure 935049dest_path_image006
 
 序列表自由内容
<110>  中南大学湘雅二医院
<120>  两种用于青光眼诊断的分子标记物、试剂盒及应用
<130>  无
<160>  6    
<170>  PatentIn version 3.3
 
<210>  1
<211>  6891
<212>  DNA
<213>  lncRNA：T267384的序列
 
<400>  1
tatttcatct tcgcattatt ttcaatactg aaggtaaatt tagatctctc tttttttctt     60
agtctggcta aaagcttgtc aattttgctt aacttttcaa aaaaccaact ttttgtttca    120
ttgatttttg tattgtttac ttcattttaa tttcatttat ttctgttctg atctttatta    180
tttcttctac taattttgga tttggtttgc tcttgttttc ctaattcttt aagatgcatc    240
attagattgt ttatttaatg taggcactat aagtttccct cttttttgat gtaggcacta    300
taacgataag cttccctctt agtactgctt ttgctgtatc ccatagattt tggtatgttg    360
tgctcccatt agcatttgtt ccaagaaatt tttaaatttc ttattaaaat gatccatcct    420
tgagaatgat ccatatgctg aggaaaagca tgtgtattcc acagccattg gatgaaatgt    480
tccgtaatta tctactggat ccatttggtc tattgtgcag attaagtctg atgtttcttt    540
attgattttt ctgcctggaa gatccatcca gtgctgaaag tggggtgtcg aagtctccag    600
ctattattgt attggggcct aactctctct ttagctcttt tttttgtttt gttttgtttt    660
gagatggagc ctggctctgt tgcccagggt ggagtgcagt ggtatgatct tggctcactg    720
caacctccgc ctcccaggtt caagctattc tcctgcctca gcctcccaag tagctgggat    780
tacagacatg tgccaccata cccggctaat tttgtatttt tagtagagat agggtttctc    840
catcttggtc aagctggtct cgaactccca acctcaggtg atccacctgc cttggcctcc    900
ccaaagtgct gggattatag gcatgagcca ctgcgcctgg ccttttttat ttttttaagg    960
aggggggggg tctcactaca ttgtctaggc tagactcaaa ctcctgggct caagtgatcc   1020
ttctgcctcg acctcctgaa atgctgtgat tacaggcatg agccaccatg actggccctg   1080
actgtgtatt ttcaaatagc ctgtcttcag gctcactaat tctttcttct gcttgatcaa   1140
ttctgctact gaaagcctct aatgcattgt tcagtatgcc aattgcattt ttagctctgg   1200
aatctcagtt tgattctttt tattttaatc tttttgttaa atgtatctga tataattttg   1260
aattccttct ctgtgttatc ttgaatttct tgagtttcct caacacagct attttgaatt   1320
ctctgcctga aaggtcacac atctctgttt ctccaggatt ggtccctggt ggctcattta   1380
gttcatttgg gaggtcatgt tttcctggat ggtgttgatg ttagtaaatg ttcttcagtg   1440
tctgggcatt gaagagtgaa gtatttattg tagtcttcac tgtctaggct tatttgtacc   1500
catccctctt agaaaggctt tccagatatt tgaaagtact tggatattgt gatgtaagct   1560
gtatctgatt tagagagcac cccaatccca gtaacactgt tttcttgcag actcatggag   1620
gtactacctt gatggtcttg gaaaagatcc agaagaattc tctgcattat caggcagaga   1680
gactcctgtt ccattctgtt actttcttcc aagcaaaaag agtctcactc tccttgttct   1740
gagccacata aagccggcgg tggagtgaca caagcatccc tgtggccacc accactatga   1800
ctgcattggg tcagacctga agccagcaca gcagtggggc ttgccaaggc tggctgtaac   1860
cacttcctgg ctactgccta tgtttgctga aggccctggg gctctacaat tagctggtgg   1920
caaagccggc caggcctgtg tccttccctt cagagccgtg aggtccccca ggccccaggt   1980
gagtccagag gtgccttttg ggaatcaggg actagagtaa aaaaacttgg aagtctagct   2040
ggtgttctat tgtactgtgg ctgagctggt attcagacca ccagatgcag tccttcccac   2100
tcttcctttc ccttcccaaa tgcagaggag cctcacccca tagccactgt caacccaggc   2160
cacgaggagt actgccaggc tacctccaat attcccttaa gcatcgaggt atcttaagtc   2220
aagcttgtgt tgaatgcttc ctgccttggg actcgccctt cagggcaatg ggttcccctc   2280
tggcctgggg caggtgcaga aatggtgtcc aacagtcaag tcctggaatc ggagacctca   2340
agagcccact tggtgcttta ccccactgtg gctgtgctgg tacctaaggt gcaagacaaa   2400
gccctcttta cttttccttc tgcttttctc aaacagaagg agttttgcct tgttgccacc   2460
acagctagta atgtgctgag tctcacctga agccagcaag tctcagaggc tcaaccaagg   2520
gccttgacat agtacctaaa tgtaactgct ggttattcag ggtcctgcca accttcagtt   2580
agcaggtgat gaatgctgcc aggactgagt ccttttcttc aaggtagtgg gtttccttct   2640
gacccatggt gtgtctagaa atgtcatctg ggaactaggg cctggaatgg gggcctcacg   2700
actcttccca gtcccctatt ctgccgcagc tgaactggta tccaagatgc aagatgaagt   2760
cctccccact cttctttctc ctctccacaa gcagaaggaa agggtctcct ttggagccac   2820
gggctgtgca gcctggagtt agggaagggg tgacgccagc acgcccttgg ctgccttagc   2880
tggtgtctca gtatgtcacc tgccctccca gttaactgcc tctgggccta gttcagcact   2940
agaactcgcc tgagatctgc agtcttcatg gcctgggctg cctttcaagt ttacttggag   3000
acccagactg tagccctcgg tggtgaggtt tgcaggaact caagtttaga ccactgggat   3060
cagtgattcc cctctgggta gggctgttat taaatgctcc ctccatgggc aggcgtcagc   3120
tgagttgggt gtggttttcc ttcctgccgt aacaggacag cactgagttc agtgcatcac   3180
agttgctgtg ttctccctcc cgtgcacgca gagatatcct cagtaccaca acacagctgc   3240
aggggcagag gggcgtcagt gattcaggac tgtttttgct atttcttcgg tgtctctttc   3300
agcattataa agttaagagc aggtactatg agtgctcacc tgatttttaa cacctcatga   3360
agttgttaaa ttggtgtcct tgtggagggg gatgatagct gcagtcttct attcctccat   3420
ttttctctgt ctcctcattt tttttttttt ttttttggag acggagtctt gctctgtcac   3480
ccaggctgga gtgcaatggc acgatctcgg ctcactgcag cctccgcctc caggttcaag   3540
cgattctcct gcctcagact cctgagtagc tgggactaca ggtgctacca tgaacagcta   3600
aatttaattt ttttaattat tatatattgt gggcgaagga ttacctaggt gccgaggcaa   3660
gagactaaag gcacaaactg tttcagtata ataaagaaaa tagaataaga atagtcataa   3720
tacaaattag atatagagat gatcgtggac aattatcaat cattattata aacattatta   3780
atcattagct tttaatatta ctctttgttg cattactaat ataacctagg aataaccagc   3840
gggtataggg tcaggtgctg aaagcacatt gtgacaagtt acctagaagg caagagctga   3900
gccttctgtc acgcccgcat aagggccgct tgagggctcc ttggtcaagc ggtaatgcca   3960
gtgtctggga agacacccac gacgtagcag accgcaaaag ggagtctcct ttccttggtg   4020
gagtcaggga acactctgct ccaccagctt cttgtgggag gctggatgtt atccaggcct   4080
gcccacagtt atccggaggc ctaaacccct ccctgtggtg ctgtgcttca gtggtcacgc   4140
tccttgtcca ctttcatgct cctccagcac tcctggttcc tctttgaagt tcgtagtaga   4200
taacggtaga agaaatagtg aaaggcttaa agtctttgac ctttcttata agtgcataga   4260
agaaaacgct gatgtatgcc accttctctc tgcttcagct acctaaaagg gaagggcccc   4320
ctgtcctatg atcacatgac ttgcttcaac ttgtcaatca cttagaagat tcaccctcct   4380
taccctgccc cctggtcttg tatgcaataa atatcagcgt gcccagccat tcgggaccac   4440
tacctgtctc cgtgtcttaa tggtagtggt cccctgggcc cagctgtttt ctctttatct   4500
ctttgtcctg tgtctttatt tattacaatc tctcatctcc gtacatggcg agaacacctg   4560
ctaagccccg taggcctcaa ctctacaata tatttatttt atattgccca ggttagtccc   4620
taactcctgg gttcaagtga tcctcccacc tcgacctccc aaagtgctat aattacaggt   4680
atgagccaca ccacacccgg ccaataatac acgtatcttt gcaagcataa acattcaggt   4740
atcctaacaa cagtctcata agtataagag ttatgagtag atgaactgta ttcataaaga   4800
aataggccaa aaagtaaaat gtattaacat atatgattgg taattgtgtg cactcaactt   4860
tataactgtg gtcatctgaa ataccaagat ggacaatata agtcttttga caaaatcaat   4920
aaaaatccgc agtgagttac caccacacat gcagttgccc aaaaagccaa gatctcaaaa   4980
tattttatct ttcacaaata tggatgtaca aaaagaagat attttattga ggaagttgca   5040
atgtttttac atacatgcac aatgcttaca caaattcagc ttcgtgataa tgcactttca   5100
tggagtcaaa tttgcaaaaa tgcataaaat gaattagaac tttctaaaca tctttataca   5160
atttatacct tcagtattaa aaatggactg aggaggccgg gcacagtggc tcacacctgt   5220
aatccctgac tttgagaggc tgaagtgggc ggatcacttg aggttaggag ttggagacct   5280
gcctggccaa catagtgaaa ccctgtctgt agtaaaaata caaaaattaa cacttgagcc   5340
caggaggcgg aggttggagt gagctgagat tgtaccactg cactccagcc tgggtgacaa   5400
agtgagaccc tgtctcaata aaaaataaaa ataaaaaaag tatgttatta tttcctgttt   5460
tattgtctaa aatggtctat gaagtgctct gttgtgtttt tatgtttctc aaatctcctt   5520
taaaaaatat aaataaagaa ttttaaagaa tttttaaaat tattttgtca agaattatat   5580
tttcaggatt ttaatcattt gggattggaa ttttggggat tttggacttc agggattttg   5640
atctttcagg atttcatcat ttgggattat aacattcaag attgtgtctt ttgggattat   5700
ggcccaaacc catgcaatgg gccagttctt tgaaaggaat agtgttccat aactcacaca   5760
aggagaaata gaaaatttga ataggcctat acctgttaaa ctaaatcaat gatttgccaa   5820
agaggtaata aggtttggac tacatttatg tcagcaaaga ggagaggaca gattaattca   5880
ttcaataagt gattgagtag atacctactg gtgtcaggca ttgttctagg cactgggaat   5940
acagcagcaa acaaaatgat tgagtagata cctactggtg tcaggcattg ttctaggcac   6000
tgggaataca gcagcaaaca aaatagatca ccccttgggg ctttcaagtg gagtatcggt   6060
gacagagtgg gtgtctgatt ggatatcggg gaaaaggaga aaggtgcgat gaggcaattc   6120
aggggtcatt ggtacctgga tttttcacct tgaacaacta agtgatatca ttggacttgt   6180
tgggtagaca aggtctaggg gacatccaga tggactcatt ttcacagcta gcaagagaga   6240
cattctgaag atcaatagta agggagaaac tggaagtaca gactttgggg tcctaagagg   6300
cagttagaac catgggagta gatgattgta taagataaat agttttgcct cttacttccc   6360
ccaccccaac aaatgaaatg gaatcagaag tgtaggagga gaactagggg aaattaggaa   6420
gtgagatttt catcagggca aaatgatgct gacggatcaa atgggacaag aaatgtgaaa   6480
aaaatcattt tacactttta tccatctatt gatggataaa atttaaccaa aattgagtca   6540
ctgaatttga tcattgatca aatttttctt actctgataa gagttttcag aaaactctga   6600
tcagagtttt cttactctga tatctcatat tcaaaaggga aagaaaatca acaacacaat   6660
aaacaacctc ctgaggaatg cagtgtaaaa gcaaggcttg gtgtgttgtt tcgcaaaaaa   6720
tcttgctttg tcagcattaa tactaacaga taacatgaga agaggcaaaa gtgttggcta   6780
aatgtaccat aactgcctgg gaatagttat ctgtagtcta tttttttttt ttctcatcta   6840
aagttcttag atgaaagaga gctgggttac tgtgaaccaa aatctcctgc t            6891
 
<210>  2
<211>  21
<212>  DNA
<213>  特异性扩增lncRNAsT267384的引物序列F
 
<400>  2
tctgggcatt gaagagtgaa g                                               21
 
<210>  3
<211>  22
<212>  DNA
<213>  特异性扩增lncRNAsT267384的引物序列R
 
<400>  3
agagggatgg gtacaaataa gc                                              22
 
<210>  4
<211>  687
<212>  DNA
<213>  lncRNA ENST00000564363序列
 
<400>  4
ttccaactac atgtctattt tttattcaat atattaaata tattaatcag aaaagtcaca     60
tactataaat ccaggaaaat acacaaatat aaatgtcaga atctgtcatt tgtcttcttc    120
ctagagtgac agtttatgta catgtggaag gagggtagga agagagagcc agcaggaggc    180
tggaaaggct gcccaggaag gcggggccca tcccttcctc ccagccccgc tcccacctca    240
agctctttct tcctgtattt acagcagcac tgggcttatt tacagcacgg cgctatgtgt    300
taaaaacggt ttatcttaca aaaaacataa aggccggggc caggctgggg gggataagac    360
aaatcgtggg gtcaccccca tgaaggcatc cccctcccca agggcttggg ggtgctctgg    420
gggtccgtgg actaaaggca catggggtgg catatgagag tgcgcatgtg cacacgtgtt    480
cccaggcagg ggtcagaagg gcagcgtgtc catgaagatc ttgtcaatga tgggtggagg    540
gggcaccaag tcctccagct tgaggtagaa gatgcgctgc aggccctggg tgcacagggt    600
ccgcagctcg ggcagtttgc ccaacagacg tgacaggcag ctggctggct ggggctcgcc    660
cgccacagct gccacgtgct ccttcag                                        687
 
 
<210>  5
<211>  21
<212>  DNA
<213>  特异性扩增lncRNAs ENST00000564363的引物序列F
 
<400>  5
acaaaaaaca taaaggccgg g                                               21
 
<210>  6
<211>  18
<212>  DNA
<213>  特异性扩增lncRNAs ENST00000564363的引物序列R
 
<400>  6
agagcacccc caagccct                                                   18
 

Claims (18)

  1. 用于青光眼诊断的分子标记物lncRNAsT267384，其序列如SEQ ID NO:1所示。
  2. 检测lncRNAsT267384表达水平的产品在制备青光眼诊断工具中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述的检测lncRNAsT267384表达水平的制剂包括：通过实时荧光定量PCR检测lncRNAsT267384表达水平的制剂。
  4. 根据权利要求3所述的应用，其特征在于，用实时荧光定量PCR 检测lncRNAsT267384表达水平的制剂包括特异性扩增lncRNAsT267384的引物。
  5. 根据权利要求4所述的应用，其特征在于，用实时荧光定量PCR 检测lncRNAsT267384表达水平的特异性扩增lncRNAsT267384的引物序列为：
    F:5'-TCTGGGCATTGAAGAGTGAAG-3’，
     R:5'-AGAGGGATGGGTACAAATAAGC-3’。
  6. 一种用于青光眼诊断的试剂盒，其特征在于，包含检测lncRNAsT267384表达水平的试剂。
  7. 根据权利要求6所述的用于青光眼诊断的试剂盒，其特征在于，包含通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂。
  8. 根据权利要求7所述的用于青光眼诊断的试剂盒，其特征在于，所述的通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂包含通过实时荧光定量PCR特异性扩增lncRNAsT267384的引物。
  9. 根据权利要求7或8所述的用于青光眼诊断的试剂盒，其特征在于，所述的通过实时荧光定量PCR检测lncRNAsT267384表达水平的试剂包含一对通过实时荧光定量PCR特异性扩增lncRNAsT267384的引物，其引物序列为：
    F:5'-TCTGGGCATTGAAGAGTGAAG-3’，
     R:5'-AGAGGGATGGGTACAAATAAGC-3’。
  10. 用于青光眼诊断的分子标记物lncRNAs ENST00000564363，其序列如SEQ ID NO:4所示。
  11. 检测lncRNAs ENST00000564363表达水平的产品在制备青光眼诊断工具中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述的检测lncRNAs ENST00000564363表达水平的产品包括：通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的制剂。
  13. 根据权利要求12所述的应用，其特征在于，用实时荧光定量PCR 检测lncRNAs ENST00000564363表达水平的制剂包括特异性扩增lncRNAs ENST00000564363的引物。
  14. 根据权利要求13所述的应用，其特征在于，用实时荧光定量PCR 检测lncRNAs ENST00000564363表达水平的特异性扩增lncRNAs ENST00000564363的引物序列为：
    F:5' ACAAAAAACATAAAGGCCGGG3’，
    R:5’AGAGCACCCCCAAGCCCT3’。
  15. 一种用于青光眼诊断的试剂盒，其特征在于，包含检测lncRNAs ENST00000564363表达水平的试剂。
  16. 根据权利要求15所述的用于青光眼诊断的试剂盒，其特征在于，包含通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂。
  17. 根据权利要求16所述的用于青光眼诊断的试剂盒，其特征在于，所述的通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂包含通过实时荧光定量PCR特异性扩增lncRNAs ENST00000564363的引物。
  18. 根据权利要求16或17所述的用于青光眼诊断的试剂盒，其特征在于，所述的通过实时荧光定量PCR检测lncRNAs ENST00000564363表达水平的试剂包含一对通过实时荧光定量PCR特异性扩增lncRNAs ENST00000564363的引物，其引物序列为：
    F:5' ACAAAAAACATAAAGGCCGGG3’，
    R:5’AGAGCACCCCCAAGCCCT3’。
     
PCT/CN2018/088324 2017-06-07 2018-05-25 两种用于青光眼诊断的分子标记物、试剂盒及应用 Ceased WO2018223848A1 (zh)

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