CN109182498A - A kind of molecular labeling and kit and the application in autoimmune disease - Google Patents
A kind of molecular labeling and kit and the application in autoimmune disease Download PDFInfo
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- CN109182498A CN109182498A CN201811116090.5A CN201811116090A CN109182498A CN 109182498 A CN109182498 A CN 109182498A CN 201811116090 A CN201811116090 A CN 201811116090A CN 109182498 A CN109182498 A CN 109182498A
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Abstract
A kind of application the present invention provides molecular labeling and kit and in autoimmune disease.The present invention is for the first time using tRF/tiRNA as point of penetration, have studied the expression of the tRF/tiRNA of autoimmune disease patient, suitable molecular labeling is filtered out, it can be used as one of the important means of diagnosis, the prognosis of autoimmune disease, to more convenient, accurately be diagnosed to the disease, so that relevant basis is laid in the treatment for disease in future.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of molecular labeling and kit and in autoimmune disease
In application.
Background technique
Non-coding RNA (non-coding RNA, ncRNA) is widely present in cell, many kinds of, is played in body
A variety of different functions.NcRNA has specifically included mRNA (message RNA, mRNA), rRNA (ribosome
RNA, rRNA), the single-stranded microRNA of transfer RNA (transfer RNA, tRNA) and some other inhibition of gene expression
(microRNA, miRNA), double-chain small disturbance RNA (smallinterfering RNA, siRNA), long-chain non-coding RNA
(longnon-coding RNA, lncRNA) etc..And with the further development of the technologies such as high-flux sequence, it has been found that one
The new ncRNA of kind, the i.e. small fragment RNA (tRNA-derived fragment, tRF) in the source tRNA.
TRF refers to that nuclease is tRNA's in specific cell or tissue or under the conditions ofs cell is forced equal
It is sheared on ring, the small fragment RNA with particular size of generation.Mature tRNA has the secondary structure of trifolium-shaped, can divide
To receive stem arm, D ring, T ψ C ring, anticodon loop and variable loop etc..In the tRF generated after shearing, precursor tRNA is derived from
3 ' leader sequences be referred to as tRF-1；It is tRF-5 from the 5 ' ends of mature form tRNA to the tRF near D ring；And mature form
TRF of the T ψ C ring of tRNA nearby to 3 ' ends is tRF-3, and the length is 14-30 nucleotide (nucleotide, nt).In addition,
It is referred to as tRNA half molecule (tRNA halves) in the tRF that the anticodon loop shearing of mature form tRNA generates.TRNA half point
Son usually generates under stress conditions, so also referred to as tiRNA (tRNA-derived stressinducedfragment),
It is divided into two kinds of 5 ' tiRNA and 3 ' tiRNA, the length is 29-50nt.
Although the specific biological function of tRF and tiRNA not yet illustrates completely, more and more evidences show that they have
There are many biological functions, and signaling molecule can be both used as in stress reaction, can also be used as the attemperator of gene expression.It is existing
It has been found that tRF and tiRNA and a variety of diseases of the mankind (such as: tumour, metabolic disease, neuropsychiatric disease and infectious disease
Deng) closely related.The composition and quantity of tRF and tiRNA is highly dependent on cell type and morbid state.Therefore, based on tRF and
The Non-Invasive biomarker of tiRNA will be with boundless prospect in the application of medical diagnosis on disease.And about tRF and
The relationship of tiRNA and autoimmune disease yet there are no all reports.Therefore, it is necessary to be studied it to expand and itself exempt from
Application in the diagnosis of epidemic disease disease.
Summary of the invention
A kind of application the object of the present invention is to provide molecular labeling and kit and in autoimmune disease.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of molecular labeling, the molecular labeling are tRF or tiRNA segment, and nucleotides sequence is classified as in SEQ ID No.1-3
It is any.
A kind of kit of diagnosis and/or the prognosis of autoimmune disease, including drawing for specific recognition molecules label
Object, molecular labeling are above-mentioned at least one.
Preferably, autoimmune disease is systemic loupus erythematosus, rheumatoid arthritis, at least one in dermatomyositis
Kind.
Kit of the reagent of the expression quantity of quantitative molecular label in diagnosis and/or the prognosis of preparation autoimmune disease
In application, molecular labeling be above-mentioned at least one.
Preferably, autoimmune disease is systemic loupus erythematosus, rheumatoid arthritis, at least one in dermatomyositis
Kind.
The beneficial effects of the present invention are:
The present invention has studied the table of the tRF/tiRNA of autoimmune disease patient for the first time using tRF/tiRNA as point of penetration
Up to situation, filtered out suitable molecular labeling, can be used as diagnosis, the prognosis of autoimmune disease important means it
One, to more convenient, accurately be diagnosed to the disease, so that relevant base is laid in the treatment for disease in future
Plinth.
Detailed description of the invention
Fig. 1 is the scatter plot of differential expression tiRNA/tRF of the invention.
Specific embodiment
The technical effect of design and generation of the invention is clearly and completely described below with reference to experiment, to fill
Ground is divided to understand the purpose of the present invention, feature and effect.
Systemic loupus erythematosus (systemic lupus erythematosus, SLE) be a kind of chronic systematicness from
Body immunological disease.Patient's self immune system generates antibodies attack own cells and tissue, leads to generation and the histologic lesion of inflammation.
The pathogenesis of SLE is still not clear, it is now recognized that it is multifactor multifaceted influence that it, which causes a disease, including immune, heredity, environment,
Sex hormone etc., all multiple cytokines are participated.The present invention is by with systemic loupus erythematosus, this is common, typically itself exempts from
Epidemic disease disease is illustrated as representative and embodiment.
Case is from the SLE patient to go to a doctor in Shenzhen people's hospital, according to " systemic lupus erythematosus diagnosis in 2010
And treatment guidelines ", the SLE classification standard recommended using American Society of Rheumatism 1997, in 11 of the classification standard, symbol
4 or 4 or more persons are closed, are infected except.Tumour and other connective tissues after being ill, the SLE patient made a definite diagnosis, then calculate SLEDAI
Scoring (SLEDAI scoring>=8 is the Lupus activity phase, and<8 be the stable disease phase), chooses the patient in the Lupus activity phase and enters reality
Test group.SLE group includes SLE patient 20, and the control group of normal health includes 20, and two groups of experimental subjects Sex, Ages etc. are basic
Condition is mutually matched, and random controls is taken to design.The inclusion criteria of patient are as follows: sign informed consent form and have complete clinic
With various other data；Exclusion criteria is not obtain informed consent or clinical imperfect with other data.
1. sequence library construction
The patient of SLE group and Normal human peripheral's venous blood (on an empty stomach) 5ml of control group are collected with EDTA anticoagulant tube, 4 is small
When interior processing sample the venous blood of collection is isolated using lymphocyte separation medium according to Ficoll density-gradient centrifugation method
Peripheral blood mononuclear cells (PBMC).The total serum IgE that PBMC is extracted using TrizolTM reagent is mentioned using Trizol method, using RNA
Purification kit carried out column purification to total serum IgE, and carried out corresponding quality testing.
Product is subjected to demethylation processing, removes end-o f-pipe -control and the acid processing that deaminizes, to filter out high quality
tRNA。
By round pcr, the library tRNA is constructed.
The expression screening of 2.SLE group and control group differential expression tRNA
Using Assemnler-3.4.4.0, Alignment 2.0,3.0 software of coverage Analysis, it is based on
The sequence alignment program of 2 algorithm of fame is assembled and is compared to data with reference to genome with h38chrM (NC-012920),
Allow the mispairing of 10 bases.Original series screen out filtration rate by pre-filtering and ratioization and are lower than lower than 40%, mapping rate
80%, the overburden depth sample data inhomogenous lower than 100X or coverage, finally screens to obtain control group and SLE group significant figure
According to finding significant differential expression tRNA.
The data of 3.tRNA degradation fragment tiRNA/tRF are analyzed
TRNA-change analyzes each tRNA and carries amino acid and do not carry the ratio of amino acid, and tRNA need to carry amino
Acid could participate in protein translation, and tRNA amino acid carrier state parses tRNA physiological and pathological function.Count codon tRNA table
Up to amount, tRNA identifies different aminoacids by anticodon, statisticallys analyze the amount of anticodon and carries the energy of different aminoacids
Power understands the corresponding anticodon expression quantity of each tRNA, specifies tRNA anticodon and RNA codon interaction relationship, distinguish
Other anticodon gene mutation, which synthesizes albumen, to be influenced.TRNA translation effect forecast and the Conjoint Analysis of mRNA express spectra are studied,
TRNA mediates signal transduction between mRNA and protein, analyzes the degree of agreement in the library tRNA and mRNA express spectra, statistics coincide and
Non- identical point helps discovery gene mutation site.TRNA degradation fragment tiRNA-5, tiRNA-3, tRF-3 and tRF-5 are counted in reality
Test the expression quantity of group with control group, quantity variance of the analysis degradation fragment in experimental group and control group.
1.tRF/tiRNA expression quantity and differential expression analysis
Fig. 1 is the scatter plot of differential expression tiRNA/tRF of the invention.As shown in Figure 1, wherein abscissa represents control group
TiRNA/tRF CPM average value logarithm, ordinate represents the logarithm of the CPM average value of the tiRNA/tRF of IgAN group
It is worth, the point inside two dotted lines represents the tRNA (totally 184) of non-differential expression, represents otherness table above upper dashed line
Up to the tRNA (totally 130) of up-regulation, the tRNA (totally 225), Pearson of differential expression downward are represented below lower dashed line
Related coefficient=0.721.Integrated Selection goes out difference from the tRNA degradation fragment that 130 up-regulated expressions and 225 lower expression
Property most significant (the nucleotide sequence of tRF-Thr-AGT-003, tRF-Tyr-GTA-011, tiRNA-Val-TAC-004 of expression
Respectively SEQ ID No.1, SEQ ID No.2, SEQ ID No.3), specifying information is as follows:
1. correlation tRF/tiRNA information of table
Wherein, FC value (Fold Change) is differential expression multiple, and p value is the p value that negative binomial distribution is examined, and s_CPM is
The logarithm of the CPM average value of SLE group tRF/tiRNA, z_CPM are the logarithm of the CPM average value of control group tRF/tiRNA,
CPM is the every million reading long numbers for reading specific tRNA in long (reads).
It can be seen from the results above that the difference that these three tRF/tiRNA segments are expressed in SLE patient and Normal group
It is heteropolar be it is significant, by being examined to expression of these three tRF/tiRNA segments in peripheral blood or some other sample
It surveys, researcher can quickly, easily diagnose autoimmune disease, it is possible to for autoimmune disease from now on
Diagnosis, prognosis a kind of effective fast diagnosis method is provided；Meanwhile whereby it can be found that with autoimmune disease
The relevant important regulating and controlling factor of occurrence and development is sought related to the treatment of this kind of disease further to disclose its physiological mechanisms
Completely new target spot provide theoretical foundation.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Belong to those skilled in the art in the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of, all answers
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection scope in claims.
SEQUENCE LISTING
<110>Shenzhen people's hospital
<120>a kind of molecular labeling and kit and the application in autoimmune disease
<130> 9
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Homo sapiens
<400> 1
aatcccagcg gtgcctcca 19
<210> 2
<211> 18
<212> DNA
<213> Homo sapiens
<400> 2
atccggctcg gaggacca 18
<210> 3
<211> 34
<212> DNA
<213> Homo sapiens
<400> 3
ggttccatag tgtagtggtt atcacgtctg cttt 34
Claims (5)
1. a kind of molecular labeling, which is characterized in that the nucleotides sequence of the molecular labeling is classified as any in SEQ ID No.1-3
Kind.
2. the kit of diagnosis and/or the prognosis of a kind of autoimmune disease, which is characterized in that the kit includes special
Property identification molecular labeling primer, the molecular labeling is at least one of claim 1.
3. kit according to claim 2, which is characterized in that the autoimmune disease is systemic red yabbi
At least one of sore, rheumatoid arthritis, dermatomyositis.
4. the reagent of the expression quantity of quantitative molecular label is in the kit of diagnosis and/or the prognosis of preparation autoimmune disease
Application, which is characterized in that the molecular labeling is at least one of claim 1.
5. application according to claim 4, which is characterized in that the autoimmune disease be systemic loupus erythematosus,
At least one of rheumatoid arthritis, dermatomyositis.
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| CN201811116090.5A CN109182498A (en) | 2018-09-25 | 2018-09-25 | A kind of molecular labeling and kit and the application in autoimmune disease |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
| US20150176073A1 (en) * | 2012-07-18 | 2015-06-25 | Exosome Diagnostics, Inc. | Use of microvesicles in diagnosis, prognosis, and treatment of medical diseases and conditions |
-
2018
- 2018-09-25 CN CN201811116090.5A patent/CN109182498A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
| US20150176073A1 (en) * | 2012-07-18 | 2015-06-25 | Exosome Diagnostics, Inc. | Use of microvesicles in diagnosis, prognosis, and treatment of medical diseases and conditions |
Non-Patent Citations (2)
| Title |
|---|
| SUNG SOO AHN等: "Serum aminoacyl-tRNA synthetase-interacting multifunctional protein-1 (AIMP1), a novel disease activity predictive biomarker of systemic lupus erythematosus", 《CLIN EXP RHEUMATOL》 * |
| 杨再兴等: "系统性红斑狼疮的分子标志物研究进展", 《临床检验杂志》 * |
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Application publication date: 20190111 |