CN107090451A - A kind of long-chain non-coding RNA uc.77 and its application - Google Patents
A kind of long-chain non-coding RNA uc.77 and its application Download PDFInfo
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Abstract
An object of the present invention is to provide a kind of related long-chain non-coding RNA of human-mouse homologous pulmonary fibrosis（Long non-coding RNA, lncRNA）, lncRNA-uc.77 is named as, its nucleotide sequence is as shown in SEQ ID No.1.LncRNA-uc.77 provided by the present invention, passes throughq-RTPCRMethod is confirmed, significantly high to be expressed in fibrosed tissue, and overexpression lncRNA-uc.77 can be by regulating and controlling its target gene ZEB2, so as to reach the purpose of influence pulmonary fibrosis label.This new lncRNA-uc.77 will further enrich pulmonary fibrosis morbidity and the research of development mechanism, and the also early diagnosis for pulmonary fibrosis and prognosis detection provides new detection target spot.
Description
Technical field
The invention belongs to biomedical professional domain, be related to a kind of new human-mouse homologous non-coding RNA uc.77 and
Application in diagnosis pulmonary fibrosis disease Reagents Drugs are prepared.
Background technology
Pulmonary fibrosis（Pulmonary fibrosis, PF）It is a kind of progressive disease, is drilled by acute and chronic interstitial diseases
Change, be the common state and pathological manifestations in a variety of interstitial lung diseases whole latter stage.With caused by all kinds of reasons of China, lung is fine
Dimensionization illness rate constantly rises, and pulmonary fibrosis turns into one of China's respiratory system principal disease, its late period and acute exacerbation shape
Ratio of the state in Severe acute disease is constantly raised, and closely related with poor prognosis.
Current clinical diagnosis pulmonary fibrosis disease mainly includes following several means：First, imageological examination：Chest high-resolution
CT（HRCT）Check that position, degree and the property of display pulmonary lesion that can be careful are sent out as special certain form of pulmonary fibrosis
The diagnosis of property pulmonary fibrosis etc. has directive significance.2nd, pulmonary function test：Pulmonary function test is also that diagnosis pulmonary fibrosis is indispensable
Few inspection project, situations such as helping to understand pulmonary ventilation and diffusion, especially dynamic lung function, which develop, can more reflect disease
The development and prognosis of disease.3rd, surgical biopsies：Clear and definite medical diagnosis on disease can be made according to lung bioplsy pathology.But current examines
Section of cutting off the hands and strategy all be difficult accomplish early diagnosis, often progression of disease to a certain extent after, patient just because symptom by
Get brighter aobvious and made a definite diagnosis and treated.Expiratory dyspnea is the most common symptom of pulmonary fibrosis.During slight pulmonary fibrosis, expiratory dyspnea is normal
Occur in aggravating activities, therefore usually ignored or mistaken diagnosis is other diseases.When pulmonary fibrosis is in progress, also sent out in tranquillization
Raw expiratory dyspnea, serious pulmonary fibrosis patients may occur in which that progressive is had difficulty in breathing.The serious consequence of lung fibrosis, causes
Normal lung tissue's structural change, function is lost.When a large amount of fibrosed tissues without gas exchanges function replace alveolar, cause oxygen
Blood can not be entered.Patient respiratory is not smooth, and anoxic, acid poisoning, disability, severe patient lethal can finally be died.Because lung is fine
The serious consequence that the dimensionization later stage is brought, therefore, early it is carried out diagnosis it is significant, early diagnosis, so as to do
To early prevention and early treatment to more preferable therapeutic effect to be obtained.But in early stage, due to the limitation of prior art,
It is other diseases often to be failed to pinpoint a disease in diagnosis even mistaken diagnosis, is easily delayed optimal treatment period, as current clinical upper respiratory system disease
One of difficult point.
Epigenetics turns into the focus and emphasis of a variety of disease researches in recent years, and is increasingly becoming pulmonary fibrosis research
One important directions.Epigenetics refers in the case where genomic dna sequence is constant, by changing DNA methylation, group egg
White modification, chromosome structure participate in a kind of reversible, heritable hereditary information of gene expression regulation.It is known that human body base
Because 98% in group not encoding proteins matter genomic DNA mostly it is transcribed generation non-coding RNA (non-coding RNA,
ncRNA).And these ncRNA are mediation and the mechanism that take part in epigenetics.According to the difference of ripe transcript size,
NcRNA can be divided into small molecule ncRNA (such as siRNA, miRNA, piRNA), moderate-length ncRNA (70-200nt) and length
NcRNA (long ncRNA, lncRNA, >=200nt).At present, that ncRNA area researches are more is small molecule ncRNA, and right
LncRNA research is also in the starting stage.Long-chain non-coding RNA（Long non-coding RNA, lncRNA）It is that a class is long
Degree is more than 200nt, the RNA itself not guarded relatively between encoding proteins and species, it is known that it may participate in X chromosome silence, gene
A variety of important biomolecule processes such as the group marking, chromatin modification, transcriptional activation, transcription interference.
The content of the invention
The present invention is by building mouse pulmonary fibrosis model, using lncRNAmicroassay technologies（Mouse lncRNA
Microarray V2.0, Arraystar.Inc, probe covers 31,423 lncRNA, 25,376 encoding genes）, open
The screening of difference lncRNA between mouse normal lung tissue, fibroid lung tissue is opened up, by sequence analysis, fibroid lung is obtained
The lncRNA that more than 5 times of the relative normal lung tissue's height expression of tissue have 513, the lncRNA of more than 5 times of low expression have 204.
What chip was detected has human-mouse homologous, difference expression gene, while being predicted to be difference lncRNA again may regulate and control
The encoding gene of target, carries out Go and Pathway bioinformatic analysis, finds a lncRNA-uc.77, its nucleotide sequence
As shown in SEQ ID No.1.Show this lncRNA high expression in pulmonary fibrosis tissue through quantitative PCR detection result, and just
Low expression in often organizing.LncRNA-uc.77 plasmid over-express vector, and transfected with human alveolar epithelial cells A549 are built, as a result
Show its expression for being obviously promoted its target gene ZEB2, therefore the corresponding alveolar epithelial cells that have impact on is converted into muscle fibre mother
The epithelium mesenchyma conversion of cell（Epithelial-Mesenchymal Transition, EMT）Process, shows as：Cell
Form generation changes, fibroblast label in induction people's alveolar epithelial cells：Vimentin、α-SMA、N-cadherin
Expression increase；Epithelial cell marker thing：E-cadherin, EpCAM, KRT-5 expression decline；Extracellular matrix label：MMP-
2nd, MMP-9 expression increase.Human bronchial epithelial cell is transfected using lncRNA-uc.77 plasmid over-express vector（HBEc）Institute
The result of acquisition is consistent with the result of A549 cell lines.
An object of the present invention is to provide a kind of related long-chain non-coding RNA of human-mouse homologous pulmonary fibrosis（Long
Non-coding RNA, lncRNA）, lncRNA-uc.77 is named as, its nucleotide sequence is as shown in SEQ ID No.1.This hair
Bright provided lncRNA-uc.77, is confirmed by q-RTPCR methods, significantly high to be expressed in fibrosed tissue；Pass through work(
Checking can be learned, its relation between target gene is obtained and regulation and control pulmonary fibrosis evolution is participated in by target gene.
This new lncRNA-uc.77 will further enrich the research of pulmonary fibrosis pathogenesis, also be examined for the early stage of pulmonary fibrosis
Disconnected and prognosis detection provides new detection target spot.
People source lncRNA-uc.77 provided by the present invention DNA sequences encoding be positioned at HOXA3 genes the 2nd includes
In sub, transcriptional orientation is opposite with HOXA3.HOXA3 genes are located at human chromosomal 7p15-p14, belong to I type hox geneses
（Hox genes）A part for A cluster genes, coding DNA combination transcription factor is the important factor for participating in tissue fibrosis process.
The directed differentiation and propagation of cell can be determined by its special " space-time " expression pattern；HOXA3 can induce epithelial cell
Migration, promotes blood vessel lncRNA-uc.77 sequences highly conserved between multiple species, there is people's mouse homologous sequence, in people's alveolar
LncRNA-uc.77 is overexpressed in epithelial cell strain A549 or human bronchial epithelial cell HBEc can not only promote its target base
Because of ZEB2 expression, additionally it is possible to which the expression of the significant albumen of inducing fibrosis changes, lncRNA-uc.77 is adjusted by participating in
ZEB2, the important function played in pulmonary fibrosis is formed provides recruit's mark and medicine for the treatment of early stage pulmonary fibrosis
Target spot.
The lncRNA-uc.77 sequences that the present invention is provided, can both be obtained by biological method, can pass through chemistry conjunction again
Obtained into method, if being prepared by chemical synthesis process, as long as its nucleotide sequence is supplied into relevant speciality company, entrust it
Synthesis.To strengthen the pharmaceutical characteristics such as its stability, bioavilability, tissue-targeting, it can be carried out thio-modification or
Methoxyl group modification etc..
The related lncRNA-uc.77 of people's pulmonary fibrosis that the present invention is provided, including with its more than 90% homologous nucleotides
Sequence, or the nucleotide sequence obtained through thio-modification or/and methoxyl group modification, pulmonary fibrosis disease is diagnosed available for preparing
Reagent.
The second object of the present invention be to provide the lncRNA-uc.77 be used for prepare diagnose or auxiliary diagnosis pulmonary fibrosis
Medicine or kit purposes.
It is preferred that, the lncRNA-uc.77 is used for the auxiliary diagnosis of pulmonary fibrosis.
The three of the object of the invention are the provision of a kind of chip agent box for being used to diagnose pulmonary fibrosis, including：It is negative
It is loaded with the detection chip of the lncRNA-uc.77 sequences.
The chip agent box also includes being used to extract the reagent needed for RNA, PCR, hybridization, colour developing etc., including but does not limit
In：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion, antibody etc., and labeled RNA sample mark
Thing and the substrate corresponding with the label.
Operation instructions and/or chip image analysis software are may also include in described chip agent box.
It is used to diagnose the kit of pulmonary fibrosis present invention also offers a kind of, including：
1）Specific primer pair for expanding lncRNA-uc.77；
2）Standard DNA template or the detection chip for being loaded with long-chain non-coding RNA described in claim 1；
3）PCR reaction systems.
Preferably, the specific primer is to including sense primer and anti-sense primer, upstream primer sequence is SEQ ID
No.2, downstream primer sequence is SEQ ID No.3.
It is further preferred that the PCR reaction systems include dNTP, PCR buffer solution and archaeal dna polymerase.
It is further preferred that the PCR reaction systems are fluorescence quantitative PCR reaction solution, fluorescent dye is included.
Further, the application method of the kit：Sense primer (10 μm of ol/L) 1.0 μ L, anti-sense primer (10 μ
Mol/L 0.3 μ L, DNA polymerase (5U/ μ L) of) 1.0 μ L, lung tissue/blood sample cDNA 0.2,19 μ L of μ L, ddH2O；Reaction
Parameter is：94 DEG C of 3min (pre-degeneration)；94 DEG C of 30sec (denaturation), 64 DEG C of 30sec (renaturation), 72 DEG C of 60sec (
Extension), the denaturation-circulation of renaturation-extension 30 times.
The four of the object of the invention are to provide a kind of method for detecting the lncRNA-uc.77, comprised the following steps：
1）Extract sample total serum IgE；
2）Prepare sample cDNA；
3）Expand lncRNA-uc.77.
The five of the object of the invention are to provide a kind of method for detecting pulmonary fibrosis, comprised the following steps：
1）Extract sample total serum IgE；
2）Prepare sample cDNA；
3）Quantitative amplification lncRNA-uc.77, and judged according to quantitative result.
It is whole blood or lung tissue to detect specimen in use, and because lung tissue is very difficult to obtain, therefore the method for the invention is also
It can be diagnosed by whole blood, and accuracy rate of diagnosis is suitable with using lung tissue, considerably increases the convenience of diagnosis, examines operation
Property and reliability.
The six of the object of the invention are to provide the lncRNA-uc.77 as the purposes of pulmonary fibrosis medicine novel targets.
Brief description of the drawings
Fig. 1 is quantitative PCR detection lncRNA-uc.77 of the present invention in pulmonary fibrosis tissue and normal control tissue
In expression of results figure.
Fig. 2 is people's alveolar epithelial cells strain A549 to transfecting lncRNA-uc.77 over-express vectors, is transferred to unloaded pEX-3
Cell line and do not transfect control cell strain carry out quantitative PCR detection lncRNA-uc.77 expression of results figures.
Fig. 3 is people's alveolar epithelial cells strain A549 to transfecting lncRNA-uc.77 over-express vectors, is transferred to unloaded pEX-3
Cell line and do not transfect control cell strain carry out quantitative PCR detection purpose controlling gene ZEB2 expression of results figures.
Fig. 4 is to transfecting people's alveolar epithelial cells strain A549 of lncRNA-uc.77 over-express vectors and being transferred to unloaded pEX-
3 cell line carries out quantitative PCR detection fibroblast label：Vimentin、α-SMA；Epithelial cell marker thing：E-
cadherin、EpCAM、KRT-5；Extracellular matrix label：The result figure of MMP-2 and MMP-9 expression.
Fig. 5 is to transfecting people's alveolar epithelial cells strain A549 of lncRNA-uc.77 over-express vectors and being transferred to unloaded pEX-
3 cell line carries out Western Blot detections prediction target gene ZEB2 and fibroblast label：Vimentin、α-
SMA、N-cadherin；Epithelial cell marker thing：The result figure of E-cadherin, connexin43 expression.
Fig. 6 is to transfecting people's alveolar epithelial cells strain A549 of lncRNA-uc.77 over-express vectors and being transferred to unloaded pEX-
3 cell line carries out Immunofluorescence test fibroblast label：Vimentin, epithelial cell marker thing：E-
The result figure of cadherin expression.
Fig. 7 is to transfection lncRNA-uc.77 over-express vectors；Unloaded pEX-3 control group is transferred to, is transferred to siZEB2's
Target gene suppression group；And lncRNA-uc.77 over-express vectors and siZEB2 people's alveolar epithelial cells strain are transferred to simultaneously
A549；Four groups of the above carries out Immunofluorescence test fibroblast label simultaneously：Vimentin, epithelial cell marker thing：
The result figure of E-cadherin expression.
Fig. 8 is to transfecting people's alveolar epithelial cells strain A549 of lncRNA-uc.77 over-express vectors and being transferred to unloaded pEX-
The cytomorphology image of 3 cell line after 72 hours.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the present invention.Those skilled in the art is from the present invention
Disclosure directly or indirectly derived all deformations, are considered as protection scope of the present invention.It is all in subordinate's embodiment
Unreceipted specific experiment condition, be the work such as Sambrook according to normal condition well known to those skilled in the art
Molecular cloning：Laboratory manual (New York：Cold Spring Harbor Laboratory Press, 1989),
Animal cell culture written by Freshney：Described in basic fundamental guide the 5th edition (John Wiley＆Sons, Inc, 2005)
Condition and experimental procedure, or according to the condition and experimental procedure proposed by manufacturer.Unless otherwise noted, following embodiments
In experiment material used, be conventional material and chemical reagent.
Embodiment 1：The clone of lncRNA-uc.77 sequences and analysis
1st, reagent
Trizol reagents are purchased from Invitrogen companies of the U.S.；MMLV reverse transcription enzyme dnas polymerase, DNA ligases, pfu
Enzyme, EcoRI enzymes, BamHI enzymes are purchased from Fermentas companies of Lithuania；Random primer is purchased from Promega companies of the U.S.；
RNeasy extraction agents box, DNA gel QIAquick Gel Extraction Kit, middle amount extraction agent box, QuantiTect SYBR Green PCR
Kit is purchased from German Qiagen companies；Mouse lncRNA Microarray V2.0 chips are purchased from upper Haikang into biology
Company；PCR primer is synthesized by Invitrogen companies.
2nd, reagent, bacterial strain, cell line and carrier
PCR-blunt-TOPO carriers are purchased from Invitrogen companies of the U.S.；BALB/c mouse, SPF (Specific
Pathogen-free, no-special pathogen) level, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
3rd, experimental method
The structure in the total cDNA libraries of 3.1 pulmonary fibrosis tissues
3.1.1 the extraction of mouse fibroid lung tissue and normal lung tissue's total serum IgE
Using Trizol reagents and according to RNeasy extraction agent box experimental procedures.The lung tissue of harvest is added into 1ml
Trizol reagents are cracked, and are inhaled and beaten several times with rifle during cracking；Per 50-100mg tissue samples, 1ml TRIZOL reagents are added,
It is homogenized with electric homogenizer；Added sample volume is no more than homogenized Trizol reagent volumes used in this sample
10%；The addition 0.2ml chloroforms in above-mentioned Trizol, vortex oscillation 10 seconds, at standing 5 minutes, 4 DEG C at room temperature,
12000rpm is centrifuged 15 minutes, draw upper strata aqueous phase move into it is new without in RNA enzyme centrifuge tubes；Add and above-mentioned upper strata aqueous phase
Isometric isopropanol, after mixing, 20 minutes is stood under the conditions of 4 DEG C, then 12000rpm is centrifuged 10 minutes at 4 DEG C, is abandoned
It is clear to precipitate；The ethanol 1ml that mass fraction is 75% is added in above-mentioned precipitation, is overturned for several times, 12000rpm centrifugations 5 at 4 DEG C
Minute, abandon supernatant；12000rpm is centrifuged 2 minutes at 4 DEG C again；Supernatant is abandoned, is dried 1~2 minute at room temperature, plus 20 μ l
Without the dissolving of RNA enzymes water, RNA extract solutions are obtained, -80 DEG C save backup；
3.1.2 reverse transcription (RT)
Following components is added in 500 μ L EP pipes：
Lung tissue extracts the μ L of RNA 10；
Random primer (10 μm of ol/L) 1 μ L；
H2O 18μL。
70 DEG C of incubation 5min, put in ice bath rapidly after mixing, stand sample-adding after 3min as follows：
The μ L of RNase inhibitor 1；
dNTP(10mmol/L)1μL；
The μ L of 5 × reverse transcriptase buffer 8；
The μ L of MMLV reverse transcriptases 1；
42 DEG C of incubations 1h, 70 DEG C of 10min inactivate reverse transcriptase, -20 DEG C of preservation samples after mixing.
3.1.3 the structure in the total cDNA libraries of pulmonary fibrosis tissue
By single-stranded cDNA obtained by the present embodiment 3.1.2 through DNA polymerases and pfu ferment treatments obtain flat terminal double link cDNA (
Operating procedure is shown in DNA polymerases, pfu enzymes operation manual).The flat terminal double link cDNA of gained is connected to PCR-blunt-TOPO
On carrier, the total cDNA libraries of pulmonary fibrosis tissue are built.
3.2 lncRNA microarray technologies and analysis
Using extracted in 3.1.1 two histioid total RNA, marked with the quick Amp labelling kits of Agilent companies
Probe hybridization on RNA, with chip；The data of chip are collected, with Agilent GeneSpring GX v12.0 software analysis numbers
According to filtering out that absolute signal value is big, group difference multiple is big, in group heterogeneous small lncRNA is expressed between sample.Using GO points
The lncRNA, i.e. lncRNA-uc.77, its core with pulmonary fibrosis process close relation are picked out in analysis and Pathway analyses
Nucleotide sequence is as shown in SEQ ID No.1.
Embodiment 2q-RTPCR identifies differential expressions of the lncRNA-uc.77 in fibroid lung tissue and normal structure
1st, using mouse fibroid lung tissue and normal lung tissue as material, fibroid lung tissue and just is extracted respectively with Trizol reagents
The total serum IgE (3.1.1 in method be the same as Example 1) of normal lung tissue.Fibroid lung total tissue RNA and normal lung tissue is total
RNA respectively takes 1 μ g, and reverse transcription reaction (3.1.2 in method be the same as Example 1) is carried out with random primer and obtains fibroid lung tissue
And total cDNA of normal lung tissue, the fibroid lung tissue of synthesis and total cDNA of normal lung tissue are quantitative PCR reaction
Template.
2nd, lncRNA-uc.77 specific primers sequence is as follows：
F:5’ –CTGTCACACTGCTCCCAAGA- 3’；R: 5’ –TCAGCCAAAGATGCTTGAAA- 3’.
3rd, PCR reaction system is：
The μ L of PCR buffer solutions (10 ×) 2.5；
dNTP(2.5mmol/L)1.0μL；
Sense primer (10 μm of ol/L) 1.0 μ L；
Anti-sense primer (10 μm of ol/L) 1.0 μ L；
The μ L of lung tissue cDNA 0.3；
DNA polymerases (5U/ μ L) 0.2 μ L；
ddH2O 19μL。
Response parameter is：94 DEG C of 3min (pre-degeneration)；94 DEG C of 30sec (denaturation), 64 DEG C of 30sec (renaturation),
72 DEG C of 60sec (extension), the denaturation-circulation of renaturation-extension 30 times.
SEQ ID NO in the sequence of RT-PCR amplification acquisitions such as sequence table：Shown in 1.
4th, experimental result
Fibroid lung tissue and normal lung tissue lncRNA-uc.77 expression are shown in Fig. 1, it can be seen that lncRNA-uc.77
The high expression in the fibroid lung tissue of several models and in normal lung tissue low expression.Test result indicates that, lncRNA-
There is notable difference in uc.77, shown by test of many times statistics in fibroid lung tissue and normal lung tissue's expression, positive
Recall rate is up to 100%, thus lncRNA-uc.77 has important biology work(in the generation evolution of pulmonary fibrosis
Can, can as fibroid lung organizational diagnosis mark, the examination as fibroid lung tissue.
Embodiment 3：LncRNA-uc.77 overexpression promotes the mesenchymal cell that turns of people's alveolar epithelial cells to grow spy
Property
1st, reagent
It is identical with embodiment 1 using conventional reagent.
2nd, bacterial strain, cell line and carrier
The transfection reagents of lipofectamine 2000 are purchased from Invitrogen companies of the U.S.；DMEM culture mediums, hyclone are equal
Purchased from Gibico companies of the U.S.；NBT (nitro blue tetrazolium, NBT) is purchased from Sigma companies of the U.S..
People's alveolar epithelial cells strain A549 is purchased from Unite States Standard type culture collection institute (ATCC)；E.coli BL2 bacterial strains are purchased from
Shanghai JiMa pharmacy Technology Co., Ltd；Carrier for expression of eukaryon pEX-3 is purchased from Shanghai JiMa pharmacy Technology Co., Ltd.
3rd, experimental method
3.1 obtain lncRNA-uc.77 sequence fragments with PCR method
3.1.1 oligo is designed, and target gene upstream and downstream primer adds EcoRI and BamHI and protection base respectively, for carrier
Subclone, oligo sequences are as follows
3.1.2 primer is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd
3.1.3 oligo is dissolved into 50 μM, oligo is taken to same volume respectively to a 1.5ml centrifuge tubes, be well mixed, be made into
oligo mix
3.1.4 the reaction of first round PCR is carried out with the oligo mix prepared,
PCR system is as follows：
oligo mix 6µl；
The μ l of 10 × Pfu Buffer (+Mg2+) 5；
dNTP 1µl；
B1294-1 1µl；
B1294-26 1µl；
ddH2O 36µl；
Pfu DNA polymerase 0.3µl。
Cycling condition：
95 DEG C of 3min (pre-degeneration)；94 DEG C of 30sec (denaturation), 55 DEG C of 30sec (renaturation), 72 DEG C of 30sec (prolong
Stretch), the denaturation-circulation of renaturation-extension 30 times.
3.1.5 the second wheel PCR reactions are carried out with B1294-1 and B1294-26, template is the product that first round PCR reacts.
PCR system is as follows：
The μ l of first round PCR primer 1；
The μ l of 10 × Pfu Buffer (+Mg2+) 5；
dNTP 1µl；
B1294-1 1µl；
B1294-26 1µl；
ddH2O 41µl；
Pfu DNA polymerase 0.3µl。
Reaction condition is identical with the first round.First round PCR can obtain being mixed with the non-single band PCR productions of target gene band
Thing mixture, then with the PCR primer of the first round be template, single target gene band can then be obtained by the second wheel PCR.
3.1.6 after the completion of PCR reactions, Agarose electrophoresis and gel extraction lncRNA-uc.77 genetic fragments are utilized.
3.2 target gene lncRNA-uc.77 are cloned into carrier pEX-3
3.2.1 digestion is carried out to uc.77 genetic fragments with EcoRI and BamHI, 37 DEG C of digestions 2 hours, digestion system is as follows：
10×Buffer 5µl；
DNA 15µl；
EcoRI 1µl；
BamHI 1µl；
ddH2O 28µl。
3.2.2 digestion is carried out to carrier pEX-3 with EcoRI and BamHI, 37 DEG C of digestions 2 hours, digestion system is as follows：
10×Buffer 5µl；
pEX-3 5µl；
EcoRI 1µl；
BamHI 1µl；
ddH2O 38µl。
3.2.3 electrophoresis, uc.77 genetic fragments and carrier pEX-3 are reclaimed with DNA gel reclaims kits.
3.2.4 the uc.77 genetic fragments and the carrier of linearisation obtained with T4 DNA ligase connection double digestions, 22 DEG C
Connection 2 hours, linked system is as follows：
T4 DNA ligase buffer 2µl；
pEX-3 2µl；
uc.77 5µl；
T4 DNA ligase 1µl；
ddH2O 10µl。
3.2.5 the preparation of competent cell：（Calcium Chloride Method）
3.2.5.1 one single bacterium colony of picking from the fresh plate in 37 DEG C of cultures 16 hours, goes to one and contains 100 ml LB
In 1 L flasks of culture medium.3 hours (rotary shaker, 300 revs/min) of culture is acutely shaken in 37 DEG C.
3.2.5.2 bacterium is aseptically transferred to one sterile, disposable, ice-cold 50
In ml PA tubes, placed 10 minutes on ice, culture is cooled to 0 DEG C.
3.2.5.3 in 4 DEG C, centrifuged 10 minutes with 4000 revs/min, reclaim cell.
3.2.5.4 nutrient solution is poured out, pipe is inverted 1 minute, the trace nutrient solution of final residual is flow to end.
3.2.5.5 every part of precipitation is resuspended with the ice-cold 0.1 mol/L CaCl2 of 10 ml, be positioned on ice bath.
3.2.5.6 in 4 DEG C, centrifuged 10 minutes with 4000 revs/min, reclaim cell.
3.2.5.7 nutrient solution is poured out, pipe is inverted 1 minute, the trace nutrient solution of final residual is flow to end.
3.2.5.8 per 50ml initial incubations thing with the ice-cold 0.1 mol/L CaCl2 of 2 ml (containing 20% glycerine)
Every part of cell precipitation is resuspended.
3.2.5.9 cell is distributed into aliquot (100 μ l/ branch), is put in -70 DEG C and freezes.
（Competent cell prepares reference：The Molecular Cloning:A Laboratory guide second edition page 55）
3.2.6 by connection product transformed competence colibacillus cell
3.2.6.1 competent cell is taken out from -70 DEG C, the centrifuge tube that will be equipped with competent cell is placed 4 minutes on ice, is treated
After competent cell thaws, 10 μ l connection products are added, content is softly mixed, placed 30 minutes in ice.
3.2.6.2 centrifuge tube is put on the rack for test tube that pre-heating is put well into 42 DEG C of water-bath, placed 90 seconds,
Centrifuge tube should not be shaken.
3.2.6.3 quickly centrifuge tube is transferred in ice bath, cell is cooled down 3 minutes.
3.2.6.4 800 μ l LB culture mediums (being free of antibiotic) are added to every centrifuge tube, then shifts centrifuge tube
To 37 DEG C of shaking tables, 250 revs/min, cultivation makes bacteria resuscitation in 45 minutes.
3.2.6.5 the cell after 200 μ l cultivations is taken to be spread evenly across containing on 50 μ g/ml Kanamycin LB flat boards.
3.2.6.6 after liquid is absorbed on grade flat board, flat board is inverted in 37 DEG C of incubators, cultivated 16 hours.
3.2.7 the picked clones bacterium colony from flat board, it is small take out plasmid and do identification choose positive colony.
3.2.7.1 from 4 independent, full bacterium colonies of picking on cultured flat board, it is placed in and (contains 50 μ g/ml containing 5ml
Kanamycin) in the test tube of LB culture mediums,
3.2.7.2 above-mentioned test tube is placed in bacterium shaking table and cultivated, 37 DEG C, 250 revs/min, cultivated 16 hours.
3.2.7.3 by cultured bacterium solution, plasmid is extracted with the small extraction reagent kit of plasmid (Tiangeng is biochemical, DP104-02),
（Plasmid extraction step refers to the small extraction reagent kit specification of plasmid）.
3.2.7.4 the plasmid extracted is subjected to double digestion identification, it is as follows per tube reaction system：
10×Buffer 1µl；
The μ l of plasmid 1；
EcoRI 0.5µl；
BamHI 0.5µl；
ddH2O 7µl。
3.2.7.5 37 DEG C of digestion, 1 hour rear electrophoresis, have the band pair that digestion is obtained in purpose band size corresponding region
The clone answered as positive colony.
3.3 recombinant plasmid sequence verifications are simultaneously largely extracted
3.3.1 take the corresponding bacterium solution of 200 μ l positive colonies to send sequencing, and remaining bacterium solution is preserved with glycerine.
3.3.2 sequencing result is compared with objective gene sequence, after normal solution is errorless, connect with the glycerine bacterium solution of preservation
Bacterium LB culture mediums, carry out a large amount of plasmid extractions, obtain the recombinant plasmid of sufficient amount.
3.4 build monoclonal human alveolar epithelial cells strain A549- lncRNA-uc.77
People's alveolar epithelial cells strain A549 is incubated to 5% CO2 37 DEG C of trainings with the DMEM culture mediums containing 5% hyclone
Support in case.The day before transfection accesses 1 × 10 in 6 orifice plates6Individual cells/well, is transfected with lipofectamine2000,
The μ g of transfection recombinant plasmid pEX3- lncRNA-uc.77 4 per hole.Cell transfecting collects cell after 48 hours.Simultaneously with zero load
Body pEX-3 transfection A549 cells (see the specifications of lipofectamine 2000), obtain control cell strain A549-pEX-3.
LncRNA-uc.77 expression quantity detection in 3.5 monoclonal human alveolar epithelial cells strains
The expression quantity of lncRNA-uc.77 in each monoclonal human alveolar epithelial cells strain is detected with conventional semi-quantitative RT-PCR, and
Using be transferred to empty expression vector pEX-3 alveolar epithelial cells strain A549-pEX-3 be used as control.
50 μ L semiquantitive PCR reaction systems, its component is：
The μ L of PCR buffer solutions (10 ×) 5
MgCl2(25mM)5μL
dNTP(2.5mmol/L)1.0μL
Sense primer (10 μm of ol/L) 0.5 μ L
Anti-sense primer (10 μm of ol/L) 0.5 μ L
Template (A549- lncRNA-uc.77 or A549-pEX-3 cDNA) 2 μ L
DNA polymerases (5U/ μ L) 0.4 μ L
ddH2O 35.6μL
All PCR response parameter is：94 DEG C of 3min (pre-degeneration)；94 DEG C of 30ec (denaturation), 64 DEG C of 30s ec are (multiple
Property), 72 DEG C of 60sec (extension), the denaturation-renaturation-extension × n cycles.Cycle-index is according to rising that PCR reacts
Hop determines that generally, specific 1-2 many compared with ski-jump of PCR reactions every time are circulated.
Fig. 2 is people's alveolar epithelial cells strain A549 to transfecting lncRNA-uc.77 over-express vectors, is transferred to unloaded pEX-3
Cell line and do not transfect control cell strain carry out quantitative PCR detection lncRNA-uc.77 expression of results figures.Fig. 2 shows, with
Transfection empty expression vector is compared, and transfects the uc.77 expression quantity of lncRNA-uc.77 over-express vectors up to more than 300 times.
People's alveolar epithelial cells strain A549 cytomorphology detections of 3.6 lncRNA-uc.77 overexpressions
Monoclonal human alveolar epithelial cells strain A549- lncRNA-uc.77 that the present embodiment 3.4 is built and it is transferred to null representation
Carrier pEX-3 cell line A549 is respectively with 1 × 106The quantity of individual cells/well is accessed in 6 orifice plates with 1mL containing 5% tire
The DMEM cultures of cow's serum, are incubated at 5% CO237 DEG C of incubators in cultivate.After 3 days, cell is checked under phase contrast microscope
Morphological change, is shown in Fig. 8.
Fig. 8 shows, compared with transfecting empty expression vector pEX-3 A549 cells, transfects recombinant plasmid pEX-3-lncRNA-
Uc.77 A549 cell overexpressions lncRNA-uc.77 can remarkably promote people's alveolar epithelial cells and turn to divide to mesenchymal cell
The morphological change of change, is converted between elongated, spindle etc. is characterized from polygonal, cobblestone sample epithelial cell morphological feature
Mesenchymal cells morphological feature, points out to enhance A549 cells to the growth characteristics of mesenchymal cell transdifferentiation.
3.7 lncRNA-uc.77 overexpressions promote people's alveolar epithelial cells to turn mesenchymal cell growth characteristics
3.6 methods describeds in cell preparation process be the same as Example 2, overexpression lncRNA- is detected using q-RTPCR methods
After uc.77, epithelial cell is converted into the epithelium mesenchyma conversion of myofibroblast（Epithelial-Mesenchymal
Transition, EMT）The expression of label：Including fibroblast label：Vimentin、α-SMA；Epithelial cell mark
Remember thing：E-cadherin、EpCAM、KRT-5；Extracellular matrix label：MMP-2、MMP-9.Such as Fig. 4.
Fig. 4 shows：After overexpression lncRNA-uc.77, hence it is evident that promote alveolar epithelial cells and be converted into muscle fibre mother carefully
The epithelium mesenchyma conversion process of born of the same parents, shows as：Induce fibroblast label in people's alveolar epithelial cells：
Vimentin, α-SMA expression increases；Epithelial cell marker thing：E-cadherin, EpCAM, KRT-5 expression decline；Extracellular base
Matter label：MMP-2, MMP-9 expression increase.This shows that there are regulation and control to make for generation development of the lncRNA-uc.77 to pulmonary fibrosis
With.
3.6 methods describeds in cell preparation process be the same as Example 2, overexpression is detected using Western Blot methods
After lncRNA-uc.77, epithelial cell is converted into the epithelium mesenchyma conversion of myofibroblast（Epithelial-
Mesenchymal Transition, EMT）The influence of the expressing quantity of label：Fibroblast label：
Vimentin、α-SMA、N-cadherin；Epithelial cell marker thing：E-cadherin；Extracellular matrix label：MMP-2 with
And the result figure of MMP-9 expression.Such as Fig. 5.
Fig. 5 shows：After overexpression lncRNA-uc.77, hence it is evident that promote alveolar epithelial cells and be converted into muscle fibre mother carefully
The epithelium mesenchyma conversion process of born of the same parents, shows as：Induce fibroblast label in people's alveolar epithelial cells：
Vimentin, α-SMA, N-cadherin expression increase；Epithelial cell marker thing：Under E-cadherin, connexin43 expression
Drop；This further demonstrates that lncRNA-uc.77 has facilitation to the generation of pulmonary fibrosis development from albumen aspect, with controlling
Treat the purposes of pulmonary fibrosis relevant disease.
3.6 methods describeds in cell preparation process be the same as Example 2, overexpression is detected using immunofluorescence method
After lncRNA-uc.77, epithelial cell is converted into the epithelium mesenchyma conversion of myofibroblast（Epithelial-
Mesenchymal Transition, EMT）The influence of label：Fibroblast label：Vimentin, epithelial cell
Label：The result figure of E-cadherin expression.Such as Fig. 6.
Fig. 6 shows：After overexpression lncRNA-uc.77, obvious change occurs for cell marker, shows as：Induce people's lung
Steep fibroblast label in epithelial cell：Vimentin expression increases；Epithelial cell marker thing：E-cadherin is expressed
Decline.
3.8 lncRNA-uc.77 are by adjusting the effect of ZEB2 target gene so as to promote turning for people's alveolar epithelial cells
Mesenchymal cell growth characteristics
3.6 methods describeds in cell preparation process be the same as Example 2, lncRNA-uc.77 over-express vectors building process is with implementation
3.1 methods describeds of 2 kinds of example,
SiZEB2 construction method：ZEB2 siRNA are purchased from Santa Cruz biotechnologies company of the U.S., article No.：sc-38641.
Use the transfection A549 cells of Lipofectamine 2000 (see the specifications of lipofectamine 2000).
People's alveolar epithelial cells strain A549 of lncRNA-uc.77 over-express vectors is transfected, unloaded pEX-3 cell is transferred to
Strain and the control cell strain not transfected, carry out quantitative PCR detection purpose controlling gene ZEB2 expression, its result is shown in Fig. 3 respectively.
Fig. 3 shows：LncRNA-uc.77 have adjusted its target gene ZEB2 expression quantity.When lncRNA-uc.77 crosses scale
After, purpose controlling gene ZEB2 expression quantity also substantially increases.
Four groups of cells are detected using immunofluorescence method：Transfect lncRNA-uc.77 over-express vectors；It is transferred to unloaded pEX-3
Control group, be transferred to siZEB2 target gene suppression group；And be transferred to simultaneously lncRNA-uc.77 over-express vectors and
After siZEB2, epithelial cell is converted into the epithelium mesenchyma conversion of myofibroblast（Epithelial-Mesenchymal
Transition, EMT）The influence of label：Fibroblast label：Vimentin, epithelial cell marker thing：E-
The result figure of cadherin expression.Such as Fig. 7.
Fig. 7 shows：LncRNA-uc.77 is by promoting target gene ZEB2 overexpression, so as to promote people's alveolar epithelium
Cell turns mesenchymal cell differentiation characteristic.After ZEB2 genes are suppressed, lncRNA-uc.77 rush fibrotic effects are entangled
Just.
Embodiment 4：LncRNA-uc.77 differential expression carries out the embodiment of examination to pulmonary fibrosis
4.1 reagents, sample and carrier
Using people's whole blood sample as material, the total serum IgE of whole blood sample is extracted (in method be the same as Example 1 with Trizol reagents
3.1.1).Whole blood sample total serum IgE is taken into 1 μ g, reverse transcription reaction is carried out with random primer (in method be the same as Example 1
3.1.2 the total cDNA of sample) is obtained, total cDNA of synthesis is the template of quantitative PCR reaction.
4.2 lncRNA-uc.77 specific primer sequence is as follows：
F:5’ –CTGTCACACTGCTCCCAAGA- 3’；R: 5’ –TCAGCCAAAGATGCTTGAAA- 3’.
4.3 PCR reaction system is：
The μ L of PCR buffer solutions (10 ×) 2.5；
dNTP(2.5mmol/L)1.0μL；
Sense primer (10 μm of ol/L) 1.0 μ L；
Anti-sense primer (10 μm of ol/L) 1.0 μ L；
The μ L of lung tissue cDNA 0.3；
DNA polymerases (5U/ μ L) 0.2 μ L；
ddH2O 19μL。
Response parameter is：94 DEG C of 3min (pre-degeneration)；94 DEG C of 30sec (denaturation), 64 DEG C of 30sec (renaturation),
72 DEG C of 60sec (extension), the denaturation-circulation of renaturation-extension 30 times.
SEQ ID NO in the sequence of RT-PCR amplification acquisitions such as sequence table：Shown in 1.Judged according to quantitative result
, judging nicety rate is up to 100%.
Embodiment 5：LncRNA-uc.77 differential expression carries out the embodiment of examination to pulmonary fibrosis
On the other hand, present invention also offers lncRNA-uc.77 in a kind of detection people's whole blood by lncRNA-uc.77 chips
Method, including step：
5.1 provide the RNA samples for being isolated from people's whole blood sample, and label is set on described RNA；
5.1.1 the method that RNA is extracted in wherein being organized from people is method well known to those skilled in the art, including Trizol
Method, it is furthermore preferred that in step 4.1.1, after RNA samples are isolated from people's whole blood sample, it is appropriate to be carried out to RNA samples
Processing, be enriched with certain length RNA, the length one between 150-250.After above-mentioned processing, utilize
These small fragment RNAs carry out follow-up hybridization, can so improve the accuracy that chip captures lncRNA.
5.1..2 those skilled in the art can be conveniently separated out the RNA with certain fragment length, such as can be using gel electricity
Swimming method is separated.It is also method well known to those skilled in the art that RNA, which is marked, its can by hybridization when add with
The method of the label of RNA specific bindings realizes that the label is such as labelling groups.Described labelling groups include
But it is not limited to：It is digoxin molecule (DIG), biotin molecule (Bio), fluorescein and its derivative biomolecule (FITC etc.), other
Fluorescence molecule (such as Cy3, Cy5), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc..These marks and its mark
Note method has all been routine techniques well-known in the art.
5.2 contact 5.1 RNA with described chip, visit the oligonucleotides on described RNA and solid phase carrier
Hybridization reaction occurs for pin, so as to form " oligonucleotide probe-RNA " binary complex on solid phase carrier；
When 5.2.1 above-mentioned RNA and lncRNA-uc.77 chips being hybridized, can first by lncRNA-uc.77 chips and
Pre-hybridization buffer carries out prehybridization；
5.2.2 the solid-phase hybridization between RNA and lncRNA-uc.77 chips of the present invention according to this area classical way
Carry out, one personnel of this area empirically easily determine relevant buffer solution, probe and concentration of specimens, prehybridization temperature, hybridization
The optimum condition of temperature and time etc..Or can also reference《Molecular Cloning:A Laboratory guide》Described in.
The label for the binary complexs that 5.3 detections 5.2 are formed, position that can be according to marking signal on lncRNA chips
Put, the acquisition of information such as intensity treats measurement information., also can be directly with fluorescence detection device (such as if amplified production is marked with fluorophor
Laser confocal scanner Scanarray 3000 etc.) obtain and treat measurement information.So that it is determined that corresponding lncRNA- in people's tissue
Uc.77 content.Judged finally according to quantitative result, judging nicety rate is up to 100%.
Sequence table
<110>Jinsong ZHANG
<120>A kind of long-chain non-coding RNA ENSMUST00000139055 and its application
<160>3
<210>1
<211>
<212>RNA
<213>homo sapiens
<400> 1
ctgcatatct agttcattaa caaatatgaa tagctaacac cagctcattt aaatagtatg 60
tcttacatga gttctcgcca cactctgttt ctgtcacact gctcccaaga atcattggct 120
cttattatac atgtaaatgg ctatcataaa aataaaaatt tcatttaaga ttgttaatga 180
cttctgcagc agtcagactt cctttaatga tcatcctctg cccctaatta aacgtattta 240
tctttcaagc atctttggct gagttctccc tcatagttga aaatctaaaa ccttaag 297
<210>2
<211>20
<212>RNA
<213>It is artificial synthesized
<400> 2
ctgtcacact gctcccaaga 20
<210>3
<211>20
<212>RNA
<213>It is artificial synthesized
<400> 3
tcagccaaag atgcttgaaa 20
Claims (10)
1. a kind of related long-chain non-coding RNA of human-mouse homologous pulmonary fibrosis, it is characterised in that its nucleotide sequence such as SEQ
Shown in ID No.1, or the nucleotide sequence homologous with the sequence more than 90%, or the sequence is repaiied through thio-modification or/and methoxyl group
Adorn obtained new nucleotide sequence.
2. long-chain non-coding RNA according to claim 1 is used for the diagnosticum or kit for preparing diagnosis pulmonary fibrosis
Purposes.
3. the kit for diagnosing pulmonary fibrosis prepared by the long-chain non-coding RNA described in a kind of claim 1, its feature exists
In, including：
1）Specific primer pair for expanding long-chain non-coding RNA described in claim 1；
2）Standard DNA template or the detection chip for being loaded with long-chain non-coding RNA described in claim 1；
3）PCR reaction systems.
4. kit according to claim 3, it is characterised in that the specific primer is to including sense primer and downstream
Primer, upstream primer sequence is SEQ ID No.2, and downstream primer sequence is SEQ ID No.3.
5. kit according to claim 3, it is characterised in that the PCR reaction systems include dNTP, PCR buffer solution
And archaeal dna polymerase.
6. kit according to claim 3, it is characterised in that the PCR reaction systems are reacted for quantitative fluorescent PCR
Liquid, includes fluorescent dye.
7. the application method of the kit described in claim any one of 3-6, it is characterised in that 10 μm of μ of ol/L sense primers 1.0
L, 10 μm of 0.3 μ L, 5U/ μ L DNA polymerases of ol/L anti-sense primers 1.0 μ L, lung tissue/blood sample cDNA 0.2 μ L, ddH2O
19μL；Response parameter is：94 DEG C of 3min of pre-degeneration；94 DEG C of 30sec, 64 DEG C of 30sec of renaturation are denatured, extend 72 DEG C
60sec, the denaturation-circulation of renaturation-extension 30 times.
8. a kind of method of long-chain non-coding RNA described in test right requirement 1, it is characterised in that comprise the following steps：
1）Extract sample total serum IgE；
2）Prepare sample cDNA；
3）Expand the lncRNA-uc.77 described in claim 1.
9. the method that the long-chain non-coding RNA described in a kind of claim 1 detects pulmonary fibrosis, it is characterised in that including as follows
Step：
1）Extract sample total serum IgE；
2）Prepare sample cDNA；
3）LncRNA-uc.77 described in quantitative amplification claim 1, and judged according to quantitative result.
10. lncRNA-uc.77 described in claim 1 is used as the purposes of pulmonary fibrosis medicine novel targets.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112088163A (en) * | 2018-03-15 | 2020-12-15 | 瓦尔希伯伦私人肿瘤研究基金会 | Micropeptides and uses thereof |
| CN113186280A (en) * | 2021-03-31 | 2021-07-30 | 温州医科大学 | Yeast target uc-with diagnostic marker for inhibiting colorectal cancer growth and application thereof |
-
2016
- 2016-02-17 CN CN201610088611.5A patent/CN107090451A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| GILL BEJERANO等: "Ultraconserved Elements in the Human Genome", 《SCIENCE》 * |
| HAO SUN等: "Integrated long non-coding RNA analyses identify novel regulators of epithelial-mesenchymal transition in the mouse model of pulmonary fibrosis", 《JOURNAL OF CELLULAR AND MOLECULAR MEDICINE》 * |
| 刘次全等著: "《核酸结构多态性》", 31 December 2000, 高等教育出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112088163A (en) * | 2018-03-15 | 2020-12-15 | 瓦尔希伯伦私人肿瘤研究基金会 | Micropeptides and uses thereof |
| CN113186280A (en) * | 2021-03-31 | 2021-07-30 | 温州医科大学 | Yeast target uc-with diagnostic marker for inhibiting colorectal cancer growth and application thereof |
| CN113186280B (en) * | 2021-03-31 | 2022-06-21 | 温州医科大学 | Target uc.77-for inhibiting colorectal cancer growth, diagnostic marker and application thereof |
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