CN105177005B - A kind of long non-coding RNA and its application - Google Patents
A kind of long non-coding RNA and its application Download PDFInfo
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Abstract
The invention belongs to genetic engineering field, more particularly to long non-coding RNA uc003hsl.1 is preparing the application in treating non-small cell lung cancer drug；Had an impact by changing long non-coding RNA uc003hsl.1 apoptosis of the expression to non-small cell lung cancer cell, propagation, drug susceptibility etc., illustrating, which reduces long non-coding RNA uc003hsl.1 expression, can promote non-small cell lung cancer cell apoptosis, suppress its propagation, strengthen its sensitiveness to chemotherapeutics.
Description
Technical field
The invention belongs to genetic engineering field, more particularly to long non-coding RNA uc003hsl.1 is non-small thin in preparation treatment
The application of born of the same parents' lung-cancer medicament.
Background technology
Genomics research shows that human genome there are about 20000 protein coding genes, about total gene number ratio
2%, remaining most of gene is transcribed into non-coding RNA (non-coding RNA, ncRNA).NcRNA can divide according to its length
For transcription initiation RNA, Piwi protein-interacting RNA, Microrna, little nucleolar RNA and long non-coding RNA (long non-
Coding RNA, lncRNA) etc..LncRNA is a kind of transcript length more than 200 nucleotides and itself not encoding proteins
RNA molecule.LncRNA be initially believed to be rna plymerase ii transcription accessory substance, be subgenomic transcription " noise ", do not have
Biological function.However, it has recently been demonstrated that they can influence base by a variety of different mechanism, in a variety of aspects
The expression of cause.There is diversity in the mode of LncRNAs controlling genes expression, show the gene expression that lncRNAs is relied on
The diversity of regulatory mechanism.In different mechanisms, lncRNAs effects are different, can not only be used for main transcription regulatory factor, again may be used
As one of common regulatory factor, regulating and controlling effect is played jointly with other components.LncRNAs is big for the level of gene expression regulation
It can be divided on body：(1) apparent modification level modulation, by modifying enzyme interacting with various chromatin, chromatin is repaiied
Decorations, change its conformation, activation or the expression for suppressing related gene.Especially in Embryonic Stages, lncRNAs participates in causing and can losing
The maintenance of allele the later stage experssion silence, Apparent character of biography, for metazoan normal development and cell differentiation extremely
Close important.(2) transcriptional level control, lncRNAs are formed by adjusting the combination and assembling of transcription factor with regulating and controlling sequence DNA
Three chain cpds, rna regulation polymerase II, transcribe the methods of disturbing and realize.(3) post-transcriptional level regulate and control, by with complementation
MRNA forms dsRNA, influences the processes such as mRNA processing, montage, transhipment, translation and degraded, so as to the expression of regulatory gene.
LncRNAs turns into the new heat of tumor research because of the latent effect that it is manifested in carcinogenic and tumor suppression approach
Point.In recent years, some lncRNAs of many studies have shown thats and corresponding human tumor are closely related.By regulating and controlling a series of biologies
Function or interference normal function, including the mode such as Transcriptional Silencing, alternative splicing, lncRNAs rise in the occurrence and development of tumour
Important function.
Show that the morbidity and mortality of lung cancer are equal in countries in the world according to the data that the World Health Organization (WHO) announces
In the country of the trend substantially risen, especially industry prosperity.In developed country, lung cancer is one of most common malignant tumour,
Arrange first, the 2nd, 3 of row women common cancer of male's common cancer.20 end of the century lung cancer have accounted for pernicious swollen
The dead first place of knurl.Non-small cell lung cancer accounts for the 80% of all lung cancer, can be divided into gland cancer, squamous carcinoma, maxicell lung cancer etc..Many lungs
Cancer patient has shifted in diagnosis, often loses operative chance, and also not good enough for the chemotherapy regimen effect of lung cancer, 5 years
Survival rate is less than 5%, needs badly and finds new, effectively to treat lung cancer method.With going deep into for genetic engineering research, scientist
Keen interest is shown to developing tumour medicine with genetic engineering.Inventor has found that lnc-uc003hsl.1 is non-small thin in treatment
There is good application prospect in terms of born of the same parents' lung cancer.
Lnc-uc003hsl.1 is the lncRNA that a length is 2216nt, is to provide normal lung tissue and lung by inventor
Cancerous tissue sample, chip is carried out by Boao Biological Co., Ltd and prepares the RNA of significantly high expression in cancerous lung tissue filtered out.
Lnc-uc003hsl.1 has found and named first have novelty by inventor.
The content of the invention
Technical purpose
It is an object of the invention to provide lnc-uc003hsl.1 to prepare the application in treating non-small cell lung cancer drug.
A kind of long-chain non-coding RNA, its nucleotides sequence are classified as Seq NO.1.
A kind of long-chain non-coding RNA is preparing the application in treating non-small cell lung cancer drug.
Lnc-uc003hsl.1 interference sequences (5 ' to 3 ')：CCAGATGGGCACTTCTAAA, see Seq NO.2
GCTGCTAGTATTGTTCTTT, Seq NO.3.
Technical scheme
Design the specific interference sequence for lnc-uc003hsl.1, using disturb the tracts of GAPDH genes as
Control, and (above carrier and fragment are synthesized by invitrogen companies) is inserted into slow virus carrier.Will be packaged slow
Viral vector infection non-small cell lung cancer cell strain A549 cells, play a part of downward lnc-uc003hsl.1 and express, after 48h
Stably transfected cell line is screened with G418.
First, lnc-uc003hsl.1 promotes the basic checking test of Treatment of non-small-cell lung cancer with chemotherapy sensitiveness
(1)：Expressions of the lnc-uc003hsl.1 in normal lung tissue and cancerous lung tissue
1. chip prepares and analysis:Prepare normal lung tissue and cancerous lung tissue mark according to the requirement of Boao Biological Co., Ltd
This, transfers to Boao Biological Co., Ltd to carry out chip preparation,Mankind long-chain non-coding RNA chip V1.0 versions complete core
Piece is analyzed.
2. chip results:Experimental data comes from Bo Ao companies, as a result as shown in Fig. 1 and form 1
3. interpretation of result:Expression quantity of the lnc-uc003hsl.1 in lung cancer is compared with having raised 32.13 times in normal lung tissue；
Prompting lnc-uc003hsl.1 plays a role in lung cancer possibly as an oncogene.
(2)：QRT-PCR analyses lnc-uc003hsl.1 is thin in normal lung epithelial cell strain 16HBE and non-small cell lung cancer
Expression quantity in born of the same parents' strain A549
1. method：Trizol reagents extract cell total rna, and qRT-PCR uses SYBR Green PCRMaster Mix
(TAKARA, Dalian, China), people GAPDH is as internal reference.
2.qRT-PCR results：As shown in Figure 2.
3. interpretation of result：Lnc-uc003hsl.1 low expressions in 16HBE, the high expression in A549, with genetic chip knot
Fruit is consistent.
2nd, lnc-uc003hsl.1 is to non-small cell lung cancer cell apoptosis, the influence of propagation, drug susceptibility
Experiment one：Cell apoptosis assay
1. flow cytomery Apoptosis：Build lnc-uc003hsl.1 slow virus interference carrier transfection A549 cells
Strain (A549/sh-lnc-uc003hsl.1), empty carrier transfection A549 cell lines (A549/lnc-NC) are set to control group.By these
Cell line kind is on 6 orifice plates, and 3 × 105Individual cells/well.Every group is given cis-platinum or docetaxel processing, flow cytometry after 48h
Survey Level of Apoptosis.
2. result determines：As shown in Figure 5,6.
3. interpretation of result：Compared with control group, transfect lnc-uc003hsl.1 slow virus interference carriers after, give cis-platinum or
There is Apoptosis and increased in docetaxel processing, and prompting, which reduces lnc-uc003hsl.1 expression, can promote non-small cell lung cancer thin
The apoptosis of born of the same parents.
Experiment two：Cell cycle is detected
1. the flow cytomery cell cycle：Build lnc-uc003hsl.1 slow virus interference carrier transfection A549 cells
Strain (A549/sh-lnc-uc003hsl.1), empty carrier transfection A549 cell lines (A549/lnc-NC) are set to control group.By these
Cell line kind is on 6 orifice plates, and 3 × 105Individual cells/well.Every group is given cis-platinum or docetaxel processing, flow cytometry after 24h
Survey the cell cycle.
2. result determines：As shown in Figure 7,8.
3. interpretation of result：After A549 transfection lnc-uc003hsl.1 slow virus interference carriers compared with control group, give suitable
There is phase cell cycle G1 and blocks increase in platinum or docetaxel processing, and prompting reduction lnc-uc003hsl.1 to express can suppress non-
The propagation of small cell lung cancer cell.
Experiment three：Cells resistance related experiment
1.MTT surveys cell IC50：Build lnc-uc003hsl.1 slow virus interference carrier transfection A549 cell lines (A549/
Sh-lnc-uc003hsl.1), empty carrier transfection A549 cell lines (A549/lnc-NC) are set to control group.By these cell line kinds
On 96 orifice plates, 2500 cells/wells.Every group is given cis-platinum or docetaxel processing, and the detection of MTT decoration methods is thin after being administered 3 days
Born of the same parents' vigor, and then calculate respective medicine IC50.
2. result determines:As shown in Figure 3,4.
3. interpretation of result:In A549 cell lines, the DDP sh-lnc-uc003hsl.1 experimental groups IC50 of processing are given：
13.05ug/ml, control group IC50 (NC)：24.87ug/ml；Give the DTX sh-lnc-uc003hsl.1 experimental groups of processing
IC50:17.92ug/ml, control group IC50 (NC):24.78ug/ml.It was observed that this phenomenon, prompts to reduce lnc-uc003hsl.1
Expression can promote non-small cell lung cancer cell apoptosis, improve cell chemosensitivity.
3rd, protein immunoblot experiment detects lnc-uc003hsl.1 to non-small cell lung cancer cell strain A549 apoptosis and week
The Mechanism Study that phase influences.
1.A549 cell lines transfect lnc-uc003hsl.1 slow virus interference carriers, empty liposome transfection (A549/ respectively
Lnc-NC) it is set to control group.Total protein of cell detection apoptotic proteins water is extracted after cis-platinum 5ug/ml, 48h are given after transfection 24h
It is flat.
2. result determines：As shown in Figure 9.
3. interpretation of result：Compared with control group, transfection lnc-uc003hsl.1 slow virus interference carrier can substantially raise egg
White bax expressions, lower the expression of anti apoptotic protein bcl -2, and prompting, which reduces lnc-uc003hsl.1 expression, can promote to swell
The apoptosis of oncocyte.Above-mentioned experimental result prompting, which reduces lnc-uc003hsl.1 expression, can promote non-small cell lung cancer cell
Apoptosis, can be as the intervention target spot of oncotherapy.
Beneficial effect
1. by lnc-uc003hsl.1 to non-small cell lung cancer cell apoptosis, increment, the influence of chemotherapy drug susceptibility,
Illustrating, which reduces lnc-uc003hsl.1 expression, can promote non-small cell lung cancer cell apoptosis；By Cyclin-dependent kinase the G1 phases so as to
Suppress cell propagation；Increase sensitiveness of the non-small cell lung cancer cell to chemotherapeutics.
2. more conventional interference lncRNAs method at present, there is the transfection of external preparation lncRNAs slow virus interference carrier
Play a part of to cell and in vivo reducing lncRNAs expression.Present invention employs former approach, this law is easy and has
Higher transfection efficiency.
Brief description of the drawings
(A is the situation of Fig. 1 gene microarray analysis normal lung tissue and long-chain non-coding RNA express spectra in cancerous lung tissue
Sample A are normal lung tissues；B is that Sample B are cancerous lung tissues, and Ritio is SampleB/Sample A signal ratio
Value).
Fig. 2 qRT-PCR survey normal lung epithelial cell strain 16HBE and non-small cell lung cancer cell strain A549lnc-
Uc003hsl.1 expressions, GAPDH are set to internal reference.
Non-small cell lung cancer cell strain A549 is for suitable before and after change lnc-uc003hsl.1 expression is surveyed in Fig. 3,4 for mtt assay
The sensitivity effects of platinum and docetaxel.Fig. 3 is that A549 cell lines carry out the IC50 after cisplatin treated；Fig. 4 is A549 cell lines
Carry out the IC50 after docetaxel processing.
Fig. 5,6 are that flow cytometry surveys A549 pairs of non-small cell lung cancer cell strain before and after change lnc-uc003hsl.1 expression
In the changes of cell apoptosis of cis-platinum and docetaxel.
Fig. 7,8 are that flow cytometry surveys A549 pairs of non-small cell lung cancer cell strain before and after change lnc-uc003hsl.1 expression
Change in the G1 phases of cis-platinum and docetaxel.
Fig. 9 protein immunoblot testing results.
Embodiment
The invention will be further elaborated by the following examples, but does not limit the present invention.
General explanation：
The experimental method of the dated actual conditions in end, is substantially all what is write according to Sambrook, J et al. in embodiment
《Molecular Cloning:A Laboratory guide (the 3rd edition)》(Molecular Cloning:A Laboratory Manual,3rdEd. Huang Peitang etc.
Translate, Science Press .2002.8) described in condition and method or enter according to the condition proposed by material supplier and method
OK, it is well known standard method that other technologies being not described in, which are corresponded to for those skilled in the art,.
The material of the present invention：Cell line, slow virus interference carrier and the culture medium referred in the application has commodity confession
Should or with other approach can be the public obtained by, they are only for example, be not to the present invention it is unique, can be respectively with other suitable
Instrument and biomaterial replace.
Embodiment 1
Lnc-uc003hsl.1 is detected to organize and the expression in cell
(1)：Expressions of the lnc-uc003hsl.1 in normal lung tissue and cancerous lung tissue：
1. chip prepares and analysis:Requirement according to the Bo Ao companies of China prepares normal lung tissue and cancerous lung tissue mark
This, transfers to Bo Ao companies to carry out chip preparation, brilliant coreMankind long-chain non-coding RNA chip V1.0 versions complete chip analysis,
The chip includes the probe of numerous LncRNAs databases latest editions and tens thousand of LncRNAs detections.The detection of the chip is former
Reason is according to lncRNA sequence lengths and architectural feature, is drawn using random primer and Ologo (dT) the reverse transcription mode being combined
Enter T7 promoters, then the linear amplification method by carrying out in-vitro transcription mediation expands to RNA, RNA linear amplification methods
Marked for sample in microarray chip analysis.The DNA that linear amplification product cDNA reverse transcriptions are obtained, then use
KlenowFragent enzymes carry out fluorescence labeling to DNA, finally realize the Oligo probes on the DNA product and chip of fluorescence labeling
Hybridization, carries out the gene expression analysis based on chip.Detected by lncRNA chips, researcher is capable of obtaining for fast high-flux
Obtain the lncRNA related to particular biological process or disease expression change.Rich biological lncRNA chips detection sample mark difficult to understand
Record a demerit journey, mainly comprise the following steps：
1) is since Total RNA sample, with T7Oligo (dT) Primer containing T7 promoter sequences and
T7RandomPrimer is primer, uses CbcScript enzymatic synthesis 1st-strand cDNA.
2) .Second strand DNA are synthesized：With RNaseH, DNA Polymerase by DNA-RNA heterozygotes
RNA chains are converted into Second strand cDNA, and synthetic dsdna simultaneously carries out DNA and crosses post purifying.
3) in-vitro transcriptions synthesis cDNA：Using double-stranded DNA as template cDNA, this step pair are synthesized using T7Enzyme Mix
RNA is expanded.
4) .cDNA is purified：The reagent such as cDNA, the salt gone out in dereaction, enzyme is purified using RNA purification columns, and cDNA is entered
Row is quantitative.
5) .cDNA reverse transcriptions：Using cDNA as template, Random Primer are primer, and CheScriptII enzymes are inverted
Record.Cross post and purify and reclaim the cDNA that cRNA reverse transcriptions obtain.
6) .cDNA fluorescence labelings：Using the cDNA products of reverse transcription as template, Random Primer are primer, use Klenow
Fragment enzymatic synthesis cDNA complementary strands simultaneously mix Cy3-dCTP or Cy5-dCTP with fluorophor.Purify simultaneously quantitative mark
Product.These can be used to chip hybridization with fluorophor DNA.
CDNA microarray goes out in cancerous lung tissue after the lncRNAs of specificity overexpression, and researcher is expanded by qRT-PCR methods
It is target DNA (lnc-uc003hsl.1) to increase DNA identify to obtain, and lnc-uc003hsl.1 primer is as follows：F
- the agcagcaacaaacaatcctga-3 ' of Primer 5 ', see SEQ NO：4, R Primer 5 '-
Ccacaagtcatttcaccaagg-3 ', see SEQ NO：5.
2. chip results:Experimental data comes from Bo Ao companies, as a result as shown in Fig. 1 and form 1.
The significant difference list of table one
3. interpretation of result:Lnc-uc003hsl.1 signals in cancerous lung tissue (Sample B) are 293.4204, normal
Signal is that 9.1301, Ratio (Sample B/Sample A) is 32.1377 in lung tissue (Sample A), represents lnc-
Uc003hsl.1 high expression in cancerous lung tissue, the low expression in normal lung tissue, the two difference are prompted up to 32.1377 times
Lnc-uc003hsl.1 plays a role in lung cancer possibly as an oncogene.
(2) it is thin in normal lung tissue cell line 16HBE and non-small cell lung cancer to analyze lnc-uc003hsl.1 by qRT-PCR
Born of the same parents' strain A549 express spectra
1. method：Trizol reagents extract cell total rna, and qRT-PCR uses SYBR Green PCR MasterMix
(TaKaRa, Dalian, China), people GAPDH is as internal reference.
2.qRT-PCR results：As shown in Figure 2.
3. interpretation of result：Lnc-uc003hsl.1 low expressions in normal lung tissue cell line 16HBE, in non-small cell lung
High expression, consistent with gene chip results in JEG-3 A549.
Embodiment 2
Build lnc-uc003hsl.1 slow virus interference carrier transfectional cells：
The specific interference sequence for lnc-uc003hsl.1 is designed, its sequence is (5 ' to 3 ')：
CCAGATGGGCACTTCTAAA, see Seq NO.2, to disturb the tract of GAPDH genes as control, and be inserted into slow disease
In poisonous carrier (above carrier and fragment are synthesized by invitrogen companies).Packaged slow virus carrier is infected into non-small cell
Lung cancer cell types cell, play a part of downward lnc-uc003hsl.1 and express, it is thin with G418 screening stable transfections after 48h
Born of the same parents are to be named as A549/sh-lnc-uc003hsl.1 (control is A549/lnc-NC).
Embodiment 3
Mtt assay detection cell propagation：
1) inoculating cell：With 0.25% Trypsin Induced single-layer culturing cell, trained with 1640 containing 10% hyclone
Nutrient solution is made into single A549/sh-lnc-uc003hsl.1 (control is A549/lnc-NC) cell suspension, 2500 thin with every hole
Born of the same parents are inoculated in 96 well culture plates, per pore volume 200ul.Be administered respectively DDP (cis-platinum, it is limited purchased from the permanent auspicious medical share in Jiangsu
Company) or DTX (docetaxel, purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov.).
2) cell is cultivated：Culture plate is moved into CO2In incubator, in 37 DEG C, 5%CO2And cultivated under relative humidities, train
Support 3 days.
3) colour generation：After cultivating 6h, 24h, 48h, 72h, 96h, addition MTT solution (5mg/ml) 20ul per hole, 37 DEG C, after
It is continuous to be incubated 4h, culture is terminated, careful inhale abandons culture supernatant in hole.150 μ l DMSO (dimethyl sulfoxide (DMSO)) vibration 10min are added,
Crystal is set fully to dissolve.
4) colorimetric：OD value (OD) is determined at 490nm wavelength, every group of 3 multiple holes, is tested in triplicate.Calculate existence
Rate.Survival rate=experimental group OD values/control group OD value × 100%.
5) result determines：By as above method by A549/sh-lnc-uc003hsl.1 (control be A549/lnc-NC) cell
Strain kind is on 96 orifice plates, 2500 cells/wells.MTT decoration methods monitor cell viability after cultivating 6h, 24h, 48h, 72h, 96h.
Interpretation of result：A549/sh-lnc-uc003hsl.1 compared with control group A 549/lnc-NC ability of cell proliferation substantially by
Suppress.It was observed that this phenomenon, prompts interference lnc-uc003hsl.1 expression to suppress cell proliferation of NSCLC, improves
Cell chemosensitivity.
Embodiment 4
Flow cytometry Annexin V/PI double-stainings survey Apoptosis：
1) cell is collected：Suspension cell is directly collected into 10ml centrifuge tube, and attached cell is first gently blown with dropper
Beat, apoptotic cell may take off wall once piping and druming, be collected into 10ml centrifuge tube, and the cell of not de- wall is disappeared with 0.02% EDTA
Change is allowed to de- wall, is (1~5) × 10 per sample cell number6, 500~1000r/min centrifugation 5min discard nutrient solution.
2) washed 1 time with incubation buffer, 500~1000r/min centrifugations 5min.
3) cell is resuspended with 100 μ l label solution, lucifuge is incubated 10~15min at room temperature.
4) 500~1000r/min centrifuges 5min sedimentation cells, and incubation buffer is washed 1 time.
5) add at 4 DEG C of fluorescent solutions and be incubated 20min, lucifuge is simultaneously vibrated frequently.
6) flow cytometer measure Apoptosis.
Such as above-mentioned method, by A549/sh-lnc-uc003hsl.1 (control is A549/lnc-NC) cell kind in 6 orifice plates
On, 3 × 105Individual cells/well.Cis-platinum or docetaxel processing are given after transfection 48h for every group, and flow cytometry is surveyed after 48h is administered
Level of Apoptosis.
As a result determine：As shown in Figure 5,6.
Interpretation of result：Compared with control group A 549/lnc-NC, A549/sh-lnc-uc003hsl.1 gives cis-platinum or more western
There is Apoptosis increase at match processing in him, and prompting, which reduces lnc-uc003hsl.1 expression, can promote non-small cell lung cancer cell
Apoptosis, improve cell chemosensitivity.
Embodiment 5
Flow cytometry cell growth cycle (PI dyeing)：
1) cell is collected：With collected by trypsinisation cell, PBS is washed twice, abandons supernatant, adds the pre-cooled ethanols of 1ml 70%
In, piping and druming is uniform, and 4 DEG C are fixed more than 12 hours.
2) ethanol is removed in PBS washings, 1000rpm, 5min, washes twice.
3) cell is resuspended in 0.5mlPBS, adds PI and RNaseA to final concentration 50 μ g/ml, 37 DEG C of warm bath 30min.
4) the Flow Cytometry Assay cycle.
The Flow cytometry cell cycle：Such as above-mentioned method, by A549/sh-lnc-uc003hsl.1, (control is
A549/lnc-NC) cell kind is on 6 orifice plates, and 3 × 105Individual cells/well.Given after transfection 48h at cis-platinum or docetaxel for every group
Reason, flow cytometry surveys the cell cycle after 24h is administered.
As a result determine：As shown in Figure 7,8
Interpretation of result：After the reduction lnc-uc003hsl.1 expression of A549 cells compared with control group, cis-platinum or more western is given
There is phase cell cycle G1 and block increase in his match processing, and non-small cell lung can be suppressed by prompting to disturb lnc-uc003hsl.1 to express
The propagation of cancer cell.
Embodiment 6
Protein immunoblot experiment detects lnc-uc003hsl.1 to non-small cell lung cancer cell strain A549 apoptosis correlation eggs
The influence of white bax, bcl-2 expression.
Specific method is as follows：
1. extract total protein of cell
2.SDS-PAGE electrophoresis
1) clean, glass plate, potsherd are installed.
2) perfusion separation gel, 37 DEG C of standing 25min are prepared.
3) perfusion concentration glue is prepared, is stored at room temperature 20min.
4) protein sample and molecular weight marker are added, each period albumen applied sample amount is 40 μ g.
5) constant pressure electrophoresis (concentration glue 80V, separation gel 120V).
1. half dry type transferring film
1) after electrophoresis terminates, gloves are worn, glue (corner cut mark is cut with reference to molecular weight marker according to destination protein molecular weight ranges
Note), cut formed objects nitrocellulose filter (corner cut mark) one and filter paper 2 is opened.
2) glue, film and filter paper 10min are soaked in transferring film buffer solution.
3) tiled from bottom to up on transferring film instrument：Filter paper, film, gel, filter paper, bubble removing is caught up with, especially pay attention to film and gel
Between prohibit stay bubble.Dry surplus liquid, 0.8mA × membrane area (cm2) electric current transferring film 1.5 hours.
2. Western blotting
1) transferring film terminates, and examines whether marker has turned on film (or Ponceaux dyeing) to judge transferring film effect.TBST
After cleaning film, 5% skimmed milk power (TBST preparations) room temperature shakes 2h.
2) primary antibody bax, bcl-2 monoclonal antibody (1 is added:1000) (cell signal companies are purchased from), 4 DEG C of closings are overnight.
3) TBST is rinsed 3 times, each 5min.
4) secondary antibody (1 is added:10000) (cell signal companies are purchased from), room temperature shakes 2h.
5) TBST is rinsed 3 times, each 10min.
3.ECL is detected
1) AB liquid mixes, and after film does not drip, tiling is thereon.
2) after reacting 5min, according to 5~30min of fluorescence intensity darkroom tabletting.
3) develop 15~30s, washing, is fixed 1~3min.
4) picture scanning and quantitative analysis.
5) as stated above, A549/sh-lnc-uc003hsl.1 (control is A549/lnc-NC), cell gives DDP
Total protein of cell detection apoptosis-related protein bax, bcl-2 expression is extracted after 5ug/ml 48h.Protein level change such as Fig. 9
It is shown.
Compared with control group, reduce lnc-uc003hsl.1 expression can obvious upregulation of apoptosis albumen bax expressions, downward
The expression of anti apoptotic protein bcl -2, prompting, which reduces lnc-uc003hsl.1 expression, can promote non-small cell lung cancer cell
Apoptosis, increase non-small cell lung cancer cell, can be as the intervention target spots of oncotherapy to the sensitiveness of chemotherapeutic.
SEQUENCE LISTING
<110>The Second Affiliated Hospital of Nanjing Medical University
<120>A kind of long non-coding RNA and its application
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 2213
<212> DNA
<213>Artificial sequence
<400> 1
ctgttagcaa tgcttcctga tgttgtgcgt ggcccttttt ggttgattct ctccaaattc 60
gggtcagctg ctgccacctg gcaaataaca gaggatatgc tgaatctcct gtccatcctt 120
gtaacgatat ccttcttaat gaaattcttc aactggctga gcaattacaa atgtcatctg 180
tccagacaca tgggcttaag gatgtctaca aaattttaga catttttgca aatgggaaaa 240
aaaatagtct tgtaaatact gaaacagatt tccatgaact ttatcctact cttggaaaga 300
aaacaattct ccttggctgc agaaatcaaa taagctgggt ttgcaatgac caaggacata 360
aatgaagatg gattgaagtg gaaaaattct gtctcccaag tgatcagtga catctgccag 420
aggtcattac agctactttt aactgtgaac agtcaccagc taaactactc acttgccaca 480
acaaaataac ctctctcaaa ataaatccag tgcatctgta tatatgtgta gatagcagca 540
acaaacaatc ctgaaacatt atttttggct gttaggtaag taaacgtgat gataattata 600
aacaacattc aaataacctt ggaccttggt gaaatgactt gtggtggcca gaatggtgca 660
acaagatgtt atttgcaagt ttttttaaga cacaaatatc tcagatacta ataatgagaa 720
taaagactgt tgaatatgaa attaaagcca agcaataatg tgccaaaaag aggcagttat 780
accagcaaat gcatctatta tgggcacacc attatataat gatggtttgc tttatgaaga 840
ctgactgtaa cccacaggat aaaataagca aaggcatagt ttctgctttc ttcctggaaa 900
aacttgttta gaagcttcat aaagaggtac agcactaatg agcattagtc aggatacagt 960
tggcatctat gtttttatgt gagcccagag ggaagaggag ccactcaaag tcttgctggc 1020
ttaaaactca agacagctgc aaccagaagt tttgttgaaa tggagacttt aaacttatgg 1080
taattactct ttctggacac tagcatgtag aaagcaattc agttaactct gcccagagga 1140
ttaccagctt tagctgtgaa aaaatgggct cccggatgta aaatcactaa aacatgagat 1200
cttgtatcca aagaggcttc aaatgatgcc ttacagaaaa cgatgctcca gatgggcact 1260
tctaaatgct aactcttcat caagtatctt tctggattca agctcaaaat taattggctg 1320
caaaatagta ggaataaaaa tcacatattt tacactttag aaaaggatat tgatgatcaa 1380
cctgcatggt gataattatg atgagatacc ccagtgattt aatgatgtta gaaagaatta 1440
aatgggagag aattgctaac agctttcttg atctcttaac tatggagatg tcattcattt 1500
atttctgggg tgaaaattat agcttgcttt ttgacattgc tgctagtatt gttctttgtt 1560
gctttaaaaa ttgtctctct ttagaaaaac tcttgagcag ttaaacagtt ttttttctga 1620
ttcatatcat tgcttttaat aacatgtaaa ggctgtgtgt agagcaaact atataaaatg 1680
agtagaaagg gcttactcat gttaattggc atccttgatg attttagttg agattcctta 1740
acatttattt tagatcacat ctttacgtaa cttatttttc ctaatgtttt ccatcgtgtc 1800
ttaaaatgat gctggtatat caggagattg cagtattata gtcatactcc ccaatcccta 1860
gaggagagga aagactaatt cttgttttaa gggcccctgg agataccttt tattaaggtt 1920
gaaaaaggtc aacacagcct gaaaataaga aaaatatata ctagcaatta ctaattttct 1980
aaatgtgtgt atctctgctg tactaatgtg tgaacaatat gtcgtgcata atactgtagc 2040
tggtcgtggt atgtcaatac attctgtgag tgtgtacagt ctgagtgatc agttttctat 2100
ttttatgtgt aaaaaaaata acttgtcgta tcccatttaa aggccaattt ctgtattcag 2160
gcaggcatat gtacatacat gaataaagcc aacagtgtgc acatgtaaat agt 2213
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
ccagatgggc acttctaaa 19
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
gctgctagta ttgttcttt 19
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
agcagcaaca aacaatcctg a 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
ccacaagtca tttcaccaag g 21
Claims (1)
1. a kind of interference sequence of long-chain non-coding RNA is quick to cis-platinum or docetaxel in preparation increase non-small cell lung cancer cell
Application in sensitive drug, the interference sequence described in it are Seq NO.2.
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| CN105969846B (en) * | 2016-05-06 | 2019-09-27 | 江苏省人民医院 | A kind of long non-coding RNA and its application in diagnosis/treatment non-small cell lung cancer |
| CN105950628A (en) * | 2016-07-12 | 2016-09-21 | 江阴市人民医院 | Long non-coding RNA (Ribose Nucleic Acid) and application thereof to diagnosis/treatment of non-small-cell lung cancer |
| CN106434868A (en) * | 2016-07-29 | 2017-02-22 | 南京医科大学第二附属医院 | Long non-coding RNA and application thereof in diagnosis and treatment on non-small cell lung cancer |
| CN108236724B (en) * | 2016-12-23 | 2022-03-18 | 复旦大学 | Application of long-chain non-coding RNA in preparation of preparation for inhibiting tumor cell metastasis |
| CN109371136B (en) * | 2018-12-28 | 2021-07-02 | 青岛泱深生物医药有限公司 | Lung adenocarcinoma-related lncRNA and application thereof |
| CN112175953B (en) * | 2020-03-13 | 2022-05-24 | 芯超生物科技(河南)有限公司 | Application of gene inhibitor in preparation of lung cancer medicine |
| CN111378664B (en) * | 2020-03-27 | 2022-06-07 | 南京医科大学第二附属医院 | Circular RNA and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Human Gene AK026379 (uc003hsl.2);GBshape;《The Genome Browser for DNA Shape》;20130614 * |
| The Long Noncoding RNA MEG3 Contributes to Cisplatin Resistance of Human Lung Adenocarcinoma;Liu et al;《PLOS ONE》;20150520;摘要，表1 * |
| 长链非编码RNA与肿瘤研究进展;朱雯等;《中国肿瘤临床》;20121231;476-480 * |
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