CN110184271A - A kind of application of mRNA-like ncRNA, the antisense RNA for inhibiting it to express and the two - Google Patents
A kind of application of mRNA-like ncRNA, the antisense RNA for inhibiting it to express and the two Download PDFInfo
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Abstract
This programme discloses a kind of mRNA-like ncRNA of genetic engineering field, and cDNA is the nucleotide sequence with one of following nucleic acid sequence: 1) DNA sequence dna of the SEQ NO:1 in sequence table；2) there is the nucleotide sequence of 90% or more homology with the DNA sequence dna of the SEQ NO:1 in sequence table；3) nucleotide sequence that can hybridize with the DNA sequence dna of the SEQ NO:1 in sequence table under high high stringency conditions, the high high stringency conditions are to hybridize at 65 DEG C in 0.1XSSC, the solution of 0.1%SDS and wash film.The application for inhibiting the antisense RNA and the two of mRNA-like ncRNA expression in prevention and/or tumor etc. is additionally provided simultaneously.MRNA-like ncRNA claimed in this programme and the antisense RNA for inhibiting it to express are of great significance for diseases such as treatment breast cancer.
Description
Technical field
The invention belongs to genetic engineering field, in particular to a kind of mRNA-like ncRNA, the antisense for inhibiting it to express
The application of RNA and the two.
Background technique
Cancer causes huge threat to human health, and about 7,000,000 people die of cancer, the annual cancer in China every year in the whole world
About 1,300,000 people of disease death, is in second in all kinds of causes of the death.Capturing cancer, mitigating patient's pain is always world's difficulty
Topic.Breast cancer is the number one Health Killer of global women, and annual 1200000 women in the whole world suffers from breast cancer, has 500,000 people to die of
The disease.And the China in the opposite area Di Fa, with the development of economy, working women the accelerating rhythm of life and eating habit
Change, which just rises year by year.Disease incidence tends to rejuvenation up to 25.48/10 ten thousand, and it is raw to seriously threaten women
Life health.As other cancers, generation, evolution, invasion and the transfer of breast cancer are the progressive accumulation that a polygenes participates in
Process, be related to numerous gene regulations, expression, mutation etc. variation.Cancer is recognized using Protocols in Molecular Biology, from molecular level
Pathology root be for clinic the effective way of effective clinical means is provided.
The gene of coding protein about 30,000 in human genome, the exon sequence of coding protein accounts for about entire base
Because of the 2% of group sequence.But recently with complete genome DNA microarray analysis rna transcription situation research shows that the gene being transcribed
Group sequence is far longer than 2%, it is estimated that about 50% genome sequence can transcribe RNA.These discoveries prove exist in organism
The RNA of a large amount of not coding protein, referred to as non-coding RNA (non-coding RNA, ncRNA).Although non-coding RNA is not
Coding protein, but more and more evidences show itself but there is different physiological roles in recent years, participate in physiology and development
Regulation of gene expression in the process, including chromosome structure, rear raw memory, transcription, RNA montage, editor, translation etc..RNA regulation
Net may decide our complicated biological natures, in the hereditary variation field in disease and unknown species between species all
It plays an important role.Functional non-coding RNA includes small RNA, miRNA with mRNA-like ncRNA (similar courier
The non-coding RNA of RNA) etc., having many researchs in the recent period has shown that with tumour correlation occurs for non-coding RNA.Wherein miRNA regulates and controls that
The expression with development, proliferation, apoptosis and stress reaction related gene is well known a bit, and they are passed through in human malignancies
Often there is unconventionality expression.The research of mRNA-like ncRNA and cancer-related is recently there has also been report, in April, 2006,
Ginger MR et al. has found a non-coding RNA PINC (Ginger relevant with cell cycle and cell survival for the first time
MR,Rosen JM,et al.A noncoding RNA is a potential marker of cell fate during
mammary gland development.Proc Natl Acad Sci U S A.2006Apr 11；103(15):5781-
6)；In July, 2006, Lin R et al. have found the non-coding RNA MALAT-1 for being up to 8.3kb, and the non-coding RNA is in liver
High expression in cancer, while also having very strong expression in human breast carcinoma, lung cancer, bladder cancer, colon cancer and cancer of pancreas, author thinks
Its marker (Lin R, Edgington TS, et al.A large noncoding RNA that can be formed as a tumor
is a marker for murine hepatocellular carcinomas and a spectrum of human
carcinomas.Oncogene.2006 Jul 31.)；After one month, Wang XS et al. then bladder cancer have found one it is complete
The non-coding RNA UCA1 of a length of 1.4kb, it is believed that it is marker gene (Wang XS, the Chen WF, et of a bladder cancer
al.Rapid identification of UCA1 as a very sensitive and specific unique
marker for human bladder carcinoma.Clin Cancer Res.2006Aug 15；12(16):4851-8).
It can be seen that mRNA-like ncRNA has also assisted in the generating process of tumour, and it is contemplated that mRNA-like ncRNA in tumour
The very important effect as miRNA is played in generation, also will be the sides such as future tumors research, prevention, diagnosis and treatment
The indispensable a part in face.
Antisense technology passes through the mRNA of Watson-Crick basepairing rule and target gene using DNA or RNA molecule
Complementation combines, and so that it is degraded by various mechanism or it is inhibited to encode the translation of albumen, to inhibit the expression of target gene.With
The afunction Journal of Sex Research method such as gene knockout is compared, and antisense technology has less investment, and the period is short, simple operation and other advantages, because
This has received widespread attention.Antisense technology includes three classes: using antisense oligodeoxynucleotide (AS-ODNs) or antisense RNA
Antisense oligonucleotides (AS-ONs) technology；Ribozyme and DNA enzymatic technology with catalytic activity；Using siRNA (siRNA)
RNAi technology.Wherein Antisense OligodeoxynucleotideTechnique Technique passes through nearly development in 20 years, has obtained gradual perfection and has started to be applied to doctor
It treats in practice.Antisense oligonucleotides can regulate and control gene expression in DNA replication dna, transcription and translation level.In DNA replication dna water
Flat, antisense oligonucleotides can be used as the inhibiting factor of DNA replication dna, in conjunction with primed RNA or acts on primer precursor and inhibits
DNA replication dna, to reduce the duplicating efficiency of DNA.In transcriptional level, antisense oligonucleotides is in conjunction with mRNA5 ' termini-complementary, resistance
Disconnected cap sequence is formed；The bonding pad of exon and introne is acted on, premessenger RNA montage is hindered；It acts on polyA and forms position
Point hinders the maturation of mRNA and its to cytoplasmic transport.It is related with its skeleton structure in the mechanism of action of translation skill: to have negative electricity
The AS-ODNs of lotus in conjunction with complementary mRNA after can activate RNase H, the latter cuts mRNA chain in RNA-DNA heteroduplex, suppression
Make its expression；Other not negatively charged AS-ONs are played a role by space steric effect.More and more antisense oligonucleotides
Acid shown in clinical test good safety and specificity inhibiting effect, although its comprehensively move towards clinic there is also
Many challenges, but may be easier to overcome compared with other field.Clinical test proves Antisense OligodeoxynucleotideTechnique Technique and conventional medicament
Combination the treatment of cancer is very effective, while Antisense OligodeoxynucleotideTechnique Technique is also proved to be effectively overcome chemotherapy
With the method for radiotherapy tolerance.
In order to which the molecular mechanism to breast cancer conducts further research, applicant utilizes suppressed subtractive hybridization technology, sieve
A batch is selected in gene highly expressed in mammary gland of mouse cancerous tissue, one of gene category is found by bioinformatic analysis
In imaginary albumen, there is very high similitude with the gene of people, cDNA overall length is 2.8kb, we temporarily name it for Z38 base
Cause.Its albumen with transmembrane domain that may encode 253 amino acid of Bioinformatics Prediction.We, which clone, has recombinated it
Open reading frame, construct its protokaryon and eukaryotic expression system respectively, but fail to give expression to the albumen.In doctor's moral company
Synthesis polypeptide, injection rabbit are prepared for polyclonal antibody, immunohistochemistry and Western are utilized in the Various Tissues of people
The methods of Blot does not detect the albumen.But by both ends PCR and sequencing, show its with mRNA feature, so I
Preliminary assert that Z38 gene is the non-coding RNA (mRNA-like ncRNA) of a similar mRNA.Pass through a variety of biology letters
Breath learns software prediction, may encode three Micrornas (microRNA/miRNA) in the sequence of Z38 2.8kb.It collects and uses glimmering
Fluorescent Quantitative PCR technology has detected the expression quantity of Z38 in 50 breast cancer tissues and cancer beside organism, as the result is shown wherein 35 mammary gland
The expression of Z38 is higher than its cancer beside organism in cancerous tissue.In order to which the function to Z38 is studied, it is (opposite that we pick HBL100
Normal cell line of mammary gland, this cell line hardly express Z38 gene), the Z38 gene containing EGF connector of building is turned
It contaminates into cell, is analyzed by the growth curve of 96 hours, determine that the cells growth activity of transfection Z38 is greater than control group.In addition
We have selected the breast cancer cell line MDA-MB-231 of expression Z38 one high, the microRNA sequence design according to prediction
Antisense RNA transfect cell, after 48 hours, compared with the control group, MTT analysis and TUNEL labeled analysis its cell as the result is shown
Vigor decline, Apoptosis increase.
Therefore, mRNA-like ncRNA claimed in the application and the antisense RNA for inhibiting it to express are for treatment
The diseases such as breast cancer are of great significance.
Summary of the invention
The invention is intended to provide the application of a kind of mRNA-like ncRNA, the antisense RNA for inhibiting it to express and the two.
One of this programme mRNA-like ncRNA, cDNA are the nucleotides sequences with one of following nucleic acid sequence
Column:
1) DNA sequence dna of the SEQ NO:1 in sequence table；
2) there is the nucleotide sequence of 90% or more homology with the DNA sequence dna of the SEQ NO:1 in sequence table；
3) nucleotide sequence that can hybridize with the DNA sequence dna of the SEQ NO:1 in sequence table under high high stringency conditions, it is described
High high stringency conditions are to hybridize at 65 DEG C in 0.1XSSC, the solution of 0.1%SDS and wash film.
Expression vector, cell line and host strain containing the mRNA-like ncRNA encoding gene belong to this hair
Bright protection scope.
It is a further object to provide the antisense RNAs and DNA that inhibit the mRNA-like ncRNA expression.
The antisense RNA provided by the present invention for inhibiting the mRNA-like ncRNA expression is that have following nucleotides sequence
The single stranded RNA or DNA of column.
1) nucleotide comprising SEQ NO:2, SEQ NO:3, SEQ NO:4, SEQ NO:5 or SEQ NO:6 in sequence table
These antisense RNAs are respectively designated as AntiZ38-1, AntiZ38-2, AntiZ38-3, AntiZ38-4, AntiZ38- by sequence
5；
2) with sequence table in SEQ NO:2, SEQ NO:3, SEQ NO:4, SEQ NO:5 or SEQ NO:6 RNA sequence
DNA sequence dna with 90% or more homology.
Expression containing AntiZ38-1, AntiZ38-2, AntiZ38-3, AntiZ38-4 or AntiZ38-5 encoding gene
Carrier, cell line and host strain all belong to the scope of protection of the present invention.
The mRNA-like ncRNA and antisense RNA the answering in prevention and/or tumor for inhibiting it to express
With.
The mRNA-like ncRNA and antisense RNA the answering in prevention and or treatment inflammation drug for inhibiting it to express
With.
The mRNA-like ncRNA and the antisense RNA for inhibiting it to express are in inflammation and Tumor in Vitro diagnosis, prognosis drug
In application.
Detailed description of the invention
Fig. 1 is that RT-PCR detects Z38 expression in human breast carcinoma and cancer beside organism；
Fig. 2 is the mRNA precursor secondary structure figure of prediction and the sequence of antisense RNA；
Fig. 3 is the structural schematic diagram of carrier for expression of eukaryon pEGFP-C1-Z38；
Fig. 4 is transfection Z38 and empty plasmid enters MTT analysis result after HBL100 cell；
Fig. 5 is transfection Z38 and BrdU labeled analysis result after HBL100 cell after empty plasmid；
Fig. 6 is that Z38mRNA expression is analyzed after transfected antisense RNA enters MDA-MB-231 cell；
Fig. 7 is that MTT analyzes result after transfected antisense RNA enters MDA-MB-231 cell；
Fig. 8 is that transfected antisense RNA enters TUNEL labeled analysis result after MDA-MB-231 cell.
Specific embodiment
Experimental method in the following example is unless otherwise instructed conventional method.
One, the acquisition of Z38 (mRNA-like ncRNA) encoding gene
Using suppressed subtractive hybridization technology, a batch is filtered out in gene highly expressed in mammary gland of mouse cancerous tissue, is passed through
Bioinformatic analysis finds that one of gene belongs to imaginary albumen, has very high similitude, cDNA with the gene of people
Overall length is about 2.2kb, we temporarily name it for Z38 gene (i.e. mRNA-like ncRNA).It tests and surveys by 5 ' and 3 ' RACE
Sequence obtains Z38 full length sequence.PCR primer sequence are as follows: 1,5 ' RACE:F, 5 '-AAGCAGTG GTATCAACGCAGAGT-3 '；R
5'-GAAATGAGGCTAAGCACACAAGC-3'；2,3'RACE:F 5'-GCA TTCTCCATCTCCTTGCAG-3'；R 5'-
AAGCAGTGGTATCAACGCAGAGT-3’。
Two, real-time quantitative PCR detection Z38 is expressed in human breast carcinoma tissue
In order to detect expression of the Z38 in human breast carcinoma, we have collected 50 breast cancer and cancer beside organism, extracting
Total serum IgE obtains cDNA as pcr template through reverse transcription.Use β-actin as internal reference, PCR primer Z38:F 5 '-
GAATTTGGATGGTCCTTCTGC-3',R 5'-ACTCTTTCCGGTTGGTGTGA-3'；β-actin:F 5 '-
GGCGGCAACACCATGTACCCT-3′,R 5′-AGGGGCCGGACTCGTCATACT-3′.PCR reaction system: SYBR GREEN
PCR Master Mix(Applied Biosystems)10μl,cDNA 20ng(5μl),primers 0.4μM(5μl)。PCR
Program: 95 DEG C of 2min, 95 DEG C of 15s and 60 DEG C of 1min 40 circulations.As the result is shown wherein in 35 breast cancer tissues Z38 table
Up to higher than its cancer beside organism.It is auspicious to see Fig. 1.
Three, software prediction microRNA precursor secondary structure and Antisense RNA sequence
The prediction of secondary structure and microRNA is carried out to Z38 full length sequence using online software MFOLD and MiRAlign
Analysis, there may be three microRNA into Z38 sequence for prediction of result, and according to this three Duan Xulie, we, which design, has synthesized six
Antisense RNA, particular sequence are shown in Fig. 2.
Four, the building of people Z38 gene eucaryon expression plasmid
In order to study the function of Z38 gene, we by round pcr using the cDNA library synthesized in embodiment 2 as template,
22bp is inserted into a terminator codon, PCR product (primers F to design primer before the initiation codon of imaginary albumen in Z38 sequence
5 '-TCATGCCACACGA TATGC-3 ' of 5 '-TGAATGCCAGAATGGATA-3 ', R) insertion pEGFP-C1 multiple cloning sites
Between BglII and KpnI, carrier for expression of eukaryon pEGFP-C1-Z38 is obtained, structural schematic diagram is as shown in Figure 3.
Five, the functional study of Z38 gene
In order to confirm the function of Z38 gene, we are marked using MTT, BrdU and TUNEL labeled analysis transfects pEGFP-
The influence of cell proliferation and apoptosis is studied after C1-Z38 and the antisense RNA by transfecting Z38 induce Z38 to degrade.
(1) in 5%CO2In incubator with DMEM complete culture solution (containing 10% newborn bovine serum, 100u/ml penicillin,
100 μ g/ml streptomysins) culture HBL100 and MDA-MB-231 cell.It is 90% or so to cell density, changes antibiotic-free without blood
PEGFP-C1-Z38 and pEGFP-C1 are each separately transfected into HBL100 cell after culture 3 hours by clear culture medium；By six antisense RNAs
MDA-MB-231 cell is each separately transfected into negative control RNA.Transfection reagent uses LipofectamineTM2000
(Invitrogen/Gibco), it and by the method prompted in product description in the transfection reagent is transfected.After 6 hours again
Antibiotic-free serum free medium is changed into DMEM complete culture solution.Transfected antisense RNA enters Z38mRNA after MDA-MB-231 cell
Expression is detailed in Fig. 6.U/ml indicates units per ml.
(2) MTT: 1. the above-mentioned cell transfected is made single cell suspension and is inoculated in 96 well culture plates；103-104 cell/
Hole, every 200 microlitres of hole culture medium total amount, 37 DEG C, 5%CO2It is cultivated 12~96 hours in incubator.2. respectively in each period
The MTT liquid (50 hole μ L/) of 2 μ g/ml is added；Continue culture 3 hours.3. being sucked out in hole after culture solution, DMSO liquid (150 μ L/ are added
Hole), culture plate is placed in microwell plate and pulls to swing and is vibrated 10 minutes on device, crystal is dissolved.4. microplate reader detects the (inspection of each hole OD value
Survey wavelength is 490nm).Record is as a result, draw cell growth curve；It is detailed in Fig. 4 and Fig. 7.
(3) BrdU is marked: having transfected the HBL100 cell of pEGFP-C1-Z38 or pEGFP-C1 plasmid 1. with 1.5 × 105/
Ml cell number is inoculated in diameter 35mm culture dish (one coverslip of interior placement), is cultivated 48 hours.2. adding before terminating cell culture
Enter BrdU (final concentration of 30 μ g/L), 37 DEG C, is incubated for 8 hours.3. abandoning culture solution, slide is washed 3 times with PBS, and methanol/acetic acid is solid
Determine 10min.It is air-dried through fixed slide, 0.3%H2O2Methanol 30min inactivating endogenous oxidizing ferment.4M HCL 10min
Denaturing nucleic acid.4. 5% standard sheep blood serum is closed.PBS washing adds 1 anti-i.e. anti-mouse BrdU monoclonal antibody (working concentration 1:500), yin
Property control plus PBS or serum incubation at room temperature overnight.5. after PBS washing, the secondary antibody (working concentration 1:2000) for fluorescence of having labelled
30min, random counter 10 under fluorescence microscope are incubated at room temperature with cell fluorescence dyestuff Hoechst (working concentration 1:2000)
Total number of cells and BrdU positive cell number in high power field calculate label index.As a result see Fig. 5.
(4) TUNEL is marked: being grasped according to the specification of In situ cell death Detection POD, kit (Roche)
Make: 1. MDA-MB-231 cell is with 1.5 × 105A/ml cell number is inoculated in 12 orifice plates (coverslip is placed in hole), culture 24
Transfected antisense RNA-C1/C2, negative control RNA are distinguished after hour.Continue to cultivate 48h；2. terminating cell culture, 4% poly first
Aldehyde room temperature fixes 20min；3. PBS is washed three times, and 10min/ times；4. confining liquid (3% H2O2Methanol) incubation at room temperature 10min；⑤
PBS is washed once；6. Permeabilisation solution (0.1% (v/v) Triton X-100,0.1% (w/v) citric acid
Sodium) 2min is placed on ice；7. 37 DEG C of TUNEL reaction mixture is incubated for 60min, fluorescence microscope after PBS is washed three times
Lower analysis, as a result as shown in Fig. 8.
Sequence table
<110>Zun Yi medical university, school district, Zhuhai
<120>application of a kind of mRNA-like ncRNA, the antisense RNA for inhibiting it to express and the two
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 987
<212> RNA
<213>Genus Homo people (Homo sapiens)
<400> 1
ggggagagag gggcgggcga gcagagggga agcggcggag caggaggagg gcgagcagcg 60
aagccagaag gaaaccggca cagcagaagc gggcagccca ccaccacaca ggcagcccca 120
ggcacagacc ggagaaacga agccagcaag aaaaccagga gaaaaaagca cgggagaaca 180
aggagaggca gagaaaagac aaagagcacc gaacaaggca caggggaggg agacgggaca 240
ccaacccaaa aacagcagga agcccaccag aaaggacaga gcagagggca caaaagggag 300
cacacaacga gcagcaggag aaaggacccg gaaaccacaa agcgggagac ccaggaccac 360
ggcggccagc caccggagag ggaggcgggg cgacggacgg cgcagccgaa gcaacccacc 420
agccacgggc acccacccgc aggcggacac gggccagaag gaggcggaag aacacccacc 480
agaaacagag ccccgacaag accgggaagg aggcccgccg gcggccgccc cacagcaggc 540
cgccccacgg gcgccacacc aaccggaaag agacaccaag aaggcaacgg ggcagagcaa 600
gaaacgccgc acaagccaaa aaaaaacgaa ccccacccca agaaagggaa accaaagaaa 660
acaaaacaca caccacaaaa cacacaaacc acaaaaaggg acaagcgagc caccaaaaaa 720
caacaggcag acagaaagaa aggaagagac gacacaaaag aaagaaaagg gagaggaagg 780
accgaaaaca agggcaagcg agaccagaaa acaaagaacg acgcaacaaa aaccaaagcg 840
ggaaacccca cgcaaaacag cacgcggaga agccagcaac cagcagaagg cgcaaagaaa 900
gccaaaaagg gaaaacaaga cacccaagca gacacaagcc acgagggaga aggcagcgga 960
cgcacggcgc acaaaaaaaa aaaaaaa 987
Claims (10)
1. a kind of mRNA-like ncRNA, it is characterised in that: its cDNA is the nucleotides sequence with one of following nucleic acid sequence
Column:
1) cDNA sequence of the SEQ NO:1 in sequence table；
2) there is the nucleotide sequence of 90% or more homology with the cDNA sequence of the SEQ NO:1 in sequence table；
3) nucleotide sequence that can hybridize with the cDNA sequence of the SEQ NO:1 in sequence table under high high stringency conditions, the Gao Yan
Careful condition is to hybridize at 65 DEG C in 0.1XSSC, the solution of 0.1%SDS and wash film.
2. a kind of antisense RNA for inhibiting mRNA-like ncRNA as described in claim 1 expression, it is characterised in that: have with
The single stranded RNA or DNA of lower nucleotide sequence:
1) nucleotides sequence comprising SEQ NO:2, SEQ NO:3, SEQ NO:4, SEQ NO:5 or SEQ NO:6 in sequence table
Column；
2) in sequence table SEQ NO:2, SEQ NO:3, SEQ NO:4, SEQ NO:5 or SEQ NO:6 RNA sequence have
The RNA sequence of 90% or more homology.
3. a kind of expression vector, cell line or host comprising mRNA-like ncRNA encoding gene as described in claim 1
Bacterium.
4. a kind of comprising the expression for the antisense RNA encoding gene for inhibiting mRNA-like ncRNA to express as claimed in claim 2
Carrier, cell line or host strain.
5. application of the mRNA-like ncRNA according to claim 1 in prevention and/or tumor.
6. the antisense RNA according to claim 2 for inhibiting mRNA-like ncRNA expression is preventing and/or is treating tumour
Application in drug.
7. application of the mRNA-like ncRNA according to claim 1 in prevention and or treatment inflammation drug.
8. the antisense RNA according to claim 2 for inhibiting mRNA-like ncRNA expression is in prevention and or treatment inflammation
Application in drug.
9. mRNA-like ncRNA according to claim 1 answering in inflammation and Tumor in Vitro diagnosis, prognosis drug
With.
10. the antisense RNA according to claim 2 for inhibiting mRNA-like ncRNA expression is examined in inflammation and Tumor in Vitro
Application disconnected, in prognosis drug.
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Citations (3)
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|---|---|---|---|---|
| WO2007013670A2 (en) * | 2005-07-29 | 2007-02-01 | Oncotherapy Science, Inc. | Gene galnt6 as breast cancer marker and small interfering rnas directed against gene galnt6 |
| WO2010065787A2 (en) * | 2008-12-04 | 2010-06-10 | Curna, Inc. | Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene |
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2019
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| WO2007013670A2 (en) * | 2005-07-29 | 2007-02-01 | Oncotherapy Science, Inc. | Gene galnt6 as breast cancer marker and small interfering rnas directed against gene galnt6 |
| US20090202543A1 (en) * | 2005-07-29 | 2009-08-13 | Oncotherapy Science, Inc. | Gene and polypeptide relating to breast cancer |
| WO2010065787A2 (en) * | 2008-12-04 | 2010-06-10 | Curna, Inc. | Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene |
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| Title |
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