CN105754999A - Has-miR-221-3p inhibition oligonucleotide sequence, recombinant adenovirus and preparation method and application thereof - Google Patents
Has-miR-221-3p inhibition oligonucleotide sequence, recombinant adenovirus and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a has-miR-221-3p inhibition oligonucleotide sequence. The has-miR-221-3p inhibition oligonucleotide sequence is one of the following sequences: sequence 1, (GAAACCCAGCTGACAATGTAGCTAAGA)n>/=5; sequence 2, (GAAACCCAGCCGACAATGTAGCTAAGA)n>/=5; sequence 3, (GAAACCCAGCGGACAATGTAGCTAAGA)n>/=5; sequence 4, (GAAACCCAGCGACAATGTAGCTAAGA)n>/=5, wherein in the four sequences, the AAGA parts can be substituted by other sequences. A recombinant adenovirus (Ad-s-miR-221-3p) is obtained by intracellular DNA (deoxyribonucleic acid) homologous recombination. The invention further provides applications of the recombinant adenovirus to treatment of non-alcoholic fatty liver diseases, wherein the applications include lowering of liver triglyceride level and blood triglyceride level and improvement of hyperlipemia and body insulin sensitivity, thereby providing a new solution for improvement of non-alcoholic fatty liver.
Description
Technical field
The invention belongs to genetic engineering, bio-medical technology field, particularly relate to one and utilize oligonucleotide string
The connection method preparation suppression oligonucleotide sequence of hsa-miR-221-3p, recombinant adenovirus and reduction liver thereof and blood are sweet
The application of oil three esters.
Background technology
Liver is the central organ of regulation organism metabolism, such as lipid metabolism, absorbs including fat synthesis, lipoprotein and divides
Secrete, fatty acid oxidation etc..Non-alcoholic fatty liver disease is a kind of without excessive drinking history, becomes with hepatic parenchymal cells fat
Property and pile up the clinical syndrome that is characterized, be modal hepatic disease, in China, sickness rate is about 25%
To 40%.Along with improving constantly of China's national life level, dietary structure constantly changes, high heat, high fat
Diet is very popular.Additionally, with the quickening pace of modern life, the increase of operating pressure, a lot of people lack exercise
Deng, cause this disease to present a rapidly rising trend, if not being controlled by, easily develop into hepatic fibrosis and hepatocarcinoma.
Liver lipid metabolism regulated and control network is extremely complex, in a large number the most relevant to liver lipid metabolism genes by
Find that gene as relevant in fatty acid synthesis: SCD-1, SREBP-1C, ACC etc. closes with triglyceride
The gene that one-tenth is relevant: PPAR γ, LXR α, DGAT1, DGAT2 etc., the gene relevant to fatty acid oxidation:
PPAR α, CPT1, MCAD, LCAD, UCP2 etc., the gene relevant to fatty acid absorption: CD36,
PPAR δ etc..
MiRNA is the endogenous non-protein coding RNA of 18 to 25 nucleotide of class, mammal and
By being attached to the 3 ' untranslated regions of target gene mRNA in human cell, the translation of suppression target protein.
Hsa-miR-221-3p is initially found in overexpression in human liver cancer cell, close with hepatocyte growth and liver regeneration
Cut is closed.Research shows, in adiposis patient adipose cell, miR-221 presents overexpression.Additionally,
In muscle differentiation and process of insulin resistance, miR-221 also plays key player.
At present, there is not yet by suppressing hsa-miR-221-3p effectively to treat non-alcoholic fatty liver disease and hyperlipemia
The correlational study of disease, insulin resistant etc..
Summary of the invention
In view of this, the invention provides a kind of suppress hepatocyte hsa-miR-221-3p oligonucleotide sequence,
Recombinant adenovirus and preparation method thereof, and reduce liver and the application of blood triglyceride, improve non-alcoholic fat
Fat hepatopath's Anomalous lipid metablism and increase insulin sensitivity.
First aspect present invention provides a kind of oligonucleotide sequence suppressing hsa-miR-221-3p, described few core
Nucleotide sequence is the one in following sequence:
Sequence 1:(GAAACCCAGCTGACAATGTAGCTAAGA)n≧5
Sequence 2:(GAAACCCAGCCGACAATGTAGCTAAGA)n≧5
Sequence 3:(GAAACCCAGCGGACAATGTAGCTAAGA)n≧5
Sequence 4:(GAAACCCAGCGACAATGTAGCTAAGA)n≧5
Or AAGA part other intervening sequences replacement gained sequence in above sequence.
Second aspect present invention provide the protokaryon containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence,
Eucaryon shuttle vector, adenovirus vector.
Third aspect present invention provides the restructuring containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence
Adenovirus and the application in preparation treatment non-alcoholic fatty liver disease product thereof.
Fourth aspect present invention provides the restructuring containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence
The preparation method of adenovirus, its step includes:
A, synthesize the oligonucleotide sequence of above-mentioned suppression hsa-miR-221-3p；
B, obtain shRNA according to step a gained oligonucleotide sequence, and be attached with shuttle vector, it is thus achieved that press down
The recombinant shuttle plasmid of hsa-miR-221-3p processed；
C, by the gained recombinant shuttle plasmid linearisation of step b, then with adenoviral backbone carrier cotransformation in vitro to sense
By in state cell, obtained the recombinant adenoviral vector of suppression hsa-miR-221-3p by homologous recombination；
D, will be with liposome transfection 293A cell after the resulting vehicle linearisation of step c, it is thus achieved that suppression hsa-miR-221-3p
Recombinant adenovirus.
The invention has the beneficial effects as follows: the invention provides the recombinant adenovirus of suppression has-miR-221-3p
(Ad-s-miR-221-3p) application in terms of non-alcoholic fatty liver disease treatment, ripe for has-miR-221-3p
Body sequence, devises the oligonucleotide comprising more than at least 5 repetitive sequences, uses intracellular DNA homology weight
Group method prepares the adenovirus that can suppress has-miR-221-3p.Suppression has-miR-221-3p prepared by the present invention
Recombinant adenovirus titre can reach 1011pfu/ml.The weight of suppression has-miR-221-3p prepared by the present invention
The application in terms for the treatment of non-alcoholic fatty liver disease of the group adenovirus includes reducing liver tg level, reduces blood
Triglyceride levels, improves hyperlipemia, increases body insulin sensitivity.Given by tail vein
Ad-s-miR-221-3p treatment db/db or DIO model mice experiment, it can be observed that: Ad-s-miR-221-3p
Treatment can significantly improve hyperlipemia, improves disorders of lipid metabolism, reduces model mice liver tg level,
Reduce blood triglyceride levels, increase mouse islets element sensitivity.Ad-s-miR-221-3p reduce liver and
Blood triglyceride aspect has obviously effect and effect, present invention finds new potential target spot, for improving
Non-alcoholic fatty liver disease and hyperlipemia, insulin resistant provide new resolving ideas.
Accompanying drawing explanation
Fig. 1 is db/db mouse liver hsa-miR-221-3p expression before and after restructuring Adenoviral Therapy.
Fig. 2 is DIO mouse liver hsa-miR-221-3p expression before and after restructuring Adenoviral Therapy.
Fig. 3 is DIO mouse liver and blood triglyceride levels before and after restructuring Adenoviral Therapy.
Fig. 4 is db/db mouse liver and blood triglyceride levels before and after restructuring Adenoviral Therapy.
Fig. 5 is DIO mouse islets element tolerance curve before and after restructuring Adenoviral Therapy.
Detailed description of the invention
First aspect present invention provides a kind of oligonucleotide sequence suppressing hsa-miR-221-3p, described few core
Nucleotide sequence is the one in following sequence:
Sequence 1:(GAAACCCAGCTGACAATGTAGCTAAGA)n≧5
Sequence 2:(GAAACCCAGCCGACAATGTAGCTAAGA)n≧5
Sequence 3:(GAAACCCAGCGGACAATGTAGCTAAGA)n≧5
Sequence 4:(GAAACCCAGCGACAATGTAGCTAAGA)n≧5
Wherein, in four kinds of sequences, AAGA part is intervening sequence, therefore in above sequence, AAGA part uses it
His intervening sequence replaces gained sequence also to have effects equivalent.
The oligonucleotide sequence of above-mentioned suppression hsa-miR-221-3p by design 5 above in relation to
Hsa-miR-221-3p maturation body sequence is connected, and introduces intervening sequence (AAGA) and prepare, and born of the same parents
Inside carry out DNA homology recombination method and prepare recombinant adenovirus.
Second aspect present invention provide the protokaryon containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence,
Eucaryon shuttle vector, recombinant adenoviral vector.
Third aspect present invention provides the restructuring containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence
Adenovirus and the application in preparation treatment non-alcoholic fatty liver disease product thereof.
More detailed, described treatment non-alcoholic fatty liver disease product is for reducing liver tg level, reduction
Blood triglyceride levels, increases the product of body insulin sensitivity.
Fourth aspect present invention provides the restructuring containing above-mentioned suppression hsa-miR-221-3p oligonucleotide sequence
The preparation method of adenovirus, its step includes:
A, synthesize the oligonucleotide sequence of above-mentioned suppression hsa-miR-221-3p；
B, obtain shRNA according to step a gained oligonucleotide sequence, and be attached with shuttle vector, it is thus achieved that press down
The recombinant shuttle plasmid of hsa-miR-221-3p processed；
C, by the gained recombinant shuttle plasmid linearisation of step b, then with adenoviral backbone carrier cotransformation in vitro to sense
By in state cell, obtained the recombinant adenoviral vector of suppression hsa-miR-221-3p by homologous recombination；
D, will be with liposome transfection 293A cell after the resulting vehicle linearisation of step c, it is thus achieved that suppression hsa-miR-221-3p
Recombinant adenovirus.
More detailed, described in step a, shRNA two ends introduce BgL2 and HindIII restriction enzyme site respectively,
Annealing forms duplex structure, and this duplex structure and the Track-U6 crossed through BgL2 and HindIII enzyme action shuttle back and forth load
Body is attached.
After obtaining the recombinant adenovirus described in step d, it is enlarged cultivating in 293A cell, and uses chlorine
Change caesium density gradient centrifugation to be purified and concentrate.The recombinant adenovirus purity obtained after purification is high, titre is high,
Meet relevant national standard.
A kind of restructuring gland the suppressing hsa-miR-221-3p present invention provided below in conjunction with specific embodiment
Virus application in terms of non-alcoholic fatty liver disease treatment is further described.
The present invention devises 4 kinds of suppression oligonucleotide sequences for hsa-miR-221-3p, as follows:
Sequence 1:(GAAACCCAGCTGACAATGTAGCTAAGA)n≧5
Sequence 2:(GAAACCCAGCCGACAATGTAGCTAAGA)n≧5
Sequence 3:(GAAACCCAGCGGACAATGTAGCTAAGA)n≧5
Sequence 4:(GAAACCCAGCGACAATGTAGCTAAGA)n≧5
Intervening sequence AAGA part in above-mentioned four kinds of sequences can replace by other sequences.
In the present embodiment, according to the above difference designing copy number, following 4 class oligonucleotides of synthesis, amount to
16 kinds of nucleotide sequences, specific as follows:
The first kind:
Sponge1-5: as shown in SEQ NO.1；
Sponge1-6: as shown in SEQ NO.2；
Sponge1-7: as shown in SEQ NO.3；
Sponge1-8: as shown in SEQ NO.4；
Equations of The Second Kind:
Sponge2-5: as shown in SEQ NO.5；
Sponge2-6: as shown in SEQ NO.6；
Sponge2-7: as shown in SEQ NO.7；
Sponge2-8: as shown in SEQ NO.8；
3rd class:
Sponge3-5: as shown in SEQ NO.9；
Sponge3-6: as shown in SEQ NO.10；
Sponge3-7: as shown in SEQ NO.11；
Sponge3-8: as shown in SEQ NO.12；
4th class:
Sponge4-5: as shown in SEQ NO.13；
Sponge4-6: as shown in SEQ NO.14；
Sponge4-7: as shown in SEQ NO.15；
Sponge4-8: as shown in SEQ NO.16.
According to above-mentioned 16 kinds of DNA sequence, design shRNA, introduce respectively at shRNA two ends BgL2 and
HindIII restriction enzyme site, annealing forms duplex structure, this duplex structure directly with through BgL2 and HindIII enzyme
The Track-U6 shuttle vector cut through is attached, it is thus achieved that the recombinant shuttle plasmid of suppression hsa-miR-221-3p.
On the basis of above, build recombinant adenovirus, and amplification culture, concentrate and purify.Technical scheme is as follows:
By the shuttle vector of above-mentioned structure, carry out linearisation with PmeI, then pass through in vitro with adenoviral backbone carrier
Electroporation cotransformation, in competent cell, obtains the restructuring gland of suppression hsa-miR-221-3p by homologous recombination
Viral vector.By this carrier with using liposome transfection 293A cell after PacI linearisation, it is thus achieved that suppression
The recombinant adenovirus of hsa-miR-221-3p.Recombinant adenovirus expands in 293A cell in a large number, and adopts
Being purified with cesium chloride density gradient centrifugation and concentrate, the recombinant adenovirus purity obtained is high, titre is high, symbol
Close relevant national standard.
The concrete operations of the present embodiment are as follows:
One, design and the sub-clone of hsa-miR-221-3p oligonucleotide fragment are suppressed
1., according to 16 kinds of miRNA sponge of 4 class of above-mentioned design, introduce BgL2 and HindIII at two ends
Restriction enzyme site, synthetic oligonucleotide fragment, join in the annealing buffer boiled, be put into 4 degree of refrigerators natural
Cool overnight.
2. take 2 microgram Track-U6 carriers, be simultaneously introduced BgL2 and the HindIII restriction endonuclease of 10 units,
And add appropriate amount of buffer solution, 37 degree of enzyme action 5 hours.Carry out cutting glue after gel electrophoresis to reclaim.
3. take 1 microlitre and reclaim product, add 7 microlitre annealed product, 1 microlitre T4DNA ligase and buffering
Liquid, 16 degree connect overnight.
Take 10 microlitres connect products, transformed competence colibacillus cell, after overnight incubation, picking monoclonal, send company
Order-checking.Sequencing result is the most completely the same with implementation sequence.
Two, suppression hsa-miR-221-3p oligonucleotide fragment builds recombinant adenovirus
1. by the shuttle vector comprising suppression hsa-miR-221-3p oligonucleotide fragment by converting, expanding training
Support, after large quantity extracting plasmid DNA, take 5 microgram high-purity shuttle plasmids, add the PmeI of 25 units
Enzyme action 5 hours.Gel electrophoresis rear cutout glue reclaims.
2. take 6 microlitres and reclaim product, add 100 nanogram adenoviral backbone carriers, join 20 after mixing micro-
Rise in BJ5183 competent cell, 2500V, 200Ohms, carry out electroporation under the conditions of 25 μ F.Then use
500 μ l preheat the resuspended thalline of LB culture medium of 37 degree and manage to aseptic EP, and 37 degree are incubated 3 to 5 minutes.Take
100 μ l coatings containing the LB resistant panel of kanamycin, 37 degree of constant temperature culture 16 hours.
3. extract recombiant plasmid, carry out plasmid after conversion and prepare in a large number, through PacI linearization for enzyme restriction, then use fat
Linearisation product is transfected in 293A cell by plastid, it is thus achieved that recombinant adenovirus.
4., after recombinant adenovirus expands in 293A cell, use cesium chloride step gradients to be centrifuged pure
Change and concentrate, finally measuring virus titer, 10 can be reached11pfu/ml。
Three, recombinant adenovirus suppression hsa-miR-221-3p reduces db/db and DIO model mice liver and blood
The application of triglyceride
1. experimental animal model prepares
Db/db mice, 4-6 week old, female, normal diet is fed, and freely drinks water, and measures body weight every other day, and 8
To 9 week old, during body weight about 28 grams, start tail vein injection recombinant adenovirus.
DIO (obesity mice of diet induced) mouse model builds: 3 week old C57BL/6 female mices, uses
D12492 high fat diet is fed, 8 to 9 week old, selects the body weight individuality higher than Normal group 20%
For Experiment on therapy.
2. the lipid-lowering therapy of animal model
Female db/db or DIO mice is respectively divided into two groups, often group 7 to 8, and single metering tail vein is respectively
Injection Ad-GFP (1 × 1010Pfu/0.2ml, normal saline) and recombinant adenovirus (1 × 1010Pfu/0.2ml,
Normal saline).
After injecting 7 days, carrying out insulin resistant experiment, the 8th day eye socket takes blood examination and surveys blood triglyceride levels,
Put to death mice, detect liver tg level.
Wherein, eye socket takes blood and uses the method plucking eyeball, and 3000rpm is centrifuged 10 minutes and separates serum, sample
Send clinical laboratory's detection triglyceride levels to hospital.
Liver takes hepatic tissue after weighing, and carries out homogenized with lysate, and 3000rpm is centrifuged 10 minutes, takes
200 microlitres send mensuration triglyceride levels to hospital.
Experimental result
It is little that 1.DIO and db/db mouse liver hsa-miR-221-3p expression is significantly higher than C57BL6 comparison
Mus.
2. db/db mouse liver hsa-miR-221-3p expression such as accompanying drawing 1 institute before and after recombinant adenovirus treatment
Showing, before and after recombinant adenovirus treatment, DIO mouse liver hsa-miR-221-3p expression is as shown in Figure 2.
In two kinds of mouse models, injection suppression hsa-miR-221-3p recombinant adenovirus group with injection Ad-GFP group compared with,
Liver hsa-miR-221-3p expression all reduces about 70%.
3. before and after recombinant adenovirus treatment, DIO mouse liver and blood triglyceride levels are as shown in Figure 3, heavy
Before and after group Adenoviral Therapy, db/db mouse liver and blood triglyceride levels are as shown in Figure 4.Two kinds of mice moulds
In type, injection suppression hsa-miR-221-3p recombinant adenovirus group with injection Ad-GFP group compared with, liver glycerol
Three esters and blood triglyceride levels all significantly reduce.
4. before and after recombinant adenovirus is treated, DIO mouse islets element tolerance curve is as shown in Figure 5, and insulin is resistance to
Shown by experimental result, injection suppression hsa-miR-221-3p recombinant adenovirus group, with injection Ad-GFP group phase
Ratio, insulin resistant is obviously improved.
The recombinant adenovirus that the present embodiment provides application in terms of improving disorders of lipid metabolism, passes through tail vein injection
Db/db or DIO mouse model, it can be observed that, the recombinant adenovirus of present invention design can significantly reduce
State liver and the blood triglyceride levels of two kinds of model mices, increase insulin sensitivity.Therefore may certify that,
The recombinant adenovirus of the suppression hsa-miR-221-3p of present invention design is reducing liver and blood triglyceride, improvement
Insulin sensitivity aspect have positive, effective, act on reliably, for treatment non-alcoholic fatty liver disease and hyperlipemia
Disease, insulin resistant provide new resolving ideas.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement etc. made, should be included in the protection of the present invention
Within the scope of.
Claims (8)
1. the oligonucleotide sequence suppressing hsa-miR-221-3p, it is characterised in that: described oligonucleotide
Sequence is the one in following sequence:
Sequence 1:(GAAACCCAGCTGACAATGTAGCTAAGA)n≧5
Sequence 2:(GAAACCCAGCCGACAATGTAGCTAAGA)n≧5
Sequence 3:(GAAACCCAGCGGACAATGTAGCTAAGA)n≧5
Sequence 4:(GAAACCCAGCGACAATGTAGCTAAGA)n≧5
Or AAGA part other intervening sequences replacement gained sequence in above sequence.
2. contain the protokaryon of the oligonucleotide sequence of suppression hsa-miR-221-3p described in claim 1, true
Core shuttle vector, adenovirus vector.
3. contain the recombinant adenovirus of the oligonucleotide sequence of suppression hsa-miR-221-3p described in claim 1
Poison.
4. the application of recombinant adenovirus as claimed in claim 3, it is characterised in that: described recombinant adenovirus
Application in preparation treatment non-alcoholic fatty liver disease product.
5. the application of recombinant adenovirus as claimed in claim 4, it is characterised in that: described treatment non-alcoholic
Fatty liver product, for reducing liver tg level, reduces blood triglyceride levels, increases body insulin quick
The product of perception.
6. the preparation method of recombinant adenovirus as claimed in claim 3, its step includes:
A, the oligonucleotide sequence of synthesis suppression hsa-miR-221-3p described in claim 1；
B, obtain shRNA according to step a gained oligonucleotide sequence, and be attached with shuttle vector, it is thus achieved that press down
The recombinant shuttle plasmid of hsa-miR-221-3p processed；
C, by the gained recombinant shuttle plasmid linearisation of step b, then with adenoviral backbone carrier cotransformation in vitro to sense
By in state cell, obtained the recombinant adenoviral vector of suppression hsa-miR-221-3p by homologous recombination；
D, will be with liposome transfection 293A cell after the resulting vehicle linearisation of step c, it is thus achieved that suppression hsa-miR-221-3p
Recombinant adenovirus.
7. preparation method as claimed in claim 6, it is characterised in that: described in step a, shRNA divides at two ends
Not Yin Ru BgL2 and HindIII restriction enzyme site, annealing formed duplex structure, this duplex structure with through BgL2
The Track-U6 shuttle vector crossed with HindIII enzyme action is attached.
8. preparation method as claimed in claim 7, it is characterised in that: obtain the restructuring gland described in step d
After virus, be enlarged in 293A cell cultivate, and use cesium chloride density gradient centrifugation be purified and
Concentrate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136224A (en) * | 2018-08-21 | 2019-01-04 | 上海交通大学医学院附属瑞金医院 | MiR-221/222 and its inhibitor are used to prepare the drug of regulation liver fat deposition, liver fibrosis and hepatocellular carcinoma |
| CN114231626A (en) * | 2021-10-14 | 2022-03-25 | 杭州师范大学 | Application of miRNA (micro ribonucleic acid) combined marker in preparation of kit for diagnosing and detecting early liver cancer |
| CN115354032A (en) * | 2022-05-05 | 2022-11-18 | 陈龙 | A549 stable transgenic cell strain inhibited by hsa-miR-130b and construction method thereof |
-
2016
- 2016-03-11 CN CN201610137218.0A patent/CN105754999B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| GENG WEI: "ROLES OF microRNAs IN FATTY LIVER LIPID ACCUMULATION", 《NATIONAL UNIVERSITY OF SINGAPORE》 * |
| JIEXIA DING ET AL: "Effect of miR-34a in regulating steatosis by targeting PPARα expression in nonalcoholic fatty liver disease", 《SCIENTIFIC REPORTS》 * |
| 闫继红等: "共沉默miR-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促成软骨分化", 《中国组织工程研究》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136224A (en) * | 2018-08-21 | 2019-01-04 | 上海交通大学医学院附属瑞金医院 | MiR-221/222 and its inhibitor are used to prepare the drug of regulation liver fat deposition, liver fibrosis and hepatocellular carcinoma |
| CN109136224B (en) * | 2018-08-21 | 2022-03-18 | 上海交通大学医学院附属瑞金医院 | miR-221/222 and inhibitor thereof for preparing medicine for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma |
| CN114231626A (en) * | 2021-10-14 | 2022-03-25 | 杭州师范大学 | Application of miRNA (micro ribonucleic acid) combined marker in preparation of kit for diagnosing and detecting early liver cancer |
| CN115354032A (en) * | 2022-05-05 | 2022-11-18 | 陈龙 | A549 stable transgenic cell strain inhibited by hsa-miR-130b and construction method thereof |
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