CN110438126A - A kind of siRNA sequence and its reaction system inhibiting Nrf2 gene expression - Google Patents
A kind of siRNA sequence and its reaction system inhibiting Nrf2 gene expression Download PDFInfo
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- CN110438126A CN110438126A CN201910526036.6A CN201910526036A CN110438126A CN 110438126 A CN110438126 A CN 110438126A CN 201910526036 A CN201910526036 A CN 201910526036A CN 110438126 A CN110438126 A CN 110438126A
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
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Abstract
The invention discloses a kind of siRNA sequences and its reaction system for inhibiting Nrf2 gene expression, belong to field of biotechnology.The siRNA sequence includes: the first positive-sense strand and the second antisense strand, the sequence of first positive-sense strand includes First ray and 3 ' the base tt held for being connected to the First ray, the First ray is as shown in SEQ IN NO:1 in sequence table, the sequence of first antisense strand includes the second sequence and 3 ' the base tt held for being connected to second sequence, and second sequence is as shown in SEQ IN NO:2 in sequence table.SiRNA sequence provided in an embodiment of the present invention can effectively inhibit Nrf2 gene expression in mRNA level in-site and protein translation level.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of siRNA sequence for inhibiting Nrf2 gene expression and its anti-
Answer system.
Background technique
Nuclear factor E2-related factor 2 (Nuclear factor E2-related factor-2, Nrf2), also known as
NFE2L2 is the adjusting maincenter of body response to oxidative stress, belongs to Cap-n-collar (CNC) family, is to carry leucine
The nuclear factor of zipper.Under normal circumstances, Nrf2 can be degraded in cytoplasm, not play physiological action.Work as machine
Body occur oxidative stress when, Nrf2 be activated and indexing enter nucleus in, into nucleus after can be with Antioxidant responsive element
(antioxidant response element, ARE) is combined and is formed Nrf2-ARE signal path, to make a series of tools in downstream
The protective II phase detoxication enzyme of organism and antioxidase gene and albumen are expressed.
In the implementation of the present invention, the inventor finds that the existing technology has at least the following problems:
It can inhibit Nrf2 gene expression there is no effective method at present.
Summary of the invention
In order to solve problems in the prior art, the embodiment of the invention provides a kind of siRNA for inhibiting Nrf2 gene expression
Sequence and its reaction system.The technical solution is as follows:
On the one hand, the embodiment of the invention provides a kind of siRNA sequence for inhibiting Nrf2 gene expression, the siRNA sequences
Column include: the first positive-sense strand and the second antisense strand, and the sequence of first positive-sense strand includes First ray and is connected to described the
The base tt at 3 ' ends of one sequence, the First ray is as shown in SEQ IN NO:1 in sequence table, the sequence of first antisense strand
Column include the second sequence and 3 ' the base tt held for being connected to second sequence, SEQ IN in second sequence such as sequence table
Shown in NO:2.
Specifically, the siRNA sequence further include: the second positive-sense strand and the second antisense strand, second positive-sense strand include
Third sequence and be connected to the third sequence 3 ' ends base tt, SEQ IN NO:3 in the third sequence such as sequence table
Shown, second antisense strand includes the 4th sequence and 3 ' the base tt held for being connected to the 4th sequence, the 4th sequence
Column are as shown in SEQ IN NO:4 in sequence table.
On the other hand, the embodiment of the invention provides a kind of reaction system for inhibiting Nrf2 gene expression, the reactants
System includes siRNA sequence as described in claim 1.
Specifically, the reaction system further include: the second positive-sense strand and the second antisense strand.
Further, the reaction system further include: the improvement Du Shi Iger containing the fetal calf serum that concentration is 10%
Culture medium.
Further, the reaction system further include: transfection reagent.
Further, the reaction system further include: serum free medium.
Further, the concentration of the siRNA sequence is 20 μm of ol/L, her lattice of the siRNA sequence, the improvement Du Shi
The volume ratio of your culture medium, the transfection reagent and the serum free medium is 3:100:800:3.
Further, the reaction system further include: phosphate buffered saline solution.
Specifically, the reaction system further include: without enzyme and sterile water.
Technical solution provided in an embodiment of the present invention has the benefit that the embodiment of the invention provides a kind of inhibition
The siRNA sequence and its reaction system of Nrf2 gene expression, siRNA sequence provided in an embodiment of the present invention, in mRNA level in-site and
It can effectively inhibit Nrf2 gene expression in protein translation level.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is the glimmering of siRNA sequence transfection cattle uterus intimal epithelium cell Nrf2 gene provided by Embodiment 2 of the present invention
Light quantitative result comparison diagram；
Fig. 2 is the knot of siRNA-1672 transfection cattle uterus intimal epithelium cell Nrf2 gene provided by Embodiment 2 of the present invention
Fruit comparison diagram.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.
Embodiment one
The embodiment of the invention provides a kind of siRNA sequence for inhibiting Nrf2 gene expression, it is suitable for inhibiting in cattle uterus
The Nrf2 gene expression of film epithelial cell, the siRNA sequence include: the first positive-sense strand and the second antisense strand, the first positive-sense strand
Sequence includes First ray and 3 ' the base tt held for being connected to First ray, SEQ IN NO:1 in First ray such as sequence table
It is shown.Specifically, the first positive-sense strand is 5 '-gcuucagugauucugaaautt-3 ', and the sequence of the first antisense strand includes second
Sequence and 3 ' the base tt held for being connected to the second sequence, the second sequence is as shown in SEQ IN NO:2 in sequence table, specifically,
First antisense strand is 5 '-auuucagaaucacugaagctt-3 '.In the present embodiment, Nrf2 gene is GenBank
Accession Number:NM_001011678.2 is from the nucleotide of 5 ' ends the 1st to the 2409th.The embodiment of the present invention one mentions
The siRNA sequence of confession synthesizes and is made freeze-dried powder by Shanghai Ying Jun Biotechnology Co., Ltd, when designing siRNA sequence, In
- 3 ' ends of siRNA sequence increase base tt, this can be improved the stability of the siRNA sequence of synthesis.
Specifically, which can also include: the second positive-sense strand and the second antisense strand, and the second positive-sense strand includes the
Three sequences and 3 ' the base tt held for being connected to third sequence, third sequence is as shown in SEQ IN NO:3 in sequence table, specifically
Ground, the second positive-sense strand are 5 '-ggagcaagauuuagaucautt-3 '.Second antisense strand includes the 4th sequence and is connected to the 4th
The base tt at 3 ' ends of sequence, the 4th sequence is as shown in SEQ IN NO:4 in sequence table, and specifically, the second antisense strand is 5 '-
augaucuaaaucuugcucctt-3'.Second positive-sense strand and the second antisense strand can be same with the first positive-sense strand and the second antisense strand
When use, for further effectively inhibit Nrf2 gene expression.
Embodiment two
The embodiment of the invention provides a kind of reaction system for inhibiting Nrf2 gene expression, which includes the present invention
The siRNA sequence that embodiment one provides.
Specifically, which can also include: the second positive-sense strand and the second antisense strand.
Specifically, reaction system further include: the improvement Du Shi Iger culture containing the fetal calf serum that concentration is 10%
Base (Dulbecco's modification of Eagle's medium Dulbecco, DMEM).
Further, which can also include: transfection reagent.When realizing, transfection reagent can be3000。
Further, which can also include: serum free medium.When realizing, serum free medium can be with
For
Further, the concentration of the siRNA sequence be 20 μm of ol/L, siRNA sequence, improvement Du Shi Eagle's medium,
The volume ratio of transfection reagent and serum free medium can be 3:100:800:3.
Further, which can also include: phosphate buffered saline solution.Phosphate buffered saline solution is as rinsing
Agent, for rinsing cell suspension.
Further, which can also include: no enzyme and sterile water.It is prepared into the siRNA sequence of freeze-dried powder
When in use, it needs to carry out brief centrifugation after taking-up, using no enzyme and sterile water (RNase-free Water) is by freeze-dried powder
It is configured to the storing liquid that concentration is 20 μm of ol/L, packing is stored in -20 DEG C~-80 DEG C of environment, and freeze-dried powder should avoid as far as possible
Multigelation, even if needing multigelation, the number suggestion of multigelation is no more than 5 times.
After transfecting cattle uterus inner membrance intimal epithelium cell using reaction system provided by Embodiment 2 of the present invention, Nrf2 is detected
The variation of gene expression amount, concrete operations are as follows:
The cattle uterus intimal epithelium cell (being purchased from ATCC cell bank) of the freezen protective in liquid nitrogen is taken out, and in 37 DEG C of water
It carries out instant in bath, by the cattle uterus intimal epithelium cell after thawing in 1200 turns of centrifugation 5min, removes cells frozen storing liquid, protect
Sediment is stayed, and the improvement Du Shi Eagle's medium for the fetal calf serum (FBS) for being 10% containing concentration is added into sediment,
Simultaneously blow repeatedly it is even cell suspension is made, by cell suspension access Tissue Culture Flask in, Tissue Culture Flask is placed in 37 DEG C and is filled
Full CO2Incubator in cultivated.
In the day before transfection by cell suspension according to 1 × 105A/hole is inoculated in 6 orifice plates, and cell suspension culture is arrived
Kilter is transfected when the cell in cell suspension grows to 70% degrees of fusion.
Take the transfection reagent of 7.5 μ L3000 are added to 125 μ L'sSerum-free
It in culture medium and mixes, obtains the first solution.
The storing liquid that the concentration containing siRNA sequence of 7.5 μ L is 20 μm of ol/L is taken to be added to 125 μ L'sIt in serum free medium and mixes, wherein siRNA sequence includes First ray, the second sequence, third sequence
Column and the 4th sequence, obtain the second solution.
Second solution is added in the first solution, and is mixed well, in incubation at room temperature 5min, obtains third solution.
After being rinsed cell suspension 2 times using phosphate buffered saline solution (PBS), 2mL is added into cell suspension and contains concentration
For the improvement Du Shi Eagle's medium of 10% fetal calf serum, third solution is then added, and is 5% containing concentration in 37 DEG C
CO2It is cultivated for 24 hours in cell incubator.
Improvement Du Shi Eagle's medium old in cell suspension is removed, what addition was new into cell suspension is containing concentration
The improvement Du Shi Eagle's medium of 10% fetal calf serum.
Respectively after transfecting siRNA for 24 hours, 48h, 72h and 96h collect cell.
Extract the RNA in cell suspension, using real-time fluorescence quantitative PCR (Polymerase Chain Reaction, it is more
Poly- enzyme chain reaction) method detection Nrf2 gene expression the case where being disturbed, when detecting, the cattle uterus of siRNA will not added
Inner membrance intimal epithelium cell is used for as internal reference in detection albumen as blank control group (control group), by β-Actine
As object of reference when expression changes.
It is as shown in Figure 1 the case where mRNA level in-site is disturbed.As shown in Figure 1, after transfection for 24 hours when, the reaction system turn
The cattle uterus intimal epithelium cell Nrf2 gene expression of the siRNA sequence of final concentration of 50nmol/L has been contaminated lower than blank control group
(control group), siRNA sequence include: the first positive-sense strand (siRNA-1209), the first antisense strand (siRNA-1209), second
Positive-sense strand (siRNA-1672) and the second antisense strand (siRNA-1672)；Also transfect final concentration of 75nmol/L's simultaneously
The cattle uterus intimal epithelium cell Nrf2 gene expression difference of siRNA-1672 is extremely significant, and the inhibitory effect of siRNA-1672
Reach 90% or so.It can be effective it can be seen that demonstrating siRNA sequence provided in an embodiment of the present invention in mRNA level in-site
Inhibit Nrf2 gene expression.
It is as shown in Figure 2 that situation is disturbed in protein translation level.The final concentration of 75nmol/L of siRNA-1672 transfection,
Compared to after transfection for 24 hours, 48h, 72h and 96h, the protein expression of cattle uterus intimal epithelium cell Nrf2 gene opens in 72h
Begin to decline, it is extremely significant in 96h difference.It can be seen that being demonstrated in protein translation level provided in an embodiment of the present invention
SiRNA sequence can effectively inhibit Nrf2 gene expression.
The embodiment of the invention provides a kind of siRNA sequence and its reaction system for inhibiting Nrf2 gene expression, the present invention
The siRNA sequence that embodiment provides, can effectively inhibit Nrf2 gene expression in mRNA level in-site and protein translation level.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Academy of Agricultural Sciences, Wuhan City
<120>a kind of siRNA sequence and its reaction system for inhibiting Nrf2 gene expression
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213>artificial synthesized (Artificial Sequence)
<400> 1
gcuucaguga uucugaaau 19
<210> 2
<211> 19
<212> RNA
<213>artificial synthesized (Artificial Sequence)
<400> 2
auuucagaau cacugaagc 19
<210> 3
<211> 19
<212> RNA
<213>artificial synthesized (Artificial Sequence)
<400> 3
ggagcaagau uuagaucau 19
<210> 4
<211> 19
<212> RNA
<213>artificial synthesized (Artificial Sequence)
<400> 4
augaucuaaa ucuugcucc 19
Claims (10)
1. a kind of siRNA sequence for inhibiting Nrf2 gene expression, which is characterized in that the siRNA sequence includes: the first positive-sense strand
With the second antisense strand, the sequence of first positive-sense strand includes First ray and the 3 ' bases held for being connected to the First ray
Tt, for the First ray as shown in SEQ IN NO:1 in sequence table, the sequence of first antisense strand includes the second sequence and company
The base tt at 3 ' ends of second sequence is met, second sequence is as shown in SEQ IN NO:2 in sequence table.
2. siRNA sequence according to claim 1, which is characterized in that the siRNA sequence further include: the second positive-sense strand
With the second antisense strand, second positive-sense strand includes third sequence and 3 ' the base tt held for being connected to the third sequence, institute
Third sequence is stated as shown in SEQ IN NO:3 in sequence table, second antisense strand includes the 4th sequence and is connected to described the
The base tt at 3 ' ends of four sequences, the 4th sequence is as shown in SEQ IN NO:4 in sequence table.
3. a kind of reaction system for inhibiting Nrf2 gene expression, which is characterized in that the reaction system includes such as claim 1 institute
The siRNA sequence stated.
4. reaction system according to claim 3, which is characterized in that the reaction system further include: the second positive-sense strand and
Second antisense strand.
5. reaction system according to claim 3, which is characterized in that the reaction system further include: be containing concentration
The improvement Du Shi Eagle's medium of 10% fetal calf serum.
6. reaction system according to claim 5, which is characterized in that the reaction system further include: transfection reagent.
7. reaction system according to claim 6, which is characterized in that the reaction system further include: serum free medium.
8. reaction system according to claim 7, which is characterized in that the concentration of the siRNA sequence is 20 μm of ol/L, institute
State siRNA sequence, the volume ratio for improveing Du Shi Eagle's medium, the transfection reagent and the serum free medium is
3:100:800:3。
9. reaction system according to claim 3, which is characterized in that the reaction system further include: phosphate-buffered salt is molten
Liquid.
10. reaction system according to claim 3, which is characterized in that the reaction system further include: without enzyme and sterile
Water.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746112A (en) * | 2021-01-28 | 2021-05-04 | 武汉市农业科学院 | SNP marker related to milk production peak days of Holstein cows in south China and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102482677A (en) * | 2009-03-16 | 2012-05-30 | 欧科库尔纳有限责任公司 | Treatment of nuclear factor (erythroid-derived 2)-like 2 (nrf2) related diseases by inhibition of natural antisense transcript to nrf2 |
| WO2018117345A1 (en) * | 2016-12-20 | 2018-06-28 | 울산대학교 산학협력단 | Selective induction of cancer apoptosis through combined inhibition of glutathione, thioredoxin, and nrf2 antioxidant |
| KR20180099540A (en) * | 2017-02-27 | 2018-09-05 | 인하대학교 산학협력단 | A Composition for Increasing Radiosensitivity of Cancer Cells |
-
2019
- 2019-06-18 CN CN201910526036.6A patent/CN110438126A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102482677A (en) * | 2009-03-16 | 2012-05-30 | 欧科库尔纳有限责任公司 | Treatment of nuclear factor (erythroid-derived 2)-like 2 (nrf2) related diseases by inhibition of natural antisense transcript to nrf2 |
| WO2018117345A1 (en) * | 2016-12-20 | 2018-06-28 | 울산대학교 산학협력단 | Selective induction of cancer apoptosis through combined inhibition of glutathione, thioredoxin, and nrf2 antioxidant |
| KR20180099540A (en) * | 2017-02-27 | 2018-09-05 | 인하대학교 산학협력단 | A Composition for Increasing Radiosensitivity of Cancer Cells |
Non-Patent Citations (8)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746112A (en) * | 2021-01-28 | 2021-05-04 | 武汉市农业科学院 | SNP marker related to milk production peak days of Holstein cows in south China and application thereof |
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Application publication date: 20191112 |