CN105087586B - It is a kind of inhibit ox MEN1 gene expressions siRNAs sequences and its application - Google Patents
It is a kind of inhibit ox MEN1 gene expressions siRNAs sequences and its application Download PDFInfo
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Abstract
The present invention relates to Animal genetics and animal molecular cytobiology, provide the siRNAs for inhibiting ox MEN1 gene expressions, the siRNAs is small double-stranded RNA, including three siRNAs, and positive-sense strand nucleotides sequence is classified as SEQ NO.4, SEQ NO.5, shown in SEQ NO.6；Its antisense strand nucleotides sequence is classified as shown in SEQ NO.7, SEQ NO.8 and SEQ NO.9；The present invention provides the siRNAs of the inhibition ox MEN1 genes containing above-mentioned positive antisense strand can effectively inhibit ox MEN1 gene expressions, can provide effective technical tool for the research of ox MEN1 gene functions, and can be used for the correlative study of ox metabolic disease.
Description
Technical field
The present invention relates to Animal genetics and animal molecular cytobiology, provide a kind of inhibition ox MEN1 gene expressions
SiRNAs sequences and its application.
Background technology
Ox MEN1 genes and people or the MEN1 very high homologies of mouse, coding albumen menin and people or the homology of mouse
It is up to 98.69% and 96.73% respectively.The achievement in research of people or mouse implies the MEN1/menin of ox in ox (milk cow) machine of adjusting
Important regulative in body eubolism, thus it is speculated that metabolic disturbance diseases (ketoacidosis, acid poisoning, fat of its unconventionality expression and ox
Liver etc.) breaking-out have substantial connection.Recent studies indicate that menin is modified by participating in gene histone epigenetics
And then the transcription of influence downstream gene, the stability that genome can be also mutually made adjustments with genome protein, participation adjusting cell
The regulatory factor in period and the division growth of regulating cell, abnormal expression and leukaemia, diabetes, (non-alcoholic) fatty liver
Etc. diseases it is closely related.Meanwhile the cancerous organ of MEN1 initiations is typically the vitals of hormone secretion, such as beta Cell of islet point
Insulin, anterior pituitary Prolactin Secretion etc. are secreted, thus more highlights menin in the machine for regulating and controlling body normal development, balancing stable state
Critical function in body metabolic process.Menin/MEN1 is used currently, many researchers of MEN1/menin have begun prospect
In the treatment of some diseases.Inhibit the expression of MEN1/menin in cell that can explore announcement menin in vitro for researcher adjustable
The specific biological activity and function of control provide effective means and tool, also provide necessity to be used for the gene therapy of disease in the future
Research foundation.
RNA interference (RNA interference) technology has been developed in recent years a kind of most having for inhibition of gene expression
One of tool of power.This technology causes the mRNA of sequence homology using the double-stranded RNA of 19-23 base or hairpin different length
Degradation can be such that the target gene of different type cell expresses and be substantially reduced, this phenomenon is conservative and extensive during evolution.
This emerging biotechnology is used to inhibit the expression of certain overexpressed genes, and the expression without influencing normal gene is being fed
RNA interference technology is established in newborn zooblast, can be used for studying the function of certain specific genes, so as to solve certain diseases
The pathogenesis of disease, and new technology and methods are provided for the treatment of disease molecular level, there is certain application value.Cause
How this realizes the expression for inhibiting MEN1/menin in cell as one of the emphasis of research using the technology.
Invention content
The present inventor provides under above-mentioned technical background and inhibits ox MEN1 gene expressions
SiRNAs, the siRNAs are small double-stranded RNA, including three siRNAs, positive-sense strand nucleotides sequence be classified as SEQ NO.4,
Shown in SEQ NO.5, SEQ NO.6；Its antisense strand nucleotides sequence is classified as shown in SEQ NO.7, SEQ NO.8 and SEQ NO.9；This
The siRNAs that invention provides the inhibition ox MEN1 genes containing above-mentioned positive antisense strand can effectively inhibit ox MEN1 gene expressions, can
Research for ox MEN1 gene functions provides effective technical tool, and can be used for the correlative study of ox metabolic disease.
The siRNAs provided by the present invention for inhibiting ox MEN1 gene expressions, the siRNAs are small double-stranded RNA, including
Three siRNAs, positive-sense strand nucleotides sequence are classified as SEQ NO.4, SEQ NO.5, shown in SEQ NO.6；Its antisense strand nucleotide
Sequence is shown in SEQ NO.7, SEQ NO.8 and SEQ NO.9.
Above-mentioned sequence is obtained by following process：
It is complete according to ox MEN1 in Genebank (Bos taurus multiple endocrine neoplasia I) gene
Whole mRNA sequence (accession NO.：NM_001076161), three target sequences are selected, and then designs and special can inhibit ox
The siRNAs of MEN1 gene expressions.
Selected target sequence is respectively:
Nucleotide sequence such as SEQ NO.1：Shown in 5 ' CCGTTGACCTGTCTCTCTA3 '；
Nucleotide sequence such as SEQ NO.2：Shown in 5 ' CCACTGTCGTAACCGCAAT3 '；
Nucleotide sequence such as SEQ NO.3：Shown in 5 ' CCGAGTGACTACACGCTTT3 '；
The ends the TT method design positive-sense strand corresponding with synthesis above three target sequence and antisense of RNA oligo are utilized later
Chain, wherein positive-sense strand nucleotide sequence are followed successively by SEQ NO.4：Shown in 5 ' CCGUUGACCUGUCUCUCUAdTdT 3 '；SEQ
NO.5：Shown in 5 ' CCACUGUCGUAACCGCAAUdTdT 3 '；SEQ NO.6：5 ' CCGAGUGACUACACGCUUUdTdT3 ' institutes
Show；
Its antisense strand sequence is the sequence with corresponding sense strand sequence reverse complemental, and chain nucleotide sequence is followed successively by SEQ
NO.7：3 ' dTdT GGCAACUGGACAGAGAGAU 5 are ' shown；SEQ NO.8：3‘dTdT GGUGACAGCAUUGGCGUUA 5
It is ' shown；SEQ NO.9：3 ' dTdT GGCUCACUGAUGUGCGAAA 5 are ' shown.
Inventor is after obtaining above-mentioned positive-sense strand and antisense strand, using which give main object of the present invention genes
Inhibit the siRNAs of ox MEN1 gene expressions, the specific method is as follows：
The sequence of the positive and negative adopted chain is dissolved as 200 μM of concentration with DEPC water respectively after RNA oligo synthesis,
Double-stranded RNA is formed by annealing；The positive and negative justice chain RNA oligo Yu annealing buffer (10 × buffer solution of equivalent later：100mM
Trisbase, 1M NaCl, 10mM EDTA) mixing, on being incubated heat block after 90 DEG C of 2min, close power supply, natural cooling annealing
To room temperature (about 25 DEG C)；It is again turned on after power supply assigns at 90 DEG C and educate 2min, cooled to room temperature can be directly used for transfecting or put
4 DEG C are placed in temporarily to preserve.
After above-mentioned annealing, you can three siRNAs double-strands are obtained, wherein：
(1)sibMEN-1：By positive-sense strand nucleotide sequence SEQ NO.4 and antisense strand nucleotide sequence SEQ NO.7 annealing
It is formed；
(2)sibMEN-2：By positive-sense strand nucleotide sequence SEQ NO.5 and antisense strand nucleotide sequence SEQ NO.8 annealing
It is formed；
(3)sibMEN-3：By positive-sense strand nucleotide sequence SEQ NO.6 and antisense strand nucleotide sequence SEQ NO.9 annealing
It is formed.
Three siRNAs double-strands of above-mentioned acquisition are further carried out cell transfecting by inventor, turn then to utilize real-time fluorescence
Quantitative RT-PCR detects ox MEN1mRNA expressions, as a result, it has been found that three siRNAs can effectively inhibit ox MEN1 genes
Transcription, and it was found that three siRNAs are mixed to form a siRNAs pool, i.e., after sibMEN-1/2/3 transfectional cells for 24 hours
Jamming effectiveness highest it is best.
In conclusion the siRNAs of the inhibition ox MEN1 genes provided by the present invention containing above-mentioned positive antisense strand can be effective
Inhibit ox MEN1 gene expressions, effective technical tool can be provided for the research of ox MEN1 gene functions, and can be used for ox metabolism disease
The correlative study of disease.
Description of the drawings
Fig. 1 is MEN1mRNA expression block diagrams after transfection siRNAs,
Fig. 2 is to analyze block diagram for MEN1mRNA expressions after sibMEN-1 and sibMEN-1/2/3；
Fig. 3 is the expression of results schematic diagram of menin albumen after transfecting sibMEN-1 and sibMEN-1/2/3；
Fig. 4 is the expression analysis block diagram of menin albumen after transfecting sibMEN-1 and sibMEN-1/2/3.
Specific implementation mode
The synthesis of the corresponding RNA sequences of embodiment 1.siRNAs
According to the RNA oligo that the sequent synthesis provided in sequence table SEQ NO.4~9 is single-stranded, equal proportion is arranged in pairs or groups two-by-two
(SEQ No.4 and SEQ NO.7；SEQ No.5 and SEQ NO.8；SEQ No.6 and SEQ NO.9) respectively with annealing buffer (10
× buffer solution：100mM Trisbase, 1M NaCl, 10mM EDTA；PH7.4 it) mixes, and then anneals and form double-strand：It is being incubated
In heat block after 90 DEG C of 2min, power supply is closed, natural cooling is annealed to room temperature (about 25 DEG C)；Power supply is again turned on to educate in 90 DEG C of taxes
After 2min, cooled to room temperature can be directly used for transfecting or being positioned over 4 DEG C of temporarily preservations.
Sequence SEQ No.4 and SEQ the NO.7 siRNA to be formed that anneal are denoted as sibMEN-1；SEQ No.5 and SEQ
The NO.8 siRNA to be formed that anneal are denoted as sibMEN-2；SEQ No.6 and SEQ the NO.9 siRNA to be formed that anneal are denoted as sibMEN-
3。
2. bovine mammary epithelial cell of embodiment transfects
Cell culture
Bovine mammary epithelial cell MAC-T is used containing in 10%FBS (Gibico, US.), dual anti-(1%) added with mycillin
In DMEM (GibicoTM, US.) culture medium, 37 DEG C, 5%CO2Under conditions of cultivate and be in exponential phase after two weeks
Cell is transfected, and cell is coated in 6- orifice plates with without dual anti-culture medium for 24 hours before transfection.
Cell transfecting
Take that growth conditions are good, the cow mammary gland epithelial cells in exponential phase, with collected by trypsinisation cell, from
It is resuspended with complete medium after the heart, after cell count, with 8.0 × 105A cells/well is uniformly inoculated in 6 orifice plates, remains cell
Transfection.Cell transfecting is divided into 5 groups：1. Control transfection groups；2. sibMEN-1 transfection groups；3. sibMEN-2 transfection groups；④
SibMEN-3 transfection groups；5. sibMEN-1/2/3 transfection groups.
Wait for that cell growth to degree of the converging about 70%-80% moment is transfected in 6 orifice plates, the concrete operation step of transfection is such as
Under：
(1) culture medium in 6 orifice plates is changed to without dual anti-, 10%FBS culture medium before transfecting.
(2) premix OPTI-MEM and s iRNAs：For every hole cell, s is diluted respectively using 150 μ l OPTI-MEM
IRNAs to final concentration of 50nM, is gently incubated at room temperature 5min after mixing.
(3) premix OPTI-MEM and Lipofectamine 2000：Per hole cell 10 μ l are diluted with 150 μ l OPTI-MEM
2000 transfection reagents of Lipofectamine, are gently incubated at room temperature 5min after mixing.
(4) mixing OPTI-MEM-s iRNAs (steps 2) and 2000 (steps 3) of OPTI-MEM-Lipofectamine, this
When total volume be 300 μ l, gently mixing and place 20min at ambient temperature, make s iRNAs and Lipofectamine 2000
Form effective compound.
(5) 300 μ l compounds are gently added dropwise in each hole, are gently shaken using crossing method after adding mixed
It is even.
(6) 6 porocyte culture plates are placed in 37 DEG C of cell incubators, complete culture is replaced medium to after 5-6 hours
Base.
(7) cell extraction total serum IgE and albumen are harvested after continuing culture 24-72 hours.
3 real-time fluorescence quantitative RT-PCR of embodiment detects ox MEN1mRNA expressions
Extract experiment each group mammary gland to specifications using RNAs imple Total RNA Kit total RNA extraction reagents box
The total serum IgE of epithelial cell, using the integrality of 1.0% plain agar sugar detected through gel electrophoresis total serum IgE, using ultraviolet spectrometry light
Spend concentration and extracting purity that meter method detects RNA under 260nm and 280nm wavelength respectively.According to PrimeScript TM RT
Total serum IgE reverse transcription is by the requirement of reagent Kit with gDNA Eraser (Perfect Real Time) kit
cDNA。
Real-time fluorescence quantitative PCR amplification is carried out as template, using SYBR Green I dye methods, utilizes TaKaRa public affairs
SYBRR Premix Ex TaqTM (Perfect Real Time) kit of department carries out glimmering in real time on 7500 instruments of ABI
Fluorescent Quantitative PCR.
The special positive-sense strand primer nucleotide sequences of MEN1 used such as Seq ID No.10：5'-gatggaggtggcatttatg
Shown in g-3 ' and MEN1 specific antisense strand primer nucleotide sequences are as shown in SEQ NO.11：5’-gatgtgctcatcccggtag
Shown in t-3 '.The internal reference gene special positive-sense strand primer nucleotide sequences of ox β-act in such as SEQ No.12：5'-
Shown in cccagcacaatgaagatcaa-3', β-actin specific antisense strand primers nucleotide sequences such as SEQ No.13：Adopted 5'-
Shown in tagaagcatttgcggtggac-3'.The analysis of MEN1mRNA expression quantity, which uses, compares Ct data method (2Δ Δ Ct) carry out.
Every group of experiment is repeated 3 times.
The results are shown in Figure 1, and the result after detection is shown：Three siRNAs can effectively inhibit turning for ox MEN1 genes
Record.After transfection for 24 hours, interference effect is best compared to the control group for sibMEN-1/2/3 transfection groups, and jamming effectiveness reaches 80%, and
SibMEN-1 transfection groups jamming effectiveness about 57%；48h after transfection, interference effect most preferably s ibMEN-1 transfection groups, interference
Efficiency reaches 59%, sibMEN-1/2/3 transfection groups jamming effectiveness about 48%；72h after transfection, interference effect is most preferably
SibMEN-1 transfection groups, jamming effectiveness reach 73%, and excess-three group jamming effectiveness is close, and about 67%.Generally speaking, s ibMEN-
1 is best with the inhibition efficiency of s ibMEN-1/2/3 and relatively stable.
Then, on the basis of this preliminary experiment, and biology repetition has been carried out for sibMEN-1 and sibMEN-1/2/3
Transfection experiment (n=3).It is for statistical analysis to result with 8.2 softwares of SAS, carry out significance test with t methods of inspection：Wherein, P
＜ 0.05 shows that there are significant difference, P<0.01 shows that there are pole significant differences.As a result as Fig. 2 is shown:After transfection for 24 hours, s
IbMEN-1, s ibMEN-1/2/3 transfection groups compared with the control group can pole significantly reduce MEN1mRNA expression quantity (P<0.01),
Jamming effectiveness respectively reaches 63%, 75%；48h after transfection, sibMEN-1, sibMEN-1/2/3 transfection group equally extremely can significantly drop
Expression quantity (the P of low MEN1mRNA<0.01), jamming effectiveness respectively reaches 55%, 50%；72h after transfection, sibMEN-1 transfection group
Compared with the control group still can pole significantly reduce MEN1mRNA expression quantity (P<0.01), jamming effectiveness 74%, s ibMEN-
1/2/3 transfection group can significantly reduce the expression quantity (P of MEN1mRNA<0.05), jamming effectiveness 62%.In general, for ox
MAC-T cells, the jamming effectiveness highest of sibMEN-1/2/3 (being mixed to form a siRNAs pool), are interfered for 24 hours after transfection
Efficiency is best, can reach 75%.
Ox MEN1 gene coded protein expressions in 4 Western blot detection transfectional cells of embodiment
SibMEN-1, sibMEN-1/2/3 are transfected respectively after MAC-T cells for 24 hours, that 48h and 72h extract cell respectively is total
Protein.Western-Blot detects the relative expression quantity of ox MEN1 coding albumen menin, using ox β-actin as internal reference egg
In vain.Mouse source β-actin antibody (AA128, the green skies) is purchased from the green skies biotechnology research institute in Beijing, and rabbit source menin is polyclonal
Antibody (A300-105A) is purchased from BETHYL, USA.HRP marks mountain sheep anti-mouse igg (A0216), goat anti-rabbit igg (A0208)
Purchased from the green skies biotechnology research institute in Beijing.Film (color scanner, BenQ SCANNER 5560) is scanned, is used in combination
The gray value of ImageJ softwares analysis purpose band, the ratio of destination protein and the gray value of internal reference albumen (β-actin), as
The albumen relative expression quantity of each processing group.
As a result as shown in Figures 3 and 4, protein expression detection display：After transfection for 24 hours, sibMEN-1, sibMEN-1/2/3 are transfected
Group compared with the control group can pole significantly reduce Menin albumen relative expression quantity (P<0.01), jamming effectiveness respectively reach 68%,
71%；48h after transfection, sibMEN-1 transfection group compared with the control group can pole significantly reduce Menin albumen expression quantity (P<
0.01) jamming effectiveness of, jamming effectiveness 64%, sibMEN-1/2/3 transfection groups does not have difference between 42%, with control group；
72h after transfection does not have difference between sibMEN-1, sibMEN-1/2/3 transfection group and control group.In general, for MAC-T
Cell, after transfecting sibMEN-1/2/3 (being mixed to form a siRNAs pool) for 24 hours, the expression efficiency of menin albumen is most occurred frequently
It can reach 71%.
As a result it is found that being detected through qRT-PCR, Western blot, 3 siRNAs in the invention can be interfered effectively
The mRNA of ox MEN1 genes is expressed, but three siRNAs are mixed to form a siRNAs pool, i.e. sibMEN-1/2/3 transfections are thin
Jamming effectiveness highest after born of the same parents for 24 hours is best, and sibMEN-1 takes second place.
Claims (1)
1. a kind of siRNAs inhibiting ox MEN1 gene expressions, it is characterised in that：The siRNAs is small double-stranded RNA, just
Adopted chain nucleotides sequence is classified as SEQ NO.4, SEQ NO.5, shown in SEQ NO.6;Its antisense strand nucleotides sequence be classified as SEQ NO.7,
Shown in SEQ NO.8 and SEQ NO.9；
In three siRNAs double-strands：
（1）sibMEN-1：It is annealed and is formed by positive-sense strand nucleotide sequence SEQ NO.4 and antisense strand nucleotide sequence SEQ NO.7；
（2）sibMEN-2：It is annealed and is formed by positive-sense strand nucleotide sequence SEQ NO.5 and antisense strand nucleotide sequence SEQ NO.8；
（3）sibMEN-3：It is annealed and is formed by positive-sense strand nucleotide sequence SEQ NO.6 and antisense strand nucleotide sequence SEQ NO.9.
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Non-Patent Citations (2)
| Title |
|---|
| Accession NO.：NM_001076161.3，Bos taurus menin 1 （MEN1）mRNA;Zimin AV;《GenBank Datebase》;20090424;全序列 * |
| Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells;Peter C. Scacheri et al.;《Proc Natl Acad Sci U S A》;20040217;第101卷(第7期);表1，图1和1894页左侧第一段 * |
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