CN105087586A - siRNAs sequence inhibiting expression of bovine MEN1 gene and application thereof - Google Patents
siRNAs sequence inhibiting expression of bovine MEN1 gene and application thereof Download PDFInfo
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Abstract
The present invention relates to animal genetics and animal molecular cell biology, and provides siRNAs inhibiting expression of a bovine MEN1 gene. The siRNAs are small double-stranded RNAs including three siRNAs; the sense strand nucleotide sequences are shown as SEQ NO.4, SEQ NO.5 and SEQ NO.6; and the antisense strand nucleotide sequences are shown as SEQ NO.7, SEQ NO.8 and SEQ NO.9. The present invention provided the siRNAs containing the above sense and antisense strands inhibiting bovine MEN1 gene can effectively suppress expression of bovine MEN1 gene, can provide effective technical tools for the study of bovine MEN1 gene function and can be applied to related study on bovine metabolic disorders.
Description
Technical field
The present invention relates to zoogenetics and animal molecular cytobiology, provide a kind of the siRNAs sequence and the application thereof that suppress ox MEN1 genetic expression.
Background technology
The MEN1 very high homology of ox MEN1 gene and people or mouse, the homology of its proteins encoded menin and people or mouse is respectively up to 98.69% and 96.73%.The MEN1/menin of the achievement in research hint ox of people or mouse, regulating the important regulative in ox (milk cow) body eubolism, infers that the outbreak of the metabolic disturbance diseases (ketoacidosis, oxypathy, fatty liver etc.) of its unconventionality expression and ox has substantial connection.Research in recent years shows, menin is by participating in genome albumen epigenetics and modify and then affecting the transcribing of downstream gene, also can make adjustments genomic stability mutually with genome protein, participate in regulating the regulatory factor of cell cycle and the division growth of regulating cell, and its abnormal expression and leukemia, diabetes, (non-alcoholic) fatty liver diseases are closely related.Simultaneously, the vitals of cancerous organ's normally hormone secretion that MEN1 causes, as islet β cell Regular Insulin, prepituitary gland Prolactin Secretion etc., thus more highlight the critical function of menin in the organism metabolism process of the normal development of regulation and control body, balance stable state.At present, many investigators of MEN1/menin have started to look forward to treatment menin/MEN1 being used for some diseases.In T suppression cell, the expression of MEN1/menin can provide effective means and instrument for investigator's external exploration announcement regulatable concrete biologic activity of menin and function, and the gene therapy also for being used for disease in the future provides necessary Research foundation.
RNA interferes (RNAinterference) technology to be one of the strongest instrument of a kind of inhibition of gene expression grown up in recent years.The double-stranded RNA of this technology employing 19-23 base or hairpin different lengths causes the mRNA of sequence homology to degrade, and the target gene of dissimilar cell can be made to express and obviously reduce, and this phenomenon is conservative and extensive during evolution.This emerging biotechnology is for suppressing the expression of some overexpressed genes; and do not affect the expression of normal gene; RNA interference technique is set up in mammalian cell; may be used for the function studying some specific gene; thus the pathogenesis of some disease can be solved; and provide new techniques and methods for the treatment of disease molecular level, there is certain using value.Therefore one of this technology expression emphasis becoming research realizing MEN1/menin in T suppression cell how is utilized.
Summary of the invention
The present inventor is under above-mentioned technical background, provide the siRNAs suppressing ox MEN1 genetic expression, described siRNAs is little double-stranded RNA, comprises three siRNAs, and its positive-sense strand nucleotides sequence is classified as shown in SEQNO.4, SEQNO.5, SEQNO.6; Its antisense strand nucleotides sequence is classified as shown in SEQNO.7, SEQNO.8 and SEQNO.9; The siRNAs that the invention provides containing the suppression ox MEN1 gene of above-mentioned positive antisense strand effectively can suppress ox MEN1 genetic expression, and the research that can be ox MEN1 gene function provides effect technique instrument, and can be used for the correlative study of ox metabolic trouble.
The siRNAs of suppression ox MEN1 provided by the present invention genetic expression, described siRNAs is little double-stranded RNA, comprises three siRNAs, and its positive-sense strand nucleotides sequence is classified as shown in SEQNO.4, SEQNO.5, SEQNO.6; Its antisense strand nucleotides sequence is classified as shown in SEQNO.7, SEQNO.8 and SEQNO.9.
Above-mentioned sequence is obtained by following process:
According to the complete mRNA sequence (accessionNO.:NM_001076161) of ox MEN1 (BostaurusmultipleendocrineneoplasiaI) gene in Genebank, select three target sequences, and then design the siRNAs of special suppressed ox MEN1 genetic expression.
Selected target sequence is respectively:
Nucleotide sequence is as shown in SEQNO.1:5 ' CCGTTGACCTGTCTCTCTA3 ';
Nucleotide sequence is as shown in SEQNO.2:5 ' CCACTGTCGTAACCGCAAT3 ';
Nucleotide sequence is as shown in SEQNO.3:5 ' CCGAGTGACTACACGCTTT3 ';
Utilize positive-sense strand and antisense strand that above-mentioned three target sequences of TT end method Design and synthesis of RNAoligo are corresponding afterwards, wherein positive-sense strand nucleotide sequence is followed successively by shown in SEQNO.4:5 ' CCGUUGACCUGUCUCUCUAdTdT3 '; Shown in SEQNO.5:5 ' CCACUGUCGUAACCGCAAUdTdT3 '; Shown in SEQNO.6:5 ' CCGAGUGACUACACGCUUUdTdT3 ';
Its antisense strand sequence is the sequence with corresponding sense strand sequence reverse complemental, and its chain nucleotide sequence is followed successively by shown in SEQNO.7:3 ' dTdTGGCAACUGGACAGAGAGAU5 '; Shown in SEQNO.8:3 ' dTdTGGUGACAGCAUUGGCGUUA5 '; Shown in SEQNO.9:3 ' dTdTGGCUCACUGAUGUGCGAAA5 '.
Contriver is after the above-mentioned positive-sense strand of acquisition and antisense strand, and utilize the siRNAs which giving main purpose gene inhibition ox MEN1 of the present invention genetic expression, concrete grammar is as follows:
The sequence of described positive and negative adopted chain, after RNAoligo synthesis, is dissolved as the concentration of 200 μMs respectively, forms double-stranded RNA by annealing with DEPC water; The positive and negative adopted chain RNAoligo of equivalent and annealing buffer (10 × damping fluid: 100mMTrisbase afterwards, 1MNaCl, 10mMEDTA) mixing, to hatch on hot block after 90 DEG C of 2min, powered-down, naturally cooling is annealed to room temperature (about 25 DEG C); Power-on is after 2min is educated in 90 DEG C of taxes again, naturally cools to room temperature, can be directly used in transfection or be positioned over 4 DEG C temporarily to preserve.
After above-mentioned annealing, three siRNAs double-strands can be obtained, wherein:
(1) sibMEN-1: annealed by positive-sense strand nucleotide sequence SEQNO.4 and antisense strand nucleotide sequence SEQNO.7 and formed;
(2) sibMEN-2: annealed by positive-sense strand nucleotide sequence SEQNO.5 and antisense strand nucleotide sequence SEQNO.8 and formed;
(3) sibMEN-3: annealed by positive-sense strand nucleotide sequence SEQNO.6 and antisense strand nucleotide sequence SEQNO.9 and formed.
Three of above-mentioned acquisition siRNAs double-strands are carried out cell transfecting by contriver further, turn and then utilize real-time fluorescence quantitative RT-PCR to detect ox MEN1mRNA expression, found that three siRNAs can effectively suppress transcribing of ox MEN1 gene, and find three siRNAs to be mixed to form a siRNAspool, i.e. the highest the best of jamming effectiveness of 24h after sibMEN-1/2/3 transfectional cell.
In sum, the siRNAs of the suppression ox MEN1 gene containing above-mentioned positive antisense strand provided by the present invention effectively can suppress ox MEN1 genetic expression, and the research that can be ox MEN1 gene function provides effect technique instrument, and can be used for the correlative study of ox metabolic trouble.
Accompanying drawing explanation
Fig. 1 is MEN1mRNA expression histogram after transfection siRNAs,
Fig. 2 analyzes histogram for MEN1mRNA expression after sibMEN-1 and sibMEN-1/2/3;
Fig. 3 is the expression of results schematic diagram of menin albumen after transfection sibMEN-1 and sibMEN-1/2/3;
Fig. 4 is that the expression of menin albumen after transfection sibMEN-1 and sibMEN-1/2/3 analyzes histogram.
Embodiment
The synthesis of the RNA sequence that embodiment 1.siRNAs is corresponding
According to the RNAoligo of the sequent synthesis strand provided in sequence table SEQ NO.4 ~ 9, equal proportion is arranged in pairs or groups between two (SEQNo.4 and SEQNO.7; SEQNo.5 and SEQNO.8; SEQNo.6 and SEQNO.9) respectively with annealing buffer (10 × damping fluid: 100mMTrisbase, 1MNaCl, 10mMEDTA; PH7.4) mix, and then annealing forms double-strand: hatching on hot block after 90 DEG C of 2min, powered-down, naturally cooling is annealed to room temperature (about 25 DEG C); Power-on is after 2min is educated in 90 DEG C of taxes again, naturally cools to room temperature, can be directly used in transfection or be positioned over 4 DEG C temporarily to preserve.
The siRNA that sequence SEQNo.4 and SEQNO.7 annealing is formed is denoted as sibMEN-1; The siRNA that SEQNo.5 and SEQNO.8 annealing is formed is denoted as sibMEN-2; The siRNA that SEQNo.6 and SEQNO.9 annealing is formed is denoted as sibMEN-3.
The transfection of embodiment 2. bovine mammary epithelial cell
Cell cultures
Bovine mammary epithelial cell MAC-T is with containing at 10%FBS (Gibico, US.), being added with in mycillin dual anti-(1%) and DMEM (GibicoTM, US.) substratum, 37 DEG C, 5%CO
2condition under cultivate two weeks after be namely in logarithmic phase cell carry out transfection, before transfection, 24h is with not coated in 6-orifice plate by cell containing dual anti-substratum.
Cell transfecting
Get the cow mammary gland epithelial cells that growth conditions is good, be in logarithmic phase, use collected by trypsinisation cell, centrifugal rear perfect medium is resuspended, after cell counting, with 8.0 × 10
5individual cells/well is evenly inoculated in 6 orifice plates, waits until cell transfecting.Cell transfecting is divided into 5 groups: 1. Control transfection group; 2. sibMEN-1 transfection group; 3. sibMEN-2 transfection group; 4. sibMEN-3 transfection group; 5. sibMEN-1/2/3 transfection group.
To treat in 6 orifice plates that Growth of Cells to degree of converging is about the 70%-80% moment and carries out transfection, the concrete operation step of transfection is as follows:
(1) before transfection, the substratum in 6 orifice plates is replaced by without substratum that is dual anti-, 10%FBS.
(2) premix OPTI-MEM and siRNAs: for every porocyte, using 150 μ lOPTI-MEM to dilute siRNAs respectively to final concentration is 50nM, gently incubated at room 5min after mixing.
(3) premix OPTI-MEM and Lipofectamine2000: every porocyte 150 μ lOPTI-MEM dilute 10 μ lLipofectamine2000 transfection reagents, gently incubated at room 5min after mixing.
(4) OPTI-MEM-siRNAs (step 2) and OPTI-MEM-Lipofectamine2000 (step 3) is mixed, now cumulative volume is 300 μ l, mixing also places 20min at ambient temperature gently, makes siRNAs and Lipofectamine2000 form effective mixture.
(5) in each hole, dropwise add 300 μ l mixtures gently, add rear employing right-angled intersection method and shake mixing gently.
(6) 6 porocyte culture plates are placed in 37 DEG C of cell culture incubators, after 5-6 hour, replace medium to perfect medium.
(7) continue to cultivate harvested cell after 24-72 hour and extract total serum IgE and albumen.
Embodiment 3 real-time fluorescence quantitative RT-PCR detects ox MEN1mRNA expression
RNAsimpleTotalRNAKit total RNA extraction reagent box is adopted to extract the total serum IgE of experiment each group of mammary epithelial cell to specifications, adopt the integrity of the plain agar sugar detected through gel electrophoresis total serum IgE of 1.0%, adopt ultraviolet spectrophotometer method under 260nm and 280nm wavelength, detect concentration and the extracting purity of RNA respectively.Be cDNA according to the requirement of PrimeScriptTMRTreagentKitwithgDNAEraser (PerfectRealTime) test kit by total serum IgE reverse transcription.
Carry out real-time fluorescence quantitative PCR amplification as template, adopt SYBRGreenI dye method, utilize SYBRRPremixExTaqTM (PerfectRealTime) test kit of TaKaRa company to carry out real-time fluorescence quantitative PCR on ABI7500 instrument.
The special positive-sense strand primer nucleotide sequences of MEN1 used is as shown in SeqIDNo.10:5'-gatggaggtggcatttatgg-3 ', and MEN1 specific antisense strand primer nucleotide sequence is as shown in SEQNO.11: shown in 5 '-gatgtgctcatcccggtagt-3 '.Internal reference gene ox β-actin special positive-sense strand primer nucleotide sequences as shown in SEQNo.12:5'-cccagcacaatgaagatcaa-3', β-actin specific antisense strand primer nucleotide sequence is as SEQNo.13: shown in adopted 5'-tagaagcatttgcggtggac-3'.MEN1mRNA expresses quantitative analysis employing and compares Ct data method (2
-Δ Δ Ct) carry out.Often group experiment repetition 3 times.
As shown in Figure 1, the result after detection shows result: three siRNAs can effectively suppress transcribing of ox MEN1 gene.After transfection, to compare control group interference effect best for 24h, sibMEN-1/2/3 transfection group, and jamming effectiveness reaches 80%, and sibMEN-1 transfection group jamming effectiveness about 57%; 48h after transfection, interference effect is it is preferred that sibMEN-1 transfection group, and jamming effectiveness reaches 59%, sibMEN-1/2/3 transfection group jamming effectiveness about 48%; 72h after transfection, interference effect is it is preferred that sibMEN-1 transfection group, and jamming effectiveness reaches 73%, and its excess-three group jamming effectiveness is close, and about 67%.Generally speaking, the suppression efficiency of sibMEN-1 and sibMEN-1/2/3 is best and relatively stable.
So, on the basis of this preliminary experiment, carried out biology for sibMEN-1 and sibMEN-1/2/3 again and repeated transfection experiment (n=3).Carry out statistical study with SAS8.2 software to result, carry out test of significance: wherein with t method of inspection, P < 0.05 shows to there is significant difference, and P<0.01 shows to there is pole significant difference.Result such as Fig. 2 shows: after transfection, 24h, sibMEN-1, sibMEN-1/2/3 transfection group extremely significantly can reduce the expression amount (P<0.01) of MEN1mRNA compared with control group, and jamming effectiveness reaches 63%, 75% respectively; After transfection, 48h, sibMEN-1, sibMEN-1/2/3 transfection group extremely significantly can reduce the expression amount (P<0.01) of MEN1mRNA equally, and jamming effectiveness reaches 55%, 50% respectively; 72h after transfection, sibMEN-1 transfection group still extremely significantly can reduce the expression amount (P<0.01) of MEN1mRNA compared with control group, jamming effectiveness is 74%, sibMEN-1/2/3 transfection group significantly can reduce the expression amount (P<0.05) of MEN1mRNA, and jamming effectiveness is 62%.In general, for ox MAC-T cell, the jamming effectiveness of sibMEN-1/2/3 (being mixed to form a siRNAspool) is the highest, and 24h jamming effectiveness is best after transfection, can reach 75%.
Embodiment 4Westernblot detects ox MEN1 gene coded protein expression level in transfectional cell
24h, 48h and 72h after sibMEN-1, sibMEN-1/2/3 respectively transfection MAC-T cell are extracted total cellular protein respectively.Western-Blot detects the relative expression quantity of ox MEN1 proteins encoded menin, using ox β-actin as internal reference albumen.Mouse source β-actin antibody (AA128, the green skies) is purchased from the green skies, Beijing biotechnology research institute, and rabbit source menin 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, Beijing biotechnology research institute.Scan film (color scanner, BenQSCANNER5560), and with the gray-scale value of ImageJ software analysis object band, the ratio of the gray-scale value of target protein and internal reference albumen (β-actin), is the albumen relative expression quantity of each treatment group.
Result as shown in Figures 3 and 4, protein expression detection display: 24h after transfection, sibMEN-1, sibMEN-1/2/3 transfection group extremely significantly can reduce the relative expression quantity (P<0.01) of Menin albumen compared with control group, and jamming effectiveness reaches 68%, 71% respectively; 48h after transfection, sibMEN-1 transfection group extremely significantly can reduce the expression amount (P<0.01) of Menin albumen compared with control group, jamming effectiveness is the jamming effectiveness of 64%, sibMEN-1/2/3 transfection group is 42%, and does not have difference between control group; 72h, sibMEN-1 after transfection, between sibMEN-1/2/3 transfection group and control group, there is no difference.In general, for MAC-T cell, after transfection sibMEN-1/2/3 (being mixed to form a siRNAspool) 24h, the expression efficiency of menin albumen is the most occurred frequently reaches 71%.
Result is known, detect through qRT-PCR, Westernblot, 3 siRNAs in this invention effectively can disturb the mrna expression of ox MEN1 gene, but three siRNAs are mixed to form a siRNAspool, i.e. the highest the best of jamming effectiveness of 24h after sibMEN-1/2/3 transfectional cell, sibMEN-1 takes second place.
Claims (3)
1. suppress a siRNAs for ox MEN1 genetic expression, it is characterized in that: described siRNAs is little double-stranded RNA, its positive-sense strand nucleotides sequence is classified as shown in SEQNO.4, SEQNO.5, SEQNO.6; Its antisense strand nucleotides sequence is classified as shown in SEQNO.7, SEQNO.8 and SEQNO.9.
2. the siRNAs of suppression ox MEN1 according to claim 1 genetic expression, is characterized in that, in described three siRNAs double-strands:
(1) sibMEN-1: annealed by positive-sense strand nucleotide sequence SEQNO.4 and antisense strand nucleotide sequence SEQNO.7 and formed;
(2) sibMEN-2: annealed by positive-sense strand nucleotide sequence SEQNO.5 and antisense strand nucleotide sequence SEQNO.8 and formed;
(3) sibMEN-3: annealed by positive-sense strand nucleotide sequence SEQNO.6 and antisense strand nucleotide sequence SEQNO.9 and formed.
3. siRNAs according to claim 1 is suppressing the application in ox MEN1 genetic expression.
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| WO2017134252A1 (en) * | 2016-02-05 | 2017-08-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antisense oligonucleotides effective to reduce the expression of menin in cancer cells of a subject |
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| CN103251958A (en) * | 2013-01-22 | 2013-08-21 | 上海市内分泌代谢病研究所 | Applications of MEN1 gene and encoding protein thereof |
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| CN103251958A (en) * | 2013-01-22 | 2013-08-21 | 上海市内分泌代谢病研究所 | Applications of MEN1 gene and encoding protein thereof |
Non-Patent Citations (2)
| Title |
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| PETER C. SCACHERI ET AL.: "Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells", 《PROC NATL ACAD SCI U S A》 * |
| ZIMIN AV: "Accession NO.：NM_001076161.3，Bos taurus menin 1 （MEN1）mRNA", 《GENBANK DATEBASE》 * |
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| WO2017134252A1 (en) * | 2016-02-05 | 2017-08-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antisense oligonucleotides effective to reduce the expression of menin in cancer cells of a subject |
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