CN102586325A - shRNA expression plasmid inhibiting CYP3A4 and construction and application thereof - Google Patents
shRNA expression plasmid inhibiting CYP3A4 and construction and application thereof Download PDFInfo
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Abstract
The invention provides an shRNA expression plasmid inhibiting CYP3A4. Three shRNA template sequences which meet requirements are obtained by eliminating a sequence with homology with other genes in a genome by Blast software according to an interference fragment designing principle and a CYP3A4 sequence; and synthesized shRNA templates in above step are cloned respectively and then subjected to ammonia benzyl resistance screening and sequencing to obtain three shRNA expression plasmids. In different cell models, both the shRNA independent and mixed-expression plasmids can well inhibit mRNA expression (by 55%) and protein expression (by 50%) of the CYP3A4 while preventing an off-target effect and realizing perfect specificity. Therefore, the three shRNA expression plasmids disclosed by the invention can serve as a specific inhibitor on the CYP3A4 to be used for screening and confirming CYP3A4 substrates and inducers.
Description
Technical field
The invention belongs to biomedicine field; Be specifically related to suppress drug metabolism enzyme CYP3A4 expresses three kinds of shRNA expression plasmids and structure; And three kinds of shRNA expression plasmids separately or mix when using CYP3A4 gene and albumen restraining effect; Can reduce the expression of CYP3A4 effectively, be used for the one-tenth property of medicine evaluation of new drug development, to improve the success ratio of medicament research and development; Be used for the clinical interactional prediction that makes medicament, to reduce or to avoid untoward reaction.
Background technology
RNA disturbs (RNA interference; RNAi) phenomenon is that a kind of the evolution gone up the conservative defense mechanism of resisting transgenic or adventitious viruses infringement; There is double-stranded RNA (the double-stranded RNA of homology complementary sequence in the transcription product mRNA that is meant endogenous or exogenous and target gene; DsRNA) this mRNA that in cell, degrades specifically; Thereby cause the effectively process of sealing of specific gene, be a kind of sequence-specific PTGS (post-transcriptional gene silencing, PTGS).
The RNAi technology is to utilize the RNAi principle; At external utilization chemistry or the synthetic siRNA of enzymatic; Also can make up plasmid or the virus expression carrier of synthetic siRNA in vivo, thereby utilize the technology of method transfectional cell causes target gene silencings such as traditional microinjection calcium phosphate method, electroporation transfection method, liposome-mediated transfection then.RNA disturbs has a lot of methods: 1. chemical synthesis; 2. siRNA expression vector; 3. dsRNA expression vector.Most siRNA expression vectors relies on a kind of in three kinds of rna plymerase iii promotors (pol III), handles one section little hairpin RNA (short hairpin RNA, shRNA) expression in mammalian cell.These three types of promotors comprise the U6 promotor and the people H1 promotor in the people source and the mouse source of well known.Why adopt RNA pol III promotor to be because it can express more microRNA in mammalian cell, and its a string through adding (3 to 6) U stop transcribing.Use this type carrier, need to order the dna single chain of 2 sections coding short hairpin RNA sequences, anneal, be cloned into the pol III promotor downstream of respective carrier.The advantage of siRNA expression vector is to carry out longer-term research, and this is can in cell, continue to suppress target gene expression because have the carrier of antibiotic marker, continues a few weeks longer even more of a specified duration.RNAi has tangible advantage as a kind of simply quick, special, efficient, economy, the predictable technology of effect, and it is more superior than antisense technology, and is simpler than gene knockout, has good application prospects.
Drug metabolism character is in the drug discovery process, and compound becomes the important indicator of the property of medicine.And drug metabolism enzyme has been played the part of important role in the drug metabolism and transformation process.Wherein the oxidative metabolism of Cytochrome P450 (CYP450) enzyme participation is the main path that medicine is eliminated.CYP3A4 is the abundantest P450 enzyme of body burden, participates in the drug metabolism more than 50% on the market.In the metabolic of medicine interacted, the interaction that is caused by the inhibition of enzyme accounted for all interactional 70%.To the research of the chemical inhibitor of CYP3A4 very extensively, but because the chemical inhibitor poor specificity, thus relative inhibitors optionally comprehensive information remain further to be inquired into.Compare with chemical inhibitor, the RNAi technology then can realize the high-level efficiency blocking-up of genetic expression, and specificity is stronger, thereby avoids the influence to other drug metabolic enzyme or transporter.RNA to CYP3A4 disturbs the shRNA expression plasmids that adopt to a certain site more at present, and adopts the shRNA expression plasmid mixture that acts on many target spots still not have report.
Summary of the invention
The purpose of this invention is to provide the shRNA expression plasmid that suppresses CYP3A4, make up through following method:
(1) in Genebank, searches the sequence (NM.017460) of CYP3A4; According to interference fragment principle of design (http://www.ambion.com/techlib/misc/siRNA_finder.html); Concrete principle is following: begin from the AUG initiation codon of transcript (mRNA); Seek " AA " two and connect sequences, and write down 19 base sequences of its 3' end, with GC content at about 45%-55% siRNA as potential siRNA target site.And according to the sequence of CYP3A4; Utilize design software; And get rid of the sequence that has homology with other genes of genome (people, mouse, rat etc.) through Blast software, and obtaining three satisfactory shRNA template sequences, action site is respectively 773,923 and 1191.
The shRNA template positive-sense strand sequence in (1) 773 site is:
5'-GATCCAGTCGCCTCGAAGATACACTTCAAGAGAGTGTATCTTCGAGGCGACTTTA-3'
The shRNA template antisense strand sequence in 773 sites is:
5'-AGCTTAAAGTCGCCTCGAAGATACACTCTCTTGAAGTGTATCTTCGAGGCGACTG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-AGTCGCCTCGAAGATACAC-3' and 5'-GTGTATCTTCGAGGCGACT-3' are respectively the justice and the antisense sequences of 773 target spots.
With sterilized water with chemosynthesis 773 justice and antisense shRNA template powder be diluted to 50 μ M respectively; Under 95 ℃ to 25 ℃ conditions of progressively lowering the temperature, be annealed into double-stranded shRNA template, be cloned into then on the pSilencer4.1 CMV of BamHI and HindIII double digestion neo carrier, transform DH5 α competent cell; Ammonia benzyl antibiotic-screening positive colony; Choose incubated overnight in some bacterium colonies that grow to the LB substratum, extract plasmid (result is referring to Fig. 2, and wherein 773 1-6:shRNA expression plasmids 773 are cloned 1-6) with plasmid extraction kit (day root); Choose 773 No. 2 evaluations of checking order, the correct back of result is in-80 ℃ of preservations.773 No. 2 called after S1.
The shRNA template positive-sense strand sequence in (2) 923 sites is:
5'-GATCCACCACGAGCAGTGTTCTCTTTCAAGAGAAGAGAACACTGCTCGTGGTTTA-3'
The shRNA template antisense strand sequence in 923 sites is:
5'-AGCTTAAACCACGAGCAGTGTTCTCTTCTCTTGAAAGAGAACACTGCTCGTGGTG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-ACCACGAGCAGTGTTCTCT-3' and 5'-AGAGAACACTGCTCGTGGT-3' are respectively the justice and the antisense sequences of 923 target spots.
With sterilized water with chemosynthesis 923 justice and antisense shRNA template powder be diluted to 50 μ M respectively; Under 95 ℃ to 25 ℃ conditions of progressively lowering the temperature, be annealed into double-stranded shRNA template, be cloned into then on the pSilencer4.1 CMV of BamHI and HindIII double digestion neo carrier, transform DH5 α competent cell; Ammonia benzyl antibiotic-screening positive colony; Choose incubated overnight in some bacterium colonies that grow to the LB substratum, extract plasmid (result is referring to Fig. 2, and wherein 923 1-6:shRNA expression plasmids 923 are cloned 1-6) with plasmid extraction kit (day root); Choose 923 No. 2 evaluations of checking order, the correct back of result is in-80 ℃ of preservations.923 No. 2 called after S2.
The shRNA template positive-sense strand sequence in (3) 1191 sites is:
5'-GATCCGCTATGCTCTTCACCGTGATTCAAGAGATCACGGTGAAGAGCATAGCTTA-3'
The shRNA template antisense strand sequence in 1191 sites is:
5'-AGCTTAAGCTATGCTCTTCACCGTGATCTCTTGAATCACGGTGAAGAGCATAGCG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-GCTATGCTCTTCACCGTGA-3' and 5'-TCACGGTGAAGAGCATAGC-3' are respectively the justice and the antisense sequences of 1191 target spots.
With sterilized water with chemosynthesis 1191 justice and antisense shRNA template powder be diluted to 50 μ M respectively; Under 95 ℃ to 25 ℃ conditions of progressively lowering the temperature, be annealed into double-stranded shRNA template, be cloned into then on the pSilencer4.1 CMV of BamHI and HindIII double digestion neo carrier, transform DH5 α competent cell; Ammonia benzyl antibiotic-screening positive colony; Choose incubated overnight in some bacterium colonies that grow to the LB substratum, extract plasmid (result is referring to Fig. 2, and wherein 1191 1-6:shRNA expression plasmids 1191 are cloned 1-6) with plasmid extraction kit (day root); Choose 1191 No. 1 evaluations of checking order, the correct back of result is in-80 ℃ of preservations.1191 No. 1 called after S3.
Another object of the present invention provides the independent or application of equal proportion mixture in the specific inhibitor of preparation CYP3A4 of three kinds of shRNA plasmids of described inhibition CYP3A4; In new drug development and the research of medicine rational Application, be used for the screening and the conclusive evidence of CYP3A4 substrate and inductor.
Three kinds of new shRNA expression plasmids that act on the CYP3A4 different loci respectively that the present invention makes up; Independent or equal proportion is mixed the expression that can suppress CYP3A4 effectively; Compare with independent effect; The equal proportion mixture effect of these three kinds of shRNA expression plasmids is stronger, and to not having influence with the expression of the very high CYP3A5 of CYP3A4 homology.Therefore, this shRNA expression plasmid mixed system can be used as the specific inhibitor of CYP3A4, is used for new drug development and the research of medicine rational Application, the screening of CYP3A4 substrate and inductor and conclusive evidence.
Application in CYP3A4 substrate conclusive evidence.In Rifampin inductive HepG2 cell (Chinese Academy of Sciences's cell bank, TCHu 72), the equal proportion mixture of shRNA plasmid of the present invention can reverse the toxic action of GA (15:1) to the HepG2 cell.To sum up explanation; In new drug development: the useful effect form of three kinds of shRNA expression plasmids that (1) the present invention makes up is the equal proportion mixture; This mixture can suppress CYP3A4 gene and proteic expression specifically, and the specificity suppressor factor that can be used as CYP3A4 uses; (2) the equal proportion mixture of the shRNA plasmid among the present invention can reduce the activity of CYP3A4, in the screening of CYP3A4 substrate and inductor and conclusive evidence, important use is arranged; (3) the equal proportion mixture of the shRNA plasmid among the present invention can be used for screening and conclusive evidence process CYP3A4 metabolism increase activity or increase toxic medicine; (4) the equal proportion mixture of the shRNA plasmid among the present invention can be used for the research of the signal path of CYP3A4 participation.
The present invention through three kinds of different cells models, detects the restraining effect of shRNA expression plasmid to CYP3A4 gene and protein expression behind the structure of accomplishing the shRNA expression plasmid.(1) at CHL (Chinese Academy of Sciences's cell bank; GNHa 2)-change in CYP3A4 wink in the cell model; The shRNA expression plasmid do separately the time spent suppress effect a little less than; And the equal proportion mixture of three kinds of plasmids reaches 60% to the inhibiting rate of CYP3A4; The equal proportion mixture of three kinds of plasmids of this application note can produce more efficiently inhibition to the genetic expression of CYP3A4, thereby has confirmed the useful effect form of shRNA expression plasmid, and therefore the shRNA expression plasmid of form of mixtures is all adopted in follow-up application; (2) at HEK293 (Chinese Academy of Sciences's cell bank; GNHu 1)-change in CYP3A4 wink in the cell model; ShRNA expression plasmid equal proportion mixture is 55% to the inhibiting rate of CYP3A4 gene; Proteic expression also has the obvious suppression effect to CYP3A4 simultaneously, and the equal proportion mixture that three kinds of shRNA plasmids have further been verified in this application is expressed CYP3A4 has significant inhibitory effect; (3) at Rifampin inductive HepG2 cell (Chinese Academy of Sciences's cell bank; TCHu 72) in; ShRNA expression plasmid equal proportion mixture is about 50% CYP3A4 gene and proteic inhibiting rate; And to not having influence with the higher CYP3A5 gene of its homology, this is applied on exogenous importing CYP3A4 result's the basis, further detects the influence of the equal proportion mixture of three kinds of shRNA plasmids to endogenous CYP3A4 gene and protein expression; Confirm that the equal proportion mixture of this shRNA plasmid obviously reduces the expression of endogenous CYP3A4, and to other and the high gene unrestraint effect of CYP3A4 homology.
CYP3A4 shRNA expression plasmid provided by the invention compares with the interference plasmid of having reported; Have: the shRNA expression plasmid that (1) the present invention makes up is compared with the miRNA plasmid; Can act on the coding region sequence of CYP3A4, the mRNA of the CYP3A4 that directly degrades, more remarkable effect; And the miRNA plasmid acts on non-coding region, to the Degradation of CYP3A4 mRNA a little less than; (2) the present invention adopts shRNA expression plasmid mixture to suppress experiment, and this mixture is to the different site of CYP3A4, and it is stronger than single plasmid to suppress effect; (3) shRNA mixing expression plasmid of the present invention is compared with chemical inhibitor; Has good specificity; To other medicines metabolic enzyme or transporter unrestraint effect, disturb thereby get rid of non-specific inhibition, the specific inhibitor that can be used as CYP3A4 uses.
Description of drawings
The PCR of Fig. 1 plasmid pGL3/CYP3A4 identifies electrophorogram.
Fig. 2 shRNA expression plasmid electrophorogram.
The two luciferase reporter genes experiments of Fig. 3 detect respectively organize the shRNA expression plasmid to the influence of the CYP3A4 genetic expression of changeing cell CHL-CYP3A4 wink (means ± SD, n=3).
The two luciferase reporter genes experiments of Fig. 4 detect shRNA expression plasmid equal proportion mixture to the influence of changeing cell CYP3A4 genetic expression HEK293-CYP3A4 wink (means ± SD, n=3).
The chemical experiment of Fig. 5 immunofluorescence detects shRNA expression plasmid equal proportion mixture changes cell CYP3A4 protein expression to HEK293-CYP3A4 wink influence.
Fig. 6 Rifampin induce HepG2 cell high expression level CYP3A4 condition (means ± SD, n=3).
Fig. 7 RT-PCR experiment detects the influence of shRNA expression plasmid equal proportion mixture to Rifampin inductive HepG2 cell CYP3A4 genetic expression.
Fig. 8 Western blotting experiment detects the influence of shRNA expression plasmid equal proportion mixture to Rifampin inductive HepG2 cell CYP3A4 protein expression.
Fig. 9 Western blotting experiment detects the influence of shRNA expression plasmid equal proportion mixture to Rifampin inductive HepG2 cell CYP3A5 protein expression.
After Figure 10 MTT experiment detected transfection shRNA expression plasmid equal proportion mixture, GA (15:1) changed the viability of Rifampin inductive HepG2 cell.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is done further explanation.Through the following example technical characterictic of the present invention is done detailed description, but do not limit the present invention.
Design contains the PCR primer of XbaI and FseI restriction enzyme site, from former reorganization carrier (PGEMT-CYP3A4) amplification CYP3A4 CDS gene, is connected among the carrier pGL3 promoter; Through transforming, choose bacterium, shake bacterium, extracting the laggard performing PCR of plasmid and identify that (result is referring to Fig. 1; Wherein 1: clone 1 plasmid PCR product, 2: clone 2 plasmid PCR products, 3: clone 3 plasmid PCR products; PCR product size is 1512bp); Get the evaluation of checking order for No. 1, the result is correct, identifies that-80 ℃ of preservations in correct back are in order to the subsequent experimental use.
The structure of embodiment 2 shRNA expression plasmids
1. CYP3A4 disturbs the screening of target site sequence and the design of shRNA template
In Genebank, search the sequence of CYP3A4; Sequence according to interference fragment principle of design (http://www.ambion.com/techlib/misc/siRNA_finder.html) and CYP3A4; Utilize design software; And get rid of the sequence that has homology with other genes of genome through Blast software, and obtain three satisfactory shRNA template sequences, be respectively 773,923 and 1191 to the site of CYP3A4.
2. the shRNA template is synthetic
Synthetic shRNA template strand prepares double-stranded shRNA template then.Be to add sterilized water in every pipe shRNA template powder, to final concentration be 50 μ M, carry out annealing reaction then, reaction system is following:
According to above-mentioned all ingredients, the mixing of adding successively.Like table 3 the PCR appearance is set and carries out annealing reaction:
3. shRNA template and BamHI are connected with linearized vector pSilencer4.1 CMV neo after the HindIII enzyme is cut, and reaction system and condition are as shown in table 4:
Through transform, choose bacterium, shake bacterium, (result is referring to Fig. 2 to extract plasmid; Wherein 773 1-6:shRNA expression plasmids 773 are cloned 1-6; 923 1-6:shRNA expression plasmids, 923 clone 1-6; 1191 1-6:shRNA expression plasmids, 1191 clone 1-6), choose 773 No. 2,923 No. 1,1191 No. 1 evaluations of checking order, result's-80 ℃ of preservations in correct back are used in order to subsequent experimental.773 No. 2 called after S1,923 No. 1 called after S2,1191 No. 1 called after S3.
The experiment of the 3 pairs of luciferase reporter genes of
embodiment detects and respectively organizes the influence of shRNA expression plasmid to the CYP3A4 genetic expression of changeing cell CHL CYP3A4 wink.
PGL3/CYP3A4 and PRL-TK reporter plasmid transfection simultaneously CHL cell, with the PRL-TK reporter plasmid as confidential reference items; When transfection shRNAs plasmid, transcribing of CYP3A4 is suppressed, and then suppresses the expression of Luciferase, and fluorescence intensity reduces.
1. transfection step is following
(1) experiment is divided into groups, and it is as shown in the table.
(2) transfection previous day, with trysinization, counting and plant to 24 orifice plates, making transfection cytogamy on same day degree is 70-90% with the CHL cell.
(3) use the DMEM substratum 50 μ l of serum-free to dilute DNA 0.8 μ g, mixing.
(4) in another pipe, use the DMEM substratum 50 μ l of serum-free to dilute Lipofectamine
TM2000Reagent 2 μ l, mixing, incubated at room 5min.
(5) with the Lipofectamine that dilutes
TM2000Reagent mixes with the DMEM substratum of the pGL3/CYP3A4 plasmid that contains dilution and shRNA expression plasmid or negative control plasmid.Incubated at room 20min.
(6) transfectional cell changes not contain the DMEM substratum of serum.
(7) every porocyte adds the mixing solutions 100 μ l that step (4) obtains, and mixing is put into incubator and hatched gently.
(8) add 10% foetal calf serum behind the 4h, continue to hatch.
2. lysing cell:
(1) fresh lysate: before facing usefulness, get an amount of 5 * Passive Lysis Buffer (PLB), be diluted to 1 * PLB with distilled water, mixing.1 * PLB can deposit several weeks at 4 ℃.
(2) cell cleans: the nutrient solution in the culture plate that inclines adds capacity PBS, gently washed cell.Washings fully inclines.
(3) lysis: add 100 μ l, 1 * PLB, mixing in every hole.Culture plate can shake 15 min on microoscillator; Petridish can directly scrape cell with cell scraper, and cell suspension is moved into 1.5 ml centrifuge tubes, and fully suspendible shook 30 seconds on the vortex vibrator.The lysing cell suspension can directly be used for luminescence assays, also can be centrifugal 30 seconds, and get supernatant and do luminescence assays.
3. prepare luminous reaction liquid:
Before the mensuration, treat that in room temperature Stop&Glo Buffer, Stop&Glo Substrate I and Luciferase Assay Buffer II dissolve mixing (attention lucifuge).Resuspended Luciferase Assay Substrate is in Luciferase Assay Buffer II; With Stop&Glo Buffer dilution Stop&Glo Substrate, prepare volume required LARII and Stop & Glo Reagent (attention lucifuge) in 50/1 ratio respectively.
4. manual luminescence assays:
Get testing sample 10 μ l and add the measuring tube bottom, get LARII 50 μ l and add the pipe bottom, knock 3 ~ 5 mixings of tube wall gently, put into instrument and measure immediately, the record luminous value is the flat light emission (RLU) of Firefly luciferase; Get Stop Glo Reagent 50 μ l and add the pipe bottom, knock 3 ~ 5 mixings of tube wall gently, put into instrument and measure immediately, the record luminous value is the flat light emission (RLU) of Renilla luciferase.
The result shows; When three kinds of shRNA expression plasmids mix with the pGL3-3A4 cotransfection respectively or in twos, all lower to the inhibition efficient of CYP3A4 mRNA, and when the equal proportion mixture of three kinds of shRNA expression plasmids and pGL3-3A4 cotransfection; Suppress efficient and reach 60% (result is referring to Fig. 3); This result shows that three kinds of shRNA plasmid equal proportion mixtures can provide more efficiently inhibition, therefore, follow-up experiment with this shRNA expression plasmid equal proportion mixture as research means.Among Fig. 3, *
P<0.05 shRNA expression plasmid group vs Negative control group, * * *
P<0.001 shRNA expression plasmid group vs Negative control group, the single inhibition result of each shRNA expression plasmid of A., the negative plasmid control group of Negative; B. the shRNA expression plasmid of various combination suppresses the result; The negative plasmid control group of Negative, S1+S2+S3 are the 1:1:1 mixture of three kinds of plasmids, and S1+S2 is the 1:1 mixture of S1 and S2; S1+S3 is the 1:1 mixture of S1 and S3, and S2+S3 is the 1:1 mixture of S2 and S3.
1. two luciferase reporter gene experiments detect shRNA expression plasmid equal proportion mixture changes cell CYP3A4 genetic expression to HEK293-CYP3A4 wink influence
Experimental procedure is referring to case study on implementation 3.Experiment grouping and system are as shown in table 7.
The result shows that shRNA expression plasmid equal proportion mixture can reduce the expression of CYP3A4 mRNA comparatively significantly, and inhibiting rate is 53%, and the result is referring to Fig. 4, * * * among the figure
P<0.001 shRNA expression plasmid group vs Negative control group, the negative plasmid control group of Negative, S1+S2+S3 are the equal proportion mixture of three kinds of plasmids.
2. the immunofluorescence chemical experiment detects shRNA expression plasmid equal proportion mixture changes cell CYP3A4 protein expression to HEK293-CYP3A4 wink influence
Plant the HEK293 cell in 24 orifice plates, transfection next day pGL3/CYP3A4, shRNA expression plasmid equal proportion mixture and Negative plasmid, methyl alcohol fixed cell behind the 48h; 0.1%Triton punching with the PBS configuration that contains 1%BSA; The 1%BSA sealing, with 4 ℃ of incubated overnight of CYP3A4 one anti-(antibody dilutes by 1:500 with the PBS that contains 1%BSA), two anti-lucifuges are hatched (antibody dilutes by 1:1000 with the PBS that contains 1%BSA); 37 ℃ of 30min, fluorescent microscope detects.
The result of immunofluorescence chemistry shows; CYP3A4 high expression level group cell is bright green under fluorescent microscope behind antibody incubation, the negative control plasmid does not have influence to the expression of CYP3A4; And the fluorescence intensity of shRNA expression plasmid equal proportion mixture group cell CYP3A4 significantly reduces; Explain that shRNA expression plasmid equal proportion mixture has suppressed the protein expression of CYP3A4, the result is referring to Fig. 5, and A is a shRNA expression plasmid equal proportion mixture group; B is the Negative control group, and C is a CYP3A4 high expressing cell group.
1. the RT-PCR experiment detects the influence of shRNA expression plasmid equal proportion mixture to Rifampin inductive HepG2 cell CYP3A4 genetic expression
At first grope Rifampin and induce the condition of HepG2 cell, when 50 μ M Rifampins acted on cell 48 hours, the expression amount of CYP3A4 is 2 times (result is referring to Fig. 6) when not inducing, and therefore adopts this condition to carry out follow-up experiment.Among Fig. 6: * * *
P<0.001 Rifampin 50 μ M induce 48h group vs vehicle control group; Vehicle is the DMSO control group; Rifampin 50 μ M 24h are that the Rifampin of final concentration 50 μ M was induced the HepG2 cell 24 hours, and Rifampin 50 μ M 48h are that the Rifampin of final concentration 50 μ M was induced the HepG2 cell 48 hours.
(1) experiment is divided into groups as shown in table 8
(2) transfection step is following
1) transfection previous day, with trysinization, counting and plant to 6 orifice plates, making transfection cytogamy on same day degree is 70-90% with the HepG2 cell after inducing.
2) use the DMEM substratum 250 μ l of serum-free to dilute DNA 4 μ g, mixing.
3) in another pipe, use the DMEM substratum 250 μ l of serum-free to dilute Lipofectamine
TM2000Reagent 8 μ l, mixing, incubated at room 5min.
4) with the Lipofectamine that dilutes
TM2000Reagent with contain the DMEM substratum that dilutes plasmid and mix.Incubated at room 20min.
5) transfectional cell changes not contain the DMEM substratum of serum.
6) every porocyte adds the mixing solutions 500 μ l that step 4) obtains, and mixing is put into incubator and hatched gently.
7) add 10% foetal calf serum behind the 4h, continue to hatch.
(3) respectively organize the mRNA of cell with the extracting of TRIzol method, cDNA building-up reactions system and PCR reaction system are as shown in table 9:
Hatch the M-MuLv Reverse Transcriptase 1 μ l that adds 200units behind the 5min for 37 ℃, 42 ℃ hatch 60min after, hatch 10min for 70 ℃, place 4 ℃ at last.
Mixing, 97 ℃ 5 minutes, ice-water bath immediately, mixing carries out the PCR reaction by following condition.
Electrophoresis result to RT-PCR is carried out gray scale scanning and semi-quantitative analysis; When shRNA expression plasmid equal proportion mixture is 35% at the inhibiting rate of effect in the time of 24 hours; And the inhibiting rate when acting on 48 hours is 47.5%, and comparatively obvious suppression effect is arranged, and the result is referring to Fig. 7.* * * among Fig. 7
P<0.001 shRNA mixing expression plasmid group vs Negative control group, A. respectively organizes cell RT-PCR electrophorogram, and 1,2 induces the high expression level group for Rifampin; 3,4 for Rifampin induce the back negative plasmid control group; 5,6 induce back shRNA expression plasmid equal proportion mixture group for Rifampin; 7,8 is the vehicle control group, and the B. semi-quantitative results is contrast with Negative, and the CYP3A4 mRNA of shRNA expression plasmid equal proportion mixture group cell expresses percentage ratio.
2. Western blot experiment detects the influence of shRNA expression plasmid equal proportion mixture to Rifampin inductive HepG2 cell CYP3A4 protein expression
Behind 48h after the transfection, collect and respectively organize cell, the PBS washing of precooling 3 times, the cell pyrolysis liquid lysing cell extracts total protein, gets 2 μ l and does protein quantification (protein quantification adopts green skies test kit BCA method).All the other add 2 * loading buffer of equivalent, and boiling water boils 5min, carries out the SDS-PAGE electrophoresis.The albumen applied sample amount is 25 μ g.Be docile and obedient preface to filter paper, gel, cellulose nitrate film, filter paper with sandwich method and put well, the electrotransfer condition is 200mA 2h, 4 ℃.Nitrocellulose filter places the TBST that contains 5% skim-milk, room temperature sealing 2h.Nitrocellulose filter places the sulculus that contains anti-CYP3A4 (an anti-TBST who contains 5% skim-milk that uses dilutes by 1:1000), and jolting, is spent the night by 4 ℃.With nitrocellulose filter TBST washing 3 times, each 10min.Nitrocellulose filter is placed the sulculus that contains SA (the two anti-TBST that contain 5% skim-milk that use dilute by 1:5000), jolting, room temperature 2h.TBST washing 3 times, each 10min.With ECL chemical illuminating reagent reaction 1min, exposure in the darkroom, development, photographic fixing, the imager capture is with QuantityOne software analysis band gray scale.
Semi-quantitative results to Western blot shows, shRNA expression plasmid equal proportion mixture effect 48 hours, and the inhibiting rate of CYP3A4 protein expression is reached 50%, and (result is referring to Fig. 8, * * *
P<0.001 shRNA mixing expression plasmid group vs Negative control group.Fig. 8 A wherein: protein electrophoresis figure, Rifampin induce negative control group of component and shRNA expression plasmid equal proportion mixture group; Fig. 8 B: semi-quantitative results is contrast with Negative, the CYP3A4 protein expression percentage ratio of shRNA expression plasmid equal proportion mixture group cell).Above shRNA expression plasmid equal proportion mixture suppresses the efficient confirmatory experiment and shows, no matter on gene still was protein level, this shRNA expression plasmid equal proportion mixture all had the obvious suppression effect to CYP3A4.
The specificity research of embodiment 6 shRNA expression plasmid equal proportion mixtures
CYP3A4 and CYP3A5 have the homology of height, and therefore, this part experiment is induced the HepG2 cell with Rifampin, adopts the method for RT-PCR, and detection shRNA mixing expression plasmid has or not influence to the genetic expression of CYP3A5.Experimental technique detects with the RT-PCR among the embodiment 5.The primer sequence of PCR is as shown in table 13.
The result shows that (result is referring to Fig. 9; Semi-quantitative results is contrast with Negative, and the CYP3A5mRNA of shRNA expression plasmid equal proportion mixture group cell expresses percentage ratio); ShRNA expression plasmid equal proportion mixture does not suppress the genetic expression of CYP3A5, has good specificity.Because this mixes three target sites of plasmid to CYP3A4, so this result has stronger cogency, and the shRNA expression plasmid equal proportion mixture of the present invention effect of not missing the target is described.Can be used as the specific inhibitor of CYP3A4, carry out other research.
The application of embodiment 7 shRNA expression plasmid equal proportion mixtures in conclusive evidence CYP3A4 substrate
The positive contrast of classical inductor Rifampin with CYP3A4.Experiment is divided into two groups, is respectively Rifampin inductive HepG2 cell transfecting shRNA expression plasmid equal proportion mixture group and Rifampin inductive HepG2 cell transfecting negative control plasmid group.Concrete operations do, in Rifampin inductive HepG2 cell kind to two 96 orifice plate.Next day is transfection shRNA expression plasmid equal proportion mixture and negative control plasmid respectively.Add the GA (15:1) and the GA (17:1) of gradient dilution behind the 48h, concentration is set to 5,10,20,40,50,80,100 (μ M), and each concentration is established 3 multiple holes (averaging), and establishing not, the dosing cell is a control group.Hatch and add MTT (5mg/mL) behind the 12h and continue to hatch 4h.Stop cultivating, inhale and abandon culture supernatant in the hole, every hole adds 150 μ l DMSO, and vibration 10min reads absorbance in ELIASA 570nm place and calculates medicine half amount of suppression IC
50
The toxicity result of GA (15:1) is referring to Figure 10, table 14.In transfection in the Rifampin inductive HepG2 cell of Negative plasmid, the IC of GA (15:1)
50Be 47.4 μ M; And in transfection in the HepG2 cell of shRNA expression plasmid equal proportion mixture the expression amount of CYP3A4 reduce, the metabolism of GA (15:1) is weakened, GA (15:1) activity reduces, IC
50Increasing, is 62.4 μ M.
This result shows that GA (15:1) is the substrate of CYP3A4, and it is produced stronger activity by the CYP3A4 metabolism, and after CYP3A4 was suppressed by shRNA expression plasmid equal proportion mixture, the active of GA (15:1) significantly reduced.Therefore, shRNA expression plasmid provided by the invention can be used for compound and become property of medicine evaluation, screening and conclusive evidence CYP3A4 substrate and inductor; Increase activity or increase toxic compound through the CYP3A4 metabolism, again according to these prediction of result compound potential drug-drug interactions.
。
< 110>Zhejiang University
< 120>shRNA expression plasmid and structure and the application of inhibition CYP3A4
<160>?9
<210>?1
<211>?1512
<212>?DNA
< 213>artificial sequence
<220>
<221>?CDS
<222>?(1)…(1512)
< 223>human-cytochrome P450 CYP3A4 cDNA sequence
<440>1
atg?gct?ctc?atc?cca?gac?ttg?gcc?atg?gaa?acc?tgg?ctt?ctc?ctg 45
gct?gtc?agc?ctg?gtg?ctc?ctc?tat?cta?tat?gga?acc?cat?tca?cat 90
gga?ctt?ttt?aag?aag?ctt?gga?att?cca?ggg?ccc?aca?cct?ctg?cct 135
ttt?ttg?gga?aat?att?ttg?tcc?tac?cat?aag?ggc?ttt?tgt?atg?ttt 180
gac?atg?gaa?tgt?cat?aaa?aag?tat?gga?aaa?gtg?tgg?ggc?ttt?tat 225
gat?ggt?caa?cag?cct?gtg?ctg?gct?atc?aca?gat?cct?gac?atg?atc 270
aaa?aca?gtg?cta?gtg?aaa?gaa?tgt?tat?tct?gtc?ttc?aca?aac?cgg 315
agg?cct?ttt?ggt?cca?gtg?gga?ttt?atg?aaa?agt?gcc?atc?tct?ata 360
gct?gag?gat?gaa?gaa?tgg?aag?aga?tta?cga?tca?ttg?ctg?tct?cca 405
acc?ttc?acc?agt?gga?aaa?ctc?aag?gag?atg?gtc?cct?atc?att?gcc 450
cag?tat?gga?gat?gtg?ttg?gtg?aga?aat?ctg?agg?cgg?gaa?gca?gag 495
aca?ggc?aag?cct?gtc?acc?ttg?aaa?gac?gtc?ttt?ggg?gcc?tac?agc 540
atg?gat?gtg?atc?act?agc?aca?tca?ttt?gga?gtg?aac?atc?gac?tct 585
ctc?aac?aat?cca?caa?gac?ccc?ttt?gtg?gaa?aac?acc?aag?aag?ctt 630
tta?aga?ttt?gat?ttt?ttg?gat?cca?ttc?ttt?ctc?tca?ata?aca?gtc 675
ttt?cca?ttc?ctc?atc?cca?att?ctt?gaa?gta?tta?aat?atc?tgt?gtg 720
ttt?cca?aga?gaa?gtt?aca?aat?ttt?tta?aga?aaa?tct?gta?aaa?agg 765
atg?aaa?gaa?agt?cgc?ctc?gaa?gat?aca?caa?aag?cac?cga?gtg?gat 810
ttc?ctt?cag?ctg?atg?att?gac?tct?cag?aat?tca?aaa?gaa?act?gag 855
tcc?cac?aaa?gct?ctg?tcc?gat?ctg?gag?ctc?gtg?gcc?caa?tca?att 900
atc?ttt?att?ttt?gct?ggc?tat?gaa?acc?acg?agc?agt?gtt?ctc?tcc 945
ttc?att?atg?tat?gaa?ctg?gcc?act?cac?cct?gat?gtc?cag?cag?aaa 990
ctg?cag?gag?gaa?att?gat?gca?gtt?tta?ccc?aat?aag?gca?cca?ccc 1035
acc?tat?gat?act?gtg?cta?cag?atg?gag?tat?ctt?gac?atg?gtg?gtg 1080
aat?gaa?acg?ctc?aga?tta?ttc?cca?att?gct?atg?aga?ctt?gag?agg 1125
gtc?tgc?aaa?aaa?gat?gtt?gag?atc?aat?ggg?atg?ttc?att?ccc?aaa 1170
ggg?gtg?gtg?gtg?atg?att?cca?agc?tat?gct?ctt?cac?cgt?gac?cca 1215
aag?tac?tgg?aca?gag?cct?gag?aag?ttc?ctc?cct?gaa?aga?ttc?agc 1260
aag?aag?aac?aag?gac?aac?ata?gat?cct?tac?ata?tac?aca?ccc?ttt 1305
gga?agt?gga?ccc?aga?aac?tgc?att?ggc?atg?agg?ttt?gct?ctc?atg 1350
aac?atg?aaa?ctt?gct?cta?atc?aga?gtc?ctt?cag?aac?ttc?tcc?ttc 1395
aaa?cct?tgt?aaa?gaa?aca?cag?atc?ccc?ctg?aaa?tta?agc?tta?gga 1440
gga?ctt?ctt?caa?cca?gaa?aaa?ccc?gtt?gtt?cta?aag?gtt?gag?tca 1485
agg?gat?ggc?acc?gta?agt?gga?gcc?tga 1512
<210>?2
<211>?28
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity upstream primer of hCYP3A4 amplification total length
<440>?2
gct?cta?gaa?tgg?ctc?tca?tcc?cag?act?t
<210>?3
<211>?30
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of hCYP3A4 amplification total length
<440>?3
gcg?gcc?ggc?ctc?agg?ctc?cac?tta?cgg?tgc
<210>?4
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity upstream primer of hCYP3A4 RT-PCR detection
<440>?4
aaa?tct?gag?gcg?gga?agc
<210>?5
<211>?20
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of hCYP3A4 RT-PCR detection
<440>?5
ttg?gga?tga?gga?atg?gaa?ag
<210>?6
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity upstream primer of hCYP3A5 RT-PCR detection
<440>?6
aaa?tct?gag?gcg?gga?agc
<210>?7
<211>?20
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of hCYP3A5 RT-PCR detection
<440>?7
ttg?gga?tga?gga?atg?gaa?ag
<210>?8
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity upstream primer of people GAPDH RT-PCR detection
<440>?8
tcc?acc?acc?ctg?ttg?ctg?tag
<210>?9
<211>?22
<212>?DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of people GAPDH RT-PCR detection
<440>?9
gac?cac?agt?cca?tga?cat?cac?t
Claims (3)
1. suppress the shRNA expression plasmid of CYP3A4, make up through following method:
(1) in Genebank, searches the sequence NM.017460 of CYP3A4; According to the interference fragment principle of design: begin from the AUG initiation codon of transcript mRNA, seek " AA " two and connect sequences, and write down 19 base sequences of its 3' end; With the siRNA of GC content about 45%-55% as potential siRNA target site; And, utilize design software, and get rid of the sequence that has homology with other genes of genome through Blast software according to the sequence of CYP3A4; Obtain three satisfactory shRNA template sequences, action site is respectively 773,923 and 1191;
(2) with sterilized water 773 justice of chemosynthesis are diluted to 50 μ M respectively with antisense shRNA template powder, under 95 ℃ to 25 ℃ conditions of progressively lowering the temperature, are annealed into double-stranded shRNA template, be cloned into then on the pSilencer4.1 CMV of BamHI and HindIII double digestion neo carrier; Transform DH5 α competent cell; Ammonia benzyl antibiotic-screening positive colony is chosen incubated overnight in some bacterium colonies that grow to the LB substratum, extracts plasmid with plasmid extraction kit; Wherein 773 1-6:shRNA expression plasmids 773 are cloned 1-6); Choose 773 No. 2 evaluations of checking order, the result is correct back in-80 ℃ of preservations, 773 No. 2 called after S1;
Wherein with 923 justice and antisense shRNA template powder and 1191 justice and antisense shRNA template powder replacement 773 justice and antisense shRNA template powder; 923 1-6:shRNA expression plasmids, 923 clone 1-6; 923 No. 2 called after S2; 1191 1-6:shRNA expression plasmids, 1191 clone 1-6,1191 No. 1 called after S3;
The shRNA template positive-sense strand sequence in 773 sites is:
5'-GATCCAGTCGCCTCGAAGATACACTTCAAGAGAGTGTATCTTCGAGGCGACTTTA-3'
The antisense strand sequence is:
5'-AGCTTAAAGTCGCCTCGAAGATACACTCTCTTGAAGTGTATCTTCGAGGCGACTG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-AGTCGCCTCGAAGATACAC-3' and 5'-GTGTATCTTCGAGGCGACT-3' are respectively the justice and the antisense sequences of 773 target spots;
The shRNA template positive-sense strand sequence in 923 sites is:
5'-GATCCACCACGAGCAGTGTTCTCTTTCAAGAGAAGAGAACACTGCTCGTGGTTTA-3'
The antisense strand sequence is:
5'-AGCTTAAACCACGAGCAGTGTTCTCTTCTCTTGAAAGAGAACACTGCTCGTGGTG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-ACCACGAGCAGTGTTCTCT-3' and 5'-AGAGAACACTGCTCGTGGT-3' are respectively the justice and the antisense sequences of 923 target spots;
The shRNA template positive-sense strand sequence in 1191 sites is:
5'-GATCCGCTATGCTCTTCACCGTGATTCAAGAGATCACGGTGAAGAGCATAGCTTA-3'
The antisense strand sequence is:
5'-AGCTTAAGCTATGCTCTTCACCGTGATCTCTTGAATCACGGTGAAGAGCATAGCG-3'
Wherein the 5'-GGATCC-3' at sequence two ends and 5'-AAGCTT are respectively BamHI and HindIII restriction enzyme site, are used for being connected with pSilencer4.1 CMV neo carrier; Sequence intermediary 5'-TTCAAGAGA-3' is the loop sequence; 5'-GCTATGCTCTTCACCGTGA-3' and 5'-TCACGGTGAAGAGCATAGC-3' are respectively the justice and the antisense sequences of 1191 target spots.
2. the application of shRNA plasmid in the specific inhibitor of preparation CYP3A4 of inhibition CYP3A4 according to claim 1 is characterized in that, is used for the screening and the conclusive evidence of CYP3A4 substrate and inductor.
3. application according to claim 2 is characterized in that, described three kinds of independent or equal proportion mixing uses of shRNA plasmid that suppress CYP3A4.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441845A (en) * | 2000-06-01 | 2003-09-10 | 株式会社大塚制药工场 | Method of detecting and quantifying human P450 molecular species and probe and kit for this method |
| CN1916174A (en) * | 2006-08-31 | 2007-02-21 | 暨南大学 | siRNA sequence of idiosyncratic silent nm23II1, and carrier and application |
| CN102281758A (en) * | 2009-01-16 | 2011-12-14 | 公益财团法人实验动物中央研究所 | Mouse having human hepatocytes transplanted therein |
-
2012
- 2012-01-19 CN CN2012100171773A patent/CN102586325A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441845A (en) * | 2000-06-01 | 2003-09-10 | 株式会社大塚制药工场 | Method of detecting and quantifying human P450 molecular species and probe and kit for this method |
| CN1916174A (en) * | 2006-08-31 | 2007-02-21 | 暨南大学 | siRNA sequence of idiosyncratic silent nm23II1, and carrier and application |
| CN102281758A (en) * | 2009-01-16 | 2011-12-14 | 公益财团法人实验动物中央研究所 | Mouse having human hepatocytes transplanted therein |
Non-Patent Citations (1)
| Title |
|---|
| JIE CHEN ET AL: "Small Interfering RNA-Mediated Silencing of Cytochrome P450 3A4 Gene", 《DRUG METABOLISM AND DISPOSITION》, vol. 34, no. 9, 31 December 2006 (2006-12-31), pages 1650 - 1657 * |
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