CN103877594A - Application of silenced EF4 protein coding gene to cancer treatment - Google Patents

Application of silenced EF4 protein coding gene to cancer treatment Download PDF

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CN103877594A
CN103877594A CN201210566942.7A CN201210566942A CN103877594A CN 103877594 A CN103877594 A CN 103877594A CN 201210566942 A CN201210566942 A CN 201210566942A CN 103877594 A CN103877594 A CN 103877594A
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秦燕
郝佳
朱萍
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Abstract

The invention provides application of a reagent silencing EF4 protein coding gene in human cancer cell to prepare medicines for inhibiting growth of the cancer cells, and more specifically, the invention provides application of a siRNA interference fragment of human EF4 protein coding gene to prepare medicines for inhibiting growth of human cancer cells.

Description

The application of EF4 protein coding gene silence aspect treatment of cancer
Technical field
The present invention relates to the application of gene silencing in treatment of cancer.Particularly, the reagent that the invention provides the EF4 protein coding gene silence making in human cancer cell is in the purposes for the preparation of suppressing in the medicine of described growth of cancer cells, more specifically, the invention provides for the siRNA interference fragment of people EF4 protein coding gene in the purposes for the preparation of suppressing in the medicine of human cancer cell growth.
Background technology
Mitochondrion is evolved by Archimycetes, has DNA, RNA and the protein-synthesizing system of oneself.Mitochondrial DNA can encode 2 kinds of rRNA, 22 kinds of tRNA, 8-13 kind respiratory chain complex protein subunit.In the mankind's mitochondrion, 13 kinds of mitochondrial proteins of mitochondrial DNA coding, 7 kinds of (ND1-4, ND4L, ND5 and ND6) albumen are the subunits on Respiratory Chain Complex I; A kind of (cytochrome b) is the subunit on Complex II I; 3 kinds (CoxI, CoxII, CoxIII) are the subunits on complex IV; Two kinds (ATP6 and ATP8) are the subunits on complex V.
The assembling of Respiratory Chain Complex I, III, IV, V requires the coordinate expression of nucleus DNA (nDNA) and mitochondrial DNA (mtDNA).On each respiratory chain complex, most of subunit is encoded by nDNA, the post translational modification of albumen process, and maturation, transposition process enters mitochondrion.Mitochondrial translation machine 55S system and prokaryote translation system 70S are similar, but it has the feature of oneself: (1) translation regulatory mechanism is relatively simple, and synthesis rate is cytoplasmic 10-20 times; (2) translation error rate is higher.Translation limit, the product limit assembling of 55S ribosome translation, causes complex subunit to have lower translation fidelity.So we have just proposed such query: thereby how 55S machine translator maintains sufficiently high translation fidelity match with Cytoplasm 80S system?
Mitochondrial respiratory chain composite I, III, IV can further be assembled into super complex (RCS), and the structure of RCS is conducive to the cross-film transmission of electronics, improve ATP combined coefficient.In human cell, the assembling of RCS is a necessary step that ensures eupnea.RCS assembling defect may cause respiratory disorder.But the research at present of the molecular mechanism of RCS assembling is not too thorough.
EF4 albumen is the most conservative albumen in all known genomes, and in prokaryote, it can the ribosome in carrying out albumen extension process be combined and causes the reversed rotation of tRNA.Meanwhile, research finds that EF4 also has GTP hydrolytic enzyme activities and chaperone activity, has reduced the error rate of protein translation.It is synthetic that research in yeast finds that EF4 can affect mitochondrial albumen under stressed condition, but in humanization cell, the research of EF4 function be have not been reported.
As from the foregoing, EF4 albumen has the congener of high conservative in human cell, its Subcellular Localization should be in the energy factory of cell---in mitochondrion, metabolism to mankind's normal cell and cancer cell has important function, for the mankind's cancer, diagnosis and the treatment of metabolic disease provide potential biomarker and target site, and provide clue for the design of related drugs.
Summary of the invention
We find that EF4 albumen has important regulating and controlling effect to mitochondrial function in human cancer cell first.The silence of EF4 protein coding gene can suppress the growth of human cancer cell effectively.
More specifically, the invention provides the following:
1. the reagent that makes the EF4 protein coding gene silence in people's cell is in the purposes for the preparation of suppressing in the medicine of described Growth of Cells.
2. according to the purposes described in 1, the sequence of wherein said EF4 protein coding gene is as shown in SEQ IDNo.1.
3. according to the purposes described in 1 or 2, the wherein said pack of the EF4 protein coding gene silence in people's cell that makes is containing the siRNA interference fragment for described EF4 protein coding gene.
4. according to the purposes described in 3, wherein said siRNA interference fragment is mixture, and described mixture comprises sequence three kinds of siRNA interference fragments as shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.
5. according to the purposes described in 4, the mixed proportion of wherein said three kinds of siRNA interference fragments is 1: 1: 1.
6. according to the purposes described in 1 or 2, wherein said people's cell is human cancer cell.
7. according to the purposes described in 6, wherein said human cancer cell is Human cervical cancer cell lines Hela cell or human lung cancer cell line H1299 cell.
Below in conjunction with specific embodiment, the invention will be further described.
Brief description of the drawings
Fig. 1: the detection of the outer function of EF4 albuminous body in humanization cell;
Fig. 2: eukaryotic cell EF4 (being once called as Guf1) mRNA horizontal expression detects;
The Subcellular Localization of Fig. 3: EF4 (being once called as Guf1) albumen in eukaryotic cell;
Fig. 4: the jamming effectiveness of EF4 in eukaryotic cell (Guf1) gene detects;
Fig. 5: the impact of the expression of humanization EF4 albumen on cell phenotype;
Fig. 6: the impact of humanized EF4 (being once called as Guf1) albumen on mitochondrial respiratory chain cpd function.
Detailed description of the invention
The detection of the outer function of EF4 albuminous body in embodiment 1, humanization cell
Whether there is similarity in order to study in humanization cell the function of EF4 albumen in EF4 albumen and prokaryote, our purification EF4 in humanization cell and the EF4 albumen in prokaryote, ribosome 70S, has carried out a series of experiment in vitro.Fig. 1 is the detection of the outer function of EF4 in humanization cell (being once called as Guf1, GTPase of unknown function 1) albuminous body.Figure 1A represents the combination experiment of humanization EF4 (being once called as Guf1) albumen and ribosome 70S, and result shows that humanized EF4 can combine with ribosome 70S; Figure 1B represents the contrast of EF4 (the being once called as Guf1) binding constant in escherichia coli EF4 and humanization cell, finds that both binding constants are similar; Fig. 1 C represents footprinting experiment, detect humanization EF4 albumen and whether can be combined with ribosome 23rRNA (G and T: sequence band, C: do not have DMS to modify, N.F: do not add the factor), arrow represents that the A1607 of 23S rRNA and A2660 site have been subject to protection, and result shows that humanized EF4 can combine with 23S rRNA; Fig. 1 D represents the contrast of EF4 (being once called as Guf1) the GTP hydrolysis rate in escherichia coli EF4 and humanization cell, GTP hydrolysis rate: each ribosome was hydrolyzed the quantity of GTP in 5 minutes, and result shows that both have similar GTP hydrolysis ability; Fig. 1 E represents pyromycin experiment, detects EF4 and whether has the function (tRNA of red-label and the block diagram: AC[that mediate tRNA reversed rotation 3h] Phe-tRNA, the tRNA of blue markings and block diagram: [ 32p] tRNA met f), result shows that humanized EF4 has the ability of catalysis tRNA reversed rotation; Fig. 1 F represents external albumen compound experiment, and reactant mixture is divided into two parts: SDS-PAGE (upper strata swimming lane, total GFP); Also virgin rubber (middle swimming lane, the activated GFP of tool).Under the condition that there is no the factor, (N.F) total GFP synthetic quantity is defined as 100%, total according to this calculating, the relative quantity (N.P.: the reactant liquor of the GFP plasmid of not encoding) of the activated GFP of tool and active fragment, result shows the generation that can promote the activated GFP fragment of more tools that adds of humanization EF4 albumen, shows that humanized EF4 albumen has the effect that maintains protein translation fidelity.
Concrete grammar:
Separation and purification EF4 (being once called as Guf1) albumen: transform and have the BL21 bacterial strain 1% of EF4 (being once called as Guf1) plasmid to inoculate in 2L LB culture medium, 37 degree are cultured to exponential phase (OD A600 approximately 0.6), be cooled to add 0.3mM isopropylthiogalactoside after 16 degree, induce the centrifugal collection of 4 degree 20 hours.Centrifugal 30 minutes of 4 degree, 15000rpm after carrying out ultrasonic bacteria breaking, supernatant is crossed nickel post, the albumen (being kept in 20mM Tris-HCl pH7.5,500mM sodium chloride) that 100mM imidazoles eluting needs, is finally further purified EF4 (being once called as Guf1) albumen with the HiLoad16/60 Superdex of GE company 75.
Separation and purification reconstruct ribosome: the ratio in 1% is by the MRE600 inoculation of incubated overnight in 4L LB culture medium, and 37 degree are cultured to exponential phase (OD A600 approximately 0.6), 4 degree 4000rpm receive bacterium for centrifugal 30 minutes.Resuspended with the Tico buffer (20mM Hepes-KpH7.6,6mM magnesium acetate, 30mM ammonium acetate, 4mM β mercaptoethanol) of 4 times of volumes, twice of fragmentation under 1000Bar pressure condition.Centrifugal 90 minutes of broken bacterium liquid 30000g, supernatant is poured in another centrifuge tube 45000g into centrifugal 24 hours, is precipitated as the ribosome of slightly carrying.The resuspended ribosome of Tico buffer, approximately 6000A 260slightly carry ribosome by sucrose density gradient centrifugation (0-40% sucrose, Tico buffer).After band centrifugation (16 hours 22000rpm, Ti15 rotor), collect 70S ribosome (24 hours 24000rpm, Ti45 rotor).10ml buffer (the 20mM Hepes-KpH7.6 that dissociates for precipitation, 1mM magnesium acetate, 200mM ammonium acetate, 4mM β mercaptoethanol) resuspended, then band centrifugation (0-40% sucrose, buffer dissociates) separate 30S and 50S ribosomal subunit (Ti15 rotor 4 is spent 22000rpm, centrifugal 17 hours), collect respectively 30S and 50S ribosomal subunit.30S and the each 1500A260 of 50S subunit are blended in to (20mM Hepes-KpH7.6,20mM magnesium acetate, 30mM ammonium acetate, 4mM β mercaptoethanol) in reunion buffer, and 40 degree temperature are bathed 4 degree after 1 hour and are hatched 10 minutes.Hatch rear ribosome sucrose density gradient centrifugation and separate (10%-40% sucrose, 18000rpm 17 hours) 30S subunit and 70S ribosome.The 70S ribosome of collecting leaves the heart 24 hours with 45Ti rotor at 4 degree 24000, and the resuspended 70S ribosome of reunion buffer is hatched 20 minutes at 40 degree, and liquid nitrogen flash freezer after subpackage is kept in-80 degree refrigerators.
Ribosome is spent and is hatched altogether 15 minutes in conjunction with the 70S ribosome of experiment: 60pmol and the EF-G of 28pmol and 25 μ MGMPPNP 37, buffer system is binding buffer liquid (20mMHepes-KOH, pH7.5,4.5mM magnesium acetate, 150mM ammonium acetate, 4mM β mercaptoethanol, 2mM spermidine and 0.05mM spermine).120 μ l samples are added on 75 μ l sucrose pads, sucrose 1.1M is dissolved in buffer: 20mM Hepes-KOH, pH 7.5,4.5mM magnesium acetate, 150mM ammonium acetate, 4mM β mercaptoethanol, 2mM spermidine, 0.05mM spermidine and 25 μ M GMPPNP, spend centrifugal 30 minutes with TLA100 rotary head (Beckman) 78000rpm 4.Supernatant sucking-off, precipitates resuspended with 20 μ l Tico buffer (20mM Hepes-KOH, pH 7.5,6mM magnesium acetate, 30mM ammonium acetate, 4mM β mercaptoethanol).Survey A260, ribosome 5pmol carries out 10%SDS-PAGE gel electrophoresis.
Footprinting experiment: experiment can be divided into 2 steps, the one, the preparation of functional complex and chemical reagent are modified; The 2nd, primer extension.
For the protected mode of research EF-G to the upper A2660 of 23S rRNA sarcin-ricin loop (SRL) site; and the protected mode of the upper A1067 of 23S rRNA GTPase-associated center (GAC); press literature method (Savelsbergh, Matassova etc. 2000) and prepare ribosome EF-GGTPFA complex.20pmol ribosome and EF-G wild type or mutant (20pmol) and 1mM GTP and 2mM FA 37 spend hatches 10 minutes altogether, reaction system is: 80mM Hepes-KOH pH 7.8,100mM ammonium chloride, 20mM magnesium chloride, then adds dimethylsphingosine (DMS) to hatch again and within 8 minutes, carries out chemical modification.
Can equally with wild type stablize ribosomal hybrid state for detecting EF-G mutant, prepare ribosome function complex (100 μ l): reactant liquor (80mM Hepes-KOH pH 7.8,100mM ammonium chloride, 20mM magnesium chloride) in add 70S ribosome (20pmol), MF-mRNA (80pmol) and Ac-[ 14c] Phe-tRNA phe(40pmol), 37 degree water-bath 20 minutes; Then add the puromycin of 1mM, 37 degree water-baths 20 minutes, make P site Ac-[ 14c] Phe-tRNA phedeacylation.Ribosome complex now adds EF-GGDPNP again, and 37 degree water-baths after 20 minutes add dimethylsphingosine 37 and spend water-bath and modify for 8 minutes.
Analyze EF-G transposition activity and also have a kind of method, determine PRE or POST state by detecting the binding site of tRNA on ribosome.(100 μ are the preparation of PRE l): at reaction buffer (80mM Hepes-KOH pH 7.8,100mM NH for ribosome function complex 4cl, 20mMMgCl 2) in add 70S ribosome (20pmol), MF-mRNA (80pmol) and tRNA fMet(40pmol), 37 degree water-bath 15 minutes; Then add Ac-[ 14c] Phe-tRNA phe(40pmol), mix rear 37 degree water-bath 30 minutes.In PRE complex, add GTP (1000pmol) and EF-G (4pmol) 37 degree water-bath 10 minutes, tRNA-mRNA is shifted.Then add dimethylsphingosine 37 to spend water-bath and within 8 minutes, carry out chemical modification.
Primer extension reaction, the rRNA of modification after the extracting of phenol/chloroform, [ 32p] primer of labelling and the rRNA of modification 90 spend water-bath and anneal for 5 minutes, then adds reverse transcription to carry out reverse transcription.After degraded rRNA, 8% degeneration glue separates.Primer extension termination site is reacted and is determined by dideoxy sequencing, and the template of reverse transcription is the rRNA of unmodified.2 independently experiments are at least done in each reaction.Press the intensity of phosphorus screen detection reaction band.
GTP hydrolysising experiment: buffer used: H-mix:80mM Hepes-K pH7.6,9mM magnesium chloride, 1320mM ammonium chloride, 16mM β mercaptoethanol, 0.5mM spermine, 20mM spermidine; Tico buffer: 20mM Hepes-K pH7.6,6mM magnesium chloride, 30mM ammonium chloride, 4mM β mercaptoethanol; Cessation reaction liquid: 100 μ l water saturation 2-butanols, 40 μ l 0.5M sulphuric acid+1.5mM sodium dihydrogen phosphate, 10 μ l 200mM sodium molybdate.
Whole reaction system is made up of three parts: 10%H-mix, the 30%H of 60%Tico and dissolving GTP 2o.Final reaction condition is: 20mM Hepes-K pH7.6,4.5mM magnesium chloride, 150mM ammonium chloride, 4mM β mercaptoethanol, 0.05mM spermine, 2mM spermidine.Ribosome and GTP enzyme are dissolved in 1*Tico buffer.In the system of 10 μ l, the concentration of ribosome in reaction system is 0.2 μ M, and the final concentration of GTPase enzyme is 0.02-0.2 μ M, [32P] γ-GTP50 μ M (50-1000dpm/pmol).After adding [32P] γ-GTP, start reaction, in 37 degree water-baths, react, require to add stop buffer cessation reaction according to reaction.
After reaction terminating, shake 1 minute, centrifugal 10 minutes of room temperature 13000rpm, 2-butanol can extract free γ-Pi, gets 80 μ l 2-butanols liquid phases in 2ml scintillation solution, shakes up rear isotope counting.
Puromycin experiment: first prepare PRE complex, the reaction system of 12.5 μ l is containing 5pmol70S ribosome, 20pmol MF-mRNA and 10pmol tRNA fMet, above reaction system within 15 minutes, make tRNA enter P site in 37 degree reactions; Then with 10pmol ofAc-[14C] Phe-tRNA phehatch 30 minutes at 37 DEG C, now reaction system cumulative volume is 25 μ l, Ac-[14C again] Phe-tRNA pheenter A site, PRE complex forms.The complex of PRE state is hatched 10 minutes at 37 degree together with EF-G (0.1mM GTP), and now reaction system cumulative volume is 35 μ l.Then add 2.5 μ l puromycins, 37 degree are hatched 5 minutes, detect isotopic content and determine the ribosomal amount of POST state.
The outer compound experiment of albuminous body: taking Poly (U) as template, [ 14c] Phe-tRNA phefor the synthetic poly of substrate ([ 14c] Phe) polypeptide chain.Reaction completes (20mM Hepes-KpH7.6,4.5mM magnesium chloride, 150mM ammonium chloride, 4mM β mercaptoethanol, 0.05mM spermine, 2mM spermidine) under the ion condition of optimizing.In conjunction with the preparation of mixed liquor, 7.5 μ l reaction systems, the 70S ribosome of 1pmol and 10 μ g poly (U)-mRNA first 37 degree is hatched 15 minutes.The charging mix of then, the above-mentioned combination mixed liquor of 5 μ l and 10 μ l preheatings (2 minutes, 37 degree) together 37 degree is hatched 5 minutes.Preparation charging mix, by 15.5pmol[ 14c] Phe-tRNA phe(specific activity1000dpm/pmol), 5nmolGTP, 3pmol EF-G, 30pmol EF-Ts and 30pmol EF-Tu mix.Synthetic poly ([ 14c] Phe) product, add after ice-cold TCA precipitation, liquid scintillation counter detects isotopic content in synthetic peptide chain.
The selection of EF4 protein function system in embodiment 2, research body
For convenient research EF4 (being once called as Guf1) albumen function in vivo, we need to select the abundant cell system of EF4 (being once called as Guf1) protein expression as object of study, we have selected the cell line (comprising cancerous cell and normal cell) of several In vitro culture, detect the expression of EF4 (being once called as Guf1) in mRNA level by PCR method.Fig. 2 represents PCR result, wherein 1299 (H1299): lung carcinoma cell (purchased from ATCC, preserving number: CRL-5803); Hela: cervical cancer cell (purchased from ATCC, preserving number: CCL-2); BR, A2: lung carcinoma cell; SY5Y: neuroma cell; 3T3: l cell; 293: HEKC.It is higher that PCR result is presented in cancer cell H1299 cell and Hela cell EF4 (being once called as Guf1) protein mRNA horizontal expression, EF4 (be once called as Guf1) albumen expression on mRNA level do not detected by the method in non-cancer cell in as 3T3 cell and HEK293 cell, illustrate that this protein expression level is not high in non-cancer cell, therefore we select the cancer cell Hela cell of EF4 (being once called as Guf1) expressed in abundance and H1299 cell as research system.
Concrete grammar:
RT-PCR experiment: use Trizol reagent to separate the total RNA of Hela cell.Using oligo (dT) 15 as primer, the total RNA of 2 μ g reverse transcription under the effect of reverse transcription is become to strand cDNA.Synthetic specific Guf1 primer (Invitrogen is synthetic):
guf1-s：5-CAAGCCCAAACTGTAGCAAA-3；
guf1-a：5-CCAGCAATCAGATAGCCCAC-3；
RT-PCR amplification condition: degeneration-94 degree, 30 seconds; Annealing-55 degree, 30s; Extend-72 degree, 30 seconds; Cycle-index: 30 times; Finally extend condition-72 degree, 10 minutes.Each PCR sample of equal volume carries out agarose gel electrophoresis (gum concentration: 1%).
The research of embodiment 3, humanization EF4 (being once called as Guf1) protein subcellular location
In order to study the Subcellular Localization of EF4 (being once called as Guf1) albumen in eukaryotic cell, we have taked Western blot and two kinds of methods of immunofluorescence.Fig. 3 is the Subcellular Localization of humanization EF4 (being once called as Guf1) albumen.Fig. 3 A represents the westernblot result of EF4 (being once called as Guf1) albuminous cell location, COXIV: the marker molecule of mitochondrion component, Actin: internal reference contrast, westernblot result shows that EF4 (being once called as Guf1) albumen and mitochondrial marker molecule COXIV occur in mitochondrion component simultaneously.Show that EF4 (being once called as Guf1) protein localization is at mitochondrion.Fig. 3 B represents immunofluorescence result (GFP: the control vector of expressing GFP, GFP-Guf1:Guf1N end labelling GFP, Guf1-GFP:Guf1C end labelling GFP, Mito Tracker is mitochondrial markers), result shows that the Guf1 of N end labelling GFP does not locate altogether with mitochondrion, Guf1 and the mitochondrial label MitoTracker of C end labelling GFP locate altogether, illustrate that the N terminal sequence of EF4 (Guf1) has affected its mitochondrion location.
Concrete grammar:
The each component of isolated cell: a dish Hela cell (purchased from ATCC, preserving number: CCL-2) that is paved with 100mm plate is discarded to waste liquid, with phosphate buffer cleaning 2 times, add 1ml mitochondrion Extraction buffer (purchased from Beijing Puli's Lay company limited), cell is scraped cell harvesting, utilizes tissue grinder by lysis, 800g, 4 centrifugal 5 minutes of degree, get supernatant, 13000g, 4 degree recentrifuge, precipitation is mitochondrion, and supernatant is Cytoplasm composition.BCA protein quantification (purchased from Thermo), respectively get 50 μ g albumen and carry out SDS-PAGE, transferring film, 5% defatted milk powder sealing, adds COXIV antibody, Actin antibody, EF4 (Guf1) antibody (purchased from CST) to detect separately.
Immunofluorescence experiment: the plasmid vector with humanization EF4 (being once called as Guf1) gene is granted by K.H.Nierhaus professor (Max-Planck-Institute for Molecular Genetics, Berlin).Coding humanization EF4 (being once called as Guf1) DNA fragmentation reacts amplification by above-mentioned plasmid by PCR.Primer sequence is:
guf1-gfp-n2-s：5-CGCCTC?GAGCATGTGGACCCTCGT-3；
guf1-gfp-n2-a：5-CCCAGGTACCATTTAGAAGATTGTGTT-3。Kpn I and Xho I site are added to the end of primer, and PCR product is connected with pEGFP-N2 plasmid.Primer sequence: guf1-gfp-c3-s:5-CGGCTCGAGTGGACCCTCGT-3;
guf1-gfp-c3-a：5-TCCGGTACCTTATTTAGAAGATTGTGTT-3。EcoRI and Xho I site are added to the end of primer, and PCR product is connected with pEGFP-C3 plasmid.2 μ g build plasmid and utilize in the instantaneous Hela of the proceeding to cell of liposome 2000 (purchased from Invitrogen company).After transfection 24 hours, in cell culture fluid, adding final concentration is the MitoTracker of 5 μ M, lucifuge reaction 15 minutes, and under laser confocal microscope, (Olympus Fluoview FV500) observes the distribution of fluorescence.
EF4 in embodiment 4, eukaryotic cell (being once called as Guf1) gene silencing efficiency detects
In order to study the function of EF4 (being once called as Guf1) gene in eukaryotic cell, we utilize the expression of the reticent EF4 of mode (being once called as Guf1) of siRNA interference, the variation of observation of cell phenotype, discloses the effect of carrying out EF4 (being once called as Guf1) gene silencing with this.The siRNA fragment (purchased from Shanghai JiMa pharmacy Technology Co., Ltd) of synthetic three sections of reticent EF4 (being once called as Guf1) gene, the mixture of three sections of siRNA (mixed proportion is: 1: 1: 1) transfection is entered to Hela cell (NC: contrast siRNA, Si:siRNA-EF4,1 group and 2 groups of two parallel sampless that represent in Hela cell) and H1299 cell, western testing result shows that Hela cell and H1299 cell transfecting disturb after the little RNA of EF4 (Guf1), and the expression of EF4 (Guf1) albumen obviously reduces (Fig. 4).The above results shows that interference effect is good, and above-mentioned disturbed condition is applied in functional study below.
Concrete grammar:
EF4 (being once called as Guf1) gene expression silence:
The RNA interfering (siRNA) of 3 EF4 (being once called as Guf1) gene is as follows:
5-GGUUCUUGAUAAAUUGCAATT-3；
5-GAAGAGCAGUUCAGAAGAATT-3；
5-GUUGUUGUUGUGAAGAUUATT-3；
Above interference fragment and contrast siRNA are all purchased from Shanghai Ji Ma company.By about 4*10 5individual H1299 cell or Hela cell are inoculated in 35mm Tissue Culture Dish, incubated overnight.The EF4-siRNA (mixture of three fragments) and the NC-siRNA (contrast) that are 100 μ M by final concentration utilize respectively lipofectamine 2000 (purchased from Invitrogen company) transfection to enter H1299 cell or Hela cell, after transfection 72 hours, utilize Western blot method on protein level, to detect the jamming effectiveness that EF4 expresses.
Embodiment 5, the research of humanization EF4 (being once called as Guf1) albumen on cell phenotype impact
Fig. 5 is the expression of humanization EF4 (being once called as Guf1) the albumen impact on cell phenotype.Fig. 5 A and 5B are illustrated in H1299 cell and Hela cell and adopt MTT method to detect the speed of growth of cell.Cell is seeded in new culture medium, measure respectively 1,2,3, cellular enzymes intensity alive after 4,5 days (the interference siRNA of Si:EF4 (being once called as Guf1), NC: contrast siRNA), the cell strain growth speed that result is presented at EF4 (being once called as Guf1) gene silencing in two kinds of cells is considerably slower than normal group cell, shows that EF4 (being once called as Guf1) gene silencing has suppressed the growth of cell; Fig. 5 C utilizes flow cytometer cell cycle to measure, by each period cell number add up, the cell number showed increased that is stuck in the G1 phase after result demonstration EF4 (being once called as Guf1) gene silencing, shows that EF4 (being once called as Guf1) gene silencing has suppressed cell division; Fig. 5 D utilizes the mitochondrial form of confocal laser scanning microscope (Hochest: nucleus is painted, MitoTracker: mitochondrion is painted), Image Display is compared with matched group, the obvious disperse of mitochondrion of EF4 (being once called as Guf1) gene silencing group, shows that EF4 (being once called as Guf1) gene silencing has disturbed maintaining of Mitochondrial Shape; Fig. 5 E is the detection that utilizes the cell oxygen expenditure speed that oxygen electrode carries out, after statistical result showed EF4 (being once called as Guf1) gene silencing, the speed of cell oxygen consumption obviously declines, and shows that EF4 (being once called as Guf1) gene silencing has suppressed mitochondrial respiratory; Fig. 5 F utilizes detection (line of red-label and the block diagram: after disturbing EF4 (being once called as Guf1) to express of the intracellular reactive oxygen yield that flow cytometer carries out, the line of density bullet and block diagram: contrast), result shows after EF4 (being once called as Guf1) gene silencing, in body, ROS content obviously rises, and shows that EF4 (being once called as Guf1) gene silencing is conducive to the generation of ROS; Fig. 5 G represents to utilize microscopic examination etoposide (etoposide, a kind of antitumor drug) nuclear shrinkage situation (pyknotic cells core occurs in arrow representative) after cell death inducing, after result demonstration EF4 (being once called as Guf1) gene silencing there is the shrinkage state of apoptosis sample in nucleus, shows that EF4 (being once called as Guf1) gene silencing has accelerated apoptotic process; Fig. 5 H uses western blot to detect the apoptotic signal path of Caspase-3 mediation.Caspase-3 and its downstream molecules PARP albumen occur that shearing band represents the activation of apoptosis pathway.Cytochrome C burst size in Cytoplasm is more, represent that apoptotic degree is more serious, result shows after EF4 (being once called as Guf1) gene silencing, the shear rate of Caspase-3 and downstream apoptosis molecule PARP thereof increases the weight of, the burst size of cytochrome C from mitochondrion increases, and equally also shows that EF4 (being once called as Guf1) gene silencing has accelerated apoptotic process.
Concrete grammar:
Growth of Cells speed detects: cell proliferation uses MTT (Thiazolyl blue) analytical method to measure: 1) by 4*10 3individual H1299 cell or Hela cell are inoculated in 96 orifice plates, and 37 degree are cultivated; 2) cell inoculation 1 day, 2 days, 3 days, 4 days, after 5 days, carefully suck culture medium, wash gently with phosphate buffer; 3) every hole adds 180 μ l fresh mediums, then (DMSO (DMSO), continues to cultivate 4 hours for 5mg/ml, i.e. 0.5%MTT to add 20 μ l MTT solution; 4) stop cultivating, carefully suck culture fluid in hole, every hole adds 150 μ l dimethyl sulfoxide, puts on shaking table (TS-8) low-speed oscillation 10 minutes, and crystal is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 490nm place; 5) zeroing hole (culture medium, MTT, dimethyl sulfoxide) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide), sets 3 multiple holes for every group.
Mitochondrial Shape is observed: EF4 (being once called as Guf1) interference group and cellular control unit are carried out to nucleus dyeing containing in the Hochest33258 that final concentration is 5 μ g/ml (purchased from Sigma) cell culture fluid, 20 minutes action time, adding final concentration is that the MitoTracker of 5 μ M carries out mitochondrion dyeing 30 minutes again, observes the variation of Mitochondrial Shape under laser confocal microscope.
Cell cycle detects: for the cycle of analysis of cells, the previous day is by 10 6individual cell is seeded in the Tissue Culture Dish of 35mm.Second day collecting cell-20 spent cell night and fixed in 70% ethanol.Phosphate buffer cleans cell 2 times, adds dyeing liquor (20g/ml propidium iodide, 50g/mlRNA enzyme) to hatch 30 minutes in 37 degree incubators.In cell, DNA quantity detects by flow cytometer.
Oxygen expenditure speed: trypsin digestion cell, the concentration of cell suspension is adjusted to 1.5*10 7individual/ml, 1.5ml cell suspension is as a reaction system.The mensuration of cell oxygen consumption regulates and controls the concentration of dissolved oxygen in reactive tank by an oxygen electrode.The fall off rate that 1.5ml does not contain oxygen in the cell culture medium of cell in contrast.
ROS measures: the mensuration of cell endoperoxides hydrogen concentration is to realize by the fluorescent probe DCFH-DA to isotope of redox-sensitive (purchased from green skies company).Collect 3*10 5individual cell, uses the cell culture medium cleaning of serum-free also resuspended, and adding DCFH-DA dyestuff to make its final concentration is 5 μ M, and cell is hatched 30 minutes in 37 degree cell culture incubators.DCF fluorescence is measured excitation wavelength by the cell sorting flow cytometer (Becton Dickinson, Flanklin Lakes, NJ, USA) of fluorescence excitation: 488nm; Emission wavelength: 535nm.
Cytochrome C discharges: 2 μ M etoposide (purchased from Sigma) process cell 24 hours, promotes the apoptotic process of cell.Processed group and untreated fish group cell clean 2 times with phosphate buffer, and trypsin digestion cell, is resuspended in (50mM potassium chloride in 50 μ l cell pyrolysis liquids by cell; 250mM glucose, 0.02%digitonin) place then centrifugal 10 minutes of 4 degree 10000g on ice 5 minutes.Supernatant comprises Cytoplasm composition, and precipitation comprises mitochondrion, and above-mentioned precipitation is used to RIPA lysate cracking (purchased from Thermo).BCA method detects protein concentration, and 50 μ g albumen carry out SDS-PAGE, utilize its content in Cytoplasm and mitochondrion of cytochrome C antibody test.
Embodiment 6, the impact of humanization EF4 (being once called as Guf1) albumen on mitochondrial respiratory chain cpd function
EF4 (being once called as Guf1) can regulating cell metabolism, and mitochondrion is an important place of cellular metabolism, has therefore detected the impact of EF4 (being once called as Guf1) albumen on mitochondrial respiratory chain cpd.Fig. 6 A is illustrated in the test experience of the ATP level of carrying out in H1299 cell, and after result shows EF4 (being once called as Guf1) gene expression silence, ATP level obviously declines; Fig. 6 B is illustrated in the test experience of the ATP level of carrying out in Hela cell, and after result shows EF4 (being once called as Guf1) gene silencing, ATP level obviously declines; Fig. 6 C represents to utilize Hela cell mitochondrial to carry out Blue-native glue and then carries out the variation of the respiratory chain complex amount of SDS-PAGE glue detection that (I, II, III, IV, V represent respectively mitochondrial respiratory chain composite I, II, III, IV, V, NC: contrast siRNA, Si:siRNA-EF4), result shows that EF4 (being once called as Guf1) gene silencing does not affect the amount of mitochondrial respiratory chain cpd; Fig. 6 D utilizes western blot by the expression (FP: Complex II subunit of antibody test mitochondrion respiratory chain complex subunit, UQCRC2: Complex II I subunit, COXIV: complex IV subunit, ATP5a: complex V subunit, Tim23: mitochondrial outer membrane protein, as negative control, Tim20: mitochondrial inner membrane albumen, as negative control, β-Actin: internal reference), result shows that EF4 (being once called as Guf1) gene silencing does not affect the expression of mitochondrial complex respiratory chain subunit; The enzyme-substrate chromogenic reaction that represents Fig. 6 E to utilize detects the each complex activity of mitochondrial respiratory chain (RCS: the super complex of respiratory chain, arrow represents the corresponding band of complex), result shows that EF4 (being once called as Guf1) gene silencing does not affect the activity of mitochondrial respiratory chain cpd, but the assembling of super complex has been subject to inhibition.
Separate mitochondria respiratory chain complex: collect 400 μ g Hela cell mitochondrials, add 40 μ l buffer A (50mM sodium chloride, 50mM imidazoles/hydrochloric acid, 1mM EDTA, pH 7.0) resuspended.Every pipe adds 12 μ l digitonin, mixes and acts on 10 minutes on ice.100000g, 4 centrifugal 30 minutes of degree, get supernatant, add 5 μ l glycerol and 6 μ l Coomassie brilliant blues.Get 40 μ l samples and carry out Blue-native PAGE separate mitochondria complex, the method document (Ilka Wittig, et al., nature 2006) that sees reference.
Mitochondrial respiratory chain complex activity detects:
Complex I activity: Blue-native PAGE separation gel is incubated in to 50mM Tris-Hcl, in pH 7.4 buffer, (contain 0.5mM NBT (Nitroblue tetrazolium chloride, chlorination nitro tetrazole), 5mM NADH (Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide)), room temperature effect 1 hour.
Complex II activity: Blue-native PAGE separation gel is incubated in to 50mM Tris-Hcl, in pH 7.4 buffer, (contain 4.5mM EDTA, 10mM KCN (potassium cyanide), 0.2mMPMS (Methylphenaziniummethylsulfate, azophenlyene Methylsulfate), 84mM succinic acid, 50mM NBT), room temperature effect 1 hour.
Complex IV activity: Blue-native PAGE separation gel is incubated in to 50mM Tris-Hcl, in pH 7.4 buffer, (contain 0.1% diaminobenzidine, 24 unit/ml catalases, 0.1% cytochrome C), 37 degree effect 3-6 hour.
Complex V activity: by clear water rinsing 10 minutes for Blue-native PAGE separation gel, the glycine buffer (pH 8.6) of putting into 0.1M acts on 1 hour.Again separation gel is put into the buffer that contains following ingredients: 35mM Tris alkali, 270mM glycine, 14mM magnesium sulfate, 5mM ATP, 0.2% silver nitrate, 37 degree effect 3-6 hour.
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Figure IDA00002637496600011
Figure IDA00002637496600021
Figure IDA00002637496600031
Figure IDA00002637496600041
Figure IDA00002637496600051
Figure IDA00002637496600061
Figure IDA00002637496600071
Figure IDA00002637496600081
Figure IDA00002637496600091
Figure IDA00002637496600101
Figure IDA00002637496600111
Figure IDA00002637496600121
Figure IDA00002637496600131
Figure IDA00002637496600141
Figure IDA00002637496600151

Claims (7)

1. the reagent that makes the EF4 protein coding gene silence in people's cell is in the purposes for the preparation of suppressing in the medicine of described Growth of Cells.
2. purposes claimed in claim 1, the sequence of wherein said EF4 protein coding gene is as shown in SEQID No.1.
3. the purposes described in claim 1 or 2, the wherein said pack of the EF4 protein coding gene silence in people's cell that makes is containing the siRNA interference fragment for described EF4 protein coding gene.
4. purposes claimed in claim 3, wherein said siRNA interference fragment is mixture, described mixture comprises sequence three kinds of siRNA interference fragments as shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.
5. purposes claimed in claim 4, the mixed proportion of wherein said three kinds of siRNA interference fragments is 1: 1: 1.
6. the purposes described in claim 1 or 2, wherein said people's cell is human cancer cell.
7. purposes claimed in claim 6, wherein said human cancer cell is Human cervical cancer cell lines Hela cell or human lung cancer cell line H1299 cell.
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