CN106047874A - Dnazyme molecule of targeted SALL4 gene and application thereof in breast cancer gene therapy - Google Patents
Dnazyme molecule of targeted SALL4 gene and application thereof in breast cancer gene therapy Download PDFInfo
- Publication number
- CN106047874A CN106047874A CN201610383360.3A CN201610383360A CN106047874A CN 106047874 A CN106047874 A CN 106047874A CN 201610383360 A CN201610383360 A CN 201610383360A CN 106047874 A CN106047874 A CN 106047874A
- Authority
- CN
- China
- Prior art keywords
- dnazyme
- cell
- sall4
- gene
- breast cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a dnazyme molecule of a targeted SALL4 gene and application thereof in breast cancer gene therapy, and belongs to the technical field of targeted genes. According to a coder SALL4 mRNA sequence (NM_020436.4), the dnazyme molecule is designed and constructed, and nucleotide sequences of the dnazyme molecule are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively. The designed dnazyme molecule is a small nucleic acid which can recognize and cut sequences of the targeted gene, has the high targeted gene sequence cutting capacity, also has the chemical stability of antisense oligodeoxynucleotide and is low in synthesis. The mRNA sequences of the targeted gene SALL are effectively recognized and efficiently cut through the dnazyme molecule, so that proliferation, migration and infiltration of the breast cancer are restrained, and finally small nucleic acid drugs for restraining SALL4 expression to achieve breast cancer gene therapy are constructed.
Description
Technical field
The invention belongs to target gene technical field, be specifically related to a kind of targeting SALL4 gene deoxyribozyme molecules and
Its application in mastocarcinoma gene is treated.
Background technology
" whole world cancer report 2014 " display, whole world cases of cancer will present swift and violent growing trend, by 2012 1400
Ten thousand people, 19,000,000 people of cumulative year after year to 2025 year, it was up to 24,000,000 people by 2035.Meanwhile, China's tumor incidence is many
Persistently rise over Nian, it has also become a social problem that must pay much attention to.2015, Cancer in China always fell ill 429.16 ten thousand
Example, the most dead 281.42 ten thousand examples.The clinical treatment of cancer currently mainly includes chemotherapy, X-ray therapy and operative treatment
Deng.But, long-term chemical treatment is easily produced blastomogenic multi-drug resistance phenomenon, X-ray therapy and operative therapy and is difficult to tumor
Thorough radical cure, simultaneously serious in above strategy toxic and side effects brings great misery to patient.Therefore, a kind of height is built
Effect, the ideas of cancer therapy of safety, have great importance for solving this mankind's persistent ailment.
Compared with conventional therapeutic strategy, gene therapy, as a kind of emerging means fundamentally treating disease, becomes
The focus of international academic community research at present, is expected to " new lover " becoming in future tumors clinical treatment.Gene therapy, refers to pass through
Exogenous gene is introduced target cell or the tissue of pathological changes by gene transfer technique, by the expression of genes of interest, corrects or compensates
Dcc gene, closes or suppresses the normal function of the gene of unconventionality expression, recovery organization or organ, thus reach therapeutic purposes
A kind of novel method for the treatment of.Mankind's SALL4 gene is the homeotic gene of Drosophila gene Drosophila spalt, is positioned at
The q13.2 of No. 20 chromosomes.This gene has 4 exons, has two kinds of hypotypes, i.e. SALL4A and SALL4B, its difference be by
Different splicing mechanism in gene internal exon 2 are caused.Generally, the expression of SALL4 is in embryo, in ripe group
It is reticent in knitting.Research in recent years shows, at breast carcinoma, pulmonary carcinoma, gastric cancer, colon cancer, hepatocarcinoma, carcinoma of endometrium, neuroglia
Many solid tumors such as tumor all detect the process LAN of SALL4.As can be seen here, SALL4 is probably a crucial oncogene,
The generation of tumor and development play an important role.Therefore, with SALL4 gene as action target spot, exploitation small-molecule chemical thing,
SiRNAs etc., reduce the expression of this gene, are expected to the novel strategy becoming in clinical therapy of tumor.
DNAzyme (Deoxyribozyme, DNAzyme) is that a class can specifically identify and cut target gene mRNA
Single-stranded DNA fragments, it is thus possible to reduce the expression of Disease-causing gene, reach prevention and the purpose for the treatment of disease, simultaneously the most not
Cellular genome can be produced any side effect.At present, by the method for manual simulation, tens kinds of deoxidation cores are synthesized
Enzyme, including 10-23 type DNAzyme, 8-17 type DNAzyme, " gun type " DNAzyme etc..Wherein, 10-23 type DNAzyme
Research is the most extensive, and it is made up of an active center and two target gene brachium conjunctivums, active center sequence high conservative,
Target gene brachium conjunctivum and target gene mRNA are specific binding by base complementrity, and then mRNA can be cut in active center, lowers
The expression of target gene.Compared with siRNA, DNAzyme has higher target gene mRNA cutting efficiency, to tumor
More effectively, efficiency of simultaneously missing the target is lower, and side effect is low, has broad application prospects for the inhibition of growth.Meanwhile, deoxidation
The chemical nature of ribozyme is deoxy-oligonucleotide, and it also has the advantage that (1) molecular weight is little, and character is the most stable, to substrate
Accessibility good；(2) catalytic efficiency and specificity are high；(3) it is readily synthesized, cheap.In a word, DNAzyme is accurate due to it
Target gene mRNA sequence recognition and cutting power, it has also become the tumour-specific inhibitor that a class is important, be expected to become future
A class novel small nucleic acids genomic medicine in therapy of tumor.
Summary of the invention
It is an object of the invention to the mRNA sequence (NM_020436.4) according to encoding human SALL4 and design and build deoxidation core
Enzyme molecule, by deoxyribozyme molecules effectively identifying and high efficiency cutting target gene SALL mRNA sequence, suppresses breast carcinoma
Propagation, migrate and infiltrate, the final class that builds expresses the small nut acids realizing mastocarcinoma gene treatment by suppression SALL4
Medicine.
Deoxyribozyme molecules employed in the present invention is 10-23 type DNAzyme, is that a class can specifically identify
And cut the Single-stranded DNA fragments of target gene mRNA, particular sequence includes: an active center and two target gene brachium conjunctivum groups
Becoming, (being made up of 15 deoxyribonucleotides, its sequence is 5 '-GGCTAGCTACAACGA-to active center sequence high conservative
3 '), target gene brachium conjunctivum (being made up of 9 deoxyribonucleotides) and target gene mRNA are specific binding by base complementrity,
Its cleavage site is 5 '-R ↓ Y-3 ' (R is formed without base pairing, and Y then must form base pairing with DNAzyme).
Shown in one of the sequence of the deoxyribozyme molecules of the present invention is following, its nucleotide sequence respectively as SEQ ID NO.1,
Shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4:
1.5’-CCGCAGTTA GGCTAGCTACAACGA TTTGCCCTC-3’
2.5’-CTTTAGGTA GGCTAGCTACAACGA CACCACAGA-3’
3.5’-CTTGGGGAA GGCTAGCTACAACGA TTATAGTTT-3’
4.5’-CTGGAAGGA GGCTAGCTACAACGA TGTGGTTTC-3’
In the present invention, deoxyribozyme molecules can increase stability, the bound phosphate groups of composition DNAzyme by modification
Can modify in the free singlet oxygen position of natural phosphodiester key section, can be wherein oxygen or methoxyl group or
Sulfur.
DNAzyme involved in the present invention can reduce the expression of SALL4 by cutting the identification of mRNA sequence,
Realize the suppression to breast carcinoma propagation by induced apoptosis and Cycle Arrest, breast cancer cell can be reduced simultaneously
Migration wetting capacity.
To sum up, the deoxyribozyme molecules designed by the present invention is a kind of small nucleic acids being capable of identify that and cutting target-gene sequence
Molecule, not only has high target-gene sequence cutting power, has the chemical stability of antisense oligonucleotide simultaneously, synthesizes cost
Low.By the high efficiency cutting to target gene SALL4mRNA sequence, DNAzyme is capable of breast carcinoma propagation, migrates and soak
The suppression of profit ability, is expected to become a class effective malignant tumor gene therapy medicament.The enforcement of the present invention, will be pernicious to solving
This mankind's persistent ailment of tumor has great importance, and will produce great economic and social benefit.
Accompanying drawing explanation
The Sketch of secondary structure figure of Fig. 1 target gene SALL4mRNA；
The Sketch of secondary structure figure of the potential target site of Fig. 2 target gene SALL4mRNA；
Fig. 3 DNAzyme is to breast cancer cell MCF7 (A), the inhibition analysis curve of MDA-MB-231 (B) cell proliferation；
Fig. 4 DNAzyme is to breast cancer cell MCF7 (A), the inhibitory action of MDA-MB-231 (B) Colony forming ability
Figure；
Fig. 5 DNAzyme inducing mammary cancerous cell MCF7 (A), the detection figure of MDA-MB-231 (B) apoptosis；
Fig. 6 DNAzyme is on breast cancer cell MCF7, the impact of MDA-MB-231 cell death related protein expression
Figure；
Fig. 7 DNAzyme affects figure to breast cancer cell MCF7 (A) and MDA-MB-231 (B) mitochondrial membrane potential；
In figure, the appearance (with arrows in figure) of JC-1 monomer represents the decline of mitochondrial membrane potential in anoxic.
Fig. 8 DNAzyme affects figure to breast cancer cell MCF7 (A) and MDA-MB-231 (B) cell cycle；
Fig. 9 DNAzyme affects figure to breast cancer cell MCF7 (A) and MDA-MB-231 (B) transfer ability；
Figure 10 DNAzyme affects figure to breast cancer cell MCF7 (A) and MDA-MB-231 (B) cellular infiltration ability.
Detailed description of the invention
Example given below is that the invention will be further described, in order to those skilled in the art is more fully
Understand the present invention.But given embodiment is it is not intended that limiting the scope of the invention, thus the technology of this specialty
Nonessential improvement and adjustment that personnel are made according to foregoing invention content also should belong to scope.
Embodiment 1
According to the mRNA sequence of people's SALL4 gene, carry out the Sketch of secondary structure under mRNA physiological condition, result such as Fig. 1；
According to DNAzyme design principle, choose corresponding target site region, design four DNAzyme oligonucleotide sequences, target site
Chosen position as in figure 2 it is shown, it is thio-modification that the two ends of every deoxyribozyme molecules are respectively arranged with 3 nucleotide, use PAGE or
HPLC purification, designed DNAzyme sequence is as follows:
1.Dz-SALL4-12:5’-CCGCAGTTA GGCTAGCTACAACGA TTTGCCCTC-3’
2.Dz-SALL4-62:5’-CTTTAGGTA GGCTAGCTACAACGA CACCACAGA-3’
3.Dz-SALL4-143:5’-CTTGGGGAA GGCTAGCTACAACGA TTATAGTTT-3’
4.Dz-SALL4-191:5’-CTGGAAGGA GGCTAGCTACAACGA TGTGGTTTC-3’
Embodiment 2
Breast cancer cell MCF7, three negative breast cancer MDA-MB-231 use containing the tire Sanguis Bovis seu Bubali that volume fraction is 10%
Clearly, 100U/mL penicillin, the DMEM cell culture fluid of 100 μ g/mL streptomycins, at 37 DEG C, volume fraction is the CO of 5%2Condition
Under cultivate and pass on.The transfection of DNAzyme uses business-like transfection reagent GoldenTransferTM-D (GT-D), with
Transfection reagent is mixed by the ratio of 2:1 (v/wt) with 4 kinds of deoxyribozyme molecules in embodiment 1, hatches 20min in 37 DEG C,
Add to cell culture system, transfect 4h with serum free culture system, and continue to cultivate in the culture medium containing serum
48h.Cells Proliferation of Human Breast Cancer suppression detection process is as follows: with 5 × 103The density of cells/well is respectively by MCF7, MDA-MB-231
Cell is inoculated in 96 orifice plates, every hole culture medium 200 μ L, preculture 24h.By GoldenTransferTM-D/ DNAzyme is combined
Thing is diluted to 1,2,3,4 μ g/mL, adds to 96 orifice plates, and with DMEM cell culture fluid, (mensuration about cell survival rate is divided into
4 kinds of GoldenTransferTM-D/ DNAzyme complex (4 curve) and GoldenTransferTM-D (1 curve),
The group only adding cell culture fluid regards as 100% survival, embodies in the drawings at 0 μ g/mL, therefore without curve, and 5 songs altogether
Line) and GoldenTransferTM-D is as negative control.Cultivating to 48h, every hole adds 20 μ L 5mg/mL tetrazolium bromide (MTT)
Solution, to final concentration of 0.5mg/mL, continues to cultivate 4h.After 4h, careful suction abandons supernatant, adds 150 μ L dimethyl sulfoxides and dissolves generation
Violet precipitate, vibration 5min after, under 492nm, detect absorbance by microplate reader.Take the meansigma methods of each parallel hole OD value,
According to following equation calculating comparative survival rate of cells:
Cell viability (%)=(Asample-Ablank)/(Acontrol-Ablank) × 100%
Wherein, AsampleFor being separately added into GoldenTransferTM-D/ DNAzyme complex and only adding
GoldenTransferTMAbsorbance after-D, AcontrolFor normally cultivating the absorbance after cell with DMEM cell culture fluid
Value, AblankFor without cultivating the absorbance after cell only adds DMEM cell culture fluid
To GoldenTransfer in researchTM-D/ DNAzyme complex carries out the dilution of variable concentrations, acts on tumor
48h detection after cell, result is as shown in Figure 3.With the increase of deoxyribozyme molecules concentration, DNAzyme is thin to two kinds of breast carcinoma
The Proliferation Ability ability of born of the same parents substantially increases.Wherein, for the deoxyribozyme molecules Dz-SALL4-12 action effect of Loop12 design
The most obvious, this DNAzyme to be substantially better than three negative breast cancer MDA-to the action effect of breast cancer cell MCF7 simultaneously
MB-231。
Embodiment 3
With 2 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.Dz-SALL4-12 is transfected 4h with the concentration serum-free medium of 3 μ g/mL, changes complete medium into
Cultivate to 48h.The trypsinization attached cell using mass body fraction to be 0.25%, and thin to collect with counts plate
Born of the same parents count.In six new orifice plates, every hole adds 3000 cells after different experiments group processes, and within every 5 days, changes liquid once, training
After supporting 14 days, abandoning cells and supernatant, fix 20min with the ice ethanol that volume fraction is 75%, the phosphoric acid of the pH7.2 of pre-cooling delays
Rush liquid (Phosphate buffer solution, PBS) to clean once, be subsequently added the crystallization that mass body fraction is 0.2%
Cell is dyeed by purple/PBS solution, 4 DEG C of dyeing 20min.Subsequently, use the PBS of pre-cooling to there is no loose colour, be inverted aobvious
Take pictures under micro mirror.
In the present invention, we evaluate the shadow of DNAzyme cell cluster Forming ability by the quantity of Colony forming
Ringing, result is as shown in Figure 4.Compared with matched group, after the transfection of DNAzyme Dz-SALL4-12, cell colony quantity significantly reduces,
And transfect nonsense DNAzyme iDz experimental group and be individually added into transfection reagent GT-D group and there is no significant change, it is seen that the present invention
Designed deoxyribozyme molecules can significantly inhibit the Colony forming ability of breast cancer cell.
Embodiment 4
With 2 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.Dz-SALL4-12 is transfected 4h with the concentration serum-free medium of 1,2,3,4 μ g/mL respectively, changes into
Complete medium is cultivated to 48h.According to making of Annexin V-FITC/PI cell apoptosis detection kit (upper sea cowry winning company)
Coherent detection is carried out: the trypsinization attached cell using mass body fraction to be 0.25%, 2000g is centrifuged 5min by description
Collect cell, with the PBS washed cell twice of pre-cooling；With 50 μ L dyeing systems, cell is dyeed, including 2 μ L
Annexin V-FITC and 2 μ L PI, hatches 30min after mixing under the conditions of 4 DEG C of lucifuges；Combining buffer with Annexin V will
System is diluted to 400 μ L, by flow cytomery Apoptosis of Breast Cancer situation.
From Fig. 5, we are it is clear that the transfection of DNAzyme all has good withering to two kinds of breast cancer cells
Die inducing effect, breast carcinoma MCF7 action effect is become apparent from simultaneously.In the case of 3 μ g/mL early apoptosis ratio i.e. up to
To 26.70%, under comparable sodium, DNAzyme induces three negative breast cancer MDA-MB-231 early apoptosis of cells to be 15.94%.
Embodiment 5
With 1 × 106MCF7, MDA-MB-231 cell is inoculated in 10cm culture dish by the density of cells/well respectively, every ware
Culture medium 10mL, preculture 24h.Transfect DNAzyme Dz-SALL4-12 4h with the condition serum-free medium of 3 μ g/mL, change
Complete medium is become to cultivate to 48h.With cell scraper by cell collect and together with culture medium supernatant collect to pre-cooling 50mL from
In heart pipe, 4 DEG C of centrifugal 5min of 12000rpm.Albumen precipitation is resuspended with the PBS of pre-cooling, 4 DEG C of centrifugal 1min of 12000rpm, abandon
Supernatant, adds the cell pyrolysis liquid RIPA and 100 × protease inhibitor PMSF of 3 times of precipitation volumes in precipitation, splits on ice
Solving 1h, wherein every 15min shake makes precipitation uniform.Subsequently with 4 DEG C of centrifugal 15min of 12000rpm, supernatant is transferred to new
In 1.5mL centrifuge tube pipe.Take 10 μ L sample, demarcate protein concentration, residue with BCA protein quantification test kit (Beijing Ding Guo company)
Sample add 5 × SDS sample-loading buffer, 100 DEG C are boiled sample 15min, and after being cooled to room temperature after boiling sample, subpackage puts into-20 DEG C of refrigerators
Preserve.Preparation mass fraction is SDS-PAGE polyacrylamide gel and the 80V prerunning 5min of 12%, during loading, every hole
40 μ g protein contents add sample, and protein sample is compressed to concentrate glue/separation gel interface by 80V voltage 20min, and change voltage is extremely
120V, about 1h rear electrophoresis is to gel lower edge.With 300mA constant current ice bath 3h, cell whole protein is gone on pvdf membrane.Take the film out
After drying, film is pressed different molecular weight split to suitable size, put into mass body fraction be 5% defatted milk powder/0.1%
Tween20/PBS solution, closes pvdf membrane 2h at normal temperatures, removes the albumen non-specific binding of membrane removal.Blot with filter paper after taking-up,
Put in antibody hybridization bag, antibody with mass fraction be 3% bovine serum albumin (Bovine serum albumin, BSA)/
PBS solution is diluted to proper proportion (SALL4 1:1000；β-actin 1:2000, antibody is purchased from Abcam company) add hybridization
In Dai, shake overnight incubation at 4 DEG C.0.1%Tween20/PBS cleans three times, and each 10min is to remove the non-specific of antibody
In conjunction with, add 3%BSA/PBS solution dilution corresponding two anti-(1:5000).0.1%Tween20/PBS cleaning three times, every time
10min is to remove two anti-non-specific binding.Finally with ECL colour reagent box (Shanghai Tian Neng scientific & technical corporation) exposure imaging.
From Fig. 6 it will be seen that in breast cancer cell MCF7 and MDA-MB-231, the effect energy of DNAzyme
Enough significantly reduce SALL4 albumen expression, transfection nonsense DNAzyme iDz experimental group and be individually added into transfection reagent
GT-D group correlative protein expression amount is held essentially constant.Meanwhile, the effect of DNAzyme can reduce cell death related protein
Pro-Caspase-3, pro-Caspase-9, the expression of cell shifting related protein MMP9 albumen.This result proves, turns
DNAzyme Dz-SALL4-12 of dye can reduce the expression of SALL4, and the SALL4 of downward have activated pro-Caspase9, has
The Caspase9 of activity and then have activated the activation of pro-Caspase-3, and then inducing cell there occurs apoptosis, i.e. DNAzyme
Induced apoptosis is carried out by activating mitochondrial apoptotic pathway.
Embodiment 6
With 2 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.Transfect DNAzyme Dz-SALL4-12 4h with serum-free medium, change complete medium into and cultivate extremely
48h.Grasp according to the operation instructions of JC-1 mitochondrial membrane potential detection kit (the green skies, Shanghai biotech company)
Make: with PBS cell once after, in culture plate add 1mL JC-1 dye working solution, at 37 DEG C, volume fraction is 5%
CO2Cell is dyeed by incubator, hatches 20min.By the incubation buffer of preheating, cell is carried out；Finally to
Culture plate adds 1mL PBS, takes pictures at fluorescence microscope.Using the cell of the inactive deoxyribose iDz of transfection as
Negative control, with the cell of the oxidative phosphorylation inhibitors CCCP pretreatment 20min of final concentration of 1 μM as positive control.
From Fig. 7 it will be seen that after DNAzyme effect, in cell mitochondrial, JC-1 monomer substantially increases, and turns
Contaminate nonsense DNAzyme iDz experimental group and be individually added into the not significant change of transfection reagent GT-D group.This result is further
Proving, the deoxyribozyme molecules designed by the present invention carrys out withering of inducing mammary cancerous cell by active cell mitochondrial apoptotic pathway
Die.
Embodiment 7
With 2 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.Transfect Dz-SALL4-12 4h with serum-free medium, change complete medium into and cultivate to 48h.According to
Cell cycle detection kit (upper sea cowry wins company) description operates: using mass body fraction is the pancreatin of 0.25%
Digestion attached cell, 2000g is centrifuged 5min, abandons supernatant, and cell uses the PBS of pre-cooling to wash twice, and volume fraction is 75%
The fixing 1h of ice-cold ethanol-20 DEG C, then washed once with cold PBS, and resuspended with the 100 cold PBS of μ L；Above-mentioned system adds
RNase solution A 20 μ L, 37 DEG C of water-bath 30min remove the RNA in cell；Add 300 μ L PI dye liquors, mix latter 4 DEG C gently and keep away
Light hatches 30min, by the Cycle Arrest situation of flow cytomery breast cancer cell.
Shown in Fig. 8, compared with matched group, the transfection of DNAzyme Dz-SALL4-12 can make breast carcinoma MCF7 cell line
G2 phase ratio is increased to 21.8% by 14.7%, makes three negative breast cancer MDA-MB-231 g2 phase retardances be increased by 13.8%
To 22%, and transfect nonsense DNAzyme iDz experimental group and be individually added into transfection reagent GT-D group g2 phase content and there is no
Significant change.This result shows, the deoxyribozyme molecules designed by the present invention can effectively realize the G2 phase of breast cancer cell line
Cycle Arrest, it is achieved the suppression to growth of tumour cell.
Embodiment 8
With 3 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.When cell in culture plate grows to the 90% of culture plate area, linearly draw with yellow rifle head
Going out " wound ", PBS is once to remove cell debris.DNAzyme Dz-SALL4-12 is transfected, after 4h with serum-free medium
Change complete medium into cultivate.Take pictures every 12h inverted microscope, and measure the spacing distance of " wound " in monolayer.
We can observe that from Fig. 9, compared with matched group, the experimental group breast carcinoma of DNAzyme Dz-SALL4-12
The migration rate of cell significantly reduces, and transfects nonsense DNAzyme iDz experimental group and be individually added into transfection reagent GT-D group
Migration rate is without significant change.This result shows, the DNAzyme designed by the present invention can significantly inhibit breast cancer cell
Transfer ability.
Embodiment 9
With 2 × 105MCF7, MDA-MB-231 cell is inoculated in 6 orifice plates by the density of cells/well respectively, and every hole is cultivated
Base 2mL, preculture 24h.Transfect DNAzyme Dz-SALL4-12 4h with serum-free medium, change complete medium into and cultivate extremely
48h.The trypsinization attached cell using mass body fraction to be 0.25%, being 1% Ox blood serum containing mass body fraction
Albuminous serum-free medium re-suspended cell also counts, to aperture be 0.8 μm Transwell cell upper room add 2 ×
104Individual cell, adds 600 μ L under Transwell and contains the DMEM culture medium that volume fraction is 10% hyclone in room,
Incubator is cultivated 24h.Cultivate to the time, with 4 DEG C of fixing 20min of ice ethanol that volume fraction is 75%.With disposably
The cell not passed through in the upper room of medical cotton stick erasing, the cell mass fraction through semipermeable membrane is the crystal violet/PBS of 0.2%
Solution dyeing 20min, after PBS three times, takes pictures with inverted microscope.
From Figure 10 we can with clear view to, compared with matched group, after the effect of DNAzyme Dz-SALL4-12 test
The cell quantity of the lower room of group significantly reduces, and transfects nonsense DNAzyme iDz experimental group and be individually added into transfection reagent GT-D
Group cell quantity there is no significant change.This result shows, the infiltration energy to breast cancer cell of the DNAzyme designed by the present invention
Power has significant inhibitory action.
Claims (2)
1. the deoxyribozyme molecules of a targeting SALL4 gene, it is characterised in that: its nucleotide sequence such as SEQ ID NO.1,
Shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.
2. the deoxyribozyme molecules of a kind of targeting SALL4 gene described in claim 1 application in mastocarcinoma gene is treated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610383360.3A CN106047874B (en) | 2016-06-02 | 2016-06-02 | A kind of deoxyribozyme molecules targeting SALL4 gene and the application in mastocarcinoma gene treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610383360.3A CN106047874B (en) | 2016-06-02 | 2016-06-02 | A kind of deoxyribozyme molecules targeting SALL4 gene and the application in mastocarcinoma gene treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106047874A true CN106047874A (en) | 2016-10-26 |
| CN106047874B CN106047874B (en) | 2019-01-18 |
Family
ID=57173176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610383360.3A Active CN106047874B (en) | 2016-06-02 | 2016-06-02 | A kind of deoxyribozyme molecules targeting SALL4 gene and the application in mastocarcinoma gene treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106047874B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046353A (en) * | 2021-04-02 | 2021-06-29 | 复旦大学附属肿瘤医院 | Deoxyribozyme probe for differential screening of specific induction triple negative breast cancer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002099090A1 (en) * | 2001-06-07 | 2002-12-12 | Johnson & Johnson Research Pty Ltd | Bcl-2 dnazymes |
| EP2322619A1 (en) * | 2009-11-17 | 2011-05-18 | Deutsches Krebsforschungszentrum | Inhibitors of centrosomal clustering |
| CN103890587A (en) * | 2011-08-31 | 2014-06-25 | 昂科赛特公司 | Methods and compositions for the treatment and diagnosis of cancer |
| CN104651406A (en) * | 2015-02-11 | 2015-05-27 | 湖南大学 | Gene silencing kit and method |
-
2016
- 2016-06-02 CN CN201610383360.3A patent/CN106047874B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002099090A1 (en) * | 2001-06-07 | 2002-12-12 | Johnson & Johnson Research Pty Ltd | Bcl-2 dnazymes |
| EP2322619A1 (en) * | 2009-11-17 | 2011-05-18 | Deutsches Krebsforschungszentrum | Inhibitors of centrosomal clustering |
| CN103890587A (en) * | 2011-08-31 | 2014-06-25 | 昂科赛特公司 | Methods and compositions for the treatment and diagnosis of cancer |
| CN104651406A (en) * | 2015-02-11 | 2015-05-27 | 湖南大学 | Gene silencing kit and method |
Non-Patent Citations (6)
| Title |
|---|
| KOBAYASHI, D ET AL.: "SALL4 is essential for cancer cell proliferation and is overexpressed at early clinical stages in breast cancer", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
| MITCHELL, A ET AL.: "Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1", 《NUCLEIC ACIDS RESEARCH》 * |
| 初秋 等: "脱氧核酶对乳腺癌细胞CyclinD1 mRNA表达的影响", 《中国妇幼保健》 * |
| 李元 等: "《基因工程药物》", 31 December 2002, 化学工业出版社 * |
| 赵家明 等: "脱氧核酶对端粒酶MNO 的切割和乳腺癌细胞凋亡相关基因表达的作用", 《第一军医大学学报》 * |
| 陈园园 等: "ALL4基因沉默对MCF-7/A细胞增殖和化疗敏感性的影响", 《中国肿瘤》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046353A (en) * | 2021-04-02 | 2021-06-29 | 复旦大学附属肿瘤医院 | Deoxyribozyme probe for differential screening of specific induction triple negative breast cancer |
| CN113046353B (en) * | 2021-04-02 | 2024-02-06 | 复旦大学附属肿瘤医院 | Differential screening deoxyribozyme probe for specifically inducing triple negative breast cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106047874B (en) | 2019-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108179194A (en) | A kind of tumor cells marker circBIRC6 and its inhibitor and purposes | |
| CN106282347A (en) | HoxC11 as biomarker preparation adenocarcinoma of lung pre-diagnostic reagent in application | |
| CN110123828A (en) | Application of the inhibitor of PRALR in the drug that resistance to taxol oophoroma is treated in preparation | |
| CN104774929B (en) | The application of diagnosis, treatment and prognosis of the 3p of miR 455 in esophageal squamous cell carcinoma | |
| CN110251529A (en) | MiR-124-3p and its analog are preparing the application in anti-breast cancer disease medicament | |
| CN105039342B (en) | SiRNA and its application of MAT2A gene expressions can be suppressed | |
| CN106420712A (en) | Application of anisomycin to preparation of medicine for preventing/treating ovarian cancer | |
| CN105732560A (en) | Genistein derivative as well as preparation method and application thereof in pharmacy | |
| CN106047874B (en) | A kind of deoxyribozyme molecules targeting SALL4 gene and the application in mastocarcinoma gene treatment | |
| CN102218146B (en) | ShRNA (Short Hairpin Ribonucleic Acid) kit for treating animal model suffered from alzheimer disease | |
| CN101705227B (en) | SiRNA for inhibiting human AP-2alpha gene expression and anti-cervical cancer application thereof | |
| CN101928747B (en) | Application of E3 ubiquitin ligase CHIP in gliomatosis cerebri disease | |
| CN105648073A (en) | Ischemic stroke screening kit and application thereof | |
| CN109745335A (en) | MiR-218 is preparing the application in mammary cancer chemotherapy drug sensitizer | |
| CN104531709A (en) | siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof | |
| CN106822898B (en) | Application of the STAT3 gene expressions in terms of improving adenocarcinoma of lung chemosensitivity is lowered in targeting | |
| CN111575384A (en) | Application of human GLT8D1 gene in clinical diagnosis and treatment of tumor | |
| CN104368012B (en) | The purposes and its related drugs of people's RPL34 gene | |
| CN108949974A (en) | E3 ubiquitin ligase ASB3 is preparing the application in cancer treatment drug | |
| CN103656673B (en) | The purposes and its related drugs of people's YWHAQ genes | |
| CN102416183B (en) | Application of micro ribose nucleic acid (RNA) (miR)-297b | |
| CN112280859B (en) | Breast cancer marker and application thereof | |
| CN102337266B (en) | siRNAs and recombinant vector for inhibiting expression of Nogo A, MAG and OMgp genes, and application of siRNAs and recombinant vector | |
| CN101787368B (en) | siRNA for restraining Z38 gene expression of human being and application thereof in preparing breast-tumor resisting medicine | |
| CN106806903B (en) | Application of the LIN28 gene expression in terms of improving Small Cell Lung Cancer chemosensitivity is lowered in targeting |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |