CN106822171B - The antisense base sequences of UBC9 inhibit the application in growth of cancer cells drug in preparation - Google Patents
The antisense base sequences of UBC9 inhibit the application in growth of cancer cells drug in preparation Download PDFInfo
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Abstract
The invention discloses the antisense base sequences of UBC9 to inhibit the application in growth of cancer cells drug in preparation.In the present invention, design has synthesized the antisense base sequences of a UBC9 specificity, and nucleotide sequence is as shown in SEQ ID NO.1.The present invention utilizes the antisense base sequences of UBC9 specificity, the UBC9 expression in the malignant cell containing double minute is reduced by the method for RNAi interference, double minute number is reduced, to reach the grade malignancy for reducing tumour cell, effectively inhibits the purpose of malignant cell growth.It is proposed of the invention is provides new magnetic target therapy scheme for the biological therapy of the malignant cell containing double minute.
Description
Technical field
The present invention relates to the new applications of the antisense base sequences of UBC9, specifically, the present invention relates to the antisenses of NSMCE2
Nucleotides sequence is listed in the application in preparation inhibition growth of cancer cells drug, belongs to technical field of cancer biotherapy.
Background technique
The UBC9 gene (Ubiquitin-conjugating enzyme) also known as UBE2I of the mankind.UBC9 gene is in the mankind
Universal high expression in tissue and organ, the biological function that the different tissues and different developmental phases of organism are played not
Together, the missing of the gene is fatal for organism.The missing of UBC9 gene will lead to the growth ability of tumour cell
Decline with invasive ability, the SUMOization of cell entirety is horizontal to be reduced, the ratio increase of apoptotic cell, cell occur endoreduplication with
Aging.Research of the UBC9 gene in malignant tumour has achieved apparent progress, and part illustrates UBC9 gene in tumour
Generation, development and prognosis in important function.
UBC9 albumen participates in the adjusting of cell cycle, the important access such as DNA damage reparation, the growth of cell, differentiation and
Apoptosis regulation etc. plays an important role.Research in recent years demonstrates UBC9 albumen in colon cancer, oophoroma, breast cancer
With high expression universal in the cancers such as lung cancer.This high expression of UBC9 albumen can be mediated by SUMOization approach and own
Approach plays an important role in the occurrence and development of tumour, as the key enzyme in SUMOization approach also increasingly by
Concern.
Double minute as malignancy marker, tumour cell proliferation and in terms of play it is important
Effect, and often carry a large amount of MYC gene on double minute.
Double minute is the outstanding feature object of the outer oncogene amplification of chromosome, is the marker of malignancy, swollen
The proliferation of oncocyte and apoptosis etc. play an important role, and usually carry oncogene and drug resistant gene thereon,
The abnormal amplification of these genes, frequently can lead to tumor drug resistance and growth ability enhancing, with the genomic instability of tumour cell,
Grade malignancy increases closely related with drug resistance.Double minute is often accompanied with malignant disease and generates, and in solid tumor
Incidence is very high.The unconventionality expression of oncogene MYC is frequently accompanied by hematological system tumor and solid tumor, for UBC9 gene
Silencing can inhibit the expression of MYC gene, and then influence the generation of tumour cell.These prompt us, and double minute may be in tumour
Evolution and deterioration degree etc. play certain effect.
The characteristics of for tumour cell, which carries out specific treatment, can be improved the sensibility and specificity of oncotherapy, have
Arrow is put, the present invention is directed to the UBC9 closely related with ovarian cancer cell and Colon and rectum gland cancer cell occurrence and development, with its antisense core
Nucleotide sequence is interfered, thin with tumour for this by inhibiting the expression of UBC9 to inhibit malignant cell growth
Double minute forms closely related important target spot and intervenes in born of the same parents, by reducing double minute number in tumour cell, to press down
The growth of malignant cell processed.
Summary of the invention
The present invention is directed to the case where current oncotherapy less effective, provides a kind of antisense base sequences by UBC9
UBC9 expression is interfered, to inhibit the malignant phenotype of the cell containing double minute, plays the role of the growth for inhibiting malignant cell.
The technical scheme is that the needle UBC9 amplification of specificity carries out RNA interference with its antisense base sequences, from
And inhibit the malignant phenotype of the cell containing double minute, play the role of the growth for inhibiting malignant cell.
Specifically, the invention adopts the following technical scheme:
1, stably transfected cell line is established
Select Proliferation of Human Ovarian Cell UACC-1598-4 and people's Colon and rectum gland cancer cell COLO320DM cell containing double minute
For research object.
(1) gene in SUMOization approach is screened using Real-time PCR；
(2) the protein expression water of selected gene is then detected in several tumor models using Western blot
It is flat；
(3) select the cancer cell lines UACC-1598-4 and COLO320DM containing double minute used thin for this experiment
Born of the same parents system；
(4) cell line of stable transfection psi-U6-shRNA-UBC9 expression vector is established；
(5) albumen silencing efficiency is detected using Western blot.
2, the influence for inhibiting UBC9 gene expression to generate double minute.
(1) it prepares metaphase chromosome caryogram and analyzes double minute number of variations situation；
(2) amplification situation that gene is carried on Real-time PCR detection double minute is utilized；
3, the influence mode and reason that detection UBC9 is stabilized double minute.
(1) situation is discharged using FISH detection micronucleus；
(2) inhibit influence of the UBC9 gene expression to DNA Damage；
4, UBC9 is inhibited to express the influence to cell behaviors.
(1) situation of change of cell growth curve inspection ability of cell proliferation is drawn；
(2) situation of change of Matrigel detection cell invasion ability.
By the studies above, present invention discover that Proliferation of Human Ovarian Cell UACC-1598-4 and people's Colon and rectum gland cancer cell line
After inhibiting UBC9 expression in COLO320DM, double minute number reduces, carries the reduction of gene magnification level on double minute, and swells
Oncocyte grade malignancy reduces.
Therefore, on the basis of the studies above, the invention proposes UBC9 (Ubiquitin-conjugating
Enzyme the sh-RNA sequence of antisense base sequences) and the antisense base sequences containing the UBC9 is reduced in preparation
Double minute number in cancer cell, while inhibiting growth of cancer cells and reducing the application in the drug of cancer cell grade malignancy.
Wherein, it is preferred that the antisense base sequences of the UBC9 are as shown in SEQ ID NO.1.
Wherein, it is preferred that the sh-RNA sequence is as shown in SEQ ID NO.2.
Wherein, it is preferred that the cancer cell is Proliferation of Human Ovarian Cell or people's Colon and rectum gland cancer cell.
Compared to the prior art, the beneficial effects of the present invention are:
The present invention utilizes the antisense base sequences of UBC9 specificity, is reduced by the method for RNAi interference containing double minute
The expression of UBC9 in malignant cell reduces double minute number, the grade malignancy of tumour cell is reduced, to effectively press down
Malignant cell growth processed.It is proposed of the invention is provides newly for the biological therapy of the malignant cell containing double minute
Magnetic target therapy scheme.
Detailed description of the invention
Fig. 1 is the expression detection of SUMOization related gene；
Fig. 2 is COLO320HSR/COLO320DM, SAE2 albumen in UACC-1598-59/UACC-1598-4 cell model
Expression；
(A) SAE2 protein expression detects；(B) SAE2 protein expression gray scale in COLO320HSR/COLO320DM cell model
Analysis chart；(C) SAE2 gray expression analysis of protein expression in UACC-1598-59/UACC-1598-4 cell model；
Fig. 3 is COLO320HSR/COLO320DM, UBC9 albumen in UACC-1598-59/UACC-1598-4 cell model
Expression；
(A) UBC9 protein expression detects；(B) UBC9 protein expression gray scale in COLO320HSR/COLO320DM cell model
Analysis chart；(C) UBC9 gray expression analysis of protein expression in UACC-1598-59/UACC-1598-4 cell model；
Fig. 4 is protein expression situation after silencing UBC9 in UACC-1598-4 cell；(A) UBC9 protein expression detects；(B)
UBC9 gray expression analysis of protein expression；
Fig. 5 is its double minute quantity situation of change after UBC9 gene silencing in UACC-1598-4 cell；
(A) karyotyping；(B) double minute quantity mutation analysis figure；
Fig. 6 is the amplification situation of high amplification gene after UBC9 gene silencing in UACC-1598-4 cell；
(A) EIF5A2 gene magnification situation；(B) MCL-1 gene magnification situation；(C) MYCN gene magnification situation；
Fig. 7 is EIF5A2 and MYCN protein expression situation after silencing UBC9 in UACC-1598-4 cell；
Fig. 8 is protein expression situation after silencing UBC9 in COLO320DM cell；
(A) UBC9 protein expression detects；(B) UBC9 gray expression analysis of protein expression；
Fig. 9 is its double minute quantity situation of change after UBC9 gene silencing in COLO320DM cell；
(A) karyotyping；(B) double minute quantity mutation analysis figure；
Figure 10 is the amplification situation of high amplification gene after UBC9 gene silencing in COLO320DM cell；
(A) CDX2 gene magnification situation；(B) MYC gene magnification situation；(C) FAM84B gene magnification situation；
Figure 11 is CDX2 and MYC protein expression situation after silencing UBC9 in COLO320DM cell；
Figure 12 is micronucleus outlet situation after silencing UBC9 in UACC-1598-4 cell；
(A) contain and the micronucleus without containing signal；(B) Interphase cells have the ratio of signal micronucleus；
Figure 13 is cellular damage situation after silencing UBC9 in UACC-1598-4 cell；
(A) cell γ-H2AX level of signal situation；(B) every kind of γ-H2AX level of signal cell proportion situation；(C)
γ-H2AX protein expression situation；
Figure 14 is the proliferative conditions of cell after silencing UBC9 in UACC-1598-4 cell；
Figure 15 is the proliferative conditions of cell after silencing UBC9 in COLO320DM cell；
Figure 16 is silencing UBC9 gene rear clone formational situation in UACC-1598-4 cell；
(A) Clone formation situation；(B) quantity mutation analysis figure is cloned；
Figure 17 is that basilar memebrane invades situation after silencing UBC9 gene in UACC-1598-4 cell；
(A) basilar memebrane invades situation；(B) cell invasion number of variations analysis chart；
Figure 18 is that basilar memebrane invades situation after silencing UBC9 gene in COLO320DM cell.
(A) basilar memebrane invades situation；(B) cell invasion number of variations analysis chart.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Related gene during embodiment 1SUMOization is screened
The present embodiment selection containing be stabilized double minute cancer cell lines COLO320DM, UACC-1598-4 and
The research object that cell line COLO320HSR, UACC-1598-59 without containing double minute is screened as early period.
1, method
(1) related gene during SUMOization is screened using the method for qRT-PCR:
1. the extraction of RNA
1ml Trizol will be added in cell in logarithmic growth phase, acts on 5 minutes on ice, cell is blown and beaten
It sucks in the EP pipe of 1.5ml.0.25ml chloroform is added, violent concussion 30 times or so, is stored at room temperature 5 minutes, 12000 revs/min,
4 DEG C are centrifuged 15 minutes；The water phase 0.5ml on upper layer is drawn into new EP pipe, isometric isopropanol precipitating RNA is added, gently
It mixes, is stored at room temperature 10 minutes, 12000 revs/min, 4 DEG C are centrifuged 10 minutes, abandon supernatant, and the ethanol washing that 1ml 75% is added is heavy
It forms sediment, 12000 revs/min, 4 DEG C are centrifuged 10 minutes, abandon supernatant, drying at room temperature 15 minutes, it is water-soluble that 20 μ l RNase-free are then added
Solution precipitating.
2. the synthesis of cDNA
Using Transcriptor First Strand cDNA Synthesis Kit kit, to specifications on
Step reverse transcription synthesizes cDNA.
③Real-Time PCR
12 important genes in known SUMOization mechanism are selected, NCBI UniGene database is retrieved, obtain 12
A gene order using Gene Runner software design and synthesizes gene-specific primer (table 1).The primer by
The synthesis of Invitrogen company.
1 primer sequence of table
Utilize 2-△△CTMethod calculate analysis, the expression of each gene is and right using the expression quantity of hACTB as internal reference
It is compared according to group, statisticallys analyze the expression variation of different genes.
(2) protein expression level of selected gene is detected in cell model using Western Blotting method:
1. the extraction of albumen
Logarithmic growth phase is grown into cell, adherent cell discards original fluid, after cleaning 2~3 times with the PBS of pre-cooling
Digestion, cell is collected into 1.5ml EP pipe, 3500 revs/min, 4 DEG C of centrifugations, and 5 minutes；The cell of suspension, which will directly be blown and beaten, to be contained
There is the media transfer of cell into 10ml centrifuge tube, 1600 revs/min, supernatant is abandoned in 4 DEG C of centrifugations after five minutes, with the PBS weight of 1ml
It is outstanding to be transferred in the EP pipe of 1.5ml, 3500 revs/min, 4 DEG C of centrifugations, 5 minutes.Visual cell, which measures, is added appropriate cell pyrolysis liquid (cell
Lysate: protease inhibitors: inhibitors of phosphatases=9:1:1), it is vortexed 15 seconds.It was vortexed later at interval of 10 minutes primary, often
Secondary 15 seconds, altogether three times.12000 revs/min, 4 DEG C of centrifugations, 30 minutes.Supernatant is collected into new 1.5ml centrifuge tube.
2. the measurement of protein concentration
Using quantification of protein kits protein concentration, experimental measuring is calculated.Albumen also can be reserved for standby in 80 DEG C of ﹣
With.
3. PAGE gel electrophoresis
The molecular size range of the UBC9 albumen and SAE2 albumen that detect in the present embodiment is respectively 18kDa and 90kDa, according to
The molecular size range of destination protein is respectively 15% and 10% separation gel using concentration, and 5% spacer gel carries out electrophoresis.
5 × loading electrophoretic buffer is added in sample to be tested, mixes, boils 5 minutes, completely after cooling, centrifugation is mixed, and is taken
Equal protein sample (50~100 μ g) loading runs spacer gel with 80V constant pressure, isolates apparent red an IOU issued by a post office to albumen marker
Voltage is adjusted to 120V when band, carries out constant pressure electrophoresis.The time of electrophoresis determines according to the molecular weight of destination protein.
4. transferring film
Gel is put into transferring film buffer after to electrophoresis and is balanced 10 minutes.Clip one is opened of the same size with gel
Pvdf membrane, being first put into impregnate 30 seconds in methanol makes its activation, places into 5 minutes removal methanol in transferring film buffer.According to from negative
The sequence of pole to anode successively puts foamed fibre pad, 3 layers of 3M filter paper, gel, pvdf membrane, 3 layers of 3M filter paper and foamed fibre pad well.
Bubble rear enclosed membrane-transferring device is discharged.(the transferring film time was by molecular weight of albumen in 40 minutes and 100 minutes for 300mA constant current ice bath transferring film
Depending on size).
5. closing
After transferring film, takes out pvdf membrane and be put into hybridizing box, wash three times with 0.1% TBS-T on room temperature shaker, often
Secondary 5 minutes.Suitable confining liquid is added later, and (0.1%TBST-T of the Reagent of Blocking containing 10%Western is molten
Liquid), room temperature closes closing in 1 hour or 4 DEG C overnight.
6. hybridizing
Be separately added into it is suitable according to the diluted anti-UBC9 antibody of 1:1000 and anti-SAE2 antibody, 4 DEG C of hybridized overnights or
Room temperature hybridizes 4 hours；TBS-T washes film three times, and 5 minutes every time.It is added later suitable diluted with fluorescence according to 1:10000
The anti-rabbit antibody of element label, is protected from light, and room temperature hybridizes 1 hour.TBS-T washes film three times, every time 5 minutes (being protected from light).
7. detecting
Film is swept using Odyssey infrared imaging system imaging system, obtains original image.Adjust time for exposure and contrast
Obtain optimal image.
2, result
(1) related gene during SUMOization is screened using the method for qRT-PCR
Fig. 1 is the expression testing result of the related gene during SUMOization, can be seen that UBC9 from Fig. 1 result
The significant and stable high expression in two different cell models of gene and SAE2 gene.
(2) protein expression level of selected gene is detected in cell model using Western Blotting method
Fig. 2 and Fig. 3 is respectively the Western of SAE2 gene and UBC9 gene in two different cell models
Blotting testing result.
It can be seen that from Fig. 2 and Fig. 3 result containing the cell COLO320DM and UACC- that double minute is more and is stabilized
The expression quantity of UBC9 albumen is high compared with cell COLO320HSR and the UACC-1598-59 cell without double minute in 1598-4 cell,
And the expression of SAE2 is without apparent difference.
Influence of the embodiment 2UBC9 antisense base sequences to double minute number and cell function in tumour cell
1, method
It selects the cancer cell lines UACC-1598-4 cell containing double minute and COLO320DM cell is this implementation
Cell line used in example:
The UBC9 albumen in cell COLO320DM and UACC-1598-4 cell that is more containing double minute and being stabilized
Expression quantity is high compared with cell COLO320HSR and the UACC-1598-59 cell without double minute, and the expression of SAE2 is without obvious
Difference.Therefore the target gene that UBC9 gene is this experiment is chosen, UACC-1598-4 cell and COLO320DM cell are this reality
Apply cell line used in example.
With contain 10% fetal calf serum RPMI-1640 culture solution, in 5%CO2In 37 DEG C of culture people's ovum in constant incubator
Nest cancerous cell line UACC-1598-4 and people's Colon and rectum gland cancer cell line COLO320DM.
(1) foundation of stably transfected cell line
1. determining puromycin the best use concentration
The UACC-1598-4/COLO320DM cell inoculation of logarithmic growth phase is in 12 orifice plates, and inoculum concentration was with second day
It is best for growing to 80%.It sets in puromycin drug concentration gradient adding hole, one pericyte of culture is all dead dense to screen
Degree determines that the best screening concentration of UACC-1598-4 cell puromycin is that 1 μ g/ml, COLO320DM cell puromycin is best
Screening concentration is 8 μ g/ml.
2. sh-RNA stable transfection interferes
RNAi silencing expression vector is constructed using psi-U6RNAi system, sh-RNA sequence is GATCCGaggaaagcatg
GaggaaagacTCAAGAGgtctttcctccatgctttcctTTTTTTGGAATT (shown in SEQ ID NO.2), carries out later
Plasmid extracts.
It is thin with second day by UACC-1598-4 the or COLO320DM cell inoculation in logarithmic growth phase in 6 orifice plates
Intracellular growth is advisable to 80% or so.10 μ l transfection reagents are added in one EP pipe
2000TransfectionReagent and 250 μ l Opti-MEM is mixed；It is added what 4 μ g said extracteds obtained in another EP pipe
Plasmid and 250 μ l Opti-MEM are mixed, are stored at room temperature 5 minutes.The liquid in two EP pipes is mixed later, room temperature after mixing
Stand 20 minutes.Transfection composite is uniformly added in the 6 orifice plates of inoculating cell.37 DEG C, 5%CO2, after culture 6 hours
Change normal RPMI-1640 culture solution into.Original fluid is changed to after 24 hours and adds 1 μ g/ml or 8 μ g/ml puromycins
Culture solution carry out stable screening.The death condition culture solution of replacement in (about 2~3 days) of visual cell, uses puromycin
(still there is cell survival in the 6 orifice plates of transfection interference plasmid but transfect base in the 6 orifice plates of empty plasmid within stable screening one week or so
When this cell-free survival, can think that stabilization is interfered successfully), the cell of survival is passed to expand in culture bottle and is cultivated.
(2) Gene silencing efficacy is detected using Western blot
UACC-1598-4 the and COLO320DM cell and control group total protein of silencing UBC9 gene are extracted, method is the same as implementation
Example 1.Anti- UBC9 antibody is diluted according to 1:1000, and anti-rabbit antibody is diluted according to 1:10000.The silencing efficiency of UBC9 gene is detected,
Selecting silencing efficiency, good experimental group does next research.
(3) silencing UBC9 gene and the influence generated after UBC9 gene silencing to double minute is probed into
1) it prepares mid-term caryogram and counts double minute number:
The cell for being in logarithmic growth phase is chosen, is added demecolcine (10~20ng/ml), 37 DEG C to continue culture 1~2 small
When.It being digested later with pancreatin, culture solution blows and beats mixing after terminating digestion, cell is collected into 10ml centrifuge tube, 1500 turns/
Point, it is centrifuged 5 minutes.Supernatant is abandoned, bullet is even, is resuspended with 1ml PBS, and supernatant is abandoned in centrifugation, plays even.37 DEG C of 9ml preheatings are added
37 DEG C of water-baths hypotonic 14 minutes (depending on concrete condition visual cell's situation), it is fresh that 1ml was added dropwise in 0.075mol/L KCL solution
The fixer (methanol: glacial acetic acid=3:1) of preparation after mixing gently, 1500 revs/min, is centrifuged 5 minutes.Supernatant is abandoned, is gently played even
Afterwards plus 10ml fixer uniformly 1500 revs/min, is centrifuged 5 minutes.Repeat it is fixed primary (for the second time can also 4 DEG C fixed
Night).Supernatant is gently outwelled, suitable fixer is added dropwise according to cell concentration.Cell suspension is dripped to 4 DEG C in advance by the suitable height of selection
On cold clean slide, room temperature natural air drying.Giemsa stock staining solution and PBS are dyed 6 minutes according to the proportional arrangement of 1:9
Afterwards, it is rinsed well with deionized water, room temperature is dried.It is observed with inverted microscope, take pictures and counts double minute number.Caryogram piece
It can freeze in 20 DEG C of ﹣, in case used in FISH experiment.
2) amplification situation of entrained gene on Real-time PCR detection double minute is utilized
1. cell genomic dna extracts
Select QIAGEN company QIAamp DNA Extract Kit kit, to specifications in experimental procedure mention
Take cell genomic dna.
The cell in logarithmic growth phase is taken, PBS is washed twice, digested later with pancreatin, and full culture medium terminates digestion, will be thin
Born of the same parents are collected into 10ml centrifuge tube, 1500 revs/min, are centrifuged 5 minutes.Supernatant is abandoned, bullet is even, is resuspended with 1ml PBS, is centrifuged, and collects
Cell is into 1.5ml EP pipe.Add 200 μ l PBS that cell is resuspended, add 20 μ l QIAGEN enzymes and 200 μ l Al buffer,
Be vortexed 15 seconds and mix, 56 DEG C water-bath 10 minutes, 200 μ l dehydrated alcohols are added later, is vortexed 15 seconds and mixes.Mixed liquor is all turned
It moves on in Dneasy Mini spin column, 8000 revs/min, is centrifuged 1 minute.The collecting pipe renewed adds 500 μ l
AW1buffer, is centrifuged 1 minute by 8000 revs/min.The collecting pipe renewed adds 500 μ l AW2buffer, 13000 revs/min, is centrifuged 3
Minute.13000 revs/min of the collecting pipe renewed is centrifuged 1 minute removal AW2buffer residual.Finally by pillar be put into 1.5ml from
In heart pipe, adds 200 μ l high pressure waters, be stored at room temperature 5 minutes.It 8000 revs/min, is centrifuged 1 minute, collected liquid is as highly concentrated
The genomic DNA of degree.Experimental demand can also be put into pillar in new 1.5ml centrifuge tube, add 200 μ l high pressure waters,
It is stored at room temperature 5 minutes.8000 revs/min, collected liquid is the genomic DNA of low concentration after centrifugation 1 minute.
②Real-time PCR
The high amplification gene carried on double minute in known UACC-1598-4 cell be respectively MCL1, MYCN and
EIF5A2 gene；The high amplification gene that double minute carries in COLO320DM cell is MYC, FAM84B and CDX2.Using Real-
The method of time PCR, that distinguishes is horizontal to MCL1, MYCN, EIF5A2, MYC, FAM84B and CDX2 gene magnification on double minute
It is detected, primer sequence is as shown in table 2.
2 primer sequence of table
(4) there are concrete modes and reason that stability has influence for double minute by detection UBC9.
1) situation is discharged using FISH detection micronucleus
1. FISH probe marks
Probe label: the 1 μ l BAC DNA (40~60ng/ μ l) extracted is mixed with 4 μ l random primers, in PCR instrument
95 DEG C, 10 minutes.It is immediately placed at later 2 minutes on ice, it is made to keep single-chain state.Be added 5 μ l fluorescein mixtures (including
1.6 μ l lack dNTPs, the 3.2 μ l Cy3/Green dUTP and 0.2 μ l Klenow fragment of T), mixing is placed on 37 DEG C of water
3 hours in bath.It is eventually adding 1 μ l Stop buffer and terminates reaction.
Probe precipitating: 9 μ l, Human CotI of ssDNA, 9 μ l, Labeled BAC DNA, 3 μ l adds water polishing to 50 μ l,
The 5 μ l of sodium acetate of 3mol/L places -80 DEG C later and precipitates 20 minutes.It 12000 revs/min, is centrifuged 10 minutes.Supernatant is sucked (to be sure not
Encounter precipitating), 75% ethyl alcohol of 110 μ l pre-cooling is added, 12000 revs/min, is centrifuged 10 minutes.Supernatant carefully is sucked, is protected from light drying
5~10 minutes.
Probe dissolution: adding 9 μ l hybridization solutions, 37 DEG C water-bath 1~2 hour.
Probe denaturation: 75 DEG C water-bath 8 minutes, be placed in immediately after 2 minutes on ice.
Probe renaturation: 37 DEG C water-bath 30 minutes.
2. FISH probe is handled
Aged at room temperature 2 days metaphase chromosome caryogram slides the zone of action has been marked into diamond pen.It is placed in 1 × PBS
Cleaning 5 minutes, is successively dehydrated in 75%, 85% and 100% ethyl alcohol each 3 minutes.The zone of action adds 100 μ l RNase to work
Liquid (100 μ g/ml), covers the PE gloves of size just, is put into wet box, and 37 DEG C are incubated for 40 minutes.After incubation, slide is put
It cleans 3 minutes, is dehydrated in 75%, 85% and 100% ethyl alcohol each 3 minutes in 2 × SSC.Drying at room temperature.Later in active region
Domain adds 100 μ l pepsin working solutions, covers PE gloves, is put into wet box, and 37 DEG C are incubated for 15 minutes.After incubation, it is placed in 1 ×
It is cleaned 5 minutes in PBS, 1% paraformaldehyde fixes 10 minutes, cleans 5 minutes in 1 × PBS, 75%, 85% and 100% ethyl alcohol
Each 3 minutes of middle dehydration, dries.Slide is put into 70% formamide of 75 DEG C of preheatings, water-bath 3 minutes.Slide is put into 4 immediately
Each 3 minutes in 2 × SSC I and 2 × SSC II of DEG C pre-cooling, it is dehydrated each 3 minutes in 75%, 85% and 100% ethyl alcohol.
3. FISH hybridizes process
Each zone of action adds 9 μ l probes, covered (20mm × 20mm), Rubber Cement glue mounting.It is put into
Wet box, 37 DEG C are incubated for 48 hours.After incubation, mountant is thrown off, slide is put into 50% formamide of 44 DEG C of preheatings, is shaken
Dynamic slide makes coverslip fall off naturally, impregnates 15 minutes.Slide is put into each 3 in 2 × SSC I and 2 × SSC II of 4 DEG C of pre-coolings
Minute, it is dehydrated each 3 minutes, dries in 75%, 85% and 100% ethyl alcohol.5 μ l DAPI are redyed, coverslip (24mm × 32mm)
Mounting.Fluorescence microscopy is under the microscope.
2) inhibit influence of the UBC9 gene expression to UACC-1598-4 DNA Damage
The coverslip of 20mm × 20mm is laid in 6 orifice plates, 1 × 10 is inoculated in each hole5A cell.Second day, in
The adhered state of microscopically observation cell.Culture medium is sucked out, is cleaned 3 times, every time 5 minutes with PBS.After cleaning PBS, it is added
4% paraformaldehyde fixes 20 minutes to cell.It is cleaned 3 times, every time 5 minutes with PBS.The TritonX- of 2ml 0.1% is added
100 (being dissolved in PBS), 4 DEG C are incubated for 10 minutes.PBS is cleaned 3 times, every time 5 minutes.The BSA solution that 2ml 4% is added (is dissolved in
PBS), it is incubated for 30 minutes for 37 DEG C.Then configured 100 μ l antibody, 4 DEG C of overnight incubations are added.It is clear with PBS after incubation
It washes 3 times, every time 5 minutes.The configured fluorescence secondary antibody of 100 μ l is added, 37 DEG C are incubated for 1 hour.PBS cleans 3 times later, and every time 5
Minute.5 μ l DAPI are redyed, coverslip mounting.Fluorescence microscopy is under the microscope.
3) influence after silencing UBC9 gene to cell function
1. drawing cell growth curve
The cell in logarithmic growth phase is taken, single cell suspension is digested to, it is 8000 thin by every hole after cell count
Born of the same parents are inoculated into 96 orifice plates, and every kind of cell sets 4 multiple holes, are placed in 37 DEG C of incubator cultures.After 24 hours, former culture in hole is discarded
100 μ l culture solutions and 20 μ l MTS solution mixtures are added in liquid, every hole, and 37 DEG C are cultivated 4 hours.(the OD=in microplate reader later
492nm) measure the light absorption value in each hole.It repeats to test at the same time daily later, records and arrange experimental data, use
GraphPad Prism 5 draws cell growth curve.
2. plate clone is tested
The cell in logarithmic growth phase is taken, single cell suspension is digested to, every hole 1200,1800 is pressed after cell count
For a cell inoculation in 6 orifice plates, every kind of cell sets 3 multiple holes, and 37 DEG C (naked eyes occur in 6 orifice plates could incubator culture two weeks or so
See between clone and cell without adhesion).Original fluid is discarded, PBS is washed twice, and 2ml methanol room temperature is added later and fixes 15 minutes.
Fixer is discarded, PBS is cleaned twice, later with Giemsa dye liquor (Giemsa stock staining solution: the PBS=1:9) dyeing 9 now prepared
Minute, slowly rinse out dye liquor, drying at room temperature.It is taken pictures after 6 orifice plates are completely dried with digital camera, is counted after counting.
3. basilar memebrane Matrigel
It takes the cell for needing number to be put in 24 orifice plates, is placed at room temperature for a period of time, add into 24 orifice plates and invasion cell
Enter 1640 culture medium of the serum-free 500 μ l of preheating, 37 DEG C incubator aquation 2 hours.After aquation, careful sucking liquid, but should not
Encounter basilar memebrane.
The cell in logarithmic growth phase is taken, single cell suspension is digested to, is centrifuged, serum-free 1640 culture medium is resuspended
Then cell carries out cell count.Cell dilution is become 3 × 10 with the 1640 culture medium containing 5%FBS4A/ml.To 24 holes
750 1640 culture mediums of the μ l containing 15%FBS are added in plate, cell is put into 24 orifice plates, but to be avoided under cell basilar memebrane
Generate bubble.500 μ l cell suspensions (3 × 10 are added into cell rapidly4A cells/well), under the microscope, in 37 DEG C of incubators
Culture 72 hours.
24 orifice plates are taken out, PBS is rinsed well, drying at room temperature, and film is fixed to 1 minute in anhydrous methanol, and washing is clean.It will
Giemsa stoste and PBS are dyed 7 minutes, washing is clean according to the proportional arrangement dyeing liquor of 1:9.PBS is dipped in cotton swab gently to wipe
The cell of film is not passed through on cell inner membrance.Room temperature is dried, and is then cut film with blade, is fixed in load glass with neutral gum
On piece, after covered light under the microscope, take pictures, counted after counting.
2, result
(1) foundation of stably transfected cell line
Select UBC9 silence efficiency 80% or more cell line as subsequent experimental materials'use, stably transfected cell line
Western Blotting testing result and gray expression analysis of protein expression it is as shown in Figures 4 and 8.
(2) the mid-term karyotyping of stably transfected cell line and the variation of double minute quantity
The mid-term karyotyping statistical chart of stably transfected cell line and double minute quantity mutation analysis figure such as Fig. 5 and Fig. 9 institute
Show, can be seen that silencing UBC9 gene from Fig. 5 and Fig. 9 result will lead to the reduction of double minute number.
(3) Real-time PCR detects the amplification situation of entrained gene on double minute
In UACC-1598-4 cell after silencing UBC9, high amplification gene EIF5A2, MCL-1 and MYCN real-time quantitative PCR
And Western Blotting testing result difference is as shown in FIG. 6 and 7, can be seen that from the result in UACC-1598-4
In cell after silencing UBC9, the expression of high amplification gene EIF5A2, MCL-1 and MYCN are reduced.
In COLO320DM cell after silencing UBC9, high amplification gene MYC, FAM84B and CDX2 real-time quantitative PCR and
Western Blotting testing result difference is as shown in Figures 10 and 11, can be seen that from the result in COLO320DM cell
After middle silencing UBC9, the expression of high amplification gene MYC, FAM84B and CDX2 are reduced.
(4) micronucleus outlet situation after silencing UBC9 gene
Figure 12 is fluorescence in situ hybridization figure and fluorescence in situ hybridization bar shaped statistical chart, be can be seen that from the result in UACC-
In 1598-4 cell after silencing UBC9 gene, increase with signal micronucleus ratio.
(5) inhibit influence of the UBC9 gene expression to UACC-1598-4 DNA Damage
Figure 13 is immunofluorescence experiment figure, signal proportion histogram and Western Blotting figure, can be with from the result
Find out that silencing UBC9 gene weakens DNA Damage.
(6) influence after silencing UBC9 gene to cell function
In UACC-1598-4 cell and COLO320DM cell after silencing UBC9 gene, cell growth curve figure point
Not as shown in FIG. 14 and 15, it can be seen that the silencing in UACC-1598-4 cell and COLO320DM cell from the result
After UBC9 gene, ability of cell proliferation is reduced.
Figure 16 is cell plates colony formation and clone's quantity mutation analysis figure, can be seen that silencing from the result
After UBC9 gene, the clonality of cell declines.
Figure 17 and Figure 18 is cell basilar memebrane invasion system lab diagram and cell basilar memebrane invasion statistical chart, can be with from the result
Cell invasion ability reduces after finding out silencing UBC9 gene.
Sequence table
<110>Harbin Medical University
<120>antisense base sequences of UBC9 inhibit the application in growth of cancer cells drug in preparation
<130> KLPI161330
<160> 2
<170> PatentIn 3.5
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
aggaaagcat ggaggaaaga c 21
<210> 2
<211> 67
<212> DNA
<213>artificial sequence
<400> 2
gatccgagga aagcatggag gaaagactca agaggtcttt cctccatgct ttcctttttt 60
tggaatt 67
Claims (1)
1. the sh-RNA sequence of the antisense base sequences containing UBC9 reduces the double minute number in cancer cell in preparation, simultaneously
Inhibit growth of cancer cells and reduce the application in the drug of cancer cell grade malignancy, wherein the cancer cell is containing double minute
Cancer cell, the sh-RNA sequence is as shown in SEQ ID NO.2；
The cancer cell containing double minute is Proliferation of Human Ovarian Cell UACC-1598-4 or people's Colon and rectum gland cancer cell
COLO320DM。
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| CN103417985B (en) * | 2013-07-16 | 2015-06-24 | 哈尔滨医科大学 | Application of antisense nucleotide sequences of ribosomal protein analogues RPL22L1 in preparing medicines capable of suppressing growth of ovarian cancer cells |
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| Title |
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| Expression analysis of Ubc9,the single small ubiquitin-like modifier(SUMO) E2 conugation enzyme,in normal and malignant tissues;Stergios J.Moschos MD et al;《Human Pathology》;20101231(第41期);摘要、第1297页"4.Discussion"部分 * |
| 调节Ubc9基因对卵巢癌细胞侵袭和迁移的影响;东梅等;《解剖科学进展》;20160131;第1卷(第22期);摘要、第17-19页 * |
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