CN110423752A

CN110423752A - SiRNA, recombinant vector, cell model and the application in anti-tumor drug of silencing Ku70 gene

Info

Publication number: CN110423752A
Application number: CN201910728595.5A
Authority: CN
Inventors: 郭志刚; 潘飞燕; 夏文
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2019-11-08

Abstract

The invention discloses a kind of siRNA of silencing Ku70 gene, recombinant vector, cell model and the applications in anti-tumor drug.The siRNA of silencing Ku70 gene is as shown in SEQ ID NO.1.SiRNA of the invention can be reduced the interaction between Ku70-Pol β, can be reduced DNA of tumor cell repair ability with the expression of silencing Ku70 in vivo.

Description

The siRNA of silencing Ku70 gene, recombinant vector, cell model and in anti-tumor drug In application

Technical field

The present invention relates to biological fields, and in particular to the siRNA of silencing Ku70 gene, recombinant vector, cell model and Application in anti-tumor drug.

Background technique

Base excision repair is to repair ROS into the cell or be exposed to base oxidations caused by certain environmental factors are attacked to damage The most important mode of wound, abasic site and SSBs equivalent damage type.BER is also acknowledged as correcting some common " main force " approach of DNA damage form, this process mainly comprise the steps that (1) identify incorrect base portion (such as 8- hydroxyl guanine) then cut off, generate an abasic site (Abasic apurinic site, AP site)；(2) A DNA chain therein is cut off at AP site；(3) incision in DNA chain polymerize new deoxynucleotide；(4) even Connect ends cut.Theoretically each step in repair process can be carried out independently, some biochemistries and structure biology Result of study show that the protein during BER is played a role in reparation approach in gradually harmonious mode, referred to as " Passing-the-Bathon " model.

According to the length for extending segment in repair process, DNA base excision reparation can be divided into short-movie section base excision and repair Multiple (Short Patch Base Excision Repair, SP-BER) and long segment base excision repair (Long Patch Base Excision Repair, SP-BER) in SP-BER, only single deoxynucleotide be incorporated into DNA chain replace by The base of damage, and for LP-BER, the deoxynucleotide being incorporated into DNA chain has 2-10.The first step usually starts at knowledge The base, including uracil, 3-MA, 8-OH-dG, formyl pyrimidine etc. not being damaged and in excision DNA.At the beginning of this Stage beginning there are a variety of glycoside hydrolases to function in body, include more than ten kinds of different glycosidases in this fermentoid.These sugar The base that the identification of glycosides hydrolysis enzyme spcificity is specifically modified, is cut off by the N- glycosidic bond between catalysis base and pentose By mistake modification or impaired base, the intermediate product DNA containing abasic site is formed, for a series of repair enzymes in downstream Processing.These glycosidases include some single function glycoside hydrolases, they only have N- hydrolysis of glycoside bond function, such as UDG (Uracil-DNA glycosylase), they are still incorporated on product DNA after the completion of catalysis protects AP site not by certainly Hair cutting promotes subsequent base excision repair reaction to carry out.In addition to this also contain some multi-functional glycoside hydrolysis in body Enzyme, such as OGG1 (8-oxoguanine glycosylase), NEIL1 (endonuclease VIII-like protein), They can not only identify and cut off the base being damaged, moreover it is possible to continue the 3 ' ends in AP site with β-or β, δ-elimination reaction is cut Disconnected DNA skeleton, generation contain 3 '-α, and the single-strand break of beta-unsaturated aldehyde or 3 '-phosphoric acid is converted into normal in downstream reaction 3’-OH。

After glycosidase is processed, AP site is formed at injury site, then APE1 (Apurinic Endonuclease 1) albumen execute BER next step: APE1 be responsible at AP site 5 ' end cutting generate 3 '-OH With 5 ' ends deoxidation ribose phosphate (5 '-deoxyribose phosphate, 5 '-dRP) as the subsequent substrate for repairing reaction.When After APE1 completes its function disengaging DNA during BER, Pol β is integrated on DNA in a manner of a kind of " relay ", Pol β's AP lyases responsible for activity cuts off 5 '-dRP structures and next polymerize a new deoxynucleotide at 3 '-OH to DNA chain In.Last connection procedure is by III α of DNA ligase (III α of DNA Ligase III α, Lig) and XRCC1 (X-Ray Repair Cross Complementing Gene 1) it completes jointly, this reparation approach is commonly known as SP-BER.When Pol β is aggregated to When the number of deoxynucleotide in DNA chain is 2-10, the DNA chain in downstream will be jacked up shape by the deoxynucleotide being newly added At a Flap structure, this structure can be by nuclease FEN1 (Flap Structure Specific Endonuclease 1) Specific recognition is simultaneously cut off, and finally completes LP-BER by DNA ligase (I Ligase I, Lig I) connection notch.

The mankind encode the assignment of genes gene mapping of Pol β in the area p11 of No. 8 chromosome, and the ID of the gene is 5432, overall length 33kb, There are 14 exons.The Pol β albumen of overall length includes two big structure domains: aminoterminal, that is, N-terminal lyase activity structural domain, this area Domain size is 8KD, about 90 amino acid；C-terminus, that is, C-terminal polymerase domain, size are about 34KD, containing about 250 ammonia Base acid, the two link together by hydrophobic domains, and there are also the combination vigor of single stranded DNA for the lyase structural domain of N-terminal.It is existing Multiple studies have shown that a variety of regulatory factors adjust the remediation efficiency of BER by being anchored and regulating and controlling Pol β.In LP-BER, FEN1 can be by promoting the strand replacement reaction of Pol β to promote LP-BER [8]；Qingming Fang's et al. research shows that HSP90 (Heat Shock Protein 90) adjusts BER reparation by adjusting the interaction between Pol β and XRCC1 Process, therefore it is also considered as the regulatory factor of a BER.

Summary of the invention

Goal of the invention: the present invention provides a kind of siRNA of silencing Ku70 gene, recombinant vector, cell model and resisting Application in tumour medicine.

Technical solution: first aspect present invention provides a kind of siRNA of silencing Ku70 gene in anti-tumor drug Using the siRNA sequence is SEQ ID NO.1.Second aspect of the present invention provides a kind of siRNA of silencing Ku70 gene, The siRNA sequence is SEQ ID NO.1.

Third aspect present invention provides the recombinant vector of the siRNA of silencing Ku70 gene a kind of, and the recombinant vector contains Above-mentioned siRNA.

Further, the recombinant vector is siRNA carrier for expression of eukaryon or siRNA prokaryotic expression carrier.

The siRNA carrier for expression of eukaryon is pFLAG-CMV4 and PLVX-IRES-Puro, and siRNA prokaryotic expression carrier is PET systemic vectors.

Fourth aspect present invention provides a kind of siRNA of silencing Ku70 gene in preparation Ku70 gene silencing cell model In application.

Fifth aspect present invention provides a kind of cell model of Ku70 gene silencing, and the cell model is by Ku70 spy Different siRNA is transfected into cell.

The cell model, the cell are human renal epithelial cell line and Human cervical cancer cell lines.

Sixth aspect present invention provides the recombinant vector of the siRNA containing silencing Ku70 gene or prepared by cell model Application in anti-tumor drug.

Seventh aspect present invention provides a kind of siRNA of silencing Ku70 gene and the siRNA of silencing Pol β gene or heavy The shRNA of silent Pol β gene combines application in preparation of anti-tumor drugs.

The utility model has the advantages that (1) is present invention discover that Ku70 in combination with Pol β, influences that cell reply DNA is single-stranded and double-strand damage It repairs, in addition, the present invention is by having found that the interaction between Ku70 and Pol β has mediated two kinds of reparation approach of NHEJ-BER Between connection, promote two kinds of DNA repair modes, due to DNA repair it is related to the drug resistance of tumour, the present invention mentions The siRNA and recombinant vector or cell model for having supplied silencing Ku70 gene, can reduce DNA of tumor cell repair ability, be used for New type antineoplastic medicine exploitation.

Detailed description of the invention

Fig. 1 is Pol β deficient cells to ETO sensitivity tests as a result, in figure, and A is that various concentration ETO handles Pol β defect MEF cell and control cell detect the survival rate statistical chart of cell afterwards for 24 hours, data repeat to test from independent three times, Middle P < 0.001 * * P < 0.01, * * *；B is that Western Blotting detects Pol β knockout effect；

Fig. 2 is that Pol β and Ku70/80 has interaction；Wherein, A is to do co-immunoprecipitation experiment with Ku70 antibody, will The albumen composition to precipitate detects Pol β and Ku70/Ku80 by Western Blotting；B is to transfect in HEK293 Flag-Ku70 does co-immunoprecipitation with Flag-M2Beads, and Western Blotting detects Pol β and Ku80；C is HEK293 Middle transfection Flag-Pol β does co-immunoprecipitation with Flag-M2Beads, and Western Blotting detects Ku70 and Ku80；

Fig. 3 is the case where Pol β is combined in vitro with Ku70/80, wherein A is to do co-immunoprecipitation method with Pol β antibody The case where detection Pol β is combined in vitro with Ku70；B is external to be co-immunoprecipitation method detection Pol β and Ku70 with Ku70 antibody In conjunction with the case where；C is to do co-immunoprecipitation method with Pol β antibody to detect the case where Pol β is combined in vitro with Ku80；

Fig. 4 is the result of variations that DNA damage causes Pol β-Ku70 to combine；Wherein, A is immune to pass through after ETO processing cell The interaction result of coprecipitation method detection Pol β and Ku70/80；B is to pass through co-immunoprecipitation after handling cell using MMS The interaction result of method detection Pol β and Ku70/80；C and D uses ETO and MMS to handle cell (same Fig. 4 of processing method respectively In A and B), Pol β, Ku70 are dyed with fluorescence antibody, then taken pictures under confocal microscope, in figure Green: Ku70, it is red: Pol β, blue: nucleus, the yellow in Merge figure: the coincidence of Ku70 and Pol β foci point, figure acceptance of the bid Ruler is 10 μm.Right side is to statistically analyze figure, P < 0.001 * * to the percentage for being greater than Ku70 and Pol β common location in 100 cells；

Fig. 5 be the fluorescent staining of Pol β and γ H2AX after HEK293 cell is handled with ETO under Laser Scanning Confocal Microscope at As figure；Green: Pol β, red: γ H2AX, blue: nucleus, the yellow in Merge figure is that Pol β and γ H2AX are overlapped Foci point, scale represents 10 μm in figure；

Fig. 6 is the reparation that Pol β strikes that drop has blocked cell to DSBs；Wherein, A is Hela cell and the control that Pol β strikes drop After Hela cell is handled with ETO, the result of Western Blotting experiment detection γ H2AX.The right is horizontal to γ H2AX Quantitative analysis statistical chart, P < 0.001 * * P < 0.01, * * *, data repeat to test from independent three times.B is that Pol β strikes drop After Hela cell and control Hela cell are handled with ETO, immunofluorescence dyeing is carried out as a result, red to γ H2AX: γ H2AX, Blue: nucleus.The right is from the independent statistics for repeating the average value of γ H2AX foci point in 50 cells in experiment three times Scheme, P < 0.001 * * P < 0.01, * * *, scale is 10 μm in figure；C is the Hela cell that Pol β strikes drop and control Hela cell with not Detecting the survival rate statistical chart of cell afterwards for 24 hours with the ETO processing of concentration, data repeat to test from independent three times, * * P < 0.01,***P<0.001；

Fig. 7 is that C-NHEJ is the major way for repairing the DSBs of ETO induction, wherein A is with 1 μM NU7441 and 10 μM 0-1.5h detection γ H2AX level is replied after RI-1 pre-processes HEK293 cell 14h, then after handling 1.5h with 10 μM of ETO, B is To the quantitative statistics figure of γ H2AX in A, P < 0.001 * * P < 0.01, * * *；

Fig. 8 is that Ku70 promotes SP-BER and LP-BER remediation efficiency as a result, A is to be weighed in vitro with purifying protein and simulation substrate Structure SP-BER, top are the schematic diagram for simulating the substrate of SP-BER, and 5 ' ends have FAM fluorescent marker；Lower part is experimental result picture, The position 20nt is the intermediate product after APE1 effect in figure, and the position 41nt is to repair product；C is with purifying protein and simulation substrate External reconstruct LP-BER, is distributed with figure A, B, D, is the quantitative analysis figure of figure A and C, P < 0.001 * * P < 0.01, * * *, system respectively Result is counted to repeat to test from independent three times.U, uracil；F, tetrahydrofuran；

Fig. 9 is the remediation efficiency for inhibiting the expression of Ku70 to can reduce BER；A is that Western Blotting verifies Ku70 Strike drop efficiency and GAP-associated protein GAP expression quantity；B is to strike the cell SP-BER efficiency of drop Ku70 lower than control group, the dosage of WCE It is successively 0 μ g, 5 μ g, 10 μ g, 20 μ g, top is the schematic diagram for simulating substrate, and middle part is SP-BER experimental result, lower part broken line Figure is the quantitative analysis for repairing product；C is to strike the cell LP-BER efficiency of drop Ku70 lower than control group, and the dosage of WCE is successively 0 μ g, 10 μ g, 20 μ g, 40 μ g, 80 μ g, top are the schematic diagrames for simulating substrate, and middle part is LP-BER experimental result, lower part line chart It is the quantitative analysis for repairing product；

Figure 10 is influence of the Ku70 to Pol β polymerase activity, and upper left quarter is the substrate for detecting Pol β polymerase activity in figure Schematic diagram, red asterisk indicate the position of FAM fluorescent marker, and lower left quarter is experimental result picture, and 20nt is the substrate not polymerizeing Position after DNA unwinding, 41nt are the positions of product.The right is the quantitative analysis figure of experimental result, and data are from independent three times It repeats to test, P < 0.001 * * P < 0.01, * * *；

Figure 11 is influence of the single-stranded degree of injury to cell MMS in Ku70 deletion cells, and wherein A is after striking drop Ku70 It is dyed after the unicellular alkaline gel electrophoresis of HEK293 and control group with PI, the comet of imaging is shot under fluorescence microscope Scheme, scale is 10 μm, Scramble in figure, the cell of transfection control siRNA；B is to repeat in experiment to from independent three times The comet tail length of 100 cells is for statistical analysis, P < 0.001 * * *.C, D is to strike drop with Ku70 after the MMS of 1mM processing Group and control group HEK293 cell 12h after, taken pictures to cell and survival rate statistics, P < 0.01 * *.

Specific embodiment

One, raw material sources

It is thin that human renal epithelial cell line HEK293 and Human cervical cancer cell lines Hela used in 1.1 present invention are purchased from ATCC Born of the same parents library, prokaryotic protein expression carrier are pET systemic vectors；Eukaryotic vector is pFLAG-CMV4 and PLVX-IRES- respectively Puro.In New England Biolabs company, Ku80 is bought in Abnova for recombinant protein UDG, Lig I, Lig III α purchase Company, other recombinant proteins are obtained by by the part 1.4.7 expression and purification.In addition, material mentioned in the present invention or reagent If preparation method is provided, it is commercially available by Reagent Company.

1.2 substrate sequence

The substrate sequence of the measurement of 1 Pol β Polymerase Activity of table and reconstruct BER

Solution and buffer needed for 1.3

(1) LB culture medium: sodium chloride (10g), yeast extract (5g), peptone (10g), ddH2O is settled to 1L.

(2) 8 × LEW Buffer (PH=8.0):

NaH₂PO₄·2H₂O (6.2404g), NaCl (14.0256g), ddH₂O constant volume is to 100mL.

(3) 4 × Elution Buffer (PH=8.0): NaH₂PO₄·2H₂O (3.1202g), NaCl (7.0128g), miaow Azoles

(6.18g), ddH2O constant volume to 100mL.

(4)2×BER Reaction Buffer

(5)2×Polymerase Reaction Buffer

Required reagent	Mother liquid concentration	The amount of required mother liquor
			Tris-HCl	1M(PH8.0)	1mL
MgCl₂·6H₂O	1M	0.2mL
			DTT	1M	004mL
NaCl	4M	0.1mL
			Glycerol	100%	2mL
ddH₂O		It is settled to 10mL

1.4 experimental method

1.4.1 cell culture

All cells are existed with the DMEM culture medium culture containing 10% serum and 1% penicillin and streptomysin in the present invention 37 DEG C, carbon dioxide volumetric concentration is in 5% constant incubator, the pallet for filling sterile water is placed in incubator bottom, with dimension Hold the air humidity of incubator.Cell secondary culture (by taking the culture dish of 10cm as an example): the density to cell growth reaches 70%- When 90% (confluent cultures ware), culture solution is discarded in superclean bench, is remained in cell of sterile 1PBS rinse with removing The serum of cell surface.37 DEG C of 1mL of 0.25% trypsin solution (EDTA containing 0.02%) is added, places in the incubator Until cell rounding discards pancreatin after brightening, addition 3mL fresh complete medium is turned with pipettor featheriness cell within 1-2 minutes Move on in the centrifuge tube of 15mL, 1000rpm is centrifuged 2min, discard rejoin after culture medium culture medium blow it is outstanding, it is more being assigned to Continue to cultivate in a culture dish.

Cell cryopreservation: by (formula is the tire ox of 50% culture medium, 40% with frozen stock solution after the cell dissociation centrifugation wait freeze Serum, 10%DMSO) it blows and hangs uniformly, it is dispensed into cryopreservation tube and is put into freezing storing box, be put into -80 DEG C of refrigerators, be transferred to liquid nitrogen afterwards for 24 hours It is saved in tank.

1.4.2 cell transfecting

(1) by taking six orifice plates as an example, first day by big ware cell dissociation, blow it is outstanding be uniformly inoculated into six orifice plates, keep cell close Degree maintained 60%-70% at second day.

Culture solution is discarded after cell is adherent within (2) second days, the DMEM culture without double antibody of the fresh serum-free of 1.5mL is added Base is put into incubator and continues to cultivate, while preparing rotaring redyeing system.

(3) two 1.5mL EP pipes are taken respectively, and 250 μ L Opti-MEM are added, 10 μ L are then added in one of pipe Liposome, be added the plasmid of 2 μ g in another pipe or (20 μM) of 10 μ L siRNA mix after stand 5min, then will contain rouge The Opti-MEM of plastid is added in the Opti-MEM containing plasmid or siRNA, mixes and stands 20min.

(4) solution of the totally 500 μ L mixed is added in six orifice plates, slowly shakes up, is put into incubator and cultivates 6h After discard culture medium, the fresh culture medium without double antibody containing 10%FBS of 2mL is added, puts back to and continues to cultivate in incubator.For matter Grain transfects, and cleavable cell receives sample after 48h；SiRNA is transfected, cleavable cell receives sample after 72h.

1.4.3 cell count

Addition culture medium is blown outstanding after super-clean bench is by cell dissociation, draws 10 μ L cell suspensions, isometric platform is added and expects Blue dye liquor is uniformly mixed.Finally mixed liquor is all added to and is counted in plate hole, is read with cell counter.Each sample chooses three A visual field counts respectively, is averaged and obtains cell concentration.

1.4.4 Western Blotting detects intracellular protein expression

(1) lytic cell: the cell that density is 90% or so is discarded into culture solution in super-clean bench, is cleaned carefully with sterile PBS Born of the same parents 2~3 times.

(2) it is added in culture dish 1mL (10cm ware) SDS protein lysate (containing 1PMSF), left and right tilts 10 times to the greatest extent repeatedly Cell surface may be made all to touch lysate, be put on ice for 10min later.

(3) cell that cracking is completed is scraped with cell scraper, is transferred in new 1.5mL EP pipe, if placing ultrasonic on ice Dry time, 10min then is centrifuged under the conditions of 12000rpm, 4 DEG C.

(4) supernatant after centrifugation is drawn onto another new 1.5mL EP pipe, as cell protein lysate.

(5) obtained protein lysate is measured into its concentration with Bradford method, the method is as follows: the first step is prepared known dense The BSA protein solution of degree simultaneously measures its absorbance value with microplate reader under 595nm wavelength, draws standard curve.Second step PBS Sample to be tested is diluted 10 times and measures absorbance value with Bradford dye liquor.Last reference standard curve, calculates sample to be tested Protein concentration.

(6) SDS-PAGE running gel is prepared before electrophoresis；The separation of suitable concentration is selected according to the size of destination protein Glue, since destination protein involved in the present invention is between 10~100KDa, resolving gel concentration all 12%.

(7) 6 × Protein loading buffer is proportionally added in the cell pyrolysis liquid sample extracted, be put into Sample 10min is boiled in boiling water.

(8) loading: the offset plate prepared in advance is correctly placed in Vertial electrophorestic tank, and 1 × running buffer is added, The comb on offset plate is extracted, toward loading hole loading 30-50 μ g protein sample.

(9) electrophoresis: opening electrophoresis apparatus, and setting voltage is 80V, is again adjusted to voltage after sample is contacted to lower layer's separation gel 120V, the bromophenol blue to be seen that arrives is from stopping electrophoresis at offset plate baseline 1/3.

(10) transferring film: 1 × trans buffer containing 20% methanol of pre-cooling is poured into transferring film slot, and transferring film slot It is placed on ice.In addition clip is spread sponge and filter paper, then the offset plate for having completed electrophoresis is taken out, gel is removed, is cut Except concentration glue, separation gel is laid on filter paper.Then the pvdf membrane after cutting out is layered on glue, drives bubble away, then successively spread Upper filter paper and sponge are put into the clip of sandwich type in the transferring film slot equipped with 1 × trans buffer and start transferring film.Transferring film ginseng Number is constant current 350mA, 90min.

(11) it closes: preparing the skimmed milk power confining liquid of 5% BSA or 5% with PBS in advance when transferring film carries out, turn After film, pvdf membrane is incubated at room temperature 2h in confining liquid.

(12) it is incubated for primary antibody: after closing, film being taken out from confining liquid, rinsed once with PBS, be put into antibody incubation In box.The dilution proportion primary antibody provided with PBS or confining liquid according to antibody specification, is subsequently poured into antibody incubation box, 4 DEG C Shaking table is incubated overnight.

(13) it is incubated for secondary antibody: after primary antibody incubation terminates, recycling primary antibody, by pvdf membrane PBST (containing 0.1%Tween) drift It washes three times, each 10min.Then secondary antibody corresponding with primary antibody attribute is diluted according to 1:10000 with confining liquid or PBS, it will Pvdf membrane is incubated at room temperature 2h.

(14) develop: after secondary antibody is incubated for, after being rinsed film three times with PBST, developing solution in drop is put into imager, colour developing Exposure, preservation of taking pictures.

1.4.5 co-immunoprecipitation

(1) lytic cell: the ice-cold PBS of cell (10cm culture dish) by density in 80%-90% is rinsed three times, is added Enter IP lysate (purchased from green skies biotechnology) of the 600 μ L containing PMSF, is placed on 4 DEG C of shaking tables and cracks 4h, then 4 DEG C, 12000rpm is centrifuged 10 minutes collection supernatants, is used for subsequent operation.

(2) taking three 1.5mL EP pipes to number respectively is 1,2,3；Take 100 μ L lysates in No. 1 pipe, addition 6 × Protein loading buffer, boils sample 10min, is labeled as input.By remaining lysate mean allocation in other two In EP pipe, (precipitated in the present invention wherein the corresponding primary antibody of 30 μ L Protein A+G Agarose and 1-4 μ g is added in No. 2 pipes When albumen with Flag label, then 10 μ L Flag-M2beads are added into lysate), while it is control group that No. 3 pipes, which are arranged, 30 μ L Protein A+G Agarose and the IgG with primary antibody equal mass are added.2, No. 3 EP pipes are placed on 4 DEG C of rotations to be incubated for Overnight.

(3) be centrifuged the IP mixture after incubation under the conditions of 4 DEG C, 2000rpm (only needs to place for Flag-M2beads On magnetic frame) pearl is separated with supernatant, abandon supernatant.

(4) pearl is resuspended with the PBS containing PMSF, and is centrifuged in 4 DEG C of rotation 10min the same terms, is repeated 3 times.

(5) add 30-40 μ L 1 × Protein loading buffer into the pearl after cleaning, sample is boiled in boiling water 10min is detected for subsequent Western Blotting.

1.4.6 immunofluorescence

(1) inoculating cell: sterilizing in alcolhol burner flame envelope after coverslip is impregnated with alcohol, be then placed in six orifice plates, then Digestion is blown the cell inoculation after hanging in six orifice plates, is evenly distributed on cell in six orifice plates, inoculum density is 50% or so.

(2) next day discards culture medium after cell is adherent, three times with PBS rinsing cell, the poly first of 1mL 4% is added Aldehyde, PBS is cleaned 3 times after room temperature fixes 15min.

(3) it punches: first preparing 0.2% TritonX-100, each hole is added 1mL, is being stored at room temperature 15min, then use PBS is rinsed three times.

(4) it closes: dissolving BSA with PBS, be configured to 3% confining liquid, 1mL is added in every hole, is incubated at room temperature 2h.

(5) be incubated for primary antibody: with confining liquid according to the dilution proportion primary antibody on antibody specification, it is slow that 50 μ L primary antibodies are added dropwise in every hole It is slow to shake, make primary antibody all covering slides, six orifice plates are being put into 4 DEG C of refrigerators, are being incubated overnight.

(6) after the completion of primary antibody is incubated for, slide PBST is rinsed three times, then dilutes fluorescence according to special ratios with confining liquid 100 μ L are added dropwise in secondary antibody, every hole, are put into magazine, are incubated at room temperature 1.5-2h, are then cleaned three times with PBST.

(7) it dyes: with PBS according to the dilution proportion DAPI dye liquor of 1:1000, taking 100 μ L to be added drop-wise on slide, room temperature is kept away Light stands dyeing 10min, then three times with PBST rinsing.

(8) mounting: taking a glass slide, drips anti-fluorescence quencher, carefully takes out slide from 6 orifice plates, uses paper handkerchief Excessive moisture is blotted, is tipped upside down on glass slide, mounting around slide is carefully coated in nail polish.

(9) it takes pictures: glass slide being put and finds the suitable visual field under the microscope, is taken pictures.

1.4.7 recombinant protein Prokaryotic expression, purification

The commercially available product such as used recombinant protein Pol β, FEN1, APE1, Ku70 is bought or is passed through in the present invention Prepared by existing method, the recombinant protein in the present invention purifies the above albumen by prokaryotic expression, specific operation step Suddenly are as follows:

(1) thallus is activated: by the strain (being purchased from hundred Si He Biotechnology Co., Ltd of Nanjing) containing protein expression vector It is placed on ice to melt from -80 DEG C of refrigerator taking-ups, is inoculated into the LB culture medium that 5mL contains corresponding antibiotic according to the ratio of 1:100 In, in 37 DEG C of constant-temperature table overnight incubations.

(2) next day cultivates the strain after activation according to the corresponding antibiotic LB that contains of the ratio access 150mL of 1:100 In base, 37 DEG C of constant-temperature table cultures.

(3) when Spawn incubation to bacterium solution OD600 is 0.5, IPTG, which is added, makes its final concentration of 1mM, inducing expression 3h.

(4) bacterium solution is discarded supernatant, leave bacterial sediment in 6000rpm, 4 DEG C of centrifugation 15min after the completion of inducing.

(5) bacterial sediment is put on ice for, 1 × LEW of 3mL Buffer is added and blows outstanding thallus, final concentration is then added For the lysozyme and PMSF of 1mg/mL, 30min is placed, is mixed every 10min primary.

(6) it balances Ni column: taking 1 × LEW of 2mL Buffer to be put into Ni column, it is allowed to flow down column naturally.

(7) ultrasonic thallus: the suspension after lysozyme lysis is ultrasonic on ice, and condition is work 3s, interval 3s, when work Between 18min.12000rpm after ultrasound, 4 DEG C of centrifugation 15min collect supernatant.

(8) supernatant is added in pillar, allows it to flow down naturally by Ni column, this process carries out on ice.

(9) after the completion of whole supernatants cross column, pillar is cleaned with 1 × LEW of 2mL Buffer, in triplicate.

(10) pillar is eluted with 1 × Elution of 1.5mL Buffer, three times, eluent is distinguished with three centrifuge tubes for elution It collects, as protein crude extract.

(11) electrophoresis: after protein crude extract addition 6 × Protein loading buffer is boiled loading progress electrophoresis, Protein purification result is observed with coomassie brilliant blue staining.

(12) after determining that purity of protein is greater than 90%, protein crude extract is fitted into semi-permeable membrane, is put into dialyzate 4 DEG C It is dialyzed overnight.The concentrate finally obtained is protein purification liquid.

(13) protein concentration after dialysis is measured with Bradford method, is saved after record in -80 DEG C of refrigerators.

1.4.8Pol β Polymerase Activity detects

We measure the Polymerase Activity of Pol β using fluorescein-labeled substrate, and particular sequence is shown in Table 1.

(1) substrate is synthesized in Jin Sirui biotech company respectively, is named as Pol β-FAM and Oppsite3+0, selected PAGE way of purification.

(2) substrate is annealed, system is as follows:

The system mixed is placed in 75 DEG C of metal baths after 10min, closing metal bath makes it be down to room temperature naturally

Up to annealed product.

(3) polymeric enzyme reaction system is prepared:

Reaction system is prepared into mixing, after 37 DEG C of reaction 30min, 20 μ L 2 × DNA loading Buffer are added

(30Mm EDTA, 90% formyl amine fuel, 0.02% xylene blue) is in 95 DEG C of placement 10min, as electrophoresis sample Product.

(4) urea-denatured glue is prepared simultaneously in polymeric enzyme reaction progress, takes 8ml denaturation glue that appropriate AP and TEMED is added Afterwards, it pours into ready offset plate, solidifies and complete after its 15min.

(5) electrophoresis: pouring into appropriate 1 × TBE buffer in electrophoresis tank, clip offset plate, with syringe by the urea in hole Careful blowout, takes 7 μ L sample loadings, constant pressure 300V, 2h in darkroom.

(6) it runs to bottom blue sideline to electrophoresis is stopped at offset plate bottom 1/3, takes out offset plate and be put into Odyssey Fluorescent bands are scanned in instrument.

(7) gray analysis is carried out to product band with Image J software, compares the activity of polymerase.

1.4.9 reconstructing base excision repair experiment

(1) take respectively Pol β-U-FAM, Pol β-U, Pol β-F-FAM, Pol β-F and complementary strand Opposite3+0 according to 1.4.8 the method for annealing annealing in respectively obtains the substrate of external albumen reconstruct BER and cell pyrolysis liquid reconstruct BER.

(2) reaction system is prepared:

A, external albumen reconstructs BER system:

B, cell pyrolysis liquid reconstructs BER system:

The F substrate of annealing	0.4μL
		dNTP Mix(2.5mM each)	0.25μL
[α-32P]-dCTP	0.1μL
		2×BER Reaction Buffer	10μL
Cell pyrolysis liquid	0-20μg
		ddH₂O	It is settled to 20 μ L

(3) by prepared reaction mixture after 37 DEG C of reaction 30min, 20 2 × DNA of μ L are added LoadingBuffer boils sample 10min at 95 DEG C.

(4) urea-denatured glue (the same 1.4.8 of method) is prepared in reaction process, and the product after reaction is taken into 7 μ L sample loadings, Electrophoresis.

(5) after electrophoresis, the substrate reactions of fluorescent marker are removed glue, with 1.4.8 method Odyssey instrument It is analyzed；For the reaction of cell pyrolysis liquid reconstruct, glue is stripped down and is laid on filter paper, with preservative film by filter paper and glue Package, is placed in advance in magazine, then X-Film film is covered on glue under dark condition, fastens magazine, put -80 DEG C of ice into Case tabletting 48h.

(6) X-Film film is taken out from -80 DEG C, is put into developer solution and impregnates 3-5min, after band is clear, places into It is fixed in fixing solution.Finally X-Film film is rinsed with clear water, naturally dry.

1.4.10 alkaline comet

Comet result uses triumphant base biology comet method DNA damage detection kit (article No. KGA240-50), specifically Operating method is shown in official website specification.

1.4.11 virus packaging

(1) cell is cultivated in 10cm culture dish to when 80-90% fusion, is inoculated with 15cm culture dish；

(2) incline culture solution, washs cell twice with 1mL D-Hank ' S solution；

(3) be added 1mL Trypsin-EDTA solution, after mixing, 37 DEG C placement 2-3 minutes；

(4) trypsin solution is carefully sucked, DMEM culture solution of the 2ml containing 10%FBS is added, it is unicellular that piping and druming forms cell Suspension；

(5) by cell suspension inoculation 15cm culture dish, DMEM culture solution of the 18ml containing 10%FBS is added, 37 DEG C after mixing 5%CO₂Overnight incubation；

(6) 15ml serum-free DMEM is added in a sterile 5ml centrifuge tube, is proportionally added into containing aim sequence LV3 shuttle plasmid and packaging plasmid (pgag/Pol, pRev, pVSV-G) mix, take and separately prop up sterile 5ml centrifuge tube, are added 1.5ml serum-free DMEM adds 300al RNAI-MAT mixing, is placed at room temperature for and after five minutes mixes two pipes, be placed at room temperature for 20-25 minutes, the culture solution in 15cm culture dish is removed, the DMEM culture solution of 8ml serum-free is added；

(7) transfection mixture is added dropwise in 15cm culture dish, the culture dish that lightly rocks back and forth to mix compound, In 37 DEG C of 5%CO₂It is incubated 46 hours in incubator；

(8) it inhales and abandons transfection liquid, DMEM culture solution of the 18ml containing 10%FBS, 37 DEG C of 5%CO are added₂Continue culture 72 hours.

Collection virus

(1) cell supernatant in culture dish is drawn onto 50m centrifuge tube, 4 DEG C, 4000rpm, 4min；

(2) after low-speed centrifugal, centrifuge tube supernatant is poured into 50ml syringe, is filtered with 0.45um filter；

(3) filtrate carries out ultracentrifugation in centrifuge, and 4 DEG C, 2000rpm, 2h；

(4) concentrate is collected into packing into shipment pipe；

(5) virus liquid dispensed is labelled, and -80 DEG C of refrigerators save.

Virus titer detection

(1) cell is cultivated in 10cm dish to when 80-90% fusion, and incline culture solution, with 3mlD-Hank ' S Solution washs cell twice；

(2) add 1ml Trypsin-EDTA solution., after mixing, carefully suck pancreas sea solution, 37 DEG C of placement 3-5 divide Clock；

(3) DMEM culture solution of the 2ml containing 10%FBS is being added, piping and druming makes cell form single cell suspension；

(4) 96 orifice plates are inoculated with by the concentration of 3 × 10 cell holes, in 37 DEG C of 5%CO after mixing₂Culture is for 24 hours；

(5) by slow virus stoste 10ul, with the DMEM training 3-5 gradient of ten times of liquid dilutions of 10%FBS (according to cellular The Polybrene of final concentration of 5ug/ml can be added in state if necessary)；

(6) culture solution in 96 orifice plates is sucked, 100 diluted virus liquids are added in every hole, while setting up blank control group, in 37 DEG C of 5%CO₂Culture is for 24 hours) the dilution virus liquid abandoned in 96 orifice plates is inhaled, the DMEM that 100uL 10%FBS is added in every hole trains liquid, (according to cell state, can separate 13-15 if necessary) is in 37 DEG C of 5%CO₂Continue to cultivate 72h；

(8) fluorecyte is counted by fluorescence microscope or FACS, calculates virus titer in conjunction with extension rate.

1.4.12 data processing and analysis

Western Blotting and the picture of immunofluorescence obtained in the present invention use Photoshop software to handle, It is analyzed using Image J software, and for statistical analysis to data with GraphPad Prism software.All experimental datas It both is from independent repetition three times to test, be indicated with Mean ± SD；Difference between group data examines detection, P < 0.05 using t Indicate that there is significant difference between group.

Two, experimental result

The Pol β defect that influence of the embodiment 1:Pol β deletion cells to ETO sensibility is constructed according to the method for 1.4.2 MEF cell line is inoculated into the ETO processing cell in six orifice plates with various concentration, 0,10 μM of concentration gradient, 20 μM, 30 μM, 40 μ M changes fresh culture medium afterwards for 24 hours, detects Cell viability with CCK8 kit.

As a result as shown in Figure 1, as shown in Figure 1A, under the ETO processing of all concentration, the cell survival rate that Pol β strikes drop is equal Lower than control group, Figure 1B is the effect knocked out with Western Blotting detection Pol β.The above result shows that Pol β missing MEF cell is more sensitive to ETO.

Embodiment 2:

It is thin with HEK293 by the method for 1.4.5 from embodiment 1 the result shows that Pol β may be related to DSBs reparation Born of the same parents have done co-immunoprecipitation experiment.Firstly, we are by Ku70 antibody, Protein A+G agarose beads, full cell pyrolysis liquid 4 DEG C be incubated overnight, Ku70 protein immunization is precipitated, then with Western Blotting detect, as shown in Figure 2.Fig. 2A table It is bright that Pol β is had found in the protein complexes being enriched with Ku70 antibody, show that Ku70 and Pol β exists and interacts, and Control group (IgG group) does not draw albumen.In order to further verify this as a result, applicant further transfects in HEK293 Western is used after full cell pyrolysis liquid is incubated overnight with 4 DEG C of Flag-M2beads after Ku70 with Flag label, 48h Blotting detection, as shown in B in Fig. 2, as a result consistent with the result that the A in Fig. 2 is indicated, there are phases by Flag-Ku70 and Pol β Interaction.Likewise, transfecting the Pol β with Flag label into HEK293, Ku70/ is then proved by co-immunoprecipitation experiment Ku80 is present in the protein complexes of Pol β, as shown in Figure 2 C.The above results show that BER albumen Pol β and NHEJ albumen Ku70/80 forms composite bulk phase interaction in vivo.

As can be seen from the above results, may lead to since Pol β, Ku70 and Ku80 are DNA repair protein, between them It crosses DNA or other protein mediations forms complex.It is direct phase interaction between Pol β and Ku70/80 to probe into With having carried out co-immunoprecipitation experiment in vitro according to the method for 1.4.5.Recombinant protein Pol β and Ku70 are incubated jointly in vitro It educates, does co-immunoprecipitation with the antibody of Pol β and Ku70 respectively.As shown in figure 3, A is coprecipitated to be immunized with Pol β antibody in Fig. 3 The case where shallow lake Experimental Research Pol β is combined in vitro with Ku70, B is to do co-immunoprecipitation experiment with Ku70 antibody to probe into Pol β in Fig. 3 The case where being combined in vitro with Ku70, C is to do co-immunoprecipitation experiment with Pol β antibody and probe into Pol β to be combined in vitro with Ku80 in Fig. 3 The case where.The results show that by Pol β can immunoprecipitation Ku70, vice versa, shows that Pol β and Ku70 can be straight in vitro Binding is closed, but Pol β cannot bind directly to form complex with Ku80 under in vitro conditions.It can be with by testing us above It draws a conclusion, there is direct interactions by Pol β and Ku70, and the combination between Ku80 is indirect.

The relationship of embodiment 3:DNA damage and the combination of Pol β and Ku70

For the relationship of further validating DNA damage and the combination of Pol β and Ku70, the present invention utilizes ETO and alkylating agent MMS handles HEK293 cell respectively, and to induce DSB and SSB, (ETO handles 1.5h, and concentration is 10 μM；MMS handles 30min, concentration For 1mM), reply after 1h with the combination between co-immunoprecipitation experiment detection Pol β and Ku70.As a result as shown in figure 4, wherein Fig. 4 Middle A is the interaction result for detecting Pol β and Ku70/80 after handling cell using ETO by co-immunoprecipitation method；Fig. 4 B For use MMS handle cell after by co-immunoprecipitation method detect Pol β and Ku70/80 interaction result.The result shows that The processing of ETO and MMS can significantly increase the combination between Pol β and Ku70/80.

In order to further verify, the cell equally handled fluorescence antibody is marked into Ku70 and Pol β, in laser co-focusing The positioning scenarios of two kinds of albumen in the cell are shot under microscope, processing method is identical as A, B method of Fig. 4, fluorescence antibody pair Pol β, Ku70 are dyed, and are then taken pictures under confocal microscope.As shown in D in C and Fig. 4 in Fig. 4, green: Ku70, red: Pol β, blue: nucleus, the yellow in Merge figure: the coincidence of Ku70 and Pol β foci point, as cell ETO After MMS processing, the common location of Ku70 and Pol β in the cell is more, prompts the combination enhancing of Ku70 and Pol β, this and Fig. 4 A It is consistent with the result of Fig. 4 B.The above result shows that Ku70 and Pol β is in DNA damage, in conjunction with reinforcement.

The relationship that embodiment 4:Pol β and DSB is repaired

Cell is handled with ETO, detects imaging results of the fluorescent staining of Pol β and γ H2AX under Laser Scanning Confocal Microscope, such as Shown in Fig. 7, from the result of Fig. 7 it was found that after ETO processing cell leads to double-strand break, Pol β-γ H2AX common location Enhancing shows to recruit and increase to the Pol β of injury site, this result further proves that Pol β participates in DSB and repairs.

In order to further verify the ability that Pol β missing can reduce reparation DSBs, we use specific containing Pol β ShRNA (SEQ ID NO.2:5 '-GGAGCTGAAGCTAAGAAATTG-3 ') and control shRNA (You Jima company synthesis Nonsense sequence) virus respectively infect Hela cell (method is shown in 1.4.11), construct Pol β stablize strike drop and control Hela it is thin Born of the same parents system.Cell is layered in 12 orifice plates, changed after handling cell 1.5h with 10 μM of ETO fresh culture medium reply 0h, 0.5h, Lytic cell after 1.5h, since γ H2AX can be used as the mark sex modification of DNA double chain damage, we use Western The case where phosphorylation form γ H2AX level that Blotting method detects histone H2AX observes cell repair double-strand damage, knot As shown in fig. 6, from Fig. 6 A it will be seen that after ETO handles cell 1.5h, the γ H2AX level of two groups of cells reaches fruit Highest level, with the increase of turnaround time, the level of γ H2AX is gradually decreased, and shows that cell repairs double-strand damage It is multiple.By comparing the level of the same time point γ H2AX of two groups of cells it can be found that Pol β protein expression is suppressed after, returning γ H2AX when multiple 0h, 1h, 1.5h is above cellular control unit.The cell of same treatment is subjected to immunofluorescence reality in Fig. 6 B It tests, obtained result is also consistent with the above results.Antibiotics resistance test also illustrates that Pol β defect Hela cell is thin compared to control Born of the same parents are more sensitive to ETO, and as shown in Figure 6 C, this shows that lowering Pol β expression has blocked cell repair DSBs.

Homologous recombination repair is the mode of intracellular another important reparation DSBs, in order to determine which access is to repair The DSBs major way of ETO induction, we have selected the specific inhibitor of c-NHEJ and HR, and NU7441 (inhibits DNA-PKcs) After RI-1 (inhibit RAD51 inhibitor) pretreatment HEK293 cell 14h, change into after fresh culture medium again at 10 μM of ETO 0h, 0.5h, 1.5h are replied after managing cell 1.5h, control group is respectively set, then lytic cell detection γ H2AX is horizontal, as a result such as Shown in Fig. 7.Compared to the pretreated control group of DMSO, after inhibiting NHEJ with NU7441, the reply speed of γ H2AX obviously subtracts Slowly, after but inhibiting HR with RI-1, the reply speed of γ H2AX is had no significant effect, illustrates that c-NHEJ is the main of reparation ETO Reparation approach.It is in complex chart 7 as a result, illustrating to strike drop Pol β inhibits c-NHEJ, caused by having blocked cell repair ETO DSBs。

Influence of the embodiment 5:Ku70 to Pol β polymerase activity

In order to study influence of the Ku70 to base excision repair process, we are obtained with the method for Prokaryotic expression, purification in vitro All albumen (specific method is shown in 1.4.7) for having arrived SP-BER and LP-BER, have designed and synthesized the simulation with fluorescent marker Substrate is reconstructed two kinds of base excision repair processes in vitro.We have synthesized the DNA substrate of the mispairing containing G/U (sequence is shown in Table 1) Simulate SP-BER process, as a result as shown in Figure 8.As shown in A in Fig. 8, UDG can identify mismatch site and cut off, and generate One abasic site is known as AP site, and then APE1 identifies the site and cuts off, in last Pol β polymerization new base by Ligase connection.We first pass through preliminary experiment and have been determined that suitable Pol β protein content is 2.5ng, then negate and answer required albumen The Ku70 (see 1.4.9) gradually increased with protein content, simulation substrate and addition 2 × BER Reaction Buffer after annealing It prepares reaction system and DNA loading Buffer is added after 37 DEG C of reaction 30min and terminates and react and boil sample 10min at 95 DEG C, Then sample is separated with urea-denatured glue, is finally taken pictures with the development of Odyssey instrument.It can be seen that from the A of Fig. 8 and work as reactant When not containing Pol β in system, length is that the substrate of 41nt is cut to 20nt, does not repair product generation.After Pol β addition There is the reparation product of 41nt, after Ku70 albumen is added into reaction system, SP-BER efficiency improves 2-5 times of (swimming lane 3,4,5), and the addition of control group BSA albumen does not influence (swimming lane 6,7,8) to SP-BER efficiency.We also use same side Method, different albumen systems have rebuild LP-BER with different simulation substrates in vitro, and substrate schematic diagram is as shown in C in Fig. 8.I Use instead G/F mispairing substrate simulation LP-BER substrate.Tetrahydrofuran (THF or F) is a kind of cyclic compound, such repairing When base mismatch, cut-off F cannot be cut off by Pol β lyase vigor, and Pol β can polymerize more than two nucleotide, will under Trip DNA chain jacks up one 5 '-Flap structure of generation and is cut off by FEN1, last ligase connection breach.Experimental result such as Fig. 8 C and D Shown, Ku70 can equally enhance the remediation efficiency of LP-BER.

Embodiment 6:Ku70 participates in the case where BER in cellular environment

Ku70 special siRNA (SEQ ID NO.1:GGGTCTGTGCCAACCTCTT) and control siRNA (sharp rich biology Synthesis nonsense sequence) it is transfected respectively to HEK293 cell and strikes the expressing quantity of drop Ku70, with relatively mild cell after transfection 72h Lysate lytic cell obtains full cell pyrolysis liquid (Whole Cell Extract, WCE), wherein containing base excision repair Then all albumen take the bottom DNA but without fluorescent marker identical as reconstruction in vitro BER sequence of a certain amount of WCE and synthesis Object prepares reaction system according to the method in 1.4.9, rebuilds BER reaction.Finally experiment knot is obtained with the method for autoradiograph As a result fruit sees Fig. 9.Utilize the method for 1.4.2 cell transfecting, after transfecting Ku70 specific siRNA, the expression quantity of Ku70 in cell About reduce 80%, and the expressing quantity of Ku80 and other BER do not influence, and see Fig. 9 A.It can see from the B of Fig. 9, with The amount of two kinds of WCE increase, the reparation product of SP-BER gradually increases, but Ku70 strikes the reparation product come down to a lower group in WCE mass phase Control group is substantially less than Deng in the case where, when the WCE of 20 μ g of maximum is added, Ku70 strikes the reparation product of the SP-BER to come down to a lower group It is only about the 60% of control group.LP-BER result in Fig. 9 C is similar with SP-BER, and in the case where equal amount WCE, Ku70 is struck Resulting reparation product of coming down to a lower group is substantially less than control group.If this result illustrates that the expression of Ku70 albumen in cell is suppressed, BER remediation efficiency can be substantially reduced, this, which further demonstrates Ku70, can enhance the conclusion of BER remediation efficiency.

Front the result shows that Ku70 can improve the efficiency of BER significantly, but the mode for participating in BER not yet illustrates.It It is preceding that experiments have shown that Ku70 can be in conjunction with Pol β, and this combination can be remarkably reinforced in the case where DNA is damaged, therefore I Speculate that Ku70 promotes its activity and enhance BER by being integrated on Pol β.Pol β is mainly that polymerase activity plays in BER Effect, therefore we have detected Pol β Polymerase Activity with the method in 1.4.8.Briefly, it first passes through preliminary experiment and determines conjunction Suitable Pol β protein content is 10ng, then the reaction substrate of the Pol β of phase homogenous quantities and the Ku70 of different quality and fluorescent marker (as shown in Figure 10,1) sequence is shown in Table and Polymerase Reaction Buffer prepares reaction system in 37 DEG C of reaction 30min DNA loading Buffer is added afterwards and terminates and reacts and boils sample 10min at 95 DEG C, then separates sample with urea-denatured glue, most It is taken pictures afterwards with the development of Odyssey instrument.From Figure 10 result it is found that in reaction system the addition of Ku70 greatly facilitated Pol β's Polymerase activity (swimming lane 4,5,6), and Ku70 itself does not have the addition of polymerase activity (swimming lane 2) and control group BSA not It can promote Pol 'beta ' activity (swimming lane 7,8,9).Comprehensive BER experiment as a result, we may safely draw the conclusion: repaired in cell base excision During multiple, Ku70 can promote its polymerase activity in conjunction with Pol β, to improve base excision repair efficiency.

The influence of embodiment 7:Ku70 depletion on cell

Drop Ku70 is struck in HEK293 cell, then carries out unicellular alkaline gel electrophoresis analysis with MMS processing cell, and Cellular control unit is set, and detection cell DNA damage degree, testing result after MMS is handled is as shown in figure 11.Unicellular alkalinity For result after gel electrophoresis as shown in Figure 11 A comet figure, the length of comet tail characterizes the degree of the DNA Damage, long Degree is longer, and representation DNA damage is more serious.We are with every group of software statistics of CASP (Comet assay software project) The length of cell comet tail, statistical result is as shown in the B of Figure 11, and after MMS handles cell, Ku70, which strikes, to come down to a lower group and control group The comet length of cell obviously increases, and shows that MMS causes DNA damage, after comparison MMS processing Ku70 strike come down to a lower group it is thin with control group Born of the same parents are higher than Scramble cellular control unit it can be found that Ku70 strikes the cellular damage degree after dropping.

In order to further prove critical function of the Ku70 in BER, after we are handled with the MMS of 1mM Ku70 strike come down to a lower group and The ratio for detecting living cells after control group HEK293 cell 12h as can be seen from the results, strikes drop shown in Figure 11 C and D The death rate is significantly greater than cellular control unit after the processing of cell reply MMS after Ku70, shows that cell is for MMS after striking drop Ku70 Sensibility increases.Complex chart 13A, B as a result, show Ku70 for cell handle MMS caused by it is single-stranded damage have important work With.

As can be seen from the above results, Ku70 combination Pol β and promote the polymerase activity of Pol β, so that Pol β be promoted to join With BER the DNA being damaged is repaired, due to tumour cell by improve DNA repair ability come repairing damage DNA reduces the chemotherapy effect of drug to develop drug resistance to chemotherapeutics.

Still can be special by Ku70 siRNA, block the expression in vivo of Ku70 gene, and further pass through Pol β special siRNA or shRNA can inhibit BER and NHEJ to repair approach simultaneously, reduce DNA of tumor cell repair ability, Reach better chemotherapy effect.

Sequence table

<110>Nanjing Normal University

<120>siRNA of silencing Ku70 gene, recombinant vector, cell model and in anti-tumor drug

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gggtctgtgc caacctctt 19

<210> 2

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ggagctgaag ctaagaaatt g 21

Claims

1. a kind of application of siRNA of silencing Ku70 gene in anti-tumor drug, the siRNA sequence is SEQ ID NO.1.

2. a kind of siRNA of silencing Ku70 gene, which is characterized in that the siRNA sequence is SEQ ID NO.1.

3. a kind of recombinant vector of the siRNA of silencing Ku70 gene, which is characterized in that the recombinant vector contains such as claim 2 The siRNA.

4. the recombinant vector of the siRNA of silencing Ku70 gene according to claim 3, which is characterized in that the recombination carries Body is siRNA carrier for expression of eukaryon or siRNA prokaryotic expression carrier.

5. a kind of siRNA of silencing Ku70 gene is preparing the application in Ku70 gene silencing cell model.

6. a kind of cell model of Ku70 gene silencing, which is characterized in that the cell model is to turn Ku70 special siRNA Dye is into cell.

7. cell model according to claim 5, the cell is human renal epithelial cell line and Human cervical cancer cell lines.

8. recombinant vector or cell model as claimed in claim 7 or 8 as described in claim 3 or 4 prepare it is antitumor Application in drug.

9. a kind of siRNA of silencing Ku70 gene and the siRNA or shRNA of silencing Pol β gene, which combine, is preparing anti-tumor drug In application.