CN103405785B - Use of gene GADD45 beta in preparation of drug for treating bladder cancer - Google Patents
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- CN103405785B CN103405785B CN201310357438.0A CN201310357438A CN103405785B CN 103405785 B CN103405785 B CN 103405785B CN 201310357438 A CN201310357438 A CN 201310357438A CN 103405785 B CN103405785 B CN 103405785B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a use of a gene GADD45 beta in preparation of drugs for treating cancers and especially provides a use of the gene GADD45 beta in preparation of a drug for treating bladder cancer. According to the invention, an over-expression vector of the gene GADD45 beta is constructed and is transferred into tumor cells. A result shows that an mRNA level of the tumor cells with the gene GADD45 beta is improved obviously; roundness of the tumor cells is improved and adherence of the tumor cells is reduced; and tumor cell activity is improved obviously by 85% and tumor cells die finally. The gene GADD45 beta has high tumor cell inhibition activity and provides base data for novel drug target discovery and a tumor-treatment drug design.
Description
Technical field
The new purposes that the present invention relates to GADD45 β gene, specifically GADD45 β gene is in the application of preparing in anti-bladder cancer medicine.
Background technology
GADD45 family is by the small molecular protein GADD45 α of three kinds of structural similarities, GADD45 β and GADD45 γ composition, meeting is significantly rising of transcriptional level under the induction in retarded growth or DNA damage agent at cell, its protein binding also activates MTK1/MEKK4 kinases, thus the p38/JNK approach that mediation activates.The increase of GADD45 level is the essential condition that cell cycle pauses, allows DNA damage to repair, and in the growth of cell and apoptosis, plays an important role.
GADD45 β is the dominant response gene of the beta induced growth inhibited of TGF-and short apoptosis, and the nucleoprotein of its coding can be with other albumen as interactions such as PCNA, p21, core histones, MEKK4 kinases and Cdk1.The signal transduction pathway of the p38MAPK activating by the beta mediated TGF-β of GADD45 plays an important role in the beta mediated tumor suppression process of TGF-, but the function of this gene in bladder cancer apoptosis of tumor cells have not been reported.
Summary of the invention
The object of the present invention is to provide the new purposes of one of GADD45 β gene.
The invention provides GADD45 β gene in the application of preparing in anti-bladder cancer medicine.Described GADD45 β gene, its nucleotide sequence is SEQ ID NO:1.
The present invention passes through to build the overexpression vector of GADD45 β gene, transfection tumor cell, and result shows: in the tumor cell after transfection, the mRNA level of GADD45 β gene all significantly raises, there is becoming circle in tumor cell, adherent property variation, cytoactive significantly reduces by 85%, and final appearance is dead.
The present invention's screening obtains the gene of an efficient inhibition tumor cell, for discovery and the tumour medicine design of new drug target provide basic data.
Brief description of the drawings
Fig. 1: mRNA and protein expression after GADD45 β overexpression: (PCDNA3.1 is empty carrier to A qPCR result; PCDNA3.1-GADD45 β is the overexpression vector with GADD45 β gene)
Fig. 2: tumor cell metamorphosis after overexpression GADD45 β: A recombiant plasmid PCDNA3.1-GADD45 β is transfected into cellular morphology after T24 cell 24h; B recombiant plasmid PCDNA3.1-GADD45 β is transfected into cellular morphology after T24 cell 48h
Fig. 3: after GADD45 β overexpression, cytoactive detects
Detailed description of the invention
Embodiment 1:GADD45 β gene inhibition urinary bladder carcinoma T24 cell line
One, the structure of recombinant expression carrier
1, the preparation of PCR product
Taking the cDNA of T24 cell RNA reverse transcription as template, full length cDNA sequence (GeneID:4616) design based on GADD45 β gene is used with the expression primer of restriction enzyme site and is carried out pcr amplification.Reaction condition is: 94 DEG C of denaturation 3min, and 94 DEG C of degeneration 30s, 60 DEG C of annealing 40s, 72 DEG C are extended 1min, totally 35 circulations, 72 DEG C of insulation 10min, primer sequence is in table 1.PCR product removal process is carried out in strict accordance with the description of the Gel Extraction Kit of OMEGA company.
Table 1GADD45 β expresses primer
2, genes of interest total length checking
The genes of interest that PCR is obtained is connected on Zero Vector, is sent to Beijing AudioCodes biotech firm and checks order.
3, the double digestion of PCR product and expression vector reaction
Choose the successful plasmid of order-checking, carry out respectively PCR product and the reaction of PCDNA3.1 double digestion in 2 eppendorf pipes, reaction system is carried out with reference to Hind III, BamH I description (NEB company).
4, glue reclaims enzyme action product
Above-mentioned double digestion product is carried out to glue recovery, and removal process is carried out in strict accordance with the description of the Gel Extraction Kit of OMEGA company.
5, being connected of genes of interest GADD45 β and PCDNA3.1
Glue containing genes of interest GADD45 β is reclaimed to product and is connected to PCDNA3.1 above, in centrifuge tube, set up following reaction system:
Mix gently, instantaneous centrifugal collection liquid, in the pipe end, is placed in 4 DEG C of connections of spending the night.
Two, the conversion of recombinant expression carrier
1, Trans-T1Phage Resistant competent cell is placed on ice;
2, the connection product that adds successively 50 μ L competent cell core 5 μ L in centrifuge tube, mixes gently, is placed in 30min on ice;
3,42 DEG C of water-bath heat shock 30s, are then placed in pipe on ice ice bath 2min fast;
4, add 500 μ L not containing antibiotic LB fluid medium, mix and be placed on 37 DEG C of 200rpm shaken cultivation 1h;
5, get the resuspended bacterium liquid of 200 μ L, evenly coat on the flat board that is placed in advance ammonia benzyl resistance in 37 DEG C of incubators incubated overnight;
Three, the checking of recombinant expression carrier
1, with the single bacterium colony of the careful picking white of the rifle head of sterilizing, rifle head is placed in to 1mL LB fluid medium (containing 0.1% ammonia benzyl), in 37 DEG C, the shaken cultivation case of 200rpm, cultivates 1h.
2, utilize the total length primer PCR of above-mentioned GADD45 β to detect bacterium liquid in 1 and whether contain recombiant plasmid, PCR reaction condition: 94 DEG C of denaturation 3min, 94 DEG C of 30s, 60 DEG C of 40s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 5min, 4 DEG C of preservations.Afterwards, by 1.5% agarose gel electrophoresis testing goal fragment, the corresponding bacterium liquid that object fragment detected is sent to Beijing AudioCodes biotech firm and checks order.
Four, Transfected Recombinant Plasmid T24 cell
The trophophase cell of taking the logarithm, is not resuspended in containing in antibiotic RPMI1640 culture medium, is inoculated in 6 orifice plates.Experiment is divided into 2 groups, matched group: PCDNA3.1 empty plasmid and EGFP-N1, experimental group: PC DNA3.1-GADD45 β and EGFP-N1.Every group of 3 repetitions.Adopt the handsome Lipofectmine2000 of company by the plasmid PCDNA3.1-GADD45 β building and EGFP-N1 cotransfection to T24 cell, concrete operations are with reference to description.
Five, GADD45 β mrna expression level after Real-time PCR detection transfection
By PCDNA3.1-GADD45 β and EGFP-N1 cotransfection T24 cell 6h, after 12h and 48h, Trizol method is extracted total RNA respectively, and step is carried out according to RNAiso Plus (TaKaRa) description.After reverse transcription, Real-time PCR detects GADD45 β mrna expression level.
Six, morphological observation
Recombiant plasmid proceeds to microscope (OLYMPUS1X71) observation of cell form after T24 cell 24h and 48h.
Seven, cytoactive after MTT detection transfection
After transfection 24h, matched group and processed group are inoculated in respectively in 96 orifice plates.Every group arranges 3 multiple holes, continues to cultivate.Cultivate after 24h, discard culture fluid, add 180 μ L fresh cultures, then to add 20 μ L5mg/mL MTT(final concentrations be 0.5mg/mL), continue to cultivate 4h.Until after the time, stop cultivating, carefully suck culture fluid in hole, every hole adds 150 μ L DMSO, and shaking table low speed jolting 10min is that bluish violet crystallization is fully dissolved, and reads absorbance at microplate reader 490nm place.Deal with data, calculates the suppression ratio of brazilin to cell, draws cell growth curve.Each sample standard deviation arranges 6 repetitions, averages as final result.
SEQUENCE LISTING
<110> University Of Shanxi
<120> GADD45 β gene is in the application of preparing in anti-bladder cancer medicine
<130> .
<160> 3
<170> PatentIn version3.5
<210> 1
<211> 483
<212> DNA
<213> T24cell
<400> 1
atgacgctgg aagagctcgt ggcgtgcgac aacgcggcgc agaagatgca gacggtgacc 60
gccgcggtgg aggagctttt ggtggccgct cagcgccagg atcgcctcac agtgggggtg 120
tacgagtcgg ccaagttgat gaatgtggac ccagacagcg tggtcctctg cctcttggcc 180
attgacgagg aggaggagga tgacatcgcc ctgcaaatcc acttcacgct catccagtcc 240
ttctgctgtg acaacgacat caacatcgtg cgggtgtcgg gcatgcagcg cctggcgcag 300
ctcctgggag agccggccga gacccagggc accaccgagg cccgagacct gcattgtctc 360
ctggtcacga accctcacac ggacgcctgg aagagccacg gcttggtgga ggtggccagc 420
tactgcgaag aaagccgggg caacaaccag tgggtcccct acatctctct tcaggaacgc 480
tga 483
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
The handsome company of <223>
<400> 2
ttaagcttat gacgctggaa gagctc 26
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
The handsome company of <223>
<400> 3
ttggatcctc agcgttcctg aag 23
Claims (1)
1.GADD45 β gene is in the application of preparing in anti-bladder cancer medicine, and the nucleotide sequence of described GADD45 β gene is SEQ ID NO:1.
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| CN201310357438.0A CN103405785B (en) | 2013-08-15 | 2013-08-15 | Use of gene GADD45 beta in preparation of drug for treating bladder cancer |
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| CN103405785B true CN103405785B (en) | 2014-10-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111000858A (en) * | 2019-05-10 | 2020-04-14 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104906596A (en) * | 2015-05-20 | 2015-09-16 | 山西大学 | Application of p50 gene in preparation of bladder cancer fighting drug |
| CN104894164A (en) * | 2015-05-20 | 2015-09-09 | 山西大学 | Construction and application of GADD45betaRNA interference report vector based on EGFP |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1758615A2 (en) * | 2004-06-11 | 2007-03-07 | Board of Regents, The University of Texas System | Combination treatment of cancer with elicitor of gene product expression and gene-product targeting agent |
| CN101011571A (en) * | 2006-03-13 | 2007-08-08 | 上海交通大学医学院 | Use of GADD45 beta protein and its inhibitor in treatment of rheumatoid arthritis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111000858A (en) * | 2019-05-10 | 2020-04-14 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
| CN111000858B (en) * | 2019-05-10 | 2021-04-09 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
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