CN105769900A - Application of BAG3 gene to preparation of bladder cancer resisting medicines - Google Patents

Abstract

The invention provides application of a BAG3 gene to preparation of anticancer medicines and particularly provides application of the BAG3 gene to preparation of bladder cancer resisting medicines. By construction of a BAG3 gene over-expression vector and transfection of cancer cells, according to experiments, mRNA level of the BAG3 gene in the cancer cells is raised remarkably after transfection, the cancer cells become irregular in form, round in shape and poor in wall attaching performance, activity of the cancer cells is reduced by 60.69%, and the cancer cells are dead finally. The efficient anticancer gene is obtained by screening to provide basis for discovery of new medicine targets and design of anticancer medicines.

Description

The application in preparation anti-bladder cancer drug of the BAG3 gene

Technical field:

The present invention relates to the new application of BAG3 gene, specifically the application in preparing small molecule anticancer drug of the BAG3 gene, particularly the application in preparation anti-bladder cancer drug.

Background technology:

Immortalization gene 3 (BAG3) relevant for BCL2 is the plasmosin of a 74kDa, and it is predominantly located at rough-surfaced endoplasmic reticulum, has been confirmed as a BCL2 associated protein in albumen interaction technique.Having an about conserved domain about 110-124 aminoacid, it can be combined by the ATP enzyme domain on heat shock protein 70 (HSP70)；Meanwhile, it can also combine with including BCL2, steroid hormone receptor, Raf1 etc..In the mankind, BAG family includes 6 members, BAG1, BAG2, BAG3, BAG4, BAG5, BAG6.BAG3, also referred to as CAIR-1 or Bis, it is possible to by a BAG domain in conjunction with different parts, repeats including WW domain and proline.As a diversification albumen, BAG3 participates in many biological processes and affects the evolution of tumor by different way.

It is relevant that the expression of BAG3 converts these processes to the existence of human cancer cell, apoptosis, motility and adhesive force angiogenesis and Epithelial and stromal.BAG3 all can be invoked in various kinds of cell type, and it can open a kind of protection mechanism when cell is under pressure.In normal cell, the expression of BAG3 is relatively low, and in many tumor cells, including in leukemia, lymphoma, myeloma, melanoma, glioblastoma multiforme, cancer of pancreas and ovarian tumor, the expression of BAG3 increases.BAG3 affects the adhesive force of tumor cell, migrates and invasion, and then affect generation and the transfer of malignant tumor in organism, but BAG3 expression in bladder cancer and acting on does not have been reported that.

We by BAG3 in transitional cell bladder carcinoma cell line after process LAN, transitional cell bladder carcinoma cell line form can be caused to change, it is contemplated that by studying the BAG3 impact on urinary bladder carcinoma T24 cell line, find BAG3 effect in transitional cell bladder carcinoma cell line, tested by process LAN, it has been found that morphologically becoming round occurs in cell, form starts irregular, adherent property is deteriorated, and then dead phenomenon occurs, therefore can provide theoretical foundation for the treatment of bladder cancer for the further investigation of BAG3 gene.

Summary of the invention:

It is desirable to provide a kind of new application of BAG3 gene.

The present invention provides the application in preparing cancer therapy drug of the BAG3 gene, the particularly BAG3 gene application in preparation anti-bladder cancer drug.The nucleotides sequence of BAG3 gene is classified as SEQIDNO:1.

The present invention is by building the overexpression vector of BAG3 gene, then cytoactive and this gene mRNA expression amount are detected after transfection tumor cell, result shows: after transfection, in urinary bladder carcinoma T24 cell line, the mRNA of BAG3 gene significantly raises, becoming round occurs in tumor cell, adherent property is deteriorated, cytoactive significantly reduces, and final dead phenomenon occur.

The present invention screens and demonstrates BAG3 gene and has the function suppressing growth of bladder cancer cells, it is possible to as the new target drone gene of bladder cancer treatment, provides foundation for designing small-molecule drug based on target gene.

Accompanying drawing illustrates:

Fig. 1: pcDNA3.1-BAG3 Vector map

The PCR result of Fig. 2: gene BAG3, in figure: 1, marker；2, the PCR primer of gene BAG3

Fig. 3: Hind III and BamHI enzyme action identify in recombinant vector pcDNA3.1-BAG3, figure: 1, marker；2, recombiant plasmid pcDNA3.1-BAG3；3, the recombinant vector pcDNA3.1-BAG3 after enzyme action

MRNA level in-site expression change after Fig. 4: BAG3 overexpression, in figure: pcDNA3.1 is empty carrier；PcDNA3.1-BAG3 is the overexpression vector with BAG3 gene

After Fig. 5: BAG3 overexpression, MTT detects cytoactive change, in figure: PC3.1 is empty carrier；PC3.1-BAG3 is the overexpression vector with BAG3 gene

Fig. 6: T24 cellular morphology change after overexpression BAG3: A recombiant plasmid pcDNA3.1-BAG3 is transfected into cellular morphology after T24 cell 24h；B recombiant plasmid pcDNA3.1-BAG3 is transfected into cellular morphology after T24 cell 48h

Detailed description of the invention:

The lethal transitional cell bladder carcinoma cell line experiment of embodiment 1:BAG3 gene overexpression

One, the structure of recombinant expression carrier

1, the preparation of PCR primer

With the T24 cell cDNA of normal saline process for template, full length cDNA sequence (GeneID:9531) design based on BAG3 gene carries out pcr amplification with the expression primer with restriction enzyme site HindIII, BamHI.Reaction condition is: 94 DEG C of denaturation 2min, 98 DEG C of degeneration 10s, 55 DEG C of annealing 30s, and 68 DEG C extend 2min, totally 35 circulations, and 68 DEG C of insulation 5min, primer sequence is in Table 1.PCR primer removal process carries out in strict accordance with the description of the GelExtractionKit of OMEGA company.

Table 1BAG3 gene expression primer

2, genes of interest total length checking

The genes of interest that PCR obtains is connected on ZeroVector, is sent to Hua Da company and checks order.Find that BAG3 gene is successfully connected on Zero carrier through order-checking.

3, the double digestion reaction of PCR primer and expression vector

Choosing the successful plasmid of order-checking, in 0.2ulEP pipe, BAG3 genetic fragment and carrier framework pcDNA3.1 are being carried out double digestion respectively, reaction system carries out with reference to HindIII, BamHI description (NEB).

4, glue reclaims digestion products

With the agarose gel electrophoresis of 1%, above-mentioned double digestion product being carried out detection and glue reclaims, removal process carries out in strict accordance with the description of the GelExtractionKit of OMEGA company.

5, the connection of genes of interest BAG3 and pcDNA3.1

Glue containing genes of interest BAG3 is reclaimed product and is connected on pcDNA3.1, centrifuge tube is set up following reaction system:

Table 2 genes of interest and carrier framework linked system

Mixing gently, brief centrifugation collects liquid in, at the bottom of pipe, being placed in 16 DEG C and overnight connect.

Two, the conversion of recombinant expression carrier

1, Trans-1T1 competent cell is placed in thaws on ice；

2, in centrifuge tube, it is sequentially added into the connection product of 100 μ L competent cell core 8 μ L, mixes gently, be placed in 30min on ice；

3,42 DEG C of water-bath heat shock 60s, are then quickly placed on ice by pipe, ice bath 5min；

4, adding 300 μ L without antibiotic LB fluid medium, mixing is placed on 37 DEG C of 150rpm shaken cultivation 1h makes bacteria resuscitation；

5, take the 150 resuspended bacterium solution of μ L, be spread evenly across and be placed in 37 DEG C of incubators on the flat board of ammonia benzyl resistance in advance, incubated overnight.

Three, the checking of recombinant expression carrier

1, with the rifle head careful picking white list bacterium colony of sterilizing, rifle head is placed in 3mLLB fluid medium (containing 0.1%Amp), 37 DEG C, 12-16h cultivated by the shaken cultivation case of 200rpm.

2, adopt centrifuging to collect bacterium solution, adopt Omega company plasmid extraction kit to extract corresponding plasmid.Operation carries out in strict accordance with description.

3. with HindIII and BamHI, extracted plasmid is carried out enzyme action, enzyme action system and program to carry out according to NEB description, in Table 3.Reaction condition is 37 DEG C, 2h.Afterwards, by the agarose gel electrophoresis testing goal fragment of 1%, the fragment length scaled off after result display enzyme action is probably at about 1700bp, length close to genes of interest, corresponding plasmid purpose fragment being detected is sent to Hua Da company check order, result display is checked order successfully, and BAG3 gene has been successfully connected on expression vector pcDNA3.1.

The double digestion system of table 3 recombinant vector detection

Four, Transfected Recombinant Plasmid T24 cell

Take the logarithm trophophase cell, be resuspended in without in antibiotic RPMI1640 culture medium, be inoculated in 6 orifice plates.Experiment is divided into 2 groups, matched group: pcDNA3.1 empty plasmid and pEGFP-N1, experimental group: pcDNA3.1-BAG3 and pEGFP-N1, often 3 repetitions of group.Adopt the so-fast transfection reagent of sun horse company by the plasmid pcDNA3.1-BAG3 built and pEGFP-N1 cotransfection to T24 cell, concrete operations reference description.

Five, BAG3mRNA expression after Real-timePCR detection transfection

After pcDNA3.1-BAG3 and pEGFP-N1 cotransfection to T24 cell 48h, Trizol method extracts total serum IgE, and step carries out according to RNAisoPlus (TaKaRa) description.After reverse transcription, Real-timePCR detects BAG3mRNA expression.Result shows: after process LAN BAG3 gene, and its mrna expression level significantly raises.

Six, morphological observation

Recombiant plasmid proceeds to microscope (OLYMPUS1X71) observation of cell form after T24 cell 24h and 48h.Result shows: transfection pcDNA3.1-BAG3 plasmid in T24 cell after, it has been found that tumor cell occurs that form is irregular, becomes round, adherent property be deteriorated phenomenon.

Seven, MTT detects Transfected cells activity

After transfection 24h, matched group and process group are inoculated in 96 orifice plates respectively.Often group arranges 3 multiple holes, continues to cultivate.After cultivating 24h, discard culture fluid, add 180 μ L fresh cultures, add 20 μ L5mg/mLMTT (final concentration of 0.5mg/mL), continue to cultivate 4h.Until after the time, terminating cultivating, carefully sucking culture fluid in hole, every hole adds 150 μ LDMSO, shaking table low speed jolting 10min, makes bluish violet crystallization fully dissolve, and reads absorbance at microplate reader 490nm place and 570nm place.Process data, calculate the brazilin suppression ratio to cell, draw cell growth curve.Each sample standard deviation arranges 6 repetitions, averages as final result.MTT result shows: transfection pcDNA3.1-BAG3 plasmid in T24 cell after, cytoactive decline reach 60.69%.

Claims (2)

1. The application in preparing cancer therapy drug of the 1.BAG3 gene.
2. The application in preparation anti-bladder cancer drug of the 2.BAG3 gene.