CN109529051A - Saliva acyltransferase recombinant plasmid and its with pemetrexed preparation inhibit bladder cancer proliferation and invasion drug in application - Google Patents
Saliva acyltransferase recombinant plasmid and its with pemetrexed preparation inhibit bladder cancer proliferation and invasion drug in application Download PDFInfo
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Abstract
The present invention discloses saliva acyltransferase recombinant plasmid and its inhibits the proliferation of bladder cancer with pemetrexed in preparation and invade the application in drug; it is by constructing a kind of plasmid for being overexpressed saliva acyltransferase ST3Gal4 gene; the growing multiplication ability of bladder cancer cell can effectively be inhibited, and be by mediating PI3K-AKT signal path to play a role to the growing multiplication of bladder cancer cell.Further, by after the recombinant plasmid transfected cell, pass through and the synergy of pemetrexed can further meet bladder cells proliferation and migration ability.Sialyl based transferase ST3Gal4 can be made as the index of bladder cancer disease.Therefore, technology of the present invention is that preparation inhibits the proliferation of bladder cancer and invade in drug to provide good application prospect, provides new target spot for the early diagnosis and therapy of bladder cancer.
Description
Technical field
The invention belongs to biopharmaceutical technologies, and in particular to a kind of overexpression sialic acid acyltransferase ST3GAL4
The plasmid of gene, and its inhibit the application in the proliferation of bladder cancer and the drug of invasion with pemetrexed synergy.
Background technique
Currently, as long as malignant tumour is to threaten one of human health and life disease.In China, the disease incidence of bladder cancer
With the death rate be in Patients with Urinary System Tumors it is highest, and disease incidence has growth trend in recent years.
Bladder cancer patients pass through surgical resection therapy after recurrence rate it is higher, and using Intravesical BCG treatment have it is very big
Adverse reaction, it is very big to body harm.Since the means therapeutic effect such as traditional operation, radiotherapy, chemotherapy is poor, side effect is big, hand
Art cooperation drug therapy is come into being, and early diagnosing more becomes the most important thing.Currently, research discovery glycosyl transferase has centainly
Tumor monitoring effect, especially saliva acyltransferase, therefore, the early diagnosis index of bladder cancer patients treatment are to influence bladder
The important factor in order of cancer therapeutic effect.
The glycosylation modified of protein is important one of protein post-translational modification mode, is more than 50% in organism
There are glycosylation modified, a variety of glycosylation modified such as sialylated, fucosylations influences of tumor cell surface for protein
Proliferation, invasion and the apoptosis of tumour cell.Glycosylation albumen and sugar chain take part in immune response, reproduction, infection, inflammation, tumour
Etc. numerous important life processes.The glycosylation of protein be carbohydrate under the catalysis of glycosyl transferase in the form of covalent bond with
The process of peptide chain link.
Sialic acid is the derivative of 9 carbon monosaccharide, is played a significant role in cell recognition, adherency, signal transduction.Especially
The change of the sialic acid glycosyltransferases expression of α 2,6 and α 2,3 connection is even more closely related with tumour generation.Sialylated modification
The glycoprotein and glycolipid structure being attached thereto are shielded, stabilizing cell membrane structure improves thermal stability, and protects cell and big
Molecule is not by the attack of enzyme and immune system, to play a part of to protect cell.
Saliva acyltransferase ST3Gal4 is the specific catalytic α 2 positioned at golgiosome, and the sialic acid in 3- connection turns
Move on to the saliva acyltransferase of N- or O- glycan chain end.It is different according to the substrate of different saliva acyltransferases, by α 2,3-
Saliva acyltransferase is divided into six kinds of ST3Gal1-ST3Gal6.The unconventionality expression of saliva acyltransferase ST3Gal4 not only with it is swollen
The malignant phenotype of oncocyte is related, and can influence the prognosis of tumor patient.However currently, saliva acyltransferase
The Study on Molecular Mechanism of effect and its directed cell migration and invasion of the ST3Gal4 in bladder cancer is still few.
Pemetrexed is in a kind of structure be containing core pyrrolopyrimidine group folic acid resisting preparation, it is intracellular by destroying
The normal metabolic processes of folate-dependant inhibit cellular replication, to inhibit the growth of tumour.In vitro study is shown, it is bent to train U.S.
Plug is able to suppress the activity of thymidylate synthetase, dihyrofolate reductase and glycinamide ribonucleotide formyl transferase, these enzymes
All it is enzyme necessary to synthesis folic acid, participates in the biological synthesis process again of thymidylic acid and fast cry of certain animals nucleotide, it is bent to train U.S.
The folate binding protein transportation system on carrier and cell membrane that plug passes through delivery folic acid enters intracellular.Once pemetrexed into
Enter into the cell, it is just converted into the form of more glutamic acid under the action of leaf acyl more glutamate synthetases.More glutamic acid retain in
The intracellular inhibitor for becoming thymidylate synthetase and glycinamide ribonucleotide formyl transferase, more glutamates are in tumour cell
M- concentration dependent process when interior presentation, and concentration is very low in normal tissue.More glutamate metabolins are in tumour cell
Increased Plasma Half-life, to also just extend action time of the drug in tumour cell.Pemetrexed is as chemotherapeutics, extensively
It is general to apply in lung cancer.However currently, Effect study of the pemetrexed in bladder cancer is still few.
Therefore, the change that associated sugars gene expression in bladder cancer is detected by the kinds of experiments method such as in vitro and in vivo, grinds
Study carefully expression of the saliva acyltransferase ST3Gal4 in bladder cancer and its influence to bladder cancer's survival rate and with
Influence after pemetrexed joint to development of bladder cancer.Want to by this research be bladder cancer early diagnosis and control
It treats and new target spot is provided.
Summary of the invention
In order to solve the above technical problems, the present invention devises following technical solution: the present invention provides a kind of saliva acyl groups
Transferase ST3Gal4 plasmid studies influence of the ST3Gal4 to biological behaviours such as growth of bladder cancer cells, proliferation and invasion；
The change by influencing ST3Gal4 gene expression is demonstrated to influence proliferation of human bladder cancer cells invasive ability；Demonstrate building
The transfection cell and pemetrexed synergy for having transfected saliva acyltransferase ST3Gal4 plasmid can further influence bladder cancer
Cell Proliferation invasive ability.
The invention discloses be overexpressed saliva acyltransferase ST3GAL4 gene plasmid inhibit bladder cancer proliferation and
Application in invasion, specific application refer to that the plasmid for being overexpressed saliva acyltransferase ST3GAL4 gene inhibits wing in preparation
Application in the proliferation of Guang cancer and the drug of invasion.
In preferred situation, for application described above, described in plasmid vector insetion sequence nucleotide sequence
As shown in SEQ ID No.1.
In preferred situation, for application described above, described in plasmid vector be pcDNA3.1 (-).
In preferred situation, for application described above, the overexpression sialic acid acyltransferase ST3GAL4 base
In the plasmid construction of cause, the label used is HA, and the restriction endonuclease used is EcoR I/Hind III.
In preferred situation, for application described above, the overexpression sialic acid acyltransferase ST3GAL4 base
The use concentration of the plasmid of cause is 100nM/L~200nM/L.Best preferred concentration is 200nM/L.
Another technical solution of the invention is, will be overexpressed the recombinant plasmid of saliva acyltransferase ST3GAL4 gene with
Pemetrexed joint, and inhibit the application in the proliferation of bladder cancer and the drug of invasion in preparation.
It is to shift the overexpression sialic acid acyl group in application as described in the above technical scheme in preferred situation
The plasmid of enzyme ST3GAL4 gene is through transfecting cell；Transfection cell and pemetrexed conglomeration is taken to act on.
In preferred situation, for application described above, the overexpression sialic acid acyltransferase ST3GAL4 base
The plasmid of cause is through transfecting cell；Take the transfection cell of logarithmic phase stable transfection plasmid and the ratio of pemetrexed mixing are as follows: transfection
Cell number is 4~6 × 106A/mL, the pemetrexed conglomeration for being 0~0.6 μM with concentration act on.Pemetrexed is most
Preferred concentration is 0.6 μM, and most preferred cell number is 5 × 106A/mL.
In preferred situation, for application described above, cell used in the transfection be urinary bladder carcinoma T24 cell line or
5637 cell of bladder cancer.
The utility model has the advantages that the present invention is using ST3Gal4 after the methods of Western-blot detection transfection in bladder cancer cell
Differential expression situation；It is detected and is overexpressed after ST3Gal4 to the pernicious table of bladder cancer cell using the methods of CCK8, Transwell
Influence, the detection of type are added after pemetrexed and are overexpressed development of bladder cancer after ST3Gal4 combines with pemetrexed
Situation.Prove the early diagnosis that the plasmid pair bladder cancer of sialic acid acyltransferase ST3GAL4 gene can be overexpressed by building
And therapeutic effect plays monitoring effect.Or utilize the plasmid transfection for being overexpressed sialic acid acyltransferase ST3GAL4 gene
Cell after combining with pemetrexed, can obviously inhibit the proliferation and invasion of bladder cancer.Indicate that the present invention will be preparation bladder cancer
There is good application prospect in terms of drug, and diagnosis and treatment offer theoretical foundation to bladder cancer patients, while being guidance
Body clinic medication provides new approaches.
Detailed description of the invention
The schematic diagram of Fig. 1: A. construction recombination plasmid.
B.Western Blot trace schematic diagram, the data MOCK in figure is blank control group, chooses two concentration gradients
For 100nM/L, 200nM/L come determine best plasmid concentration detect T24 and 5637 bladder cancer cells be overexpressed ST3Gal4 matter
Protein expression level after grain, from protein level, the cell that transfection is overexpressed ST3Gal4 plasmid is apparently higher than without transfection
T24 and 5637 bladder cancer cells；
C.Western Blot trace Image Lab analysis chart, the data MOCK in figure are blank control group, choose two
Concentration gradient is that 100nM/L, 200nM/L detect T24 and 5637 bladder cancer cells and are being overexpressed to determine best plasmid concentration
Protein expression level after ST3Gal4 plasmid, from protein level, the cell that transfection is overexpressed ST3Gal4 plasmid is obviously high
In T24 and 5637 bladder cancer cells without transfection.
Fig. 2: CCK8 schematic diagram, the data NC schemed in A, B is Normal group, and MOCK is blank control group, chooses two
Concentration gradient be 100nM/L, 200nM/L come determine best plasmid concentration detect be overexpressed ST3Gal4 after T24 (Fig. 6 A) and
The proliferation of 5637 two kinds of (Fig. 6 B) bladder cancer cell lines is horizontal, from the point of view of proliferation level, it was demonstrated that overexpression ST3Gal4 bladder cancer
Ability of cell proliferation weakens.
Fig. 3: Transwell schematic diagram, the data NC in figure is Normal group, and MOCK is blank control group, chooses two
A concentration gradient be 100nM/L, 200nM/L come determine best plasmid concentration detect be overexpressed ST3Gal4 after T24 and 5637 liang
The invasion of kind of bladder cancer cell lines are horizontal, from invasion degree, it was demonstrated that bladder cancer cell is invaded after overexpression ST3Gal4
Reduced capability.
Fig. 4: CCK-8 schematic diagram, the data NC in figure be it is normal it is p- shine group, MOCK is blank control group, choose two it is dense
Degree gradient be 100nM/L, 200nM/L come determine best plasmid concentration detect be overexpressed ST3Gal4 after HepG2 liver cancer cell lines
Proliferation and invasion it is horizontal, compared with bladder cancer, hepatoma cell proliferation ability and invasive ability after being overexpressed ST3Gal4 increase
By force.
Fig. 5: Transwell schematic diagram, the data NC in figure is Normal group, and MOCK is blank control group, chooses two
A concentration gradient is 100nM/L, 200nM/L thin to detect HepG2 liver cancer after overexpression ST3Gal4 to determine best plasmid concentration
The proliferation of born of the same parents system and invasion are horizontal, the hepatoma cell proliferation ability and invasive ability compared with bladder cancer, after being overexpressed ST3Gal4
Enhancing.
Fig. 6: CCK-8 schematic diagram, figure A, B in 0 μM of data be control group, have chosen two concentration gradients be 0.3 μM,
0.6 μM is detected to determine the Drug level of best pemetrexed to bladder normal epithelium cell SV-HUC-1 (Fig. 6 A) and bladder
The influence of cancer cell T24 (Fig. 6 B), from proliferation level, the pemetrexed of dosing influences very low, inhibition on normal cell
The ability of proliferation of human bladder cancer cells is stronger.
Fig. 7: CCK-8 schematic diagram, the data MOCK in figure are control group, after ST3Gal4 transfection with 0.3 μM of various concentration,
0.6 μM of pemetrexed synergy come detect bladder cancer cell lines proliferation and invasion it is horizontal, the results show that ST3Gal4 joins
After cooperation is used, inhibit the ability of proliferation of human bladder cancer cells stronger.
Fig. 8: Transwell schematic diagram, the data MOCK in figure are control group, after ST3Gal4 transfection with various concentration 0.3
μM, 0.6 μM of pemetrexed synergy come detect bladder cancer cell lines proliferation and invasion it is horizontal, the results show that ST3Gal4
After synergy, inhibit the ability of proliferation of human bladder cancer cells stronger.
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
Embodiment 1
Authorized company of the present invention constructs the over-express vector of ST3Gal4, and the Gene ID of ST3Gal4 is 6484
(BC010645), kind is source of people, and insertion size is 1002bp, and using pcDNA3.1 (-) carrier, size 5428bp is used
Label be HA, the restriction endonuclease used be EcoR I/Hind III.Such as Figure 1A.
The nucleotide sequence of ST3Gal4 insetion sequence is as shown in SEQ ID No.1；The nucleotide sequence of pcDNA3.1 (-)
As shown in SEQ ID No.2.
Bladder cancer cell is set to be overexpressed ST3Gal4 plasmid by liposomal transfection teclmiques.It is bis- using EcoRI/Hind III
The positive colony of digestion and DNA sequencing identification ST3Gal4 recombinant expression carrier, sequencing reaction are proud that biotechnology has by Nanjing bar
Limit company provides and completes, and sequencing result is consistent completely with expected results.
ST4Gal4 sequence:
Positive-sense strand: 5'-TGAATCTGCCCACTTCGACC-3'
Antisense strand: 5'-TTCGCACCCGCTTCTTATCA-3'
In the present embodiment, the overexpression plasmid obtained using the above method of the present invention building chooses two concentration gradients
100nM/L and 200nM/L, CCK8 and Transwell are experiments have shown that overexpression ST3Gal4 plasmid concentration is 200nM/L to wing
The inhibitory effect of the invasion of Guang cancer cell multiplication and transfer ability is obvious (Fig. 2,3).Selection two is dense in water-soluble pemetrexed
0.3 μM and 0.6 μM of gradient of degree, CCK8 and Transwell experiments have shown that 0.6 μM of pemetrexed to the inhibiting effect of bladder cancer most
It is obvious.However the pemetrexed of various concentration does not have much affect to the proliferation of normal Urothelial Cell and invasion and (schemes
6)。
Embodiment 2
One, cell culture
(1) cell recovery
It is removed from liquid nitrogen the cell frozen, is placed in PE gloves, is shaken in 37 DEG C of water-baths to its thawing, it is then fast
Speed transfers them in 5mL centrifuge tube, and culture medium 3mL is added, and 1000rpm is centrifuged 4min.Cell is sunk with the culture medium of preheating
It forms sediment after being resuspended, is transferred in Tissue Culture Flask, is put into incubator and cultivates.
(2) cell change liquid and passage
Human bladder Urothelial cell system (SV-HUC-1) F12/DMEM culture medium is cultivated, and 10% tire is contained in culture medium
Cow's serum and dual anti-(penicillin 100U/mL, 100 μ g/mL of streptomysin).Human bladder cancer cell line (RT4) is cultivated with MCCOY ' S5A
Base is cultivated, and 10% fetal calf serum is contained in culture medium, dual anti-(penicillin 100U/mL, 100 μ g/mL of streptomysin).Human bladder cancer
1640 culture medium cultures of cell line (T24,5637) contain 10% fetal calf serum, dual anti-(penicillin 100U/mL, chain in culture medium
100 μ g/mL of mycin).All cells are in 37 DEG C, 5%CO2, 90% humidity the same terms under cultivate.According to the growth shape of cell
State changes a not good liquor for 2-3 days.When cell density is up to 80%~90%, had digestive transfer culture or subsequent is carried out with 0.25% trypsase
Experiment.
(3) cell is collected and is frozen
Selection is in the cell of logarithmic growth phase, for 24 hours plus renews culture medium before freeze-stored cell, 0.25% trypsase into
Row digestion, 1000rpm is centrifuged 4min after terminating digestion, and 900 μ L serum are resuspended cell precipitation, are transferred in cryopreservation tube, is added 100
μ L DMSO is frozen.Cryopreservation tube is put into freezing storing box to be placed in after -80 DEG C of refrigerators are stood overnight to be transferred in liquid nitrogen and is protected for a long time
It deposits.
Two, plasmid extracts
(1) the ST3Gal4 bacterium solution provided for company is taken to be put into LB liquid medium 37 DEG C, 140rpm, shake culture 1h；
(2) 20 μ L are taken to be evenly coated on the solid medium of Amp resistance, 37 DEG C of overnight incubations；
(3) single colonie is chosen to be seeded in the fluid nutrient medium of 3mLAmp resistance, 37 DEG C, 140rpm, shake culture is overnight, after
Its whole is inoculated in the fluid nutrient medium of 200mLAmp resistance, 37 DEG C, 140rpm, shake culture 3h；
(4) it spends endotoxin plasmid extraction kit and extracts plasmid.
Three, cell transfecting
(1) T24 and 5637 cells for taking logarithmic phase, with 5 × 10 after counting4The cell concentration of/mL is laid in 6 orifice plates, training
It supports to the adherent rear progress subsequent experimental of cell；
(2) clean cell with PBS, change it is double without culture medium Nature enemy 6h after, transfected；
(3) it takes the ST3Gal4 of 4.0 μ g to be overexpressed in the plasmid addition bis- no culture mediums of 50 μ L respectively to mix well；
(4) 5 μ L liposomes are added in the bis- no culture mediums of 50 μ L and are mixed well, be placed at room temperature for 5min；
(5) after being sufficiently mixed the mixed liquor in (3) and (4), it is incubated at room temperature 20min, forms compound to it；
(6) mixture in (5) is added dropwise in culture dish to be transfected, in 37 DEG C, 5%CO2, 90% humidity condition
Lower culture 6h, changes normal incubation medium culture；
(7) in 37 DEG C, 5%CO2, cultivate under conditions of 90% humidity, detected for subsequent experimental；
Transfecting the cell used is T24 cell and 5637 cells, and label transfection group is experimental group, and NC is Normal group
(being added without liposome, be added without plasmid), MOCK are blank control group (liposome being added, be added without plasmid), and selection two is dense
Degree gradient is 100nM/L, 200nM/L to determine best plasmid concentration.
Four, ST3Gal4 stablizes the screening for the monoclonal cell strain being overexpressed
(1) G418 is screened:
After experimental group ST3Gal4, MOCK and Par. (T24 and 5637 cells without any processing) transfect 48h, trained in original
It supports addition 700ug/mL G418 in base to continue to cultivate and screen, the complete cell death of Par. group when screening the 8th day, and
ST3Gal4 and MOCK group has fraction cell survival, transfects successful cell after about 14 days and starts to be proliferated, continues to cultivate to it.
(2) cell count:
Cell suspension is counted using cell counting board, the total number of cells in count plate four big lattice, if cell pressure
Line then only calculates left side and top, cell concentration calculation formula are as follows: and cell concentration (a/mL)=tetra- big lattice total number of cells/4 ×
104× extension rate.
(3) monoclonal cell screens:
It takes above-mentioned cell using limiting dilution assay, makes 150/mL of cell suspension cell density, every hole in 96 orifice plates
It is observed under the microscope after the cell suspension of 10uL is added, and records the hole serial number containing individual cells, be added and contain in every hole
The complete medium of 350ug/mL G418 continues to cultivate, and when cell Proliferation is more than the 1/3 of bottom hole, digestion is transferred to 24 orifice plates
It is interior, it is incubated in culture dish until expanding, freezes conservation after expanding culture.
Five, cell protein extracts experiment
(1) the big ware for cultivating T24 and 5637 cells is placed on ice, discards culture medium；
(2) three times using PBS cleaning；
(3) RIPA and PMSF is mixed with the ratio of 100:1, is added in ware, 300 μ L is added in a ware；
(4) cell is hung and taken after standing 15-20 minutes on ice；
(5) it is centrifuged using low-temperature and high-speed centrifuge, centrifugal force 16000g, 15min, 4 °；
(6) precipitating is discarded, supernatant is left and taken；
(7) the loading buffer of supernatant volume 1/4 is added；
(8) boiling 10min, 4 ° of preservations, in case subsequent experimental are denaturalized.
Six, Western Blot is tested
(1) 10% SDS-PAGE glue (glue is concentrated in 7mL separation gel, 3mL) is prepared, is carried out after protein sample (30 μ g) loading
Electrophoresis 1h30min, constant current 40mA；
(2) albumen is gone into constant current 200mA on pvdf membrane, 1h30min after pvdf membrane activates 30s with methanol；
(3) pvdf membrane 3h is shaken with 5% skimmed milk power room temperature slowly to close；
(4) primary antibody ST3Gal4 (1:1000, Protein tech) is configured with TBST；GADPH (1:1000, Protein
Tech), 4 DEG C of overnight incubations；
(5) after TBST washes film 10min × 3 time after taking out, be added dropwise horseradish enzyme peroxide label goat antirabbit secondary antibody (1:
3000), it is incubated at room temperature 1h；
(6) it after TBST washes film 10min × 3 time, is detected using chemiluminescence detection system.
Bio-Rad ChemiDoc MP Imaging System shines, and histogram is drawn in the analysis of Image lab software.
Seven, cytoactive detection (CCK-8 method)
(1) by control group, (5637 cell number of bladder cancer is 5 × 104A/mL, T24 cell number are 5 × 104A/mL) and it is real
It tests group (bladder cancer cell for being overexpressed ST3Gal4/ST3Gal6/MOCK) cell suspension 100uL to be inoculated in 96 orifice plates, every group
5 multiple holes are set, are cultivated in incubator, cultivation makes its adherent overnight；
(2) after respectively at inoculation 0,6,12,18 and for 24 hours, every hole adds CCK-8 reagent 10uL, and oscillation mixes, avoids blistering,
2h is incubated in incubator；
(3) microplate reader detects the absorbance at 450nm wavelength；
(4) independent three times to repeat to carry out statistical analysis after testing.
As seen from Figure 2, after being overexpressed ST3Gal4 plasmid, the increasing through CCK8 method detection bladder cancer cell T24 and 5637
Ability is grown, it is found that proliferative capacity weakens compared with the control group for it.
Eight, cell transwell is tested
(1) by the 5637 and T24 cell in logarithmic growth phase, (Control is Normal group, and MOCK is empty vectors
Group), with 0.25% trypsin digestion, 1000rpm/5min carries out cell weight with double no culture mediums (serum-free, without double antibody)
It is outstanding；
(2) cell counting board counts cell；
(3) normal incubation medium of 500 μ L is added in each hole of 24 orifice plates, is put into the cell transwell；
(4) it is added and contains 1 × 105A cell it is double without 200 μ L of culture medium in the cell transwell, avoid bubble from producing
It is raw；
(5) in 37 DEG C, 5%CO2Under the conditions of cultivated, T24 cultivate 15h, 5637 culture 30h；
(6) cell is taken out, PBS is washed 3 times, the fixed 15min of methanol；
(7) it is washed 3 times with PBS；
(8) with cotton swab that upper ventricular cell sassafras is net；
(9) 0.1% violet staining 30min；
(10) it is washed 3 times with PBS, natural air drying saves；
(11) it is observed with inverted microscope, randomly chooses 10 different visuals field, counted, statistical result.
As seen from Figure 3, after being overexpressed ST3Gal4 plasmid, bladder cancer 5637 and T24 are detected with Transwell method
Cell invasion ability, finding it, invasive ability weakens compared with the control group.
Nine, ST3Gal4 plasmid is overexpressed for the anticancer effect of other cancers
Hepatocellular carcinoma H22 is employed herein and has done CCK-8 and Transwell experiment, as a result, it has been found that being overexpressed
ST3Gal4 plasmid promotes the proliferation and invasion (Fig. 4,5) of hepatocellular carcinoma H22.
By having literature search, it has been found that the cancers such as breast cancer (Milde-Langosch K, Karn T, Schmidt
M, zu Eulenburg C, Oliveira-Ferrer L, Wirtz RM, Schumacher U, Witzel I, Sch ü tze D, M
üller V:Prognostic relevance of glycosylation-associated genes in breast
Cancer.Breast Cancer Research and Treatment 2014,145 (2): 295-305), in cancer cell
The expression quantity of ST3Gal4 is higher than the ST3Gal4 expression quantity in normal cell, and in bladder cancer cancer cell ST3Gal4 expression quantity
It is lower than the ST3Gal4 expression quantity in normal cell, it is exactly the opposite with the data of existing literature.
Ten, add pemetrexed synergy after cell transfecting
It (1) is 0 μM (1640 culture mediums of 100mL) by water-soluble pemetrexed concentration dilution with 1640 complete mediums,
0.3 μM (joined 15 μ g water solubility pemetrexeds in 1640 culture mediums of 100mL), 0.6 μM (in 1640 culture mediums of 100mL
It joined the water-soluble pemetrexed of 30 μ g)；
(2) the T24 cell of logarithmic phase stable transfection plasmid is taken, cell number is 5 × 106A/mL is separately added into 1mL thereto
Containing there are three 1640 complete mediums of the pemetrexed of concentration gradient and 3mL；
(3) in 37 DEG C, 5%CO2, cultivate 6h under conditions of 90% humidity, change normal incubation medium culture；
(4) in 37 DEG C, 5%CO2, cultivate under conditions of 90% humidity, for subsequent CCK-8 and Transwell experiment inspection
It surveys.Such as Fig. 7, the result of CCK-8 experiment is it can be seen that 0.6 μM of training is added after being overexpressed the T24 cell of plasmid ST3Gal4 in transfection
Beautiful Qu Saiyu others experimental group is compared, and the proliferation of the T24 cell of bladder cancer can be preferably inhibited；Such as Fig. 8, Transwell
0.6 μM of pemetrexed is added after can be seen that the T24 cell for transfecting overexpression plasmid ST3Gal4 for the result of experiment and others are real
It tests group to compare, can preferably inhibit the invasion of the T24 cell of bladder cancer, thus demonstrate transfection cell and pemetrexed combination
There is good inhibiting effect to bladder cancer cell.
Sequence table
<110>saliva acyltransferase recombinant plasmid and its with pemetrexed preparation inhibit bladder cancer proliferation and invasion medicine
Application in object
<120>Dalian Medical Univ
<130> 2015
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1002
<212> DNA
<213>ST3Gal4 is inserted into nucleotide sequence
<400> 1
atggtcagca agtcccgctg gaagctcctg gccatgttgg ctctggtcct ggtcgtcatg 60
gtgtggtatt ccatctcccg ggaagacagg tacatcgagc ttttttattt tcccatccca 120
gagaagaagg agccgtgcct ccagggtgag gcagagagca aggcctctaa gctctttggc 180
aactactccc gggatcagcc catcttcctg cggcttgagg attatttctg ggtcaagacg 240
ccatctgctt acgagctgcc ctatgggacc aaggggagtg aggatctgct cctccgggtg 300
ctagccatca ccagctcctc catccccaag aacatccaga gcctcaggtg ccgccgctgt 360
gtggtcgtgg ggaacgggca ccggctgcgg aacagctcac tgggagatgc catcaacaag 420
tacgatgtgg tcatcagatt gaacaatgcc ccagtggctg gctatgaggg tgacgtgggc 480
tccaagacca ccatgcgtct cttctaccct gaatctgccc acttcgaccc caaagtagaa 540
aacaacccag acacactcct cgtcctggta gctttcaagg caatggactt ccactggatt 600
gagaccatcc tgagtgataa gaagcgggtg cgaaagggtt tctggaaaca gcctcccctc 660
atctgggatg tcaatcctaa acagattcgg attctcaacc ccttcttcat ggagattgca 720
gctgacaaac tgctgagcct gccaatgcaa cagccacgga agattaagca gaagcccacc 780
acgggcctgt tggccatcac gctggccctc cacctctgtg acttggtgca cattgccggc 840
tttggctacc cagacgccta caacaagaag cagaccattc actactatga gcagatcacg 900
ctcaagtcca tggcggggtc aggccataat gtctcccaag aggccctggc cattaagcgg 960
atgctggaga tgggagctat caagaacctc acgtccttct ga 1002
<210> 2
<211> 5427
<212> DNA
<213>pcDNA3.1 (-) nucleotide sequence
<400> 2
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatct gcagaattcc 960
accacactgg actagtggat ccgagctcgg taccaagctt aagtttaaac cgctgatcag 1020
cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 1080
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 1140
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 1200
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 1260
cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa 1320
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 1380
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 1440
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 1500
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 1560
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 1620
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 1680
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 1740
gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 1800
gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 1860
tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 1920
cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 1980
tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 2040
cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 2100
atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc 2160
aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 2220
cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 2280
caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 2340
gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 2400
ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 2460
tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 2520
tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 2580
agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 2640
actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg 2700
cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 2760
tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 2820
tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 2880
cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg 2940
gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc 3000
gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc 3060
ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct 3120
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 3180
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg 3240
tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 3300
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 3360
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 3420
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 3480
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 3540
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 3600
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 3660
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3720
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 3780
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 3840
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 3900
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 3960
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4020
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4080
ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4140
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4200
gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4260
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4320
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 4380
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 4440
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 4500
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 4560
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 4620
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 4680
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 4740
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 4800
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 4860
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 4920
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 4980
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 5040
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 5100
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 5160
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 5220
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 5280
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 5340
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 5400
tttccccgaa aagtgccacc tgacgtc 5427
Claims (10)
1. being overexpressed application of the plasmid of saliva acyltransferase ST3GAL4 gene in the proliferation and invasion for inhibiting bladder cancer.
2. the plasmid for being overexpressed saliva acyltransferase ST3GAL4 gene inhibits the proliferation of bladder cancer and the drug of invasion in preparation
In application.
3. application according to claim 1, it is characterised in that: the nucleotide sequence of the plasmid vector insetion sequence is such as
Shown in SEQ ID No.1.
4. application according to claim 3, it is characterised in that: the plasmid vector is pcDNA3.1 (-).
5. application according to any one of claims 1 to 4, it is characterised in that: the overexpression sialic acid acyl group transfer
In the plasmid construction of enzyme ST3GAL4 gene, the label used is HA, and the restriction endonuclease used is EcoR I/Hind III.
6. application according to claim 1, it is characterised in that: the overexpression sialic acid acyltransferase ST3GAL4 base
The use concentration of the plasmid of cause is 100nM/L~200nM/L.
7. the recombinant plasmid for being overexpressed saliva acyltransferase ST3GAL4 gene is combined with pemetrexed, inhibit bladder in preparation
Application in the proliferation of cancer and the drug of invasion.
8. application according to claim 7, it is characterised in that: the overexpression sialic acid acyltransferase ST3GAL4 base
The plasmid of cause is through transfecting cell；Transfection cell and pemetrexed conglomeration is taken to act on.
9. application according to claim 8, it is characterised in that: the ratio of the transfection cell and pemetrexed mixing are as follows:
Transfecting cell number is 4~6 × 106A/mL, the pemetrexed conglomeration for being 0~0.6 μM with concentration act on.
10. application according to claim 9, it is characterised in that: cell used in the transfection be urinary bladder carcinoma T24 cell line or
5637 cell of bladder cancer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110151778A (en) * | 2019-04-02 | 2019-08-23 | 大连医科大学 | A kind of composition that anti-prostate cancer invasion are shifted and its application in terms of prostate cancer diversion medicaments are treated in preparation |
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| WO2014193999A2 (en) * | 2013-05-28 | 2014-12-04 | Caris Science, Inc. | Biomarker methods and compositions |
| CN108610414A (en) * | 2018-02-12 | 2018-10-02 | 北京大学 | IgG epitopes and its application as target spot |
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| CN110151778A (en) * | 2019-04-02 | 2019-08-23 | 大连医科大学 | A kind of composition that anti-prostate cancer invasion are shifted and its application in terms of prostate cancer diversion medicaments are treated in preparation |
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