CN109680005A - Application of the WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell - Google Patents
Application of the WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell Download PDFInfo
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- CN109680005A CN109680005A CN201910035278.5A CN201910035278A CN109680005A CN 109680005 A CN109680005 A CN 109680005A CN 201910035278 A CN201910035278 A CN 201910035278A CN 109680005 A CN109680005 A CN 109680005A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The present invention provides a kind of application of WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell, it is related to technical field of molecular biology, it is had shown that by the research of inventor, the expression of WDR12 gene and the classification of glioma are positively correlated, and the knockout of WDR12 gene all has significant impact to the increment of glioma cell, apoptosis and cell cycle.Therefore, WDR12 gene can be used as a specific gene target spot of brain glioblastoma cell inhibition and apoptosis, and then provide a kind of new theoretical basis for the exploitation of the target therapeutic agent of glioma.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to a kind of WDR12 gene in brain glioblastoma cell
Inhibit and the application in apoptosis.
Background technique
Glioma is a kind of tumour for being common in brain, and about 33% brain tumor is glioma, it is initiated by encirclement
With the Deiter's cells for supporting cerebral neuron, including astroglia, oligodendroglia and ependymocyte this three
The tumour occurred on class cell.Currently, since macromolecular drug is good at leading to examining for glioblastoma through blood-brain barrier
Disconnected and treatment method is all less desirable, and therapeutic effect is bad, and high recurrence rate, life cycle is short, therefore the treatment of glioma
The problem and hot research problem of neurosurgery clinical treatment were become already.
The biological nature of glioma depends on inherent gene alteration, finds reliable tumor marker gene and to brain
The characteristics such as glioma proliferative, invasion, differentiation judge, and are Diagnosing Gliomas, prognosis evaluation and determining effectively treatment
The important prerequisite of measure.Therefore, in the case where existing therapeutic scheme is ineffective, spy of the urgent need to glioma
Specific gene target spot is researched and developed, and then seeks to provide base for the gene diagnosis of glioma and targeting small molecule treatment
Plinth becomes very necessary and urgent.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of WDR12 gene answering in the inhibition and apoptosis of brain glioblastoma cell
With, through inventor study discovery WDR12 gene pairs glioma generation and progression in play an important role, in turn
A kind of new theoretical basis is provided for the target therapeutic agent exploitation of glioma.
A kind of application of the WDR12 gene provided by the invention in the inhibition and apoptosis of brain glioblastoma cell.
Further, the WDR12 gene inhibits the specific gene target spot with apoptosis as brain glioblastoma cell.
Further, the genebank Serial No. NC_000002.12:c202 912226- of the WDR12 gene
202878919Homo sapiens chromosome 2,GRCh38.p12Primary As sembly。
Further, the WDR12 gene source is in mammal (including people).
Further, the brain glioblastoma cell is in vitro culture glioma cell；
Preferably, the brain glioblastoma cell strain is U251, U373, U87 or A172.
Further, the WDR12 gene high expression in brain glioblastoma cell.
Further, the shRNA of lentivirus mediated knocks out WDR12 gene and significantly inhibits glioma in vitro
And promote Apoptosis.
Further, the shRNA of the lentivirus mediated knocks out the construction step of WDR12 gene are as follows: design WDR12-
ShRNA sequence, and the slow virus of expression WDR12-shRNA is constructed to inhibit the expression of WDR12, for WDR12-shRNA
Most effective target sequence: the sequence of 5'-CCTATTTC CCATGATTCCTTCATA-3', Scr-shRNA are 5'-
GTAATACGGTTAT CCACGCG-3'。
Further, the WDR12 gene inhibits glioma and promotes Apoptosis to be to pass through the cell cycle
Glioma cell is adjusted.
Further, in the cell cycle, the albumen of WDR12 gene expression passes through the phase interaction with EZH2 albumen
With, the P21 factor of adjusting cell cycle middle and upper reaches and the H3K27me factor in downstream, and then adjust tumor proliferation.
Compared with prior art, the invention has the benefit that
The present invention provides a kind of application of WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell.Pass through basis
Research has shown that the expression of WDR12 gene and the classification of glioma are positively correlated, increasing of the knockout of WDR12 gene to glioma cell
Value, apoptosis and cell cycle all have significant impact, it follows that WDR12 gene can be used as brain glioblastoma cell inhibition
With a specific gene target spot of apoptosis, and then for the target therapeutic agent of glioma exploitation provide theoretical basis.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
WDR12 genetic immunization stained slice figure in the glioma that Fig. 1 provides for the embodiment of the present invention 1；
The mRNA high of WDR12 is expressed in glioma in the TCGA database that Fig. 2 provides for the embodiment of the present invention 1；
Fig. 3 is the WDR12 gene that provides of the embodiment of the present invention 1 in glioma cell line U251, U373, U87 and A172
MRNA express figure；
Fig. 4 is the albumen for the WDR12 gene expression that the embodiment of the present invention 1 provides in U251, U373, U87 and A172
Protein electrophoresis figure；
Fig. 5 is the detection figure that the WDR12 gene that the embodiment of the present invention 2 provides knocks out in U251；
Fig. 6 is the continuous five days monitoring cell Proliferation feelings of CellomicsArrayScan VTI that the embodiment of the present invention 2 provides
Condition figure；
Fig. 7 is that the mtt assay that the embodiment of the present invention 2 provides detects cell proliferative conditions figure；
Fig. 8 is the Annexin V flow cytometer detection figure that the embodiment of the present invention 3 provides；
Fig. 9 is the Caspase3/7 detection figure that the embodiment of the present invention 3 provides；
Figure 10 is the RNA-seq gene variation thermal map that the embodiment of the present invention 4 provides；
Figure 11 is the genescreen figure of the possibility that the embodiment of the present invention 4 provides and WDR12 interaction；
Figure 12 is PLK, EZH2, CDK1, FOX1 and the UBE2C gene that provide of the embodiment of the present invention 4 in shWDR12
MRNA expression figure；
Figure 13 is protein electrophoresis figure of the PLK and EZH2 albumen that provides of the embodiment of the present invention 4 in shWDR12；
Figure 14 is the immunocyte fluorescent staining figure of the WDR12 that the embodiment of the present invention 5 provides and EZH2 interaction；
Figure 15 is the Western that the WDR12 that the embodiment of the present invention 5 provides and EZH2 interaction adjusts the upstream and downstream factor
Bloting electrophoresis detection figure.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The present invention provides a kind of application of WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell.The WDR12
The Serial No. of gene: NC_000002.12:c202912226-202878919 Homo sapiens chromosome 2,
GRCh38.p12Primary Assembly。
The high expression in brain glioblastoma cell of WDR12 gene described in embodiment 1
(1) immunohistochemistry shows that the expression of WDR12 gene and glioma grading are positively correlated
Human glioblastoma is divided into I-IV grades by the World Health Organization (WHO), is classified the progress with glioma, treatment and prognosis
It is highly relevant.The pathological grading of glioma is generally divided into low level and high-level.Low grade glioma (LGG) includes I and II grades,
Usually there is better prognosis, and advanced glioma (HGG) includes III level and IV grades, usual prognosis is poor.
As shown in Figure 1, inventor is by passing through the table of Immunohistochemical Evaluation WDR12 gene to glioma sample
It reaches.From in Fig. 1 it should be apparent that differential expression of the WDR12 gene in different grades of glioma is obvious, WDR12 base
Because being apparently higher than the expression in Low grade glioma (LGG) in the expression in advanced glioma (HGG), WDR12 gene is shown
Expression and glioma Pathological degree between there are significant differences.
In the preferred embodiment of the present invention, the WDR12 system that immunohistochemistry is sliced in samples of human glioma
Preparation Method are as follows:
A, glioma samples cell is dehydrated: 50% ethyl alcohol (90min) → 70% ethyl alcohol (90min) → 80% ethyl alcohol
(90min) → 95% ethyl alcohol (90min) → 100% ethyl alcohol (60min) → 100% ethyl alcohol (60min).
B, transparent :+1/2 dimethylbenzene (60min) of 1/2 100% ethyl alcohol (60min) → dimethylbenzene (60min) → dimethylbenzene
(60min)。
C, it embeds: 1:1 dimethylbenzene/paraffin (60min) → paraffin I (60min) → paraffin II (60min).
D, be sliced: paraffin slicing machine carries out 5 μ m thicks
E, it dewaxes: toasting 30 minutes organization chips in 60 DEG C of insulating boxs and be placed in dimethylbenzene and impregnate 15 minutes；Replacement two
It is impregnated again after toluene 15 minutes；Dimethylbenzene: ethyl alcohol=1:1 is mixed to be impregnated 10 minutes in liquid；Dehydrated alcohol (10min) → 95% ethyl alcohol
(10min) → 85% ethyl alcohol (10min) → 75% ethyl alcohol (10min) → distilled water (10min)；
F, it closes: configuring fresh 3%H2O2 with distilled water or PBS, room temperature is closed 10 minutes；
G, antigen retrieval: high fire heating 0.01M sodium citrate buffer (pH6.0) is to after boiling by group in micro-wave oven
It knits chip to be put into, low fire maintains 20 minutes；
H, close: 10% serum (TBS preparation) is closed 30 minutes；
I, it dyes: inhaling and abandon serum, do not wash and corresponding primary antibody overnight incubation is added；Primary antibody is recycled, TBS is washed 2 times, every time 5 points
Clock；Secondary antibody is added, is incubated at room temperature 60 minutes；TBS is washed 4 times, every time 5 minutes；VULCAN FAST RED CHROMOGEN is added
Kit2 is dyed 15 minutes, is terminated；DAB dyeing is added, until showing light yellow, is put into distilled water and terminates reaction；
J, be dehydrated mounting: in 75% ethyl alcohol, leaching is set 5 minutes；It is impregnated 5 minutes in 85% ethyl alcohol；5 points are impregnated in 95% ethyl alcohol
Clock；It is impregnated 5 minutes in dehydrated alcohol；Dimethylbenzene: ethyl alcohol=1:1 is mixed to be impregnated 5 minutes in liquid；It is impregnated again after dimethylbenzene 5 minutes；More
It changes and is impregnated again after dimethylbenzene 5 minutes；After taking-up, 30ul neutral gum is added dropwise, with coverslip mounting；
K, it takes pictures.
(2) WDR12 gene overexpression in samples of human glioma is shown by TCGA database analysis
As shown in Fig. 2, inventor includes cancer gene group picture (TCGA) and genotype tissue expression (GTEx) by analysis
The WDR12 gene expression of data set, compared with normal cerebral tissue's control, WDR12 gene is presented in glioma and is raised, and in glue
It is significantly raised in matter blastoma (GBM).
(3) WDR12 gene high expression in U251, U373, U87, A172 cell strain.
There are causality, inventions between generation or progress in order to study WDR12 gene expression and human glioma
People has detected the expression of the WDR12 gene in glioma cell line U251, U373, U87 and A172.
As shown in figure 3, aobvious by the PCR result of the mRNA expression of the WDR12 in U251, U373, U87, A172 cell strain
Show, the high expression in glioma cell line of WDR12 gene.
As shown in figure 4, by protein electrophoresis, WDR12 gene is height in U251, U373, U87 and A172 as the result is shown
Expression.
In the preferred embodiment of the present invention, the extracting method of mRNA are as follows:
A, the culture medium in culture plate is sucked, is washed 1 time with pre-cooling PBS；
B, the Trizol (TakaRa) of 4 DEG C of pre-coolings, the every 200 μ L of hole of 6 orifice plates is added, ice chest stands 5min；
C, each hole is blown and beaten with pipettor, collects the sample in every hole and is transferred in EP pipe, the static 5min of ice chest；D, 1 is pressed:
Chloroform is added in 5 ratio, is mixed by inversion, and stands 4 DEG C of centrifugation 15min of 12000g after 3min；
E, transfer supernatant is added isometric isopropanol precipitating RNA, is stored at room temperature in the EP pipe of another no RNA enzyme
4 DEG C of centrifugation 10min of 12000g after 10min；
F, isopropanol is carefully discarded, 4 DEG C of centrifugation 10min of 600 μ L 75% ethanol washing RNA, 10000g are added；
G, ethyl alcohol is sucked, room temperature is dried, and it is mixed that pyrocarbonic acid diethyl ester (diethylpyrocarbonate, DEPC) liquid is added
It is even, detect RNA concentration.
In the preferred embodiment of the present invention, reverse transcriptase polymerase chain reaction (reverse
Transcriptasepolymerase chain reaction, RT-PCR) method are as follows:
RT reaction system and condition
In the preferred embodiment of the present invention, real-time quantitative PCR (real-time quantitative PCR)
Method be
A. kit specification is pressed, following component is prepared:
B. PCR reaction is carried out by the following conditions, totally 40 circulation, collects fluorescence signal and analyzes result.
95℃ 15s
50℃ 15s
72℃ 20s
In the preferred embodiment of the present invention, the side of the extraction of U251, U373, U87, A172 cell strain total protein
Method are as follows: cleaned cell one time with the PBS of pre-cooling, 50 μ l lysates and 1 μ l PMSF (phenylmethylsulfonyl fluoride) is added.It stands on ice
20min is centrifuged under the conditions of 4 DEG C: 12000g is centrifuged 10min.
In the preferred embodiment of the present invention, the method for bicinchoninic acid (BCA) reagent method measurement protein concentration
Are as follows: according to sample size, add 1 volume BCA reagent B liquid to prepare BCA working solution by the BCA reagent A liquid of 50 volumes, after mixing well
Avoid light place, with 96 orifice plates, 200 μ l are added in every hole, and standard items are added in 96 orifice plates according to 0,1,2,4,8,12,16,20 μ l,
Then it is supplied with the PBS of pre-cooling to 20 μ l.3 multiple holes are arranged in each concentration.2 μ l volume samples are added, are supplied with pre-cooling PBS
3 multiple holes are arranged in 20 μ l.37 degree of placement 30min.Measure A576.Standard curve is drawn according to absorbance value, calculates the egg of sample
White concentration.
In the preferred embodiment of the present invention, the method for Westerning Blot detection are as follows:
A, BCA method measures sample protein concentration, and sample applied sample amount (20 μ g/10 μ l) is consistent；
B, separation gel and concentration glue are prepared；
C, electrophoresis: 10 μ l samples are added in each well, and 80V electrophoresis 30min is electric after sample enters separation gel
Pressure is adjusted to 120V electrophoresis 60min, until bromophenol blue enters separation gel bottom end；
D, transferring film: using wet turn of method, first shift to an earlier date 3min activation pvdf membrane with methanol, then successively from cathode to anode
It is placed for the sequence of sponge, filter paper, gel, PVDF, filter paper, sponge, in the transferring film buffer constant current 300mA low temperature electric of pre-cooling
Turn 90min；Whole process is all in ice water.
E, after the completion of transferring film, film is placed in the culture dish with 5% (w/v) skimmed milk power of TBST configuration, at room temperature
90min is closed, is washed 3 times with TBST later, each 5min；
F, primary antibody is incubated for: with primary antibody diluted primary antibody, then 4 DEG C of overnight incubations are being shaken with appropriate TBST buffer
It is washed 3 times on bed, each 10min；
G, secondary antibody is incubated for: being diluted secondary antibody with TBST, is incubated for 1h at room temperature, is then washed on shaking table with appropriate TBST buffer
3 times, each 10min；
H, it chemiluminescence and first uses: by fresh BeyoECLplus work drop on film, it is ensured that working solution cover film
On, fluorescence signal is acquired with ALLIANCE4.7 instrument.
2 WDR12 clpp gene of embodiment subtracts the proliferation for affecting glioma cell in vitro
(1) building of shWDR12 gene
Building shWDR12 gene simultaneously obtains total protein and RNA.Using Real-time PCR (qPCR) and western trace
To assess the shWDR12 gene expression of mRNA and protein level.
As shown in figure 5, the horizontal of WDR12 albumen significantly reduces in the cell of WDR12-shRNA, this display slow virus
ShRNA strategy effectively inhibits WDR12 gene in the expression of protein and mRNA level in-site.
In the preferred embodiment of the present invention, the shRNA of the lentivirus mediated knocks out the building of WDR12 gene
Step are as follows:
1) it connects: 20 μ l reaction systems is prepared according to Fermentas T4 DNA Ligase specification, by double-stranded DNA
Oligo is connected with the carrier of linearisation.In 16 DEG C of reaction 1h-3h, connection product is named as psc45764, and it is real to carry out conversion later
It tests.
2) it converts: connection product being converted into competent escherichia coli cell, Detailed operating procedures are as follows:
10 μ l connection product psc45764 are added in 100 μ l competent escherichia coli cells, ice bath 30min.42℃
Heat shock 90sec, ice bath 2min.The LB liquid medium of 500 μ L antibiotic-frees is added, 200rpm is in 37 DEG C of shaking table shake cultures
1hr.It takes 150 μ l bacterium solutions to be uniformly applied on the LB solid medium containing Amp, is incubated overnight in 37 DEG C of incubators.
3) the PCR identification of positive colony
A, primer: being directed to the most effective target sequence of WDR12-shRNA: 5'-CCTATTTCCCATGATTCCTTCATA-3',
The sequence of Scr-shRNA is 5'-GTAATACGGTTATCCACGCG-3'
B, PCR amplification: according to the form below prepares 20 μ l PCR reaction systems, is template with sterile pipette tips picking single bacterium colony, into
Row PCR amplification, reaction condition are as follows: 94 DEG C of 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 22 circulations；72℃5min.PCR knot
Shu Hou takes 5 μ l products, 1% agarose gel electrophoresis test strip.Then, to positive colony sequencing interpretation of result.
C, plasmid extraction: will be sequenced correct bacterium solution switching in the LB liquid medium of 150ml antibiotic containing Amp, and 37
DEG C shaking table shake culture is stayed overnight.Plasmid, the plasmid of quality inspection qualification are extracted according to EndoFree Maxi Plasmid Kit specification
Into down stream train.
(2) shWDR12 influences the increment of glioma cell in vitro
Lasting proliferation signal is the important symbol of cancer；The shadow subtracted to glioma is struck in order to study WDR12
It rings, we assess cell Proliferation with two different detection methods to reduce subjective factor.
First, as shown in fig. 6, inventor uses continuous five days monitoring cell Proliferations of CellomicsArrayScan VTI.
These the results show that with Scr-shRNA processing cell compared with, with WDR12-shRNA handle cell 48 hours after cell Proliferation
Start to significantly reduce.In addition, WDR12-shRNA is on the restricted influence of proliferation, process is dramatically increased at any time, knocks out WDR12 glue
Matter oncocyte, cell Proliferation reduce.
Second, as shown in fig. 7, inventor detects cell proliferative conditions by mtt assay, find the 3rd, 4 after cell definite value,
5 days, WDR12 struck the growth rate for subtracting cell significantly lower than Scr-shRNA cell, and knocking out WDR12 glioma cell activity reduces.
In the preferred embodiment of the present invention, the method for the MTT detection are as follows:
A, by after each experimental group cell tryptase enzymic digestion in logarithmic growth phase, complete medium is resuspended into cell suspension,
And it counts.
B, speed being grown according to cell and determines bed board cell density (majority is 2000cell/well), every group of 3-5 is repeated,
Bed board quantity (such as detection 5 days, then spreading 5 96 orifice plates) is determined according to experimental design.
C, after uniformly completing, after cell precipitates completely, the cell density of each experimental group is observed under the microscope, such as
Fruit Density inhomogeneity then fixes one group, and finely tuning the amount of other group of cell, (such as: discovery Con group cell is more, reduction for bed board again
Cell concentration bed board again), it is put into cell incubator and cultivates.
D, since after bed board second day, the MTT Yu Kongzhong of 20 μ L 5mg/mL is added in 4h before culture terminates, without changing liquid.
E.4h culture solution is sucked after completely, is careful not to sop up the first a ceremonial jade-ladle, used in libation particle of orifice plate bottom, 100 μ LDMSO is added to dissolve first
A ceremonial jade-ladle, used in libation particle.
F. oscillator vibrates 2-5min, and microplate reader 490/570nm detects OD value.
G. data statistic analysis.
In the preferred embodiment of the present invention, the cell number destination party at each time point is recorded using Celigo detection
Method are as follows:
A, by after each experimental group cell tryptase enzymic digestion in logarithmic growth phase, complete medium is resuspended into cell suspension,
It counts；
B, speed is grown according to cell and determines that (most cells bed board number is set as 2000cell/ to bed board cell density
well).Every group of 3 multiple holes, cultivating system are 100 holes μ L/, need to ensure that every hole addition cell number is consistent during bed board, 37 DEG C,
5%CO2 incubator culture；
C, since after bed board second day, daily Celigo detection read plate is primary, continuous detection read plate 3-5 days；
D, by adjusting the input parameter of analysis settings, the band in scanning orifice plate every time is accurately calculated
The quantity of the cell of green fluorescence；Statistics drawing is carried out to data, draws 5 days cell Proliferation curves.
3 shWDR12 of embodiment has the function of promoting Apoptosis in vitro
(1) Annexin V flow cytometer detection glioma cell apoptosis
As shown in figure 8, inventor assesses the glioma of WDR12-shRNA processing using flow cytometer and AnnexinV
The apoptosis of cell.Compared with Scr-shRNA group, the significant increase of percentage of WDR12-shRNA group apoptosis glioma cell, these
The result shows that WDR12 has anti-apoptotic effect in glioma cell, knocks out WDR12 glioma cell line and promote and wither
It dies.
In the preferred embodiment of the present invention, the method for the Annexin V flow cytometer detection apoptosis are as follows:
A, drug-induced apoptosis when coverage rate is about 70% is grown to when each experimental group 6 orifice plates cell
B, if attached cell, then cell also needs to collect in supernatant.Pancreatin digestion, complete medium are resuspended outstanding at cell
Liquid is collected in same 5mL centrifuge tube with supernatant cell, and every group sets three multiple holes (, cell number enough as the upper machine cell number of guarantee
Mesh >=5 × 105/ processing).
C, 1300rmp is centrifuged 5min, abandons supernatant, and the D-Hanks (pH=7.2~7.4) of 4 DEG C of pre-coolings washs cell precipitation.
D, 1 × binding buffer washing cell precipitation is primary, and cell is collected in 1300rmp, 3min centrifugation.
E, cell precipitation is resuspended in 200 μ 1 × binding of L buffer.
F, 10 μ L Annexin V-APC dyeing is added, room temperature is protected from light 10-15min.
G, according to cell concentration, 400-800 μ 1 × binding of L buffer, upper machine testing are added.
(2) Caspase3/7 is detected
As shown in figure 9, Caspase3/7 is played an important role in apoptosis process.It internal or external withers some
It dies under signal function, Caspase3/7 activity is activated, such as the activity by influencing endonuclease, it is made to shear nucleosome
Between DNA, promote Apoptosis.Therefore inventor can detecte Apoptosis situation by detection Caspase3/7 activity.With
Scr-shRNA group is compared, and WDR12-shRNA group Caspase3/7 activity increases, these are the result shows that in glioma cell
WDR12 has anti-apoptotic effect, knocks out the expression that WDR12 promotes Caspase3, enhances its apoptotic effect.
In the preferred embodiment of the present invention, the method for the Caspase3/7 detection are as follows:
A, using CO2 incubator culture cell 3-5 days (according to the specific speed of growth of cell) at the inoculation of 96 orifice plates, 37 DEG C.
B, Caspase-Glo3/7 buffer and Caspase-Glo3/7 freeze-dried powder 18-22 DEG C of (room temperature) environment is placed in wait for
Equalized temperature.Then 10mlCaspase-Glo3/7 buffer is added in the brown bottle equipped with Caspase-Glo3/7 substrate, whirlpool
Rotation or repeatedly overturn until substrate be completely dissolved, formed Caspase-Glo reaction solution.
C, after cell count, concentration of cell suspension is adjusted at room temperature to 1 × 104 cells/well, and aim cell is not negative
Property control cell be added according to every 100 μ l of hole in 96 new orifice plates, while one group of celliferous empty map group is set and (training is only added
Support 100 hole μ l/ of base).
D, 100 μ lCaspase-Glo reaction solutions are added in every hole, replacement pipette tips are paid attention in sample-adding, strictly avoid intersecting dirty
Dye.It was mixed being placed on rocker machine added with the culture plate of cell with the revolving speed jog of 300-500rpm 30 minutes.Then according to thin
18-22 DEG C of born of the same parents' situation incubation at room temperature 0.5-3 hours (being advisable with 1-2 hours).
E, it is analyzed using Instrument measuring signal strength data.
4 WDR12 of embodiment inhibits the proliferation function of cell by cell proliferation signals access
(1) RNA-seq gene variation volcano figure
As shown in Figure 10, obtaining WDR12 by upper reconciliation downward variation gene number progress volcano map analysis may cause
Other 683 other genes change.
(2) gene that may be interacted with WDR12
As shown in figure 11, Figure 11 is the genescreen figure that possible interact with WDR12, passes through these bases in RNA-seq
The expression quantity of cause is than more significant.
(3) KEGG signal path is analyzed
By the analysis of signal path KEGG caused by WDR12 low expression it is found that WDR12 may take part in proliferation to regulate and control glue
Matter oncocyte plays a role.
(4) PLK, EZH2, CDK1, FOX1 and UBE2C low expression in shWDR12
As shown in figure 12, the PCR knot of the mRNA expression by PLK, EZH2, CDK1, FOX1 and UBE2C in shWDR12
Fruit shows, PLK, EZH2, CDK1, FOX1 and UBE2C gene low expression in shWDR12.WDR12 downward can cause PLK,
EZH2, CDK1, FOX1 and UBE2C mRNA are lowered.
(5) PLK and EZH2 albumen low expression in shWDR12
As shown in figure 13, by protein electrophoresis as the result is shown PLK and EZH2 gene expression albumen in shWDR12
For low expression, WDR12 downward can cause the protein expression of PLK1 and EZH2 to reduce.
5 WDR12 of embodiment adjusts the proliferation of glioma by interacting with EZH2
(1) by immunocyte fluorescent staining, show that WDR12 and EZH2 interacts
As shown in figure 14, the immunocyte fluorescent staining figure for WDR12 and the EZH2 interaction that Figure 14 is, WDR12 may
It adjusts performance jointly with EZH2 and inhibits glioma cell effect.
In the preferred embodiment of the present invention, prove that WDR12 may be with EZH2 by immunocyte fluorescent staining
The method of interaction are as follows:
A, culture plate is taken out out of incubator, is rinsed cell 2 times with suitable 1xPBS is pre-chilled；
B, 300 μ l, 4% paraformaldehyde, fixed 30min is added in every hole；
C, paraformaldehyde is abandoned, appropriate 1xPBST is added, is cleaned 3 times, each 5min；
D, 1xPBST is sucked, 200 μ l super BB are added in every hole, close 1h；
E, confining liquid is sucked, 1xPBST is added, is cleaned 3 times, each 5min；
F, it is added and uses the diluted primary antibody of super BB, 16h is incubated in Cool Room 4 DEG C；
G, primary antibody is sucked, 1xPBST is added, is cleaned 3 times, each 5min, secondary antibody and DAPI is added, is incubated at room temperature 90min；
H, secondary antibody is sucked, 1xPBST is added, is cleaned 3 times, each 10min；
I, mountant mounting is taken pictures under fluorescence microscope.
(2) as shown in figure 15, by Western bloting detection show the albumen of WDR12 gene expression by with
The interaction of EZH2 albumen adjusts the P21 factor of cell cycle middle and upper reaches and the H3K27me factor in downstream, and then plays tune
Save the effect of tumor proliferation.
In conclusion the present invention is proved by basic research, the expression of WDR12 gene and the classification of glioma are positively correlated,
The knockout of WDR12 gene all has significant impact to the increment of glioma cell, apoptosis and cell cycle, it follows that
WDR12 gene can be used as a specific gene target spot of brain glioblastoma cell inhibition and apoptosis, and then be glioma
Target therapeutic agent exploitation provides theoretical basis.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of application of WDR12 gene in the inhibition and apoptosis of brain glioblastoma cell.
2. application according to claim 1, which is characterized in that the WDR12 gene as brain glioblastoma cell inhibit and
The specific gene target spot of apoptosis.
3. application according to claim 1, which is characterized in that the genebank Serial No. NC_ of the WDR12 gene
000002.12:c202912226-202878919Homo sapiens chromosome 2,GRCh38.p12Primary
Assembly。
4. application according to claim 1, which is characterized in that the WDR12 gene source is in mammal；
Preferably, the WDR12 gene source is in people.
5. application according to claim 1, which is characterized in that the brain glioblastoma cell is that in vitro culture glioma is thin
Born of the same parents；
Preferably, the brain glioblastoma cell strain is U251, U373, U87 or A172.
6. application according to claim 1, which is characterized in that the WDR12 gene high expression in brain glioblastoma cell.
7. application according to claim 6, which is characterized in that the shRNA of lentivirus mediated knocks out WDR12 gene in vitro
It significantly inhibits glioma and promotes Apoptosis.
8. application according to claim 7, which is characterized in that the shRNA of the lentivirus mediated knocks out WDR12 gene
Construction step are as follows: design WDR12-shRNA sequence, and the slow virus of expression WDR12-shRNA is constructed to inhibit WDR12's
Expression, for the most effective target sequence of WDR12-shRNA: 5'-CCTATTTCCCATGATTCCTTCATA-3', Scr-shRNA's
Sequence is 5'-GTAATACGGTTATCCACGCG-3'.
9. application according to claim 1, which is characterized in that the WDR12 gene inhibits glioma and promotees
It is that glioma cell is adjusted by the cell cycle into Apoptosis.
10. application according to claim 9, which is characterized in that in the cell cycle, the albumen of WDR12 gene expression
By the interaction with EZH2 albumen, the P21 factor of cell cycle middle and upper reaches and the H3K27me factor in downstream are adjusted, in turn
Adjust tumor proliferation.
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