CN108441488A - A kind of deubiquitinating enzymes for stablizing BMI1 in glioma - Google Patents
A kind of deubiquitinating enzymes for stablizing BMI1 in glioma Download PDFInfo
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Abstract
The invention belongs to disease treatment field more particularly to a kind of deubiquitinating enzymes for stablizing BMI1 in glioma, and it is ubiquitin-specific protease 22 to remove ubiquitin enzyme（USP22）, strike subtract USP22 expression can by reduce BMI1 levels inhibit glioma transplantable tumor formation.
Description
Technical field
The invention belongs to disease treatment field more particularly to a kind of deubiquitinating enzymes for stablizing BMI1 in glioma.
Background technology
Glioma is most common encephalic primary tumo(u)r.For many years, although conventional therapeutic strategy include operation, radiotherapy and
Chemotherapy, but the median survival time is almost without being improved.There are sub-fraction cells to be referred to as glioma stem cells in glioma,
These cells have been widely regarded as the main reason for glioblastoma drug resistance.Therefore, targetedly targeted therapy, especially
Being intended to eliminate the targeted therapy of glioma stem cells will be of great significance to the progress for the treatment of and the improvement of prognosis.
More comb gene B cell specificity moloney murine leukemia virus integration sites 1 (BMI1) are that more combs inhibit compound
The core member of object 1 (PRC1) mainly participates in the regulation and control of cell development differentiation by epigenetic modification.Multinomial research table
It is bright, the BMI1 often high expression in tumour, and lead to tumour, such as colorectal cancer and adenocarcinoma of lung.BMI1 is one in glioma
A dryness relevant molecule.It can not only promote proliferation, migration and the invasion of differentiated glioma cell, moreover it is possible to it is dry to adjust glioma
Self-renewing, apoptosis and the immunogenicity of cell.However, the mechanism that BMI1 is over-expressed in glioma is not yet by complete
Solution.
Ubiquitin-specific protease 22 (USP22) is the member that ubiquitin enzyme (dub) is removed in mammal, it includes one
A N-terminal zinc finger domain is used for substrate interaction and a C-terminal ubiquitin-specific peptase domain.As mankind's spt-ada-gcn5- second
The component part of acyltransferase (SAGA) compound, USP22 can participate in gene transcription regulation by epigenetic modification.This
Outside, USP22 can also promote the stability of protein by cracking the ubiquitin label in substrate, to prevent proteolysis from dropping
Solution.A member in the cancer mortality label that USP22 is made of 11 genes；Therefore, it is a kind of stem cell mark that it, which is pushed off,
Remember object, is widely studied in cancer.However, in glioma, the effect of USP22 and mechanism, especially to tumour dryness
It influences, is still largely unknown.
Invention content
In view of this, present invention finds one kind removing ubiquitin enzyme, which is characterized in that described to go ubiquitin enzyme be in glioma
Middle stable BMI1.
Further, described to go ubiquitin enzyme for ubiquitin-specific protease 22 (USP22).
Further, the USP22 and BMI1 positive correlations on protein level, and without obvious relation on transcriptional level.
Further, the USP22 and BMI1 maintains glioma dryness by the activation of oncogenic signals and promotes tumour hair
It is raw.
The another aspect of this paper provides the patient that USP22 is especially over-expressed with BMI1 in treatment glioma
The application of aspect.
Further, the USP22 strike subtract it is rear caused by the diameter of tumour ball reduce.
Further, it is described strike subtract USP22 expression can by reduce BMI1 levels inhibit glioma transplantable tumor formation.
Herein, it has been found that expression of the expression of USP22 in dryness sample glioma tumor ball is than in differentiated glue
Expression in matter oncocyte will obviously increase.It strikes after subtracting USP22 expression, the decrease of glioma dryness is by lowering BMI1 albumen
Level, rather than the transcriptional level of BMI1 is realized.The clinical analysis of samples of human glioma also indicates that USP22 and BMI1 is in egg
Positive correlation on white level, and without obvious relation on transcriptional level.USP22 can be with BMI1 interactions and upon translation in level
Stablize the level of BMI1.In glioma cell and clinical sample, USP22 and BMI1 can regulate and control jointly as POSTN, HEY2,
PDGFRA, ATF3 etc. and the relevant series of genes of glioma dryness.In tumor formation in nude mice, strike subtract USP22 expression it is same
Sample can inhibit the generation of glioma by lowering BMI1 expression.These discoveries not only show that USP22 is a kind of new of BMI1
Type removes ubiquitin enzyme, but also there are a USP22-BMI1 signal shafts, maintain glioma dry by the activation of oncogenic signals
Property and promote tumour.Therefore, targeting USP22 may be the patient for treating glioma and especially being over-expressed with BMI1
A kind of promising strategy.
Description of the drawings
Fig. 1：Strike the expression decrease glioma dryness for subtracting USP22.(A) compared to the cell of serum adhere-wall culture, USP22 exists
Higher is expressed in the tumour ball of serum-free stem cell culture.(B) it is verified using Western blot in U251 and U87MG cell lines
The validity of shRNA.(C, F) USP22 strikes the representative configuration figure for subtracting group and negative control group tumour ball.(D, G) USP22, which strikes, to be subtracted
The average diameter of the tumour ball of group is significantly smaller than and negative control group.(E, H) detects USP22 to colloid by limiting dilution assay
The influence of tumor stem cells self-renewal.After USP22 strikes and subtracts, in U251 and U87MG cells, at least one tumour is needed to form
Cell number needed for ball obviously increases.In U251 cells from 51.3 to 119.5, P=0.038 and in U87MG cells from
50.9 to 146.6, P=0.01.Student t- are examined.* p ＜ 0.05, * * ＜ 0.01, * * * ＜ 0.001.Engineer's scale=25 are micro-
Rice.
Fig. 2：It is by maintaining the expression of BMI1 to realize that USP22, which can maintain glioma dryness part,.(A, B) is two
USP22 strikes the protein level for subtracting and having lowered BMI1 in kind cell line.(C, D) strikes in two kinds of cells subtracts USP22 hardly shadows
Ring the mRNA level in-site of BMI1.(E) after demonstrating transduction pTAT-HA-BMI1 membrane-spanning proteins with Western blot in U251 cells
Expression (f) with pTAT-HA-BMI1 membrane-spanning proteins can restore because BMI1 strike subtract it is rear caused by the diameter of tumour ball reduce.Ratio
Tumour ball caused by ruler=50 micron (G) can largely be restored with pTAT-HA-BMI1 membrane-spanning proteins after USP22 strikes and subtracts
Diameter reduce.Engineer's scale=50 micron, student t are examined.***p<0.001.
Fig. 3：USP22 can deubiquitination stabilization BMI1 albumen.(A) GFP-BMI1 and Flag-USP22 wild plasmids or
Flag-USP22C185S enzymatic activitys are mutated deletion plasmid cotransfection HEK293FT cells together, with Flag tag antibodies by exempting from
Epidemic disease elutriation goes the interaction between detection USP22 and BMI1 genes.(B) HA-ub plasmids, GFP-BMI1 plasmids and
Cotransfection HEK293FT is thin together for Flag-USP22 wild plasmids or Flag-USP22C185S enzymatic activitys mutation deletion plasmid
Born of the same parents.Expression is immunoprecipitated with GFP tag antibodies detection BMI1, the poly ubiquitination level of BMI1 is detected with HA tag antibodies.
(C) GFP-BMI1 plasmids and Flag-USP22 wild plasmids or Flag-USP22C185S enzymatic activitys are mutated deletion plasmid together
Cotransfection HEK293FT cells.Effect with western blot analysis USP22 to BMI1 stability.In USP22 enzymatic activity mutation groups,
GFP-BMI1 protein levels significantly reduce；However, the BMI1 levels reduced can be restored by proteasome inhibitor MG132.
(D) GFP-BMI1 plasmids and Flag-USP22 wild plasmids or Flag-USP22C185S enzymatic activitys are mutated deletion plasmid together
Cotransfection HEK293FT cells.After protein synthesis inhibitor cycloheximide CHX processing, in different time sections difference Flag and
GFP tag antibodies remove the protein level of detection USP22 and BMI1.As a result show and transfected enzymatic activity mutation deletant plasmid group
It compares, after having transfected wild type USP22 plasmids, the half-life period of exogenous BMI1 significantly extends (E, F) and transfected in U251 cells
Negative control shRNA and USP22shRNA.After blocking the synthesis of endogenous egg with CHX, with Western blot detection endogenous BMI1
Protein expression level.Each time point BMI1 expressions are measured.Later, line chart is depicted.
Fig. 4：USP22 and BMI1 only has correlation on protein level.(A) as shown by arrows, USP22 and BMI1 is not only total
It is positioned at the nucleus of U251 with U87MG cells and expression and distribution in also similar core.(B) pass through Human protein
The analysis of the USP22 and BMI1 transcriptional levels of 153 patients with gliomas in Atlas databases finds USP22 and BMI1 in base
Because of non-correlation on transcriptional level.P ＞ 0.05.(C) typical Western groups of the USP22 and BMI1 in different patients with gliomas is shown
It is intended to.Engineer's scale=100 micron (D) are in 30 glioma clinical samples, and USP22 and BMI1 is on protein level at positive
It closes.Pearson correlation coefficients r=0.7438, p ＜ 0.0001.
Fig. 5：USP22 and BMI1 genes can participate in adjusting that many and glioma is pernicious and the relevant downstream base of dryness jointly
Cause.(A, B) Vean diagram, which is shown in USP22 and strikes to subtract to organize to strike with BMI1, to be subtracted in group, and 506 at least occur 2 times or more expressions and change
The difference up-regulation gene of change or the intersection situation of difference down-regulated gene, wherein that lowers jointly has 320, that raises jointly has
186.(C) the KEGG accesses enrichment analysis for the differential gene that 506 are changed jointly.(D, F) has chosen in 506 genes
9 carry out qRT-PCR verifications (E) from (D) and (F) with glioma is pernicious or dryness is closely related target gene to chip results
In again pick 4 verifications that immunoblot experiment is further carried out with the relevant target gene of glioma dryness.(G) it is marked in clinic
The expression of USP22, BMI1 and above-mentioned 4 dryness associated downstream target gene are detected in this tissue with Western blot.
Con：It is right, GBM：Glioblastoma.As a result it shows compared with normal cerebral tissue, USP22, BMI1 and 4 target genes are in colloid
It expression pattern in blastoma tissue and is consistent in U251 cell lines.
Fig. 6：Strike subtract USP22 expression can by reduce BMI1 levels inhibit glioma transplantable tumor formation.(A) nude mice skin
Lower injection USP22 stablizes the U87MG cells for striking the U87MG cells subtracted or transfecting negative control shRNA and carrys out transplanting in construct
Tumor model.(B) schematic diagram after transplantable tumor excision.(C) since after transplanting the 8th day, USP22 strikes the transplantable tumor volume for subtracting and organizing and opens
Beginning and control group have the reduction of significant difference.After transplanting 28 days, gross tumor volume strikes in USP22 subtracts group and control group
Size is respectively 183.50 ± 21.12 cubic millimeters and 674.73 ± 86.62 cubic millimeters (* * * P ＜ 0.001).(D) the 28th
Two groups of tumor weights are carried out statistical comparisons by it, it is found that USP22 strikes and subtracted group weight and significantly reduce.(E) two groups of transplanting oncocytes
Representative haematoxylin eosin stains schematic diagram, engineer's scale=25 micron.(F) two groups of transplantable tumor groups are detected respectively with Western blot
Endogenous BMI1 expressions in knitting.GAPDH is as internal reference.(G) BMI1 expression is struck in USP22 subtracts group and is substantially reduced, poor
It is different that there is statistical significance, * P ＜ 0.05.(H) the mechanism pattern figure of a kind of USP22-BMI1 signal shafts proposed, i.e. USP22 are logical
It crosses deubiquitination and stablizes BMI1 to promote the oncogenic activation of downstream gene to promote glioma dryness and progress.
Fig. 7：USP22 is overexpressed and related with glioma prognosis mala in glioma.(A) USP22 is in normal brain activity group
Knit the representative immunohistochemistry schematic diagram in the clinical samples of human glioma with different stage.Amplification factor, 200 ×.(B)USP22
The quantization (C) of Immunohistochemical Expression level in the clinical samples of human glioma of normal cerebral tissue and different stage is at normal group
It knits, compare the expression of USP22 in Low grade glioma and High Grade Gliomas.(D) it detects and compares using Western blot
Compared with the USP22 endogenous expression levels in normal astroglia system HA and various standardization glioma cell lines.GAPDH
For internal reference.(E) according to Immunohistochemical Expression as a result, patient is divided into two groups of USP22 high expression and low expression.With Kapp orchid-
The survival analysis that meyer's method has carried out between two groups compares, and as a result shows that USP22 high expression (H) group wants Overall survival to show
It writes and is shorter than USP22 low expressions (L) group, p=0.0069.
Fig. 8：USP22, which strikes, subtracts rear dryness decrease to inhibit the migration and invasion of glioma cell.(A, B) USP22, which strikes, to be subtracted
The transcriptional level of dryness gene Oct4 and Nanog is caused to decline in glioma U251 cells afterwards, along with differentiation marker
The rising of GFAP and TuJ1 transcriptional levels.(C) disease of U251 and U87MG cell infections negative control shRNA and USP22shRNA
Poison.After 72 hours, wound healing assay is carried out to study influences of the USP22 to cell migration.Four are randomly selected under microscope
The visual field, it is for statistical analysis to the mobility of each condition and time point.Statistical result is shown in (D), as a result shows that USP22 strikes and subtracts
The mobility of U251 and U87MG cells reduces (p ＜ 0.05) afterwards.(E, F) U251 and U87MG cell infection negative controls shRNA
With the virus of USP22shRNA.After 72 hours, Transwell Matrigels are carried out to study influences of the USP22 to cell invasion.
The cell check figure randomly selected under microscope in 10 visuals field is for statistical analysis, the results show that USP22 strike subtract rear U251 and
The invasion rate of U87MG cells reduces (p ＜ 0.05).
Specific implementation mode
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated,
But present disclosure is not limited solely to following embodiment.
Applications of the embodiment 1USP22 in terms for the treatment of glioma
1. clinical sample
30 clinical glioma samples are the patients year to perform the operation in Xiang Ya hospitals neurosurgery from 2013 to 2015
Middle acquisition.5 normal cerebral tissues are to cut off in decompression operation to obtain from the cranium brain cerebral lobe part of craniocerebral trauma brain edema patient
's.The acquisition of all samples and degree of correlation are obtained for the agreement of patient, and obtain batch of Xiang Ya Hospital Ethical Committees
It is accurate.
2. cell culture
The established glioblastoma cell line comprising LN229, U251, U87MG, A172 and HA are normal star-like thin
Born of the same parents are to be purchased from Cell Bank of Chinese Academy of Sciences.The authenticity of these tumor cell lines is verified by Short tandem repeatSTR analysis
's.All cell lines cultivate (Gibco, USA) in DMEM culture mediums, are aided with 10% (v/v) fetal calf serum (Gibco), 100u/
Ml penicillin (Gibco), 100U/ml streptomysins (Gibco) are containing 5% CO2Incubator in cultivate.Glioma is dry thin
The culture solution of born of the same parents includes serum-free DMEM/F12 (Gibco) culture medium, the basic fibroblast growth factor U.S. of 20ng/ml
), Peprotech the epidermal growth factor (sigma, the U.S.) of 20ng/ml, and 20 μ g/ml B27 confactors (Life
Technologies)。
3. virus packaging and slow-virus transfection.
Oligonucleotide is annealed, and be cloned into carrier LV3 (pGLVH1/GFP+Puro) (Shanghai GenePharma, in
State), to generate the specific shRNA of expression plasmid.Slow-virus transfection process has been described in research before.
The external structure of 4.pTAT-HA-BMI1 and transduction.
The structure and transductive process of cell-penetrating peptides have been described in research before.In a typical process
In, the DNA sequence dna for encoding BMI1 is expanded from the mankind BMI1 expression structures (Addgene, USA, BC011652) of an overall length
Increase.Later, DNA sequence dna is inserted on pTAT-HA carriers.The size and purity of protein are by immunoblotting assay, weight afterwards
Histone is added in neurosphere culture solutions with 0.2 μM of concentration and hatches 2h.It is analyzed with Western blot methods
TAT fusion proteins wear membrane efficiency.
5.Tumorsphere forms analysis
During one is typical, every group 1 × 104Free cell contain serum-free medium 60 millimeters are low to stick training
It supports and is cultivated in ware；Then, the formation of tumour ball is observed.In 10-14 days, 30 visuals field are randomly selected under the microscope, and right
The diameter of tumour ball is for statistical analysis.
6. limiting dilution is tested
Monolayer adherence cell or tumour ball are broken down into individual cells and with the serum-free medium of 0.2ml in 96 orifice plates
On cultivated.Last cell dilution range is from 120 cells/wells to 1 cells/well.At first 7 days, just newly add within every 2 days
The serum-free medium of 0.025ml.Then, calculate under different dilutions not at the percentage of ball.Draw the tropic, meter
X-axis values of intercept is calculated, this represent the cell quantities formed in each hole needed at least one tumour ball.
7. the migration and invasion of tumour.
The step of tumor migration and intrusion, shows in Doc.S1.
8. quantitative RT PCR analysis
The process of quantitative RT PCR analysis has been described in research before.Select HPRT1 as experiment
Internal reference.PCR primer sequence is listed in table S2.
9. immunoblotting
The process of western blot analyses has been described in research before.The antibody used is USP22
(ab4812), POSTN (ab172615), ATF3 (ab200655) are bought from Abcam；GFP (M20004) from Abmart,
Flag (M20008) and HA (M20003)；PDGFRA(sc-21789),α-Tubulin(sc-53646),β-Actin(sc-
47778), and GAPDH is from Santa Cruz (sc-47724)；BMI1 (66161-1-Ig) and HEY2 (10597-1-AP) come from
Proteintech。
10. immune precipitation
After exogenous plasmid transfection HEK293FT cells 48h, cell is collected and the karyorhexis for being dissolved in a freezing delays
In fliud flushing (the Nonidet P40 of the Tris-HCl (pH 8.0), the NaCl of 150mmol/l, 1% of 50mmol/l), and add 2%
(v/v) CPI and NaVO3.Lysate is collected, centrifuged 15 minutes with the speed of 14000g in 4 DEG C of environment and collects supernatant.
Antibody coupling albumin A+G agaroses magnetic bead (Sigma) is added in the sample；Later, sample is fully rotatable mixed in 4 DEG C of refrigerators
Even hatching 7-9h.With 1 milliliter of ice solution wash buffer 4 times, sample buffer is boiled 2 minutes, release immunoprecipitates albumen.
11. the stability analysis of protein
After every group by sowing equal cell culture 24, sample handle with the cyclohexylamine (CHX) of 10mg/ml corresponding
Time, and protein stability is analyzed.In order to analyze contribution of the proteasome pathway in BMI1 degradations, Wo Men
GFP-BMI1 is transfected in HEK293FT cells and is screened with puromycin, cell culture 72h, and MG132 is used before extracting takes albumen
(sigma) 6h is handled.
12. immunohistochemical staining (including IHC)
Tumor tissues are fixed with 10% formaldehyde, then carry out paraffin embedding and slice.Concrete operation step is before
It is described in research.Positive tumor cell percentage is 0 (0%-25%), 1 (26%-50%), 2 (51%-75%), 3
(76%-100%).Staining power is divided into:0 (feminine gender), 1 (weak), 2 (moderates) or 3 (strong), last dyeing synthesis are commented
Divide and staining power is multiplied by using positive cell ratio integral to calculate.That is, (-)=0, (+)=1-3, (++)=4-6,
(+++)=7-9.
13. immunofluorescence, haematoxylin and eosin stains.
The step of immunofluorescence has been described in research before.The main antibody used be USP22 (ab4812,
) and BMI1 (66161-1-Ig, Proteintech) Abcam.Laser Scanning Confocal Microscope (Japanese Olympic this) shooting figure is used in combination
Piece.For haematoxylin eosin stains, tumor sample is made to 4 μm of paraffin section, is then dyed, light microscope is used in combination
(German Lycra) shoots picture.
14. ubiquitination is analyzed in body
By GFP-BMI1, cotransfection arrives pCDNA3.1+-HA-ub WT and usp22WT/C185S plasmid together
In HEK293FT cells.These cells are after transfecting 42h, then carry out the processing of 10 μM of MG132 (sigma) of 6h；Later, carefully
Born of the same parents are dissolved.Extract proteins sample is used in combination the agar magnetic bead (Biotool, Switzerland) with anti-HA to carry out immunoprecipitation analysis.
15. zooscopy
By negative control and the stable U87MG cells for subtracting USP22 expression that strike with 2.0 × 106The concentration of/200 μ l is planted respectively
It plants at the both sides flank of the SCID nude mices of 54 week old.After 4 weeks, mouse is condemned to death, and tumour is collected.Gross tumor volume (V) is certainly
It is fixed that by measuring longest diameter (a) and shortest diameter (b) according to formula V (mm3)=(b) 2, ×/2 calculates.All mouse experiments
All it is to be carried out according to the institutional guidelines of government.All experiments on mice are obtained for Ethics Committee of Shanghai Communications University
Approval.
16. microarray analysis
RNA is extracted by TRIzol reagents (Invitrogen), and is hybridized to the Agilent people of Shanghai Ou Yi companies
(8*60K designs ID on genoid expression analysis platform:039494).Using Genespring GX softwares (Agilent
Technologies) for statistical analysis and data normalization.The gene of 2 times of variations is considered as being regulated and controled by USP22 and BMI1
's.Use 6.7 (http of DAVID version://david.abcc.ncifcrf.gov/) by gene shine to KEGG accesses
On.The microarray data reported herein has been filed in gene expression Omnibus (number GSE46059).
17. statistical analysis
All experiments are all triplicate, are at least repeated three times, unless otherwise indicated.Data are expressed as average ± mark
It is accurate poor.Compared using two groups of means of GraphPad PRISM softwares pair and carry out student t inspections, calculates the statistics between two groups
Learn meaning.In being related to the experiment that multiple groups are compared, with ANOVA variance analyses.Chi-square Test is carried out with SPSS softwares, is counted
Calculate the statistical significance of multivariate liner regress analysis.Gene expression correlation is analyzed using Pearson correlation coefficient.Statistical significance is with star
Number (*) is indicated.P value<0.05 is statistically significant for difference, i.e. * p<0.05, * * p<0.01, * * * p<0.001.
As a result
1. in glioma cell, the knockout of USP22 reduces tumour dryness.
It is a kind of cancer stem cell marker in view of USP22 is estimated to be, we have inquired into it to glioma dryness
It influences.The differentiation that we compare undifferentiated stem-like cell Tumor sphere cell sum with Western blot detections first is adherent thin
USP22 expression in born of the same parents.We have found that USP22 is expressed more rich (Figure 1A) in the dryness tumour ball of U251 and U87MG cells.
Expression of the expression in U251 and U87MG cells in view of USP22 is relatively high, we select both cells to carry out RNA
Interference experiment (figure S1D).Two shRNA are certified as effectively, we select more effective shRNA-2 to carry out follow-up function
It tests (Figure 1B).Then, the influence we have studied USP22 to tumour balling-up ability.The result shows that USP22 clpp genes subtract it is aobvious
Work reduces the diameter of the tumour ball of U251 (Fig. 1 C and D) and U87MG cells (Fig. 1 F and G), we have also carried out limiting dilution
Experiment, to assess influences of the USP22 to GSCs self-renewal capacities.We have found that after knocking out USP22, it is thin in U251 and U87MG
The cell number needed for a tumour ball is generated in born of the same parents to obviously increase (Fig. 1 e, H).Analyzed by qRT-PCR, USP22 strikes subtract after
The decrease of glioma dryness can be using partial interpretation as the Transcription inhibition and differentiation marker of dryness related gene Oct4 and Nanog
The transcriptional activation (figure S2A and B) of GFAP and Tuj1.These discoveries show have the weight that stem cell USP22 maintains glioma
It acts on and malignant tumour.
USP22 maintains the dryness of glioma to be realized by adjusting BMI1 levels.
In view of BMI1 is potential target spots of the USP22 in other cancers, it is intended to study whether BMI1 can mediate
Influences of the USP22 to glioma stem cells.First, we have studied translations of the USP22 to BMI1 in U251 and U87MG cells
Afterwards with the influence of the expression on transcriptional level.USP22 significantly reduces BMI1 protein levels (Fig. 2A and B) after knocking out, but almost not
It is horizontal (Fig. 2 C and D) to influence BMI1mRNA.Then, we have purified the TAT-BMI1 albumen that can wear film, in U251 cells
Middle progress function saves experiment (Fig. 2 E).First, we are made by detecting BMI1 and striking the tumour ball in the U251 cells subtracted and save
With, it is determined that the bioactivity (Fig. 2 F) of TAT-BMI1 albumen.Later, TAT-BMI1 albumen addition USP22 strikes to subtract U251 thin
Born of the same parents (Fig. 2 G).Compared with the control group, USP22 strikes the average tumor diameter for subtracting and organizing and is substantially reduced, but by by TAT-BMI1 albumen (figure
2G) reversed.These results indicate that BMI1 has mediated influences of the USP22 to glioma stem cells.
USP22 deubiquitinations BMI1 makes its stabilization.
Although BMI1 expression can be maintained by USP22, exact mechanism is unclear.In order to whether examine USP22
Can interact with BMI1, we in HEK293FT cells respectively by exogenous GFP-BMI1 and wild type USP22 and
Catalytic activity saltant type USP22 (C185S) cotransfection.After GFP-BMI1 is immunoprecipitated, detect relevant WT-USP22 and
CI-USP22.This result shows that between USP22 and BMI1 interaction it is unrelated with the deubiquitination enzymatic activity of USP22 (scheme
3A).It whether is influenced by the form of deubiquitination to what BMI1 was expressed to further study USP22, we are in HEK293FT
Wild type USP22 or saltant type USP22 and GFP-BMI1 and HA-Ub are co-expressed in cell.It is immune in GFP-BMI1
After precipitation, it is observed that the ubiquitination (lane 1, Fig. 3 B) of BMI1.However, almost being eliminated after transfected wild-type USP22
BMI1 ubiquitinations, but the ubiquitination (lane 2 and lane 3, Fig. 3 B) that cannot eliminate BMI1 of saltant type.In order to test
Whether USP22 can stablize BMI1 albumen by antagonism ubiquitin-proteasome pathway, we in HEK293FT cells by wild or
Saltant type USP22 and GFP-BMI1 are co-expressed, and then observe GFP-BMII1 degradations.We have found that with wild group of USP22 group phase
Than the GFP-BMI1 protein levels of mutation USP22 groups are decreased obviously (lane 2 and lane 3, Fig. 3 C)；And this decline can be with
It is reversed by proteasome inhibitor MG132 (the 4th article, Fig. 3 C).The synthesis of new albumen is blocked using protein synthesis inhibitor CHX
Afterwards, in HEK293FT cells, the GFP-BMI1 of transfection empty vectors group degrades rapidly (Fig. 3 D, upper figure).And it is mutated with transfection
Group USP22 is compared, and wild type USP22 significantly extends GFP-BMI1 half-life period (Fig. 3 D, figure below).In order to detect endogenous BMI1
Whether also by regulation and control similar USP22, after being handled with protein synthesis inhibitor, we struck in U251 cells subtracted it is endogenous
Property USP22, it is found that the stability of endogenous BMI1 also weakens (Fig. 3 e and F).These the result shows that, USP22 can be with BMI1 phases
Interaction, and BMI1 protein degradations are prevented by deubiquitination BMI1.
USP22 is only related in protein level to BMI1.
Find that USP22 can stablize BMI1 albumen in glioma cell line before, but do not influence the transcription of BMI1, then I
Also explore horizontal upper correlativity after the transcription and translation of USP22 and BMI1 again in clinical glioma sample.It is logical first
Crossing immunofluorescence dyeing, it has been found that USP22 and BMI1 is not only distributed in the nucleus of U251 and U87MG cells, but also
There is expression and distribution in similar core (Fig. 4 A).By to human protein's collection of illustrative plates (http://www.proteinatlas.org/)
In 153 patients with gliomas Gene Transcript Analysis, we are not it is again seen that USP22 has correlation with BMI1 on transcriptional level
(Fig. 4 B).On the contrary, the immunohistochemical analysis by analyzing 30 clinical samples, it is found that it is significant the two has on protein level
Correlation (Fig. 4 C and D).
USP22 and BMI1 adjusts the various genes relevant with glioma dryness and malignant phenotype jointly.
In view of the expression correlation of USP22 and BMI1, we have studied the gene expression profiles downstream in glioma
Correlation.Using differential expression microarray analysis, USP22 and BMI1 regulates and controls 506 differential genes jointly in U251 cells, this
The variation of a little at least 2.0 times of genes.In these genes, there are 320 genes to be lowered, 186 genes by up-regulation (Fig. 5 A and
B).The path analysis of 506 modulated genes shows there is significant enrichment in several core signal accesses, such as common up-regulation
Gene be enriched on MAPK and neural activity ligand receptor access more, and common down-regulated gene is in smell transduction signal access
Upper enrichment more (Fig. 5 C).We therefrom pick 9 and have been reported and glioma dryness and the relevant gene use of malignant phenotype
QRT-PCR has carried out Microarray results the verification in mRNA level in-site, it was confirmed that the accuracys of microarray results (Fig. 5 D and F,
Table S3).Then, we by Western blot in above-mentioned 9 genes and the albumen of relevant 4 genes of glioma dryness
Expression has carried out further verification, and the mRNA variations of the variation and microarray data that find protein level are consistent (Fig. 5 E).Most
Afterwards, we further have detected the expression of 4 selected target spots in clinical normal or tumor sample, find these candidate downstream genes
Change also with microarray verification result it is consistent (Fig. 5 G).In conclusion USP22 and BMI1 can adjust extensively jointly it is a series of
It is related to glioma dryness and the gene of malignant phenotype.
By reducing the expression of internal BMI1, USP22 can inhibit the oncogenic function of glioma.
After we confirm effects of the USP22 in glioma in vitro experiment, it is intended to further confirm it
Carcinogenesis in vivo experiment.Subcutaneous Xenograft nude mice tumor formation model is carried out by using U87MG cells, we have found that
Striking for USP22 subtracts the growth that can significantly inhibit tumour (Fig. 6 a and B).The size of tumour and volume are all aobvious after USP22 knockouts
Writing reduces (Fig. 6 c and D).It is shown in after USP22 strikes and subtract in addition, carrying out HE dyeing to xenograft tumor, tumor cell number is also shown
It writes and reduces (Fig. 6 e, black arrow).In order to confirm influences of the USP22 to BMI1 in vivo, transplantable tumor between we compare two groups
Middle BMI1 albumen gray scale.USP22, which strikes, to be subtracted a group BMI1 protein levels and is substantially less than control group (Fig. 6 F and G, * p<0.05).Therefore, I
Propose the working model of a USP22-BMI1 signal shaft, USP22 makes BMI1 stablize using its deubiquitination enzymatic activity, and
By adjusting various downstream gene such as POSTN, the oncogenic transcription activation of HEY2, PDGFRA and ATF3, to promote glioma dryness
Occur with tumour.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not limited by embodiment
System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, replacement, simplification should be
Equivalence replacement mode, is included within the scope of the present invention.
Claims (7)
1. one kind removing ubiquitin enzyme, which is characterized in that described to go ubiquitin enzyme that stablize BMI1 in glioma.
2. removing ubiquitin enzyme as described in claim 1, which is characterized in that described to go ubiquitin enzyme for ubiquitin-specific protease 22
（USP22）.
3. removing ubiquitin enzyme as claimed in claim 2, which is characterized in that the USP22 and BMI1 positive correlations on protein level,
And without obvious relation on transcriptional level.
4. removing ubiquitin enzyme as claimed in claim 3, which is characterized in that the activation that the USP22 and BMI1 passes through oncogenic signals
To maintain glioma dryness and promote tumour.
5. USP22 is in treatment glioma especially with the application in terms of the patient of BMI1 overexpressions.
6. application as claimed in claim 5, which is characterized in that the USP22 strike subtract it is rear caused by the diameter of tumour ball reduce.
7. application as claimed in claim 5, which is characterized in that it is described strike subtract the expression of USP22 can be horizontal by reducing BMI1
Inhibit the formation of glioma transplantable tumor.
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Citations (4)
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|---|---|---|---|---|
| CN101928747A (en) * | 2010-08-31 | 2010-12-29 | 中国人民解放军第二军医大学 | Application of E3 ubiquitin ligase CHIP in gliomatosis cerebri disease |
| WO2011031844A1 (en) * | 2009-09-09 | 2011-03-17 | Advanced Micro Devices, Inc. | Tessellation engine and applications thereof |
| CN107607727A (en) * | 2017-10-27 | 2018-01-19 | 上海交通大学医学院附属仁济医院 | Applications of the H3K23ac in diagnosis of glioma |
| CN108203732A (en) * | 2017-10-27 | 2018-06-26 | 上海交通大学医学院附属仁济医院 | Applications of the TRIM24 in diagnosis of glioma |
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2018
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|---|---|---|---|---|
| WO2011031844A1 (en) * | 2009-09-09 | 2011-03-17 | Advanced Micro Devices, Inc. | Tessellation engine and applications thereof |
| CN101928747A (en) * | 2010-08-31 | 2010-12-29 | 中国人民解放军第二军医大学 | Application of E3 ubiquitin ligase CHIP in gliomatosis cerebri disease |
| CN107607727A (en) * | 2017-10-27 | 2018-01-19 | 上海交通大学医学院附属仁济医院 | Applications of the H3K23ac in diagnosis of glioma |
| CN108203732A (en) * | 2017-10-27 | 2018-06-26 | 上海交通大学医学院附属仁济医院 | Applications of the TRIM24 in diagnosis of glioma |
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