CN105214077A - The application of USP33 in tumor - Google Patents
The application of USP33 in tumor Download PDFInfo
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Abstract
The present invention relates to the application of USP33 in tumor, relate to USP33 more specifically and detect mark and the application of USP33 in tumour medicine exploitation as tumor prognosis.Data display provided by the invention, in kinds of tumors patient, USP33 expression declines bad relevant to tumor prognosis; And USP33 albumen is by regulation and control Slit-Robo signal path and then inhibition tumor cell transfer.Therefore, USP33 can detect mark as tumor prognosis, and USP33 can also be applied to the exploitation of tumour medicine simultaneously, and the test-and-treat for tumor provides new method and scheme.
Description
Technical field
The present invention relates to medical art, be specifically related to be used as the USP33 of tumor markers and the application in preparation treatment lung-cancer medicament thereof.
Background technology
Pulmonary carcinoma is modal cancer, annual global death about 1,380,000 people, and its 5 years survival rates are about 15% simultaneously.Although there is numerous treatment meanss at present, comprise operation, radiotherapy, chemotherapy and targeted therapy even combined therapy between them, its effect is all barely satisfactory.Pulmonary carcinoma can be divided into small cell lung cancer SCLC and nonsmall-cell lung cancer NSCLC.Nonsmall-cell lung cancer, comprises squamous cell carcinoma, adenocarcinoma, does not break up large cell carcinoma, adenosquamous carcinoma and bronchial gland carcinoma; Small cell lung cancer, is made up of the minicell of uniformity, typical in Herba bromi japonici sample.NSCLC accounts for the case of about 80%.The treatment of current pulmonary carcinoma mainly to be carried down the Staging System moved based on TNM based on tumor morphology with by tumor lympha.In pulmonary carcinoma, many antioncogenes are found, and comprise TP53, P16, LKB1/STK11, NF1, RASSF1, APC, BRG1, PTEN and RB.Various tumor susceptibility gene is also found recently, but, suppress the endogenetic mechanisms of the Infiltration and metastasis of pulmonary carcinoma still to be known little about it.
Recent report display, neuron guide molecule is considered to relevant to the Infiltration and metastasis process of tumor.Slit gene is found at first in fruit bat, and its product is a secretory protein family, serves as the effect of neural guide molecule.Nearest research shows that slit gene participates in infecting of dissimilar cancer cell and shifts.Slit albumen is by realizing its function with the combination of a single pass transmembrane albumen Roundabout (Robo).One of albumen of regulation and control Slit-Robo intracellular signaling effect is ubiquitin-specific protease 33 (USP33).As one of the member of ubiquitin-specific protease family, USP33 finds that protein binding as substrate is to VHLE3 ligase at first.In Slit signal path regulation and control center line position Colaesce axon guidance, USP33 is considered to required.
In addition, slit signal is realizing in its function suppressing breast cancer cell to move, and USP33 also participates, and this shows that USP33 may play a significant role in invasive cancer and transfer.But how USP33 regulates and controls the mechanism of slit intracellular signaling and unclear in pulmonary carcinoma.
Summary of the invention
Technical problem to be solved by this invention is, provides the USP33 application in pulmonary carcinoma, particularly USP33 preparing the application in test-and-treat tumour medicine.
For solving the problem, the technical scheme that first aspect present invention provides is: the application of USP33 in tumor, and USP33 detects mark and the application of USP33 in tumour medicine exploitation as tumor prognosis.
Preferably, USP33 detects mark as tumor prognosis, and described tumor comprises pulmonary carcinoma, breast carcinoma, melanoma and acute myelocytic leukemia.
Preferably, USP33 is preparing the application in tumour medicine, and described tumor is pulmonary carcinoma.
Preferably, USP33 is as cancer target.
Preferably, USP33 is by the transfer of regulation and control slit-Robo signal path inhibition tumor cell.
Second aspect present invention provides a kind of diagnostic kit, and this test kit contains detection USP33 protein substance, and described USP33 protein sequence is SeqNo.1.
Third aspect present invention provides a kind of test kit, and this test kit contains the material detecting USP33 gene, and described USP33 gene order is SeqNo.2.
Fourth aspect present invention provides a kind of purposes of diagnostic kit, for detecting tumor and tumor prognosis USP33 expresses.
Fifth aspect present invention provides a kind of tumors pharmaceutical combination, and this pharmaceutical composition contains: the albumen of USP33 gene and/or its coding, and medically acceptable pharmaceutic adjuvant.
Preferably, described tumor is pulmonary carcinoma.
In this research, inventor proves that Slit albumen suppresses the transition process of lung carcinoma cell to need USP33 to participate in.USP33 regulates the level of Robo receptor in lung carcinoma cell.This and USP33 have obvious difference (Yuasa-Kawadaetal., 2009a, b) at the model of action of neuron and non-cancerous cells.In addition, USP33 plays the part of the role of tumor inhibitor in lung carcinoma cell, because the lower survival rate of the prognosis of the low expression of USP33 and patients with lung cancer presents very strong dependency relation.
In cancerous lung tissue, USP33 expresses and significantly declines.High USP33 expresses better relevant with prognosis.USP33 can improve Robo1 stability, and is that the migration of slit albumen inhibition tumor cell is necessary.These discoveries show, USP33 is the crucial participant that pulmonary carcinoma occurs, and is hopeful as a new lung cancer for prognosis mark.
USP33 provided by the invention can regulate the level of Robo receptor in lung carcinoma cell, improves Robo1 protein stability, and is that the migration of slit albumen inhibition tumor cell is necessary.Therefore USP33 as the important component for the treatment of tumor, particularly lung-cancer medicament, can combine other cancer therapy drug co-administration.In addition, USP33 can also detect mark as lung cancer for prognosis, and the test-and-treat for tumor, particularly pulmonary carcinoma provides new method and scheme.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described:
Fig. 1 shows the expression of mRNA in 25 groups of pulmonary carcinoma and non-lung cancer tissue samples, and Y-axis is expression, and GAPDH gene does reference, * * * P<0.001.
USP33 expression in Fig. 2 immunohistochemical staining detection of lung cancer and check sample.Wherein, a and c is the Strong positive signals detecting that USP33 expresses in the normal tissue, and b is the USP33 positive expression example in adenocarcinoma, and d is squamous cell carcinoma USP33 positive expression example.
The quantitative analysis of Fig. 3 pulmonary carcinoma sample (N=25) and contrast normal structure sample (CTRL) (N=25) USP33 protein expression situation.
The computational methods of this USP33-SABC mark are as follows: USP33 SABC score=(percentage ratio of positive tumor cell) × staining signals intensity.In pulmonary carcinoma, USP33 expressing quantity is significantly lower than non-lung cancer tissue (* *, P<0.01).
Fig. 4 finds that from the data base announced the expression of USP33 in patients with lung cancer sample declines by analysis; Wherein,
Fig. 4 A is USP33 genovariation and expression in TCGA data base pulmonary carcinoma sample.Light blue expression Homozygous deletions; Green expression somatic mutation; Pink represents up-regulated; Blue expression down-regulated expression.The calculating data that these results rely on from cancer C BIO portal website ((
http:// www.oncomine.org) .)
Fig. 4 B is USP33 amino acid mutation situation in TCGA data base pulmonary carcinoma sample.
Fig. 4 C is for utilizing Oncomine (http://www.oncomine.org). analyze the expression of patients with lung cancer USP33 gene in five groups of data bases.In five groups of data bases, pulmonary carcinoma sample is compared with non-tumor sample, and the expression of USP33 occurs obviously to decline.LUAD: adenocarcinoma of lung; LuSC: squamous cell lung carcinoma, Ctrl: contrast.
Fig. 5 is USP33 changes in gene expression situation in different tissues sample.
Fig. 6 shows in various cancers data base and shows low expression USP33 gene and mean shorter life cycle; Specific as follows
Fig. 6 A is TCGA lung cancer data storehouse AgilentG4502A_07_3 data, n=91 in n=91, USP33high in USP33low; P=0.0436,
Fig. 6 B is TCGA lung cancer data storehouse IlluminaHiSeq_RNASeqV2 data, n=106 in n=106, USP33high in USP33low; P=0.0267,
Fig. 6 C is the data in GSE3141 lung cancer data storehouse is analyzed by KM (http://kmplot.com/analysis/).N=83 in n=28, USP33high in USP33low; P=0.031,
Fig. 6 D is the data in GSE31210 lung cancer data storehouse is analyzed by KM (http://kmplot.com/analysis/).N=113 in n=113, USP33high in USP33low; P=0.034,
Fig. 6 E is n=61 in n=61, USP33high in TCGA lung cancer data storehouse USP33low; P=0.0188,
Fig. 6 F is that lung cancer data Cooley KM to analyze in USP33low n=553 in n=562, USP33high; P=0.034,
Fig. 6 G is n=50 in n=145, USP33high in TCGA melanoma data base USP33low; P=0.0453,
Fig. 6 H is TCGA acute myelocytic leukemia data base, n=82 in n=77, USP33high in USP33low; P=0.0282.
Fig. 7 A and 7B is respectively H1299 cell after transfection siRNA or siUSP33 with picture and analysis of accounts figure after contrast or the process of slit albumen, does not have significant change at the 3rd day and the 4th day matched group and slit processed group cell quantity.
Fig. 8 is that USP33 regulates slit to suppress lung carcinoma cell transfer by ubiquitination catalytic site; Wherein, Fig. 8 A is the H1299cells wound healing assay picture of transfection control siRNA or siUSP33; Fig. 8 B and Fig. 8 E represents cell migration distance.Fig. 8 C show Western test prove siUSP33 instead of contrast siRNA can suppress the expression of USP33 gene in H1299 cell.Fig. 8 D is that wild type USP33 or sudden change USP33 (C163A) are used to comfort contrast or slit contrast.After producing cut, the cell at edge will move towards right side, and figure is different time points collection after cut, and white dotted lines represents 0 hour edge of wound.Fig. 8 F is that Western verifies that USP33 wild type and mutant gene (C163A) are at H1299 cells.
Fig. 9 shows USP33 and Robo1 and interacts, and affects the stability of Robo1 in lung carcinoma cell.Wherein, the interaction of Fig. 9 A USP33 and Robo1 in H1299 cell.In contrast and slit group, utilize contrast IgG or Robo1 antibody to carry out co-immunoprecipitation experiment.The albumen that immunoprecipitation obtains utilizes USP33 antibody to carry out Westernblotting detection,
Fig. 9 B shows transfection Robo-HA, and the H1299 of Flag-ubiquitin, CtrlsiRNA or siUSP33 carries out ubiquitination experiment.After processing 6h through MG132 (20 μm of ol/L), utilize HA antibody to carry out co-immunoprecipitation experiment, afterwards Westernblotting detection carried out to obtained albumen,
Fig. 9 C shows transfection Robo-HA, and the USP33 of wild type USP33 or sudden change and the H1299 cell of untransfected carry out ubiquitination situation.After processing 6h through MG132 (20 μm of ol/L), utilize HA antibody to carry out co-immunoprecipitation experiment, afterwards Westernblotting detection carried out to obtained albumen,
Fig. 9 D and Fig. 9 E shows at transfection siRNAsUSP33#1, USP33#2, and after contrast siRNAs, Westernblot detects the expression of USP33 and Robo1 in H1299 cell,
Fig. 9 F processes different time with CHX (50 μ g/mL) after showing H1299 cell transfecting siUSP33 or contrast siRNA, and then detects the total amount in cell of Robo1.
Fig. 9 G, Fig. 9 H process 6h (what do not process contrasts) with MG132 (20 μm of ol/L) after showing 1299 cell transfecting siUSP33 or contrast siRNA, carry out Westernblot detection afterwards.
Detailed description of the invention
Below in conjunction with accompanying drawing, the specific embodiment of the invention is described.
One, material
1. laboratory animal or material source and process
Cancerous lung tissue and non-tumor lung tissue are from the sample collected after the diagnosis of The Fourth Military Medical University's Hospital Pathological Department room, and all sample standard deviations obtain patient's informed consent and in accordance with the guilding principle of associated mechanisms and country.As following table 1 shows pulmonary carcinoma sample distribution situation.
The tumor tissues sample of excision is used liquid nitrogen cryopreservation rapidly, in order to extract RNA.
Cancerous lung tissue sample fixedly spends the night through 10% neutral formalin solution, specimens paraffin embedding slices, carries out h and E dyeing according to standard scheme, carries out immunohistochemical analysis.
Table 1 pulmonary carcinoma sample distribution
H1299 cell (from ATCC cell bank), HEK293 cell (from ATCC cell bank), with the DMEM culture medium culturing of 10% (v/v) hyclone, 50mg/ml penicillin/streptomycin, temperature is 37 DEG C, and gas concentration lwevel is 5%.
2, medicine and reagent:
2.1 medicine
USP33 antibody is purchased from PROTEINTECHGROUP company, and Robo antibody is purchased from PROTEINTECHGROUP company, and beta-actin antibody is purchased from PROTEINTECHGROUP company; Cycloheximide available from Sigma; MG132 available from Sigma.Trizol is purchased from Invitrogen company
2.2 reagent
Lipofectamine2000 transfection reagent is purchased from Invitrogen company; The siRNAs of USP33 (#1:5'-UCUCGACAGUGGCUUAAUUAA-3',
#2:5'-GGAUUCAGUUGGUGAAAUUAC-3') and negative control siRNA (5 '-CGUACGCGGAAUACUUCGATT-3 ') synthesized by Shanghai JiMa pharmacy Technology Co., Ltd; SYBRgreenPCRmix purchased from ThermoFisherScientific company,
2.3 instrument
Ultrospec5300pro spectrophotometer is purchased from GE company; ScanScopeXT scanner purchased from American Aperio company, real-time fluorescence quantitative PCR instrument is purchased from ABI company.
Grouped data represents with mean plus-minus standard deviation.T inspection (two samples) or ANOVA (more than two samples) carries out statistical significance analysis.Survival Analysis, draws analytical curve and uses Prism software and log-rank to analyze.At least in triplicate, 95% confidence level of p<0.05 is meaningful in all experiments.
Two, experimental technique and interpretation of result
1, the expression of USP33 in lung carcinoma cell and non-tumor lung tissue cell.
1.1 analyze USP33mRNA expression by quantitative RT-PCR method, and compared with non-tumor lung tissue sample, USP33 expresses and significantly reduces (P<0.001) (Fig. 1).
Trizol is used to extract RNA total in cancerous lung tissue, Ultrospec5300 spectrophotometer is utilized to measure the amount of RNA and purity, from RNA sample, genomic DNA is removed with DNA digestion enzyme I, synthesize cDNA first chain with 5 microgram RNA for template oligomer primers (dT) and reverse transcriptase (M-MLVRT), utilize the StepOne real-time PCR of ABI to carry out amplification the primer as follows:
USP33(5'-TGTGATGCTTAGGCAAGGAG-3',SeqNo.3,
And 5'-GGCCCTCCACCATAAATAGA-3'), SeqNo.4;
Robo1(5'-GCATCGCTGGAAGTAGCCATACT-3',SeqNo.5
And 5'-CTAGAAATGGTGGGCTCAGGAT-3') SeqNo.6;
GAPDH(5'-GGAGCGAGATCCCTCCAAAAT-3',SeqNo.7
And 5'-GGCTGTTGTCATACTTCTCATGG-3'), SeqNo.8.Amplification program:
95 DEG C, 15 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds, totally 40 circulations, utilized 2
-Δ Δ CTmethod calculates.
1.2 next, and inventor detects USP33 protein expression situation (specificity see the anti-USP33 of Fig. 2) in sample by immunohistochemical staining method.
Fixed through 10% formalin by cancerous lung tissue sample, the tumor biopsy of paraffin-embedded 5 micron thickness carries out immunochemical analyses.First dewax, rehydration, heats 20 minutes in citric acid (pH value 6.0) microwave oven.After antigen retrieval, by 3% hydrogen peroxide treatment 10 minutes, close endogenous peroxidase activity, then hatch with lowlenthal serum 30 minutes obstructives.USP33 primary antibodie, to section 4 DEG C of overnight incubation, then hatches biotin antibody, room temperature 30 minutes.Finally, cut into slices through 3,3-diaminobenzidine dyeing observation.Painted section is through ScanscopeXT digitized.Represent USP33 protein expression height with numerical value, in each section, at least choose the average percent determining positive tumor cell after five random areas amplify × 200.USP33 intensity scores is as follows: 0, negative; 1+, weak; 2+, gentle 3+, acutely.Positive tumor cell, then the percentage ratio of staining power amplify generation USP33-immunohistochemical staining mark.
In normal lung tissue, USP33 staining signals indication range is 73.33 ~ 300.00 (meansigma methods is 198.18), and the staining signals change in those lung cancer samples is from 0.00 to 266.00 (meansigma methods is 142.38).USP33 expression in lung cancer sample is starkly lower than non-tumor lung tissue (P<0.01, Fig. 2 and 3).
What reason 1.3 caused by investigate the change that in patients with lung cancer, USP33 expresses, and inventor analyzes the data of 554 examples in cBioPortal cancer gene group data base (http://cbioportal.org).Patients with lung cancer survival rate and USP33 gene expression correlation use TCGA cancer databases and online KM-Plotter data base to analyze.
In 9 routine cases of lung cancer, USP33mRNA lowers, and 2 these gene delections of routine cases of lung cancer, this gene mutation of somatic cell (Fig. 4 A and 4B) appears in other 9 examples.It should be noted that inside the catalyst structure domain that three USP33 sudden change is positioned at USP33 (Fig. 4 B).Therefore, these cases of lung cancer about 4%, the expression showing Homozygous deletions or somatic mutation or mRNA is lowered.In addition, somatic cell USP33 missense mutation is found (Fig. 5) in the sample of adenocarcinoma of lung (4.3-6.2%) and squamous cell lung carcinoma (3.4%).Analyze (http://www.oncomine.org/) the data set of USP33 express spectra in Oncomine data base further, this data base is containing 5 groups of independence lung cancer sample mRNA gene table data.It should be noted that compared with control sample, in all 5 groups of data (Fig. 4 C), the USP33mRNA expression in lung cancer sample significantly reduces.In sum, in these lung cancer samples, the downward of USP33 expression has the concordance of height.
2, the expression of USP33 and clinical effectiveness
In order to study USP33 expression and clinical between relation, inventor is analyzed associating between the expression of USP33 with survival of patients phase by disclosed micro-array chip data.Inventor generates Kaplan-Meier (KM) survival curve data and online KM-Plotter data base from cancer gene picture group spectrum CancerGenomeAtlas (TCGA) data.For often organizing data, according to the expression of USP33 gene, patients with lung cancer is divided into two groups.Kaplan-Meier is used for the life span difference of analysis and evaluation USP33 high expressed and the low expression group of USP33.The data base of all four pulmonary carcinoma, higher USP33 expression is with patient longer life cycle (Fig. 6 A-6D).
Inventor analyzes the relation of cancer patient life cycle of USP33 expression and other types further.Result shows, in the cancer of other types, comprise breast carcinoma (Fig. 6 E and 6F), melanoma (Fig. 6 G) and acute myeloid leukemia (AML, Fig. 6 H), shorter life cycle is within the specific limits with the low expression level of USP33.These data show, in multiple human cancer, USP33 plays the role of tumor suppressor gene.
3, USP33 participates in the suppression lung carcinoma cell transfer that Slit signal mediates
Inventor's research in the past shows, in different tumor cells, (comprising pulmonary carcinoma and breast carcinoma), slit expressing quantity obviously declines, and slit plays important effect in anti-curing cancers further develops.Existing result of study also proves simultaneously, and in pulmonary carcinoma, USP33 participates in the intracellular signaling of slit.In order to explore the concrete mechanism of action of USP33 in lung carcinoma cell, inventors have investigated it and how regulating and controlling Slit signal path thus the transfer suppressing lung carcinoma cell.
3.1 restructuring Slit2 produce and wound healing assay
By merging the slit2 gene transfection HEK293 cell having c-myc label, produce the Slit2 full-length proteins with c-myc label.Cell is cultivated in the DMEM culture medium of 5% hyclone.From culture supernatant purification Slit2 albumen.Compare not have the HEK293 cell conditioned medium of transfection.
When carrying out wound healing assay, H1299 cell kind scribbled on collagen protein photochemical etching grid slide, these slides are placed in 35mm culture dish.Once cell aggregation, generate wound area with the careful cell scraping monolayer of aseptic 10ul pipette tip.Rinse plate hole with PBS and remove disengaging cell, then cultivate at matched group with containing in Slit2 culture medium.Within 0 hour, form rear 13-16 hour with wound, under inverted microscope, take ten pictures.The wound moving forward formation by the distance measuring cell initial position carrys out the level of quantization cell migration.
Prove that Slit albumen significantly can reduce the cell migration of H1299 lung carcinoma cell by cell wound healing assay.And this inhibitory action is knocked out rear disappearance (Fig. 8 A-8C) at USP33 by specific siRNA (siUSP33), such result shows that USP33 have impact on the migration of lung carcinoma cell by slit signal path.It should be noted that no matter be act on H1299 cell with slit process H1299 cell or siUSP33 in these experiments, all do not affect propagation or the cell cycle (see Fig. 7) of H1299 cell simultaneously.
Whether in order to detect, to be the ubiquitin activity of going of USP33 mediated this function, and inventor uses USP33 mutant to test.Inventor constructs USP33-C163A that is gone the point mutation of ubiquitination catalytic site, wild type (Wt) USP33 or mutant C163A-mutantUSP33 (Mt) is transformed H1299 cell, afterwards with containing the culture medium of Slit or not cultivating containing the control medium of Slit, can find out from experimental result deposits in case at Slit, and the sudden change (C163A) of USP33 can increase the migration of H1299 cell.Inventor can draw thus: the ubiquitin active structure domain that goes of USP33 albumen plays an important role to suppressing the transport of lung carcinoma cell.
4, USP33 regulates the stability of Robo1 albumen
Existing report display, in breast cancer cell, the effect of USP33 is the distribution changing Robo1 albumen in cell, is re-assigned to cell surface, and can not affects the aggregate level of Robo1 albumen by Robo1Robo1 in cell.So in lung carcinoma cell, whether USP33 has similar function.
Inventor have detected the interaction (Fig. 9 A) of USP33 and Robo1 by co-immunoprecipitation method, USP33 gene is lacked in H1299 cell, under MG-132 (inhibitor of Ubiquitin-proteasome systerm) exists, the ubiquitination level of Robo1Robo1 increases (Fig. 9 B).Process LAN wild type USP33 gene (instead of mutant C163A of catalytically inactive), the ubiquitination level of Robo1 reduces (Fig. 9 C).Unexpectedly, lower USP33 in H1299 cellular expression levels by siRNA method, Robo1 protein expression level also reduces, this (Fig.9D contrary to the result in breast cancer cell; Yuasa-Kawadaetal., 2009a).Contrary, the USP33 of process LAN wild type, instead of the USP33 of C163A sudden change, in H1299 cell, Robo1 expression increases (Fig. 9 E).
The stability of Robo1 albumen how is maintained in order to study USP33, inventors performed following experiment: with contrast siRNA (CTRL) or siUSP33 transfection H1299 cell, use cycloheximide (CHX, protein synthesis inhibitor) cell to be carried out to the process of different time sections simultaneously.Robo1 protein expression level in cell pyrolysis liquid is detected by Western blotting (WB) analysis.After cycloheximide process 6 hours, Robo1 level declined; Process after 12 hours, compared with the siRNA of contrast, in the H1299 cell of transfection siUSP33, Robo1 albumen is almost degradable, and such experimental result shows that the half-life (Fig. 9 F) of Robo1 is shortened in the downward of USP33 expression.By siUSP33 reduce Robo1 protein level phenomenon can the experiment (Fig. 9 G) that suppresses by proteasome inhibitor MG-132, show that in H1299 cell Robo1 degraded realizes mainly through Ubiquitin-proteasome system.These results show, in lung carcinoma cell, USP33 can improve Robo1 protein stability, stop Ubiquitin-proteasome to its Degradation.
Three, interpretation of result
The evidence that the result of study of inventor provides shows that USP33 is a new member in pulmonary carcinoma, regulates slit signal path.Inventors have demonstrated that the down-regulated expression of USP33 is relevant to clinical effectiveness in pulmonary carcinoma.USP33 suppresses lung carcinoma cell migration by regulating the activity of slit signal path.In addition, USP33 improves Robo1 protein stability by Profilin degrading enzymatic activity.These results disclose USP33 in lung carcinoma cell, mediate the previously unknown effect of slit-robo signal one.
USP33 participates in many cell processes, and the research first time of inventor proves that USP33 is a kind of lung cancer related gene, and this gene expression in lung cancer tissue sample reduces.This downward USP33 expression is in pulmonary carcinoma many microarray datas storehouse, and confirmed by real-time quantitative RT-PCR and immunohistochemical analysis.The high expressed of USP33 is better relevant to patients with lung cancer prognosis.Mankind USP33 gene is positioned at No. 1 chromosome p31.1, about has this region deletion allele of 50% in nonsmall-cell lung cancer.In this region, some tumor suppressor genes are determined.Such as, be positioned at DnaJ-like heat shock protein (HLJ1) propagation that this region has been identified as suppressing lung carcinoma cell, Anchorage Independent grows, and tumor occurs, cell transfer and invasion and attack.CAMP-deopendent protein kinase β (PRKACB) is lowered in nonsmall-cell lung cancer (NSCLC) tissue, and the rise of PRKACB can prevent the development of nonsmall-cell lung cancer.In this research, the result of study of inventor shows, USP33 is the crucial participant that pulmonary carcinoma occurs, and can be as one of prediction lung cancer development potential prognosis biomarker.
Nearest research shows, the change that in ubiquitin protein family, different genes is expressed is relevant to dissimilar cancer, comprises pulmonary carcinoma etc.Such as, USP44 is lowered in human lung adenocarcinoma.Patient USP44 expression of enzymes level is lower shows that Overall survival will significantly reduce.Find that this goes to reset in ubiquitin (BAP1) homozygote and disappearance at lung cancer cell line.USP1/UAF1 can as a kind of oncogene, because the inhibitor of USP1/UAF1 and cisplatin play synergism in suppressing nonsmall-cell lung cancer cisplatin-resistant cell to be bred.Patent application research of the present invention shows: USP33 may be a kind of new antioncogene in pulmonary carcinoma.
In Human Lung Cancer, the low expression of Slit2 albumen is not good relevant to the state of an illness prognosis of advanced lung cancer and patient.The mice of Robo1 defect shows bronchiolar epithelium hypertrophy and focal hypertrophy, the pathological characteristic that namely early stage of lung cancer is relevant.In the research of inventor, Slit2 albumen significantly suppresses the migration of lung carcinoma cell, supports that slit-Robo signal path has inhibitory action to pulmonary carcinoma.
The research of inventor proves, slit suppresses breast cancer cell migration to depend on the effect of USP33, and in lung carcinoma cell, USP33 and Robo1 interacts, and is that slit suppresses the migration of lung carcinoma cell necessary.The modification of ubiquitin mediation plays a key effect (CiechanoverandSchwartz, 1994) in the stability of Function protein.But USP33 and Robo1 interacts, and is that the Robo1 that slit mediates is redistributed to required for plasma membrane, but does not affect the level of total Robo1 albumen.
Show in this research, USP33 is by suppressing ubiquitin-proteasome pathway thus stablizing the expression of Robo1 albumen in lung carcinoma cell.USP33 regulates and controls the conduction of slit signal in lung carcinoma cell, and this needs the enzymatic activity of USP33, because after the sudden change of USP33 catalytic site (C163A), slit no longer suppresses lung carcinoma cell to move.What is interesting is, in cBioPortal pulmonary carcinoma sample genomic database, determined 4 catalyst structure domain missense mutation at USP33.Comprehensively can show the application for a patent for invention research of breast carcinoma, in different cancerous cell, USP33 can by different mechanism regulating slit intracellular signaling.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not by the restriction of above-mentioned example; what describe in above-mentioned example and description just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (10)
- The application of 1.USP33 in tumor, is characterized in that: USP33 detects mark and the application of USP33 in tumour medicine exploitation as tumor prognosis.
- 2. application according to claim 1, is characterized in that: USP33 detects mark as tumor prognosis, and described tumor comprises pulmonary carcinoma, breast carcinoma, melanoma and acute myelocytic leukemia acute myelocytic leukemia.
- 3. application according to claim 1, is characterized in that: USP33 is preparing the application in tumour medicine, and described tumor is pulmonary carcinoma.
- 4. application according to claim 3, is characterized in that: USP33 is as cancer target.
- 5. application according to claim 3, is characterized in that: USP33 is by the transfer of regulation and control slit-Robo signal path inhibition tumor cell.
- 6. a diagnostic kit, this test kit contains the material detecting USP33 albumen, and described USP33 protein sequence is SeqNo.1.
- 7. a diagnostic kit, this test kit contains the material detecting USP33 gene, and described USP33 gene order is SeqNo.2.
- 8. a purposes for the diagnostic kit of claim 6 or 7, for detecting tumor and tumor prognosis USP33 expresses.
- 9. be used for the treatment of the pharmaceutical composition of tumor, this pharmaceutical composition contains: the albumen of USP33 gene and/or its coding, and medically acceptable pharmaceutic adjuvant.
- 10. pharmaceutical composition according to claim 9, is characterized in that described tumor is pulmonary carcinoma.
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Date of cancellation: 20220128 Granted publication date: 20190205 Pledgee: Gulin sub branch of Ningbo Yinzhou Rural Commercial Bank Co.,Ltd. Pledgor: ZHEJIANG ASCLEPLUS BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020330000160 |
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Effective date of registration: 20220222 Address after: No. 129-8, Chuanjian South 1st Road, Jinniu District, Chengdu, Sichuan 610081 Patentee after: Sichuan asikeli Biotechnology Co.,Ltd. Address before: 215123 a2-425, bio nano Industrial Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province Patentee before: ASCLEPIUS (SUZHOU) TECHNOLOGY COMPANY GROUP Co.,Ltd. Effective date of registration: 20220222 Address after: 215123 a2-425, bio nano Industrial Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province Patentee after: ASCLEPIUS (SUZHOU) TECHNOLOGY COMPANY GROUP Co.,Ltd. Address before: 315000 Gulin Section 2, Yinxian Avenue, Gulin Town, Haishu District, Ningbo City, Zhejiang Province Patentee before: ZHEJIANG ASCLEPLUS BIOTECHNOLOGY Co.,Ltd. |