CN107058596A

CN107058596A - A kind of mark related to glioblastoma diagnosis and its application

Info

Publication number: CN107058596A
Application number: CN201710464798.9A
Authority: CN
Inventors: 符达; 马雨水; 吕中伟; 卢改霞
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2017-06-19
Filing date: 2017-06-19
Publication date: 2017-08-18

Abstract

The present invention relates to a kind of mark related to glioblastoma diagnosis, belong to technical field of molecular biology, specifically, the mark is TNC genes and its expression product.The present invention also provides the application of the mark.Its advantage is shown：Genomics and proteomic assays that the present invention is integrated to GBM, are analyzed by gene expression analysis and LC MS/MS protein spectrum based on genetic chip and RNA sequencings, it is found that TNC genes are raised in mRNA level in-site and protein level.Survival analysis has been done to TNC, it is found that its overexpression is relevant with patient's GBM prognosis mala, has illustrated that TNC can be as GBM biomarkers and potential therapy target.

Description

A kind of mark related to glioblastoma diagnosis and its application

Technical field

It is a kind of mark related to glioblastoma diagnosis specifically the present invention relates to technical field of molecular biology Will thing and its application.

Background technology

GBM is initially named as glioblastoma multiforme, is exactly attempting the significance difference of the different cells of description intra-tumor Different, this also reflects the heterogeneity of molecule.In the case that genome-based technologies can specify GBM characterization of molecules, one simple Clinical picture just remolded GBM as a kind of this conception of species of single disease.Clinically, most of glioblastomas are (about 90%) it is primary, only minority is to be in progress (Secondary cases GBM) from Low grade glioma.Primary and Secondary cases GBMs tables Reveal entirely different characterization of molecules, for example, EGF-R ELISA (EGFR) and phosphatase tensin homologue (PTEN) it is often abnormal in primary GBMs, and TP53 is abnormal in Secondary cases GBMS.Although Secondary cases GBMs is very rare, only 5% to the 10% of GBMs is accounted for, but its unique characterization of molecules and clinical process let us recognize that GBM is a kind of heterogeneous disease Disease.

GBM is the cancer of cancer gene group collection of illustrative plates (TCGA) project first choice research, and nature magazines are delivered within 2008 First result of study that TCGA is sent, it is in terms of number variation, gene expression, DNA methylation is copied to GBM characterization of molecules Multi-angle has been carried out to analyze on a large scale.Except common somatic mutation, including TP53, PTEN, NF1, EGFR, ERBB2, RB1, PIK3R1, PIK3CA etc., TCGA also found that some new conspicuousnesses make a variation, such as NF1, PARK2 homozygous deletion, with And AKT3 amplification etc., and it was found that these hereditary variations are mainly enriched in three signal paths：(a) receptor tyrosine kinase/ RAS/ phosphatidylinositol3 3 kinases (RTK/RAS/PI3K) signal path (abnormal in 88% patient GBM)；(b) p53 signals lead to Road (abnormal in 87% patient GBM)；And (c) R/B signals path (abnormal in 78% patient GBM).

The achievement in research of TCGA and other research groups depicts the molecule landscape map of a width GBM：EGFR,MET, The isogenic amplification of PDGFRA, MDM4, MDM2, CCND2, PIK3Cad and CDKN2A/B, CDKN2C, PTEN, RB1, NFKB1A etc. The missing of gene；And p53, PTEN, NF1, Rb1, IDH1 and IDH2 have the mutation of different frequency in body cell, such as EGFR is extracellular The mutation in domain, including EGFR variation III (EGFRvIII).With the progress of genome-based technologies, some new hereditary changes are sent out Show, including there is somatic mutation, and FGFR3/TACC fusion bases in 20% glioblastoma in Wnt signal conditioners FAT1 The discovery of cause.Although these new hereditary changes may be uncommon, it is cognitive that they can extend our biological information And there may be very big clinical value.

By the analysis to TCGA gene expression datas, GBM points are four hypotypes by Verhaak et al., i.e. Neural, Proneural, mesenchymal, classical hypotype, each hypotype have entirely different molecular changes.Proneural Hypotype feature is that have abundant PDGFRA, CDK6, CDK4 and MET to change, and is mutated with frequently IDH1；Classical Hypotype feature is with EGFR amplifications, and PTEN and CDKN2A missings；Mesenchymal hypotype features be NF1, TP53 and CDKN2A mutation and/or missing.It is finally neural hypotypes, its hereditary feature is still not clear.

The content of the invention

The purpose of the present invention is that there is provided a kind of new application of TNC genes for deficiency of the prior art.

To achieve the above object, the present invention is adopted the technical scheme that：TNC genes as diagnosis marker prepare it is pernicious Application in diagnosis of glioma product.

Application of the expression product of TNC genes as diagnosis marker in glioblastoma diagnostic products are prepared.

Further, the expression product of TNC genes is Tenascin C proteins.

Application of the reagent of detection TNC genes or its expression product in glioblastoma diagnostic products are prepared.

Further, described glioblastoma is glioblastoma multiforme.

Further, described glioblastoma diagnostic products are used for diagnosis or the Index for diagnosis of glioblastoma.

Further, described diagnostic products are kit, chip or test paper.

The invention has the advantages that：

Genomics and proteomic assays that the present invention is integrated to GBM, by based on genetic chip and RNA surveys The protein spectrum analysis of the gene expression analysis and LC-MS/MS of sequence, it is found that TNC genes are raised in mRNA level in-site and protein level. Survival analysis has been done to TNC, it is found that its overexpression is relevant with patient's GBM prognosis mala, has illustrated that TNC can be marked as GBM biologies Will thing and potential therapy target.

Brief description of the drawings

Accompanying drawing 1：Clustering (the p for the mRNA that 45 5 times of differences change in 15 LGGs<0.01).

Accompanying drawing 2：Clustering (the p for the gene that 20 5 times of differences change in 7 GBM<0.01).

Accompanying drawing 3：21 5 times of differences change gene (p in 22 LGGs<0.01) clustering.

Accompanying drawing 4：25 5 times of differences change gene (p in 27 GBM<0.01).

Accompanying drawing 5：The GO analyses of 148 genes.Main bioprocess is：Bioprocess positive regulation, metabolic process is just To regulation and control, cell processes positive regulation, bioprocess negative regulation, the positioning of single creature body；Main molecular function is：Appraise and decide Position, acceptor is combined, and protein transport activity, mRNA3 '-non-translational region AU- enrichment regions are combined, cell cycle process；Main is thin Born of the same parents' component is：Cytosolic part, cytoplasm, intracellular organelle, polymer composite, organelle.

Accompanying drawing 6：KEGGPathway is analyzed.Five primary signal pathways are：Oxytocins signal transduction pathway, hemp of driving in the wrong direction Signal path, is carried secretly round the clock, vascular smooth muscle contraction, glutamic acid nerve synapse.

Accompanying drawing 7：SRING on-line analyses.Important albumen includes PLK4, CDK6, PRDM10 and MEF2C.

Accompanying drawing 8：15 albumen for significantly lacking of proper care.That raises includes HEXB, NES；That lowers includes MBP, HSPA12A.

Accompanying drawing 9：16 are adjusted the consistent molecule of trend above and below gene with albumen aspect.

Accompanying drawing 10：NES, HEXB and HSPA12A, MBP relative expression quantity in GBM.QRT-PCR results show NES, HEXB Expression significantly up-regulation；HSPA12A, MBP expression are notable to lower.

Accompanying drawing 11：NES IHC coloration result schematic diagrames.

Accompanying drawing 12：The GO analyses of 693 albumen.Primary biological process is：The generation of energy and metabolism premise, passes through oxidation Energy, cellular respiration, the metabolism of purine nucleotides, ATP metabolic process are derived from effect；Main molecules function is：NADH is aoxidized Reductase activity, nadh dehydrogenase activity, redox active, catalytic activity, cation transport atpase activity；Main cell Component is：Myelin, cell external capsule, excretion body, bounded membrane vesicle, mitochondria.

Accompanying drawing 13：KEGG is analyzed.Major avenues of approach is：Oxidative phosphorylation, Huntington's disease, Parkinson's, alzheimer ' Silent disease, metabolic pathway.

Accompanying drawing 14：STRING protein interaction analysis.Important albumen includes NDUFV2, ENO2andATP2A1.

Accompanying drawing 15：10 with 693 differential proteins be common differential gene/albumen molecule.

Accompanying drawing 16：14 common moleculars.Including HSPA12A, SNCA, GNAO1, SERPINA3, CAPG, ANXA1, SOD2, TNC, PYGL, CD44, TGFBI, GBP1, SERPINH1, NES.The NES wherein raised, TGFBI and the HSPA12A of downward, SNCA Also appear in simultaneously in Figure 15 and Fig. 9.

Accompanying drawing 17：Gene expression joint mass spectral analysis.36 difference molecules are always obtained, wherein 25 are raised, under 11 Adjust.

Accompanying drawing 18：GO is analyzed.Primary biological process includes：Electron transport chain, chemical balance, response to traume, cellular respiration, Drug response；Main molecules function includes：Protein binding, albumen composition is combined, with reference to structural molecule activity, collagen With reference to；Main cell composition includes：Excretion body, part extracellular region, bounded membrane vesicle, extracellular region, actin cytoskeleton.

Accompanying drawing 19：KEEG Pathway are analyzed.Major avenues of approach：Parkinson's, metabolic pathway, drive in the wrong direction hemp signal path, Oxidative phosphorylation, nerve signal transmission path.

Accompanying drawing 20：Protein interaction analysis.Important molecule includes STAT3, CD44.

Accompanying drawing 21：There is situation about being mutated in normal cerebral tissue and GBM in 14 peptide fragments of 14 protein.

Accompanying drawing 22：The ratio of the protein of different mutation counts.There are more than 10 mutation counts in MYH11, FN1, SYNM.

Accompanying drawing 23：There is the schematic diagram of SAAVs amino acid sites in 3 albumen (MYH11, FN1, SYNM).

Accompanying drawing 24：19 mutains (9.5%) are enriched on Focal adhesion paths, and the protein of mutation is with red Collimation mark goes out.

Accompanying drawing 25：SOAT1 and NES is our GEO gene expression chips, TCAG gene expression chips and mass spectrum mutation identifications Common molecule；APOB is that TCAG is sequenced, TCGA gene expression chips and mass spectrum mutation identify common molecule.

Accompanying drawing 26：Survivorship curve shows that NES up-regulation is relevant with shorter OS, imply that poor prognosis.

Accompanying drawing 27：Survivorship curve shows that TNC up-regulation is relevant with shorter OS, imply that poor prognosis.

Embodiment

The embodiment that the present invention is provided is elaborated below in conjunction with the accompanying drawings.

Embodiment 1

Various techniques known in the art detect the expression of TNC genes.

Embodiment 2

Various techniques known in the art detect the expression product (such as Tenascin C proteins) of TNC genes.

Embodiment 3

1. materials and methods

1.1 tissue of patient samples

We have collected 8 pairs of glioblastomas (GBM) and pairing through surgical excision and according to newest WHO classification The flesh tissue sample of normal cerebral tissue；Sample standard deviation comes from the People's Hospital of Ganzhou City.All patients sign informed consent form, should Item operation is permitted by Ethics Committee of the People's Hospital of Ganzhou City.

1.2 preparation of reagents

UA buffer are formulated：Urea 48.048g, Tris 1.2114g, add 100ml water, adjust pH=8.5

IAA：0.005M IAA are dissolved in UA (13.875mg IAA are dissolved in 1.5ml UA)

200mM DTT:30.86mg DTT are dissolved in 1ml 100mMNH₄HCO₃In

ABC/ ammonium hydrogen carbonate：0.05M ABC are dissolved in H₂(395.3mg is dissolved in 100ml ddH in O₂O)

50mM NH₄HCO₃：50ul 1M NH₄HCO₃(79.06mg is dissolved in 1ml ddH₂O 950ul ddH) are added₂O

SDT cracks formula of liquid:SDS 4g, DTT 1.54g, Tris 1.21g, HCl adjust pH=7.6

2.1 LC-MS/MS protein spectrums are analyzed

2.1.1 preparation of samples (following operation is in progress on ice)

1. 8 couples of GBM and normal cerebral tissue's sample are cut into fine grained chippings (about 1mm³), and use PBS；

2. 20-50mg is organized (containing about 2-5mg albumen), SDT lysates (4%SDS, 0.1M DTT, 0.1M are added Tris-HCl, pH 7.6) 400ul, 4 DEG C of low temperature homogenate 90s；

3. 3min is boiled in 100 DEG C of water-baths；

4. ultrasound 10 times, condition：80w, each ultrasound 10s, is spaced 15s；

5. 16,000g centrifugation 10min, take supernatant (should not be drawn onto precipitation, rather few not many)；

6. fluorescence spectrophotometry protein concentration；

2.1.2 mass spectrum sample pre-treatment (FASP method, the progress in 10K super filter tubes)

1. 20-200ug albumen adds 30ul SDT lysates (should not increase in 30ul SDT, it is possible to block filter core, such as Concentration is low, and graded adds SDT lysates and UA, can not once be added beyond 30ul)；100ul UA, 600rpm are added, is mixed Instrument mixes 1min, and 14,000g centrifugation 15min, specific centrifugation time depends on the circumstances, and from dry, notices that liquid can not in centrifuge tube Excessively；

2. adding 200ul UA, 14,000g centrifugation 15min remove liquid in centrifuge tube；

3. adding 100ul IAA, 600rpm blending instrument mixes 1min, dark place is incubated 20min；

4. 14,000g centrifuges 10min；

5. adding 100uLUreabuffer, 14000g centrifugation 10min are repeated twice；

6. adding 100uL 50mM ABC into super filter tube, 14000g centrifugation 10min are repeated twice；

7. add the ABC (pancreatin of the 40uL 50mM g pancreatin of μ containing 1-4：Albumen is 1:50-1:100) 1min, is vibrated；

8. super filter tube is put into 37 DEG C of water-bath and digests 16-18h；

9. super filter tube is transferred in new collecting pipe, 14,000g centrifugation 10min collect the peptide fragment that ultrafiltration is got off；

10. adding 50uLABC into super filter tube, 14,000g centrifugation 10min collect the peptide fragment that ultrafiltration is got off；

11. freezed after -80 DEG C of freezings of mixtures of polypeptides；

12. mixtures of polypeptides is through solid-phase extraction column desalting processing；

2.1.3 nano-LC-MS/MS is analyzed

Lyophilized mixtures of polypeptides is dissolved in 0.1% formic acid solution again, uses the professional mass spectrums of LTQ Orbitrap Velos Instrument identifies mixtures of polypeptides.Easy nano LC system instrument parameters are set to：Full scan scope is 300-2000amu (1 Individual micro scanning), relying on two grades of scannings afterwards for 20 signals, (sweep length is 3amu, the collision of 35% normalized energy, dynamic row Except 1min).

2.1.4 data analysis

Initial data is analyzed using Maxquant softwares (version 1.3.0.5), and searching database is Swissprot people Protein Data Bank (www.uniprot.org).Search argument is：Trypsase, 1% cuts rate by mistake, and enzyme site is 2 by mistake, it is female from Protonatomic mass error is 0.05Da, and the fixed urea that is modified to methylates modification Carbamidomethy (C), without variable modification.

Search condition：Pancreatin digests, and maximum omits restriction enzyme site 1, and peptide fragment Mass accuracy ± 0.1, MS/MS is high in quality Exactness ± 0.1, fixed modification Carbamidomethyl (C).The peptide sequence that artificial sequencing is obtained passes through EMBL (http:// Dove.embl-heidelber.de/blast2/ non-redundant proteins sequence library nrdb95) is searched for, parameter is set to write from memory Recognize value.

2.2 Quantitative Real-time PCR

2.2.1 total serum IgE is extracted

1. tissue plus 1ml Trizol cracking per 50mg-100mg, the volume of tissue, which is not to be exceeded, adds Trizol volumes 10%, fully it is homogenized using homogenizer；Homogenate is in (15 DEG C~30 DEG C) placement 5min of room temperature；

2.RNA is separated：By 1:0.2(TRIzol:Chloroform) volume ratio add chloroform, cover tightly, firmly acutely concussion 15 seconds, Centrifuged 15 minutes under the conditions of room temperature placement 3-5min, 12000rpm, 4 DEG C；Above-mentioned mixed liquor can be divided into three-phase after centrifugation, under Layer for phenol chloroform phase, the colourless aqueous layer in intermediate layer and upper strata.It is careful to take out EP pipes, by transparent aqueous phase of the upper strata containing RNA It is transferred in new EP pipes；

3) RNA precipitate：Add the ratio of 0.5ml isopropanols according to every 1ml Trizol, add isopropanol, mix, room temperature is put Put 10min.12,000rpm, 10min is centrifuged under the conditions of 4 DEG C, RNA precipitate formation jelly sinks to ttom of pipe.

4.RNA is washed：Supernatant is abandoned, is 1 according to Trizol and proportion of ethanol：1 adds 75% ethanol, is washed, and is vortexed Mixing；4 DEG C, centrifuge 5min under the conditions of 7500rpm, abandon supernatant；It is placed in drying at room temperature precipitation.It is careful not to allow RNA to be completely dried, It is difficult dissolving after being completely dried because of RNA.

5.RNA is redissolved：RNA is dissolved again with the DEPC water treated.

Note：Mouth mask, gloves need to be put on when extracting RNA, special RNase free pipette tips and centrifuge tube, centrifugation are used Machine needs precooling in advance.

2.2.2 RNA is quantified and purity detecting

Quantitative analysis is carried out to above-mentioned RNA using micro-spectrophotometer, its concentration and purity is detected.A260/A280 ratios Value is the important indicator of purity detecting, typically in 1.8-2.0, less than the pollution that this value indicates RNA or other impurities, after quantifying RNA samples can carry out RT-PCR or to be stored in -80 DEG C of refrigerators standby.

2.2.3 cDNA synthesis

Reverse transcription, which is carried out, by TaKaRa companies PrimeScript TM RT-PCR kits specification synthesizes cDNA, it is following Component prepares RT reaction systems (operating on ice)：

Reverse transcription reaction condition：42℃10min；95℃2min；16℃forever

CDNA solution plus 80 μ l H that reaction is obtained after terminating₂O (20 μ l → 100 μ l), makes final concentration of 10ng/ μ l, Proceed qRT-PCR reaction or -20 DEG C freeze it is standby.

2.2.4 qRT-PCR

All mRNA primers of this experiment and internal reference are designed by Shanghai Sheng Gong biotech firms to be synthesized.

Real-time fluorescence quantitative PCR primer sequence

By the operating procedure on TaKaRa companies SYBR Premix Ex Taq Kit specifications, real time fluorescent quantitative is carried out PCR is detected.It is loaded on ice, reaction system is as follows：

Reaction condition is as follows：

Melting curve analysis, judges product purity：After reaction terminates, RQ manager analysis software statistical dispositions are obtained Ct values, and judge according to melting curve the purity of product.

2.2.5 data analysis and processing

The Ct values (C represents Cycle, and T represents Threshold) of each test objective gene and internal reference are obtained, Ct values are represented Fluorescence intensity in each reacting hole reaches the period undergone during the threshold value of setting.Circular is as follows：

△ Ct=target gene Ct values-house-keeping gene GAPDH Ct values

△ △ Ct=target gene △ Ct- sample for reference (i.e. control group) target gene △ Ct

Relative expression quantity=2^-△△Ct

2.3 immunohistochemical staining

1. dewaxing and aquation：Conventional xylene dewaxes, graded ethanol aquation；

2.PBS is rinsed 3 times, each 5min；

3. antigen retrieval：Antigen is repaired with 0.01M citrate buffer solutions (pH6.0) high pressure 2min, room temperature is cooled to；

4.PBS is washed 3 times, each 5min；

5. in 3%H₂0₂10min is incubated in-methanol solution, with deactivating endogenous peroxydase activity；

6.PBS is washed 3 times, each 5min；

7. closing：Normal 5% lowlenthal serum is added dropwise, is incubated at room temperature 30min, gets rid of surplus liquid；

8.PBS rinses 3 times and removes serum deprivation, and primary antibody (PBS is used as negative control instead of primary antibody), 4 DEG C of refrigerator overnights is added dropwise；

9. next day PBS is washed 3 times, each 5min；

10. the secondary antibody of horseradish peroxidase (HRP) mark is added dropwise, 30min is incubated at room temperature；

11.PBS is washed 3 times, each 5min；

12. SP (Streptavidin-peroxidase) is added dropwise, 10min is incubated at room temperature；

13.PBS is washed 3 times, each 5min；

14. colour developing：DAB colour developing 5-10min, microscope judges dye levels, distillation washing color development stopping；

15. redye：Haematoxylin redyeing, 1% hydrochloride alcohol breaks up several seconds；

16. running water rinses 5min；

17. conventional be dehydrated transparent, neutral gum mounting, microscopy.

2.4 bioinformatic analysis

We downloaded from GEO databases two groups of gliomas full-length genome expression modal data (GSE45921 and GSE51146 hierarchical cluster analysis), and with MeV 4.7.1 softwares has been done to differential gene.Gene ontology (Gene Ontology, GO)((http://www.geneontology.org/) according to biological approach (Biology Process), molecular function (Molecular Function) and cellular localization (Cellular Location) are annotated and classified to gene, are used to Evaluate differential gene is enriched in which function monoid in GBM.KEEG Pathway have recorded the interaction of molecules among cell Network and specific biological specific version, are used to evaluate which signal path differential gene is enriched in.Protein Interaction carries out on-line analysis (http by STRING://string-db.org/).

2.5 statistical analysis

All statistical analyses use IBM SPSS Statistics20 (IBM Corp.；Armonk, NY, USA) statistics Software kit carries out data processing.Expression quantity is presented with mean ± standard deviation.Independent samples t test carries out group difference comparison, The comparison in difference of rate between Chi-square Test group.Kaplan-Meier curves determine the overall survival of each group, as a result with logarithm Rank tests is compared.P ＜ 0.05 are considered as statistically significant.

3. result

The analysis of 3.1 GEO database genetic chips

We are analyzed using the chips of Affymetrix U133plus 2.0 to 22 gliomas and pairing normal cerebral tissue Data (the GEO dataset of the full-length genome expression pattern analysis of progress:), GSE45921 including the colloid of 15 low levels Knurl (LGGs) and 7 glioblastomas (GBM).

As a result find：Relative to the normal cerebral tissue of pairing, there are 492 more than 2 times differences to change (Fold in 15 LGGs change≥2；P<0.05) mRNA, wherein 367 up-regulations, 125 downwards；Relative to the normal cerebral tissue of pairing, 15 There are 45 more than 5 times differences to change (Fold change >=5 in LGGs；P<0.01) mRNA, wherein 35 up-regulations, including SFRP2, RPE65, CD44, MYC etc.；10 downwards, including ARG1, ENC1, PSG5 etc..Clustering is done with MeV4.7.1 softwares (Fig. 1).

Relative to the normal cerebral tissue of pairing, have in 7 GBM 657 more than 1.5 times differences change (Fold change >= 1.5；P<0.05) differential gene, wherein 133 up-regulations, 524 downwards；Relative to the normal cerebral tissue of pairing, in 7 GBM There are 20 more than 5 times differences to change (Fold change >=5；P<0.01) mRNA, wherein 8 up-regulations, 12 are downward.On That adjusts includes TOP2A, GFAP, SOD2, CDCA7L, GDF8, ID3, XIST, CDC2；That lowers includes VIPR1, CAMK2A, GJB6,RYR2,CNTNAP2,SCEL,SLC8A2,CREG2,MFSD4,CACNG3,NEUROD6,CABP1.It is soft with MeV 4.7.1 Part does clustering (Fig. 2).

In addition, we also analyze what another set was carried out using the chip of Shanghai Bo Xing companies BiostarH-140s × 32 Full-length genome chip of expression spectrum data (GEO dataset:), including 49 gliomas and the normal brain activity group of pairing GSE51146 Knit, wherein 22 Low grade gliomas (LGGs), 27 glioblastomas (GBM).

As a result find：Relative to the normal cerebral tissue of pairing, there are 399 more than 2 times differences to change (Fold in 22 LGGs change≥2；P<0.05) mRNA, wherein 332 up-regulations, 67 are downward；Relative to the normal cerebral tissue of pairing, 22 There are 21 more than 5 times differences to change (Fold change >=5 in LGGs；P<0.01) mRNA, wherein 17 up-regulations, including ITSN2,RPS6KC1,SB92,KCMF1,THAP3,WIPI49,SND1,ITGA5,YES1,BHMT2,PP1553,LRRTM2, COL1A2,SNX16,SLCO1B1,PAF400,UVRAG；4 are to lower, including RTN1, ATXN10, NAP1L3, DMBT1.With MeV 4.7.1 softwares do clustering (Fig. 3).

Relative to the normal cerebral tissue of pairing, there are 609 more than 1.5 times differences to change (Fold change in 27 GBM ≥1.5；P<0.05) mRNA, wherein 481 up-regulations, 128 are downward；Relative to the normal cerebral tissue of pairing, 27 GBM In there are 25 more than 5 times differences to change (Fold change >=5；P<0.01) mRNA, wherein 23 up-regulations, including RPS6KC1, YWHAG, RXRB, TRRAP, TOMM22, HTT, SMARCAL1, UTP15, COL3A1, ITGA5, TIMP1, CCL2, TRP2, EHD3, RANBP2, SR140, B3GALNT1, PHF10, UMPS, DHCR7, METTL5, ZDHHC2, TPPP2；2 downwards, Including TAF10, PLP1.Clustering (Fig. 4) is done with MeV 4.7.1 softwares.

By contrasting the mRNA (Affymetrix that 1.5 times of differences of GBM and normal cerebral tissue in two groups of genetic chips change In chip 657 with 609 in BiostarH chips), it has been found that the gene that 148 co expressions change.

3.2 GO, KEGG Pathway and protein interaction analysis

In order to judge differential gene is mainly enriched in which function monoid and metabolic pathway, we are carried out to 148 genes GO (Fig. 5) and KEGG Pathway (Fig. 6) analyses and protein interaction analysis (Fig. 7).

3.3 protein spectrums are analyzed

We have carried out the sequencing of holoprotein group to 8 GBM and its pairing normal cerebral tissue using nano-LC-MS/MS. Each sample has done 3 repetitions.

As a result find：Relative to the normal cerebral tissue of pairing, there are 693 more than 1.5 times differences to change (Fold in 8 GBM change≥1.5；P<0.05) differential protein, wherein 500 up-regulations, 193 are downward；Wherein there are 15 changes most notable Differential protein (Fold change >=10；P<0.01), 6 up-regulations, including TMX1, ACOX1, HEXB, GORASP2, SYNM, NES；9 downwards, including MBP, CNP, PLP1, MYLPF, TUBA4A, SNCG, HSPA12A, GPD1, DMTN.Use MeV 4.7.1 Software does clustering (Fig. 8).

In 693 differential proteins, there are 16 common molecules, and upper down regulation trend compared with 148 differential genes Unanimously (Fig. 9), including NES, HEXB, CNN3, H2AFZ, STAT3, RPS28, ELAVL1, ILF3, ARPC5, EIF2S2, MCTS1, NDUFS2, NDUFA10, CALM1, HSPA12A, MBP, the change for illustrating these protein levels are probably because gene alteration causes 's.

In 16 common differential gene/albumen, we have chosen NES, the HEXB most significantly raised and most significantly lower HSPA12A, MBP carried out qRT-PCR (Figure 10) and IHC (Figure 11, NES IHC results) checking, as a result confirm that these are poor It is consistent with chip/mass spectral results that expression trend is lowered in the upper mediation of allogene/albumen.

3.4 GO, KEGG Pathway and protein interaction analysis

Then, the albumen that our 693 1.5 times of differences to above-mentioned GBM and normal cerebral tissue change, has carried out GO (figures 12), KEGG Pathway (Figure 13) and protein interaction analysis (Figure 14).

3.5 TCGA database gene expression analysis

We have downloaded GBM gene expression (IlluminaHiSeq) sequencing result of TCGA databases, comprising 172 samples, wherein 5 normal cerebral tissues, 167 GBM tissues.Relative to normal cerebral tissue, have 2881 in 167 GBM Individual more than 1.5 times difference changes (Fold change >=1.5；P<0.05) differential gene.What 2881 1.5 times of differences changed In gene, there are 10 to change albumen with 693 1.5 times of differences for common differential gene/albumen, including SNCG, SNCA, SPTB, CKMT1A, MYH1, MYH4, F2, HIST1H1B, TGFBI, LMNB1 (Figure 15).

We have also downloaded another set microarray data GBM gene expression (TCGA_ in TCGA databases GBM_exp_u133a), 539 samples are contained, wherein 10 normal cerebral tissues, 529 GBM tissues.Relative to normal brain activity group Knit, there are 298 1.5 times of differences to change (Fold change >=1.5 in 529 GBM；P<0.05) gene.298 1.5 times poor In the gene of different change, it is common differential gene/albumen (Figure 16) to have 14 to change albumen with 693 1.5 times of differences.

In a word, we utilize gene expression and protein spectrum Conjoint Analysis, and 36 common difference molecules have always been obtained (Fold change≥1.5；P<0.05) GNAO1, SNCG, SNCA, SPTB, CKMT1A, MYH1, MYH4, NDUFA10 are included, CALM1,HSPA12A,MBP,SERPINA3,CAPG,ANXA1,SOD2,TNC,PYGL,CD44,TGFBI,GBP1,SERPINH1, NES,F2,HIST1H1B,LMNB1,HEXB,CNN3,H2AFZ,STAT3,RPS28,ELAVL1,ILF3,ARPC5,EIF2S2, MCTS1, NDUFS2, wherein 25 up-regulations, 11 downwards.GEO- genetic chips/common molecular of protein spectrum 16, TCGA- bases Because of chip/protein spectrum 14, TCGA- sequencings/protein spectrum 10.Wherein two-by-two common molecule have 4 (up-regulation TGFBI, NES and the SNCA, HSPA12A lowered), without three kinds of common molecules (Figure 17).

3.6 GO, KEGG Pathway and protein interaction analysis

We have been GO (Figure 18), KEGG Pathway to this 36 molecules lacked of proper care in mRNA level in-site and protein level (Figure 19) and protein interaction analysis (Figure 20).

The identification of 3.7 monamino acids variation (SAAVs)

In order to analyze Single amino acid mutations from mass spectral results, we utilize the mutation result that TCGA_GBM extrons are sequenced (http://cbio.mskcc.org/cancergenomics/pancan_tcga/) construct GBM monamino acid variation (SAAVs) database.

We search for obtained mass spectral results, including 4834 albumen in Swissprot people's standard protein database 36858 peptide segment informations；Average 7.6 peptide fragments/each albumen.And we by mass spectral results in the SAAVs data newly built Re-searched in storehouse, obtained the mutant peptide segment data of 8 pairs of GBM samples, including 23405 peptides of 2515 albumen Segment information；Average 9.3 mutation peptide fragment/each albumen.Compared with data of normal people storehouse, the SAAVs databases that we build are searched Rope obtains 3884 mutation peptide fragments of 897 new protein, average 4.3 mutation peptide fragment/each albumen.

There is (normal group in 8 normal cerebral tissues in 14 peptide fragments for wherein having 14 protein in only one of which sample Knit middle mutation probability 12.5%), and occurred in that at least six GBM in 8 GBM once mutation (mutation probability in GBM >= 75%) (Figure 21).

335 peptide fragments of 186 protein only occur in that mutation in GBM, average 1.8 mutation peptide fragment/each albumen (from 1-48 bars peptide fragment)；There is SAAVs, mutation count in one peptide fragment of 150 protein (80.6%) only one of which GBM samples (Numbers ofMutation, NM) was equal to for 1 (mutation count is equal to the GBM samples × mutational site for occurring being mutated)；20 albumen (10.8%) mutation count is equal to 2；18 albumen (7.0%) mutation counts are 2-10；3 albumen (1.6%) (MYH11, FN1, SYNM) there are more than 10 mutation counts, NMs ＞ 10 (Figure 22) there are 48,16 and 14 NMs respectively；3 albumen (MYH11, FN1, SYNM) there are SAAVs amino acid sites as shown in the figure (Figure 23).

Compared with normal cerebral tissue, a total of 200 protein occurs in that the mutation of probability >=75% in above-mentioned GBM.It is right KEGG pathway analyses are done in these mutation, and discovery has 19 protein (9.5%) on Focal adhesion pathway (Figure 24).

The protein of this 200 mutation and 1.5 times of difference bases of GBM chip results (7+27) in our 37 GEO databases Because comparing, there are 6 common molecules；Compared with TCGA-GBM-U133a 529 GBM expression chips, 1.5 times of differential genes, have 3 common molecules；1.5 times of differences of IlluminaHiSeq sequencing results are organized with TCGA-GBM-U133a 167 GBM Gene is compared, and has 8 common molecules.

A total of 14 of the common molecular that mass spectrum mutation analysis is obtained with gene expression difference genescreen, 8 up-regulations, 6 It is individual to lower, including APOB, RUFY1, CEACAM5, LDB3, KRT82, C1QL3, SOAT1, COL6A3, ACIN1, NES, TNC, CILP, IKBIP, HK3 (Figure 25).

3.8 survival analysis

We have always been obtained 36 difference molecules, have been utilized base using gene expression and the quantitative Conjoint Analysis of protein spectrum Because of expression and amino acid mutation Conjoint Analysis, 14 common moleculars are obtained, wherein NES and TNC go out in two groups of Conjoint Analysis It is existing.We are further to the two molecules in our 27 GBM chip of expression spectrum data and 529 GBM chip of expression spectrum numbers According to having done survival analysis in (15 patients are lost to follow-up, only analyze 514 GBM).

As a result show：NES in our 27 GBM chip of expression spectrum data and 514 GBM chip of expression spectrum data, Expression is higher, and patient's GBM life span is shorter (P=0.012 and 0.018) (Figure 26)；27 GBM express spectras of the TNC at us In chip data and 514 GBM chip of expression spectrum data, higher patient's GBM life span shorter (P=0.036) (figure is expressed 27)。

4. discuss

Glioblastoma (GBM) is the most common primary malignancy brain tumor of adult, is also one of most fatal human cancer. The genomics and proteomic assays integrated herein to GBM, is newly shown in understand that GBM pathogenesis provides a bit Solution, and some potential therapy targets are identified, provide molecular basis for the new more effective treatment method of exploitation.

By the gene expression analysis based on genetic chip and RNA sequencings and based on Liquid Chromatography-tandem Mass skill The protein spectrum analysis of art (LC-MS/MS), we identify 36 and point lacked of proper care occur in mRNA level in-site and protein level Son, includes TGFBI, the NES of up-regulation, and the SNCA, HSPA12A lowered.And we are analyzing two groups of GEO microarray datas Middle to find, the differential gene of GBM and normal cerebral tissue, and LGGs and the differential gene of normal cerebral tissue repeat few, this explanation It is two kinds of entirely different lifes to develop into Low grade glioma (LGGs) or glioblastoma (GBM) from normal Glial cells Thing process, this also complies with our clinical observation, i.e., the GBM of most of (about 90%) is primary, only less than 10% It is by Low grade glioma is in progress.

Simultaneously, it has been found that imbalance gene, GBM is had been found to including TGFBI, NES, SNCA, HSPA12A etc. And played an important role in other tumours.Transcription growth factor induced gene (TGFBI, Transforming growth factor, Beta-induced), coding one kind can combine I, II and IV collagen types contain arginine, glycine and aspartic acid The albumen of (RGD, L-arginine, glycine, and L-aspartic acid) sequence, it is logical by TGF-β signal in GBM Road up-regulated expression；Nestin (NES) gene, a member of coding intermediate filament protein family, nestin, mainly in nerve Cell is expressed, and is proved to be a GBM prognostic marker；Synapse nucleoprotein α (SNCA, synuclein alpha) is cynapse A member of nucleoprotein family, in brain expressed in abundance, it is overexpressed what can be mediated by tumor necrosis factor α (TNF-alpha) Signal path promotes the apoptosis of human glioma cell line U373 cells；HSPA12A(heat shockprotein FamilyA (Hsp70) member 12A), it is a member of heat-shock protein family, be reported the hair claimed with stomach cancer in Chinese Han Population Sick risk is relevant, and it is overexpressed the prognosis mala correlation of the hepatocellular carcinoma related to early stage HBV.

Some other important molecule predicted by bioinformatic analysis, including CD6, CD44 and STAT3 etc., also there is phase Close research and show that it plays an important role in the morbidity of GBM and other tumours.Signal transduction and transcriptional activators 3 (STAT3, Signal transducer and activator oftranscription-3), be a member of stat protein family, by GBM is reported in, is played an important role in development and invasion and attack.A kind of cell surface for having a variety of isomers of CD44 gene codes is sugared Albumen, participates in cell-cell interaction, cell adhesion and migration, is proved to be overexpressed in the kinds cancer including GBM, swollen Played an important role in oncocyte malignant progression, such as cell migration, tumour growth and vascularization.CDK6 is in GBM by small general The effect driving tumour cell cycle of elementization dressing agent 1 (SUMO1) changes, and promotes tumour progression.CDK4 and CDK6 selectivity Targeted inhibition agent, such as Abemaciclib have been reported in solid tumor, including breast cancer, non-small cell lung cancer, neuroglia It is safely and effectively in blastoma and other solid tumors.

One root problem of protein science is exactly that the change for identifying which albumen coded sequence can cause protein expression The change of level.Because standard database can not identify the peptide fragment of mutation from mass spectral results, we utilize COSMIC data GBM extrons sequencing result constructs monamino acid variation database (SAAVs) in storehouse.By analyzing mass spectrometric data, Wo Menjian The albumen of 200 high mutation rates is defined, with reference to gene expression analysis, it has been found that 14 molecules have changing for mRNA level in-site simultaneously Become, wherein NES and TNC are again while there is the change of protein level.We have further done survival analysis to NES and TNC, find it Overexpression it is relevant with patient's GBM prognosis mala, illustrate that NES and TNC are likely to become GBM biomarkers and potentially controlled Treat target spot.

Certainly, deeper into genome analysis can produce more rich and deeper molecular isoform, but how It is our key issues to be solved therefrom to find drug target.A kind of method is to carry out predictive molecule using bioinformatics tools Function.Carro et al. application bioinformatics reverse-engineering algorithm be found that from TCGA GBM databases two transcription because Sub- C/EBP β and STAT3, they can adjust mesenchyma expression pattern, and have obvious antitumor action.Sumazin et al. is with separately A kind of method of biological information driving develops a kind of multivariate analysis tools (HERMES), can from full-length genome transcription and The candidate targets of microRNA effects are systematically inferred in the comprehensive analysis of miRNA spectrums.In this way, they disclose one MicroRNA regulated and control networks after individual abundant transcription, identify central role of the PTEN missings in tumour growth is promoted, i.e., Pass through the regulating networks of microRNA dependences.Genovese et al. using another algorithm disclose one it is new, by MiR-34a regulations, the signal path that TGF (TGF)-β is relied on.Therefore, predicted using bioinformatics tools Disease occur in key molecule, the functional verification of Binding experiment model has begun to produce a series of with clinical value It is theoretical.

Another method, concentrate on assess the core signal path that is converged of aberrant molecules and they to downstream effector Influence.Brennan et al. is to (including the translation in known signal path of 57 signal protein molecules in 20 GBM tissue samples Modified protein afterwards) analyzed, as a result disclose three signal paths：EGFR subgroups, with EGFR activation and PI3K signal enhancings With the characteristics of；(b) PDGF subgroups, feature is PDGFR signal paths；(c) NF1 subgroups, signal effect device is indefinite downstream.It is right Analysis shows EGFR, PDGFRA, NF1 of TCGA gene expression hypotypes change are Uncrossed each other, and these find all It is the three core signal paths (RTK/RAS/PI3K, p53 and RB1 path) defined based on TCGA.

In a word, comprehensive genomics and proteomic assays are pathogenesis and the new treatment side of exploitation for understanding GBM Method provides new molecular basis.And amino acid mutation analysis is possible to expand us to protein mutant in other cancers Understanding.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as Protection scope of the present invention.

SEQUENCE LISTING

<110>Shanghai Tenth People's Hospital

<120>A kind of mark related to glioblastoma diagnosis and its application

<130> /

<160> 4

<170> PatentIn version 3.3

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gaactggact gtggcattga ga 22

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cacaaagcga ctggatgaac c 21

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Claims

Application of the 1.TNC genes as diagnosis marker in glioblastoma diagnostic products are prepared.
Application of the expression product of 2.TNC genes as diagnosis marker in glioblastoma diagnostic products are prepared.
3. application according to claim 2, it is characterised in that the expression product of TNC genes is Tenascin C proteins.
4. application of the reagent of detection TNC genes or its expression product in glioblastoma diagnostic products are prepared.
5. according to any described applications of claim 1-4, it is characterised in that described glioblastoma is that polymorphy colloid is female Cytoma.
6. according to any described applications of claim 1-4, it is characterised in that described glioblastoma diagnostic products are used to dislike The diagnosis of property glioma or Index for diagnosis.
7. according to any described applications of claim 1-4, it is characterised in that described diagnostic products be kit, chip or Test paper.