CN108893535A - Detection of osteosarcoma lung metastasis related gene mutation based on blood circulation exosome RNA and application thereof - Google Patents
Detection of osteosarcoma lung metastasis related gene mutation based on blood circulation exosome RNA and application thereof Download PDFInfo
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- CN108893535A CN108893535A CN201810768414.7A CN201810768414A CN108893535A CN 108893535 A CN108893535 A CN 108893535A CN 201810768414 A CN201810768414 A CN 201810768414A CN 108893535 A CN108893535 A CN 108893535A
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Abstract
The invention relates to a detection method of a metastatic osteosarcoma molecular target, a gene combination and a kit. Its advantages are: the research discovers for the first time that 1) for metastatic osteosarcoma, a molecular characteristic spectrum obviously related to metastasis is discovered based on extracellular vesicle complete transcriptome sequencing, and the molecular characteristic spectrum has the advantages of being noninvasive, dynamic tracking and the like; 2) a large number of tumor biological markers are enriched in the metastatic bone and meat extracellular vesicles, and the method has important significance for clinically evaluating tumor metastasis and detecting drug targets. The invention designs a gene combination containing 158 genes, according to which the osteosarcoma lung metastasis related driving gene and drug target gene can be dynamically monitored after RNA extraction and purification by clinical patient blood exosome.
Description
Technical field
The present invention relates to technical field of clinical medicine, specifically, being the diagnoses and treatment molecular target of osteosarcoma with lung transfer
Body fluid detection.
Background technique
Survival rate is less than 20% after Lung metastases occur for osteosarcoma, poor prognosis.But it is sent out since osteosarcoma with lung shifts early stage
Existing difficulty, biopsy extraction is difficult, there are the reasons such as heterogeneity for Pulmonary metastasis focuses, and clinic is for metastatic bone sarcoma molecular target at present
Detection there is no effective ways.This research is quasi- to be explored by external, experiment in vivo based on the sequencing detection of outer vesica (EV) transcript profile
A possibility that mutation of osteosarcoma with lung metastasis related gene and molecular target.
Excretion body (exosome) is the vesica corpusculum secreted by a variety of living cells, wherein a variety of containing protein and RNA etc.
Key component, excretion body plays an important role in a variety of physiology and pathologic process, including malignant tumour.
Chinese patent literature CN107144688A discloses CD19 positive excretion body as molecular labeling and examines in preparation tumour
Application in disconnected kit.Chinese patent literature CN107119015A discloses a kind of excretion body in the drug of preparation treatment lung cancer
In application.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of diagnostic kit of osteosarcoma with lung transfer.
Second object of the present invention is to provide a kind of based on blood circulation excretion body RNA detection osteosarcoma with lung transfer phase
Correlation gene mutation and the assortment of genes of drug target.
To achieve the above object, the technical solution adopted by the present invention is that：MRNA expresses ratio conduct in extracellular vesica RNA
Application of the diagnosis marker in the diagnostic kit of preparation detection bone and flesh tumor metastasis.
Diagnosis of the mRNA mutant proportion as diagnosis marker in preparation detection bone and flesh tumor metastasis tries in extracellular vesica RNA
Application in agent box.
Further, the bone and flesh tumor metastasis is osteosarcoma with lung transfer.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that：One kind being based on blood circulation excretion body RNA
Detect osteosarcoma with lung metastasis related gene mutation and drug target the assortment of genes, the assortment of genes include drug target gene,
Gene, Meta gene are driven, drug target gene is：ABL1,AKT1,ALK,AR,ATM,BRAF,BRCA1,BRCA2,CCND2,
CDK4、CDK6、CDKN2A、CHEK2、CTNNB1、DNMT3A、EGFR、ERBB2、ERBB3、ERCC2、ESR1、FBXW7、FGF3、
FGF4、FGFR1、FGFR2、FGFR3、FLT3、GNA11、GNAQ、HRAS、IDH1、IDH2、JAK2、KIT、KRAS、LRP1B、
MAP2K1、MDM2、MET、MYD88、NF1、NF2、NOTCH1、NOTCH2、NPM1、NRAS、PDGFRA、PIK3CA、PLCG2、
PTCH1、PTEN、RAD51C、RB1、RET、SERPINB3、TP53、TSC1、TSC2、VEGFA、ROS1、PDGFRB、MTOR、
RAF1、NTRK1、NTRK2、NTRK3、DPYD、FLCN、SMARCA4、ARID1A、CCND1、DDR2、ERBB4、RNF43、MLL、
FANCA、SMO、PTPRD、TPMT、JAK1、PIK3R1、HDAC2、STK11、RICTOR、B2M、PIK3CB、MAP3K11、KDR、
CCNE1、AHR、PIP5K1A、IGF1R、PGR、EPHB2、EPHA4、SYK、HDAC9、EPHA1、NCOR2、APC、ADCY1、TRIO、
PIK3C2B,CCND3,ATR,PCSK6,BLM,PTPRF,CAD,EZH2,ARAF；Drive gene：RECQL4,CDKN2B,WWOX,
ATRX、DLG2、PIM1、MYC、TWIST1、RUNX2、CDC5L、COPS3、VEGFR1、VEGFR3、SRC、LSAMP、SHH、IRS1、
GLI1,WRN,SFPQ,MUTYH,BAP1,NUMA1,MDC1,BUB3；Meta gene：MUC3A,MUC6,MUC4,MUC16,
COL5A3、COL18A1、MMP15、CACNA1S、GPC1、CSPG4、MMP3、COL1A1、FN1、SPARC、COL11A2、
COL27A1、LAMA3、MMP2、ITGA10、ITGB6、ITGB3、ITGA11。
The invention has the advantages that：
1, this research is found for the first time 1) for metastatic bone sarcoma, based on the full transcript profile sequencing of extracellular vesica and traditional group
Pathological examination is knitted compared to having no obvious bias, and there are noninvasive, the advantages such as dynamically track.2) in the extracellular vesica of metastatic bone and flesh
Enriched tumor marker, for clinically assessing metastases, detection drug target is of great significance.
2, it includes the assortment of genes including 158 genes that the present invention, which devises a kind of,, can be by facing according to this combination
Bed blood samples of patients excretion body purification, RNA shifts associated drives gene for osteosarcoma with lung after extracting and drug target gene moves
State monitoring.
Detailed description of the invention
Attached drawing 1 is experimental design and research object.A：Experimental design uses paired samples, chooses two Patients with Osteosarcoma and turns
Before moving and after transfer outside (primary tumor excision) two pairs of osteosarcoma isologous cell lines (transfer, non-diverting) derived cell vesica carry out into
The enrichment of one step, detection；B and C, compared with non-diverting cell line MNNG, homologous transferable cell line 143B cell invasion ability
It significantly improves (C1), visible lung's multiple nodules after Patients with Osteosarcoma (H1) transfer.
Attached drawing 2 is extracellular vesica separating-purifying and characterization identification.A：Extracellular vesica is seen under Electronic Speculum after negative staining；B：Carefully
Extracellular vesica median diameter is located at 150nm or so；C：Extracellular vesicle surface marks CD81, and Alix, CD9 are positive, cell fragment
It interferes surface markers Calnexin negative, prompts without obvious cellular debris；D：Unit serum (cell conditioned medium) intracellular vesicle RNA
Concentration it was found that, vesica RNA (EV-RNA) concentration obviously rises after transfer.
Attached drawing 3 is that EV-RNA composition compares.A：The interior EV-RNA concentration of unit serum (cell conditioned medium) it was found that, after transfer
Vesica RNA (EV-RNA) concentration obviously rises；B-F：Transferable (after transfer) (with _ M ending) extracellular vesica and it is non-diverting (turn
Before shifting) (with _ N ending), extracellular vesica is compared, and EV-RNA composition ratio, which has, to be substantially change, and mRNA is expressed in EV-RNA after transfer
Ratio (A), mutant proportion (D) rise.Its cover mRNA type (C) and sequencing depth (E, F) before transfer with nothing after transfer
Notable difference.
Attached drawing 4 is the research of EV-RNA express spectra.Clustering prompt transferable (after transfer) extracellular vesica with it is non-diverting
(before transfer) extracellular vesica is compared, and there are notable difference (upper lefts) for EV-RNA expression figure.
Attached drawing 5 is the research of EV-RNA express spectra.The enrichment analysis prompt of GSEA function, difference are mainly enriched with remaining PI3K-AKT
And PRE_NOTCH access.
Attached drawing 6 is the research of the EV-RNA transcript profile spectrum of mutation.Thermal map compares prompt, and transferable (after transfer) (with _ M ending) is thin
For extracellular vesica compared with non-diverting (before transfer) (with _ N ending) extracellular vesica, EV-RNA mutational load is significantly raised；With public affairs
Osteosarcoma mutated gene compares in database Cosmic altogether, prompts a large amount of coincidence mutated genes and potential driving gene mutation.This
In a little driving genes, fractional mutations have potential drug target spot (table)：If patient 2 is mutated with FGFR1, targeted drug
In the current Early trials research of Lucitanib.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1
Method：Collect MG63.2 (can Lung metastases), MG63 (can not Lung metastases) and HOS/143B (can Lung metastases), HOS/
(only primary tumor) before two pairs of osteosarcoma cell line supernatants of MNNG (can not Lung metastases) and two Patients with Osteosarcoma Lung metastases, turn
(only metastatic tumor) serum after shifting.Pass through vesica outside ultracentrifugation separation, purifying cells.By NTA, transmission electron microscope,
WesternBlot characterizes excretion body；By RNA extracting in EV, build behind library carried out using Hi-seq2500 microarray dataset it is complete
Transcript profile deep sequencing (>30X)；By genome alignment tumour relevant mutational site, potential tumor driving gene and is analyzed
Know drug target.
The separation of 1 ultracentrifugation and purifying：Be centrifuged blood plasma under 3000g low-speed conditions, be centrifuged 15 minutes, removal dead cell and
Big cell fragment；Cell conditioned medium is filtered with 0.44- μm of filter screen (Millipore), is further removed small thin
Born of the same parents' fragment；Under the Ultracentrifugation conditions of 100000g, centrifuged overnight precipitates the excretion body in plasma supernatant；It uses
The excretion body precipitating after centrifugation is resuspended in the PBS of 20ml or so, washs to excretion body, again under the conditions of the hypervelocity of 100000g,
Centrifugation 70 minutes, precipitates the excretion body in blood plasma；Weight finally is carried out to the excretion body after precipitating using the PBS of 200 μ l
It is outstanding.
2. excretion body is identified：The measurement of nanometer particle size is carried out to excretion body sample 2ul using NTA instrument；Negative staining examination is added dropwise
Agent passes through transmission electron microscope observing excretion volume morphing after standing 2-3 minutes at room temperature；Using BCA method to protein concentration into
Row is quantitative, is measured at 462nm wavelength.Standard protein curve is made, the result of measurement is calculated；With protein sample：
4xSDS=4：1 ratio is mixed, and protein sample is placed in dry bath and is heated, and condition is 95 DEG C, 10 minutes；:According to inspection
The size for surveying the molecular weight of albumen, configures the protein electrophoresis glue of various concentration；The albumen loading in every hole is calculated according to protein concentration
The protein content of amount, general loading is 20 μ g loadings, and the condition of electrophoresis is：Voltage 100V, 20 minutes, are adjusted into again later
120V, 70 minutes；Transferring film after closing, film is completely submerged in the skim milk of 5% concentration prepared with TBS-T, at a slow speed item
It is mixed under part, according to the size of the molecular weight of detection albumen, is in advance cut film；By the pvdf membrane transfer after cutting
Into incubation box；According to the introduction of antibody specification, a certain proportion of primary antibody dilution is added, mixing overnight is incubated in 4 DEG C of refrigerators
It educates, carries out washing film using TBST, according to the source of primary antibody, the secondary antibody of corresponding different genera is added, is incubated for 60 at room temperature
Minute, developing solution is prepared, developing solution is added on film, development carries out protein immunoblot in 4000 Color Appearance System of LAS,
Identify CD63, CD9, Alix, the markers such as Calnexin.
3. excretion body transcript profile extracts Jian Ku and the sequencing of two generations：Trizol cracking is added in excretion weight suspension sample
Liquid carries out 10~15min of cracking to excretion body at normal temperature, then uses Agilent4200TapeStation, Qubit instrument
It carries out sample and carries out quality inspection, the sample that quality inspection passes through is carried out with 1ug initial amount with TruSeq RNALibrary Prep Kitt
Library construction, and library quality inspection is carried out again with Agilent2200TapeStation, by the library of quality inspection, use Single
Read Flow Cell carries out HiSeq 2500 according to the instruction of HiSeq2500User Guide and operates the computer, and runs
The full transcript profile deep sequencing of Paired-end (2 × 100) standard sequencing program progress (>30X)；Sequencing program operation finishes, right
The data obtained carries out bioinformatic analysis.By genome alignment tumour relevant mutational site, analyzes potential tumor and drive base
Cause and known drug target spot.
As a result：We are successfully separated the outer vesica of the circulating cells extracted in cell strain culture supernatant and human serum, shift bone
Sarcoma is compared with non-diverting osteosarcoma, and EV-RNA content is higher (1.5~2.5 times) in unit supernatant/serum.NTA,
The characterization results such as Western Blot and transmission electron microscope prompt EV meets typical morphological feature, and purity is ideal, and no heteroproteose cell is dry
It disturbs.By to Metastatic Osteosarcoma compared with non-diverting osteosarcoma EV-RNA sequencing data, it has been found that：1) with non-diverting osteosarcoma
It compares, mRNA content is higher in the excretion body of metastatic bone sarcoma source, and wherein encoding mutant gene mRNA content is more up to closely
10 times (2250 point mutation vs240 point mutation).2) by detecting totally 2406 point mutation and Cosmic common data 1.2 ten thousand
The point mutation of osteosarcoma samples source is compared, it has been found that the two height phase in the features such as genome distribution, composition and Katagis
Seemingly.On the contrary, to measure point mutation point mutation similarity related to chondrosarcoma and other soft tissue sarcomas poor by EV-RNA.3)EV-
RNA measures point mutation and is concentrated mainly on NOTCH, mTORC1, and RTKs related pathways and cell-ECM, cell-microenvironment are related
In icam gene family.4) mutation is subjected to drug target analysis, it has been found that two known medicine targets：KRAS(p.G12S
P.A59T, H/143B cell line) and FGFR1 (p.S158L, patient two) and multiple potential medicine targets.Relative medicine includes ground west
His shore, pazopanib, Imatinib etc..
Conclusion：This research finds 1) based on the full transcript profile sequencing of extracellular vesica and to pass metastatic bone sarcoma for the first time
System histopathology result, which is compared, has no obvious bias, and has noninvasive, the advantages such as dynamically track.2) the extracellular capsule of metastatic bone and flesh
Enriched tumor marker in steeping, for clinically assessing metastases, detection drug target is of great significance.
Embodiment 2
1) from oncoKB, IntoGen, Cosmic tri- collections are currently known the gene mutation of tumour associated drives and medicine target
Point mutation.2) according to our previous experiments results filter out in body fluid excretion body RNA group can stable detection, and in metastatic bone and flesh
The gene pathway raised in tumor.3) finally being devised according to intersection of the two includes the assortment of genes including 158 genes.
According to this combination, associated drives are shifted for osteosarcoma with lung after can extracting by clinical patients blood excretion body purification, RNA
Gene and drug target gene carry out dynamic monitoring.
Drug target gene：ABL1,AKT1,ALK,AR,ATM,BRAF,BRCA1,BRCA2,CCND2,CDK4,CDK6,
CDKN2A、CHEK2、CTNNB1、DNMT3A、EGFR、ERBB2、ERBB3、ERCC2、ESR1、FBXW7、FGF3、FGF4、FGFR1、
FGFR2、FGFR3、FLT3、GNA11、GNAQ、HRAS、IDH1、IDH2、JAK2、KIT、KRAS、LRP1B、MAP2K1、MDM2、
MET、MYD88、NF1、NF2、NOTCH1、NOTCH2、NPM1、NRAS、PDGFRA、PIK3CA、PLCG2、PTCH1、PTEN、
RAD51C、RB1、RET、SERPINB3、TP53、TSC1、TSC2、VEGFA、ROS1、PDGFRB、MTOR、RAF1、NTRK1、
NTRK2、NTRK3、DPYD、FLCN、SMARCA4、ARID1A、CCND1、DDR2、ERBB4、RNF43、MLL、FANCA、SMO、
PTPRD、TPMT、JAK1、PIK3R1、HDAC2、STK11、RICTOR、B2M、PIK3CB、MAP3K11、KDR、CCNE1、AHR、
PIP5K1A、IGF1R、PGR、EPHB2、EPHA4、SYK、HDAC9、EPHA1、NCOR2、APC、ADCY1、TRIO、PIK3C2B、
CCND3、ATR、PCSK6、BLM、PTPRF、CAD、EZH2、ARAF。
Drive gene：RECQL4,CDKN2B,WWOX,ATRX,DLG2,PIM1,MYC,TWIST1,RUNX2,CDC5L,
COPS3、VEGFR1、VEGFR3、SRC、LSAMP、SHH、IRS1、GLI1、WRN、SFPQ、MUTYH、BAP1、NUMA1、MDC1、
BUB3。
Meta gene：MUC3A,MUC6,MUC4,MUC16,COL5A3,COL18A1,MMP15,CACNA1S,GPC1,
CSPG4、MMP3、COL1A1、FN1、SPARC、COL11A2、COL27A1、LAMA3、MMP2、ITGA10、ITGB6、ITGB3、
ITGA11。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (4)
1. mRNA expresses ratio as diagnosis marker in the diagnostic reagent for preparing detection bone and flesh tumor metastasis in extracellular vesica RNA
Application in box.
2. mRNA mutant proportion detects the diagnostic reagent of bone and flesh tumor metastasis as diagnosis marker in preparation in extracellular vesica RNA
Application in box.
3. application according to claim 1 or 2, which is characterized in that the bone and flesh tumor metastasis is osteosarcoma with lung transfer.
4. a kind of genome based on blood circulation excretion body RNA detection osteosarcoma with lung metastasis related gene mutation and drug target
It closes, which is characterized in that the assortment of genes includes drug target gene, driving gene, Meta gene, and drug target gene is：ABL1,
AKT1、ALK、AR、ATM、BRAF、BRCA1、BRCA2、CCND2、CDK4、CDK6、CDKN2A、CHEK2、CTNNB1、DNMT3A、
EGFR、ERBB2、ERBB3、ERCC2、ESR1、FBXW7、FGF3、FGF4、FGFR1、FGFR2、FGFR3、FLT3、GNA11、
GNAQ、HRAS、IDH1、IDH2、JAK2、KIT、KRAS、LRP1B、MAP2K1、MDM2、MET、MYD88、NF1、NF2、NOTCH1、
NOTCH2、NPM1、NRAS、PDGFRA、PIK3CA、PLCG2、PTCH1、PTEN、RAD51C、RB1、RET、SERPINB3、TP53、
TSC1、TSC2、VEGFA、ROS1、PDGFRB、MTOR、RAF1、NTRK1、NTRK2、NTRK3、DPYD、FLCN、SMARCA4、
ARID1A、CCND1、DDR2、ERBB4、RNF43、MLL、FANCA、SMO、PTPRD、TPMT、JAK1、PIK3R1、HDAC2、
STK11、RICTOR、B2M、PIK3CB、MAP3K11、KDR、CCNE1、AHR、PIP5K1A、IGF1R、PGR、EPHB2、EPHA4、
SYK、HDAC9、EPHA1、NCOR2、APC、ADCY1、TRIO、PIK3C2B、CCND3、ATR、PCSK6、BLM、PTPRF、CAD、
EZH2,ARAF；Drive gene：RECQL4,CDKN2B,WWOX,ATRX,DLG2,PIM1,MYC,TWIST1,RUNX2,CDC5L,
COPS3、VEGFR1、VEGFR3、SRC、LSAMP、SHH、IRS1、GLI1、WRN、SFPQ、MUTYH、BAP1、NUMA1、MDC1、
BUB3；Meta gene：MUC3A,MUC6,MUC4,MUC16,COL5A3,COL18A1,MMP15,CACNA1S,GPC1,CSPG4,
MMP3、COL1A1、FN1、SPARC、COL11A2、COL27A1、LAMA3、MMP2、ITGA10、ITGB6、ITGB3、ITGA11。
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110527729A (en) * | 2019-09-23 | 2019-12-03 | 中山大学附属第一医院 | The small osteosarcoma with lung transfer of molecules marker of one group of detection and application |
| CN111060585A (en) * | 2020-01-02 | 2020-04-24 | 上海交通大学医学院附属瑞金医院 | Plasma exosome body spectrum peak and application thereof |
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| CN111733188A (en) * | 2020-07-28 | 2020-10-02 | 华南农业大学 | Method for promoting skeletal muscle development, inhibitor for promoting skeletal muscle development and application |
| CN111733188B (en) * | 2020-07-28 | 2021-11-23 | 华南农业大学 | Method for promoting skeletal muscle development, inhibitor for promoting skeletal muscle development and application |
| CN113881768A (en) * | 2021-06-15 | 2022-01-04 | 上海长征医院 | Gene for osteosarcoma typing and osteosarcoma prognosis evaluation and application thereof |
| CN113881768B (en) * | 2021-06-15 | 2023-10-03 | 上海长征医院 | Gene for osteosarcoma typing and assessing osteosarcoma prognosis and application thereof |
| CN116259360A (en) * | 2023-03-16 | 2023-06-13 | 中国人民解放军空军军医大学 | Identification and characteristic gene set of hyperproliferative tumor subgroup in lung adenocarcinoma and application |
| CN116259360B (en) * | 2023-03-16 | 2024-02-09 | 中国人民解放军空军军医大学 | Identification and characteristic gene set of hyperproliferative tumor subgroup in lung adenocarcinoma and application |
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