CN107356461A - Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared - Google Patents
Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared Download PDFInfo
- Publication number
- CN107356461A CN107356461A CN201710348306.XA CN201710348306A CN107356461A CN 107356461 A CN107356461 A CN 107356461A CN 201710348306 A CN201710348306 A CN 201710348306A CN 107356461 A CN107356461 A CN 107356461A
- Authority
- CN
- China
- Prior art keywords
- bckdk
- cell
- expression
- mek
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100037607 [3-methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial Human genes 0.000 title claims abstract description 120
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 45
- 238000003745 diagnosis Methods 0.000 title claims abstract description 10
- 239000000090 biomarker Substances 0.000 title claims abstract description 5
- 101000739853 Homo sapiens [3-methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial Proteins 0.000 title abstract 5
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 63
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims abstract description 56
- 206010018338 Glioma Diseases 0.000 claims abstract description 11
- 208000032612 Glial tumor Diseases 0.000 claims abstract description 10
- 108010016038 (3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase Proteins 0.000 claims description 116
- 230000026731 phosphorylation Effects 0.000 abstract description 20
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 20
- 238000001514 detection method Methods 0.000 abstract description 11
- 201000010099 disease Diseases 0.000 abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 230000003993 interaction Effects 0.000 abstract description 6
- 230000004048 modification Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 5
- 238000004393 prognosis Methods 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 3
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 230000010534 mechanism of action Effects 0.000 abstract description 2
- 239000007787 solid Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 76
- 230000014509 gene expression Effects 0.000 description 61
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 29
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 29
- 229940124647 MEK inhibitor Drugs 0.000 description 19
- 230000012010 growth Effects 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 13
- 238000001262 western blot Methods 0.000 description 12
- 208000029742 colonic neoplasm Diseases 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 9
- 231100000673 dose–response relationship Toxicity 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 238000011580 nude mouse model Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- 102100031480 Dual specificity mitogen-activated protein kinase kinase 1 Human genes 0.000 description 7
- 101710146526 Dual specificity mitogen-activated protein kinase kinase 1 Proteins 0.000 description 7
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 5
- 230000010307 cell transformation Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 201000010989 colorectal carcinoma Diseases 0.000 description 5
- 101150042537 dld1 gene Proteins 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 102000020233 phosphotransferase Human genes 0.000 description 5
- 238000010837 poor prognosis Methods 0.000 description 5
- 102100035352 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial Human genes 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 101000597665 Homo sapiens 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial Proteins 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 208000029824 high grade glioma Diseases 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 239000012742 immunoprecipitation (IP) buffer Substances 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000000021 kinase assay Methods 0.000 description 4
- 201000011614 malignant glioma Diseases 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 3
- 102100030840 AT-rich interactive domain-containing protein 4B Human genes 0.000 description 3
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 3
- 101000792935 Homo sapiens AT-rich interactive domain-containing protein 4B Proteins 0.000 description 3
- 102000001253 Protein Kinase Human genes 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 150000005693 branched-chain amino acids Chemical class 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000012151 immunohistochemical method Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 108060006633 protein kinase Proteins 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- OBKXEAXTFZPCHS-UHFFFAOYSA-N 4-phenylbutyric acid Chemical compound OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 description 2
- 101710105312 Branched-chain-amino-acid aminotransferase Proteins 0.000 description 2
- 101710097328 Branched-chain-amino-acid aminotransferase, cytosolic Proteins 0.000 description 2
- 101710194298 Branched-chain-amino-acid aminotransferase, mitochondrial Proteins 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 2
- 241001062009 Indigofera Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 101710158343 Probable branched-chain-amino-acid aminotransferase Proteins 0.000 description 2
- 101710199693 Putative branched-chain-amino-acid aminotransferase Proteins 0.000 description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 description 2
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 2
- -1 Sibutramine Hydrochlorides Chemical class 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- AZPBDRUPTRGILK-UHFFFAOYSA-N benzotriazol-1-ium-1-ylidenemethanediamine;4-methylbenzenesulfonate Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1.C1=CC=C2N(C(=N)N)N=NC2=C1 AZPBDRUPTRGILK-UHFFFAOYSA-N 0.000 description 2
- 230000005907 cancer growth Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 210000001842 enterocyte Anatomy 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 230000006920 protein precipitation Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 206010038038 rectal cancer Diseases 0.000 description 2
- 201000001275 rectum cancer Diseases 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000005748 tumor development Effects 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108010046716 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) Proteins 0.000 description 1
- 241000208140 Acer Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 108050009223 Branched-chain aminotransferases Proteins 0.000 description 1
- 102000001967 Branched-chain aminotransferases Human genes 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102100032606 Heat shock factor protein 1 Human genes 0.000 description 1
- 101000867525 Homo sapiens Heat shock factor protein 1 Proteins 0.000 description 1
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 description 1
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 description 1
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000002701 cell growth assay Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 230000004736 colon carcinogenesis Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229950009215 phenylbutanoic acid Drugs 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229960004425 sibutramine Drugs 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/44—Sample treatment involving radiation, e.g. heat
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses BCKDK downstream direct interaction molecule, its phosphorylation modification site and its application as biomarker in tumor diagnosis kit is prepared, belongs to biology and medical diagnosis on disease field.Present invention finds the New function of BCKDK molecules and specific mechanism of action, specify that specific phosphorylation modification site and corresponding substrate, and directly detects clinical tissue sample using existing BCKDK antibody, develops its new application.It there is no the clinical practice for BCKDK Molecular Detections on the market at present, the present invention lays a solid foundation for the tumour especially early diagnosis of colorectal cancer and glioma and prognosis prediction treatment.
Description
Technical field
The invention belongs to biology and medical diagnosis on disease field, and in particular to BCKDK Molecular Detections and anti-BCKDK antibody are in tumour
Development and application in diagnostic kit.
Background technology
Colon cancer morbidity is number three in global male cancer, is number two in female cancer, and increases newly every year
Colorectal cancer patients are up to 1.4 million peoples【1】.The colorectal cancer data analysis in the U.S. in 2017 shows that colorectal cancer is at me
This adds the incidence of disease highest in aboriginal and Black people, and the incidence of disease in asian ancestry and Pacific Ocean islander is minimum【2】.China's knot is straight
Intestinal cancer fatal rate is number five in tumor mortality rate, and colorectal cancer fatal rate standardizes tumor mortality rate in men age
Middle rising is most fast【3】, in addition, though colorectal cancer is considered as largely a kind of disease of old people as other tumours,
Principal pathogenetic population ages are more than 60 years old, and up to 62%, but the patient for still having about 10% is less than 45 years old, wherein about
0.1% patient is less than 5 years old【4】.Therefore the fact that so cruel is faced, in order to preferably treat colorectal cancer, colorectal cancer
Pathogenesis still need to further study.
In the past few decades, scientist achieves great successes on the research of Colorectal Cancer mechanism.
RAS-RAF-MEK-MAPK【5】、PI3K-AKT-mTOR【6】、Wnt/β-catenin【7】And JAK-STAT3【8】Lead to Deng signal
Road abnormal activation and apoptotic signal path extra-inhibitory【9】It is closely related with the generation of colorectal cancer.In clinical treatment,
For EGFR targeted drug, such as：Cetuximab, Victibix and Gefitinib, make the nothing of part colorectal cancer patients
Be in progress life span extension, and quality of life is greatly improved【10】.But due to EGFR in itself【11,12】Or downstream molecules
Mutation activation is such as：KRAS【13】、BRAF【14】、PIK3CA 【15】、PTEN【16】Deng the resistance produced to targeted drug, treatment
Effect is poor.Downstream targets of the MEK as RAF, there is the key effect played the part of and integrate simultaneously shunting signal in growth signals path.
In colorectal cancer, MEK gene mutation is not common, therefore, with the exploitation of the targeted inhibition agent for MEK gradually by weight
Depending on having two kinds of mek inhibitor Sibutramine Hydrochlorides at present and being used to treat melanoma for Buddhist nun for Buddhist nun and Kao Bi, and (be directed to Wei Luofeini
BRAF mutation inhibitor) combination it is better【17,18】.Find HSF1 as being in researchs such as Tang Zijian in 2016
Before MEK direct downstream target, ERK is considered as MEK unique direct downstream target always【19】.Due to tumour
Heterogeneous and polygenic mutation feature, the generation of colorectal cancer regulate and control this classical growth signals with the presence or absence of new signaling molecule
Path needs further research.
Discovered in recent years, tumor metabolic are closely related with tumor development【20-22】.Branched-chain amino acid
(Branched-chain amino acids, BCAA) catabolism is anomaly existed in a variety of diseases of the mankind【23-25】.Side chain
Aminotransferase (Branched-chain amino transferase, BCAT) activity raises extremely in kinds of tumors,
It is glioma, colorectal cancer and the efficiency index of medulloblastoma classification and gene expression characteristicses judgement【26,27】；But
There is a document report, the expression of the BCAT in prostata tissue is higher than the expression in prostate cancer tissue【28】.Therefore,
Effect of the BCAA catabolism in tumour need to be studied further.Branched-chain alpha-keto acid dehydrogenase kinases (Branched-chain α-
Keta acid dehydrogenase kinase, BCKDK) be BCAA catabolism a crucial negative regulation kinases, pass through
The α subunits of phosphorylation branched-chain alpha-keto acid dehydrogenase (Branched-chain α-keta acid dehydrogenase, BCKDH)
Suppress its activity【29】.Research shows BCKDK and Huntington chorea syndrome【30】, maple sugar diabetes【23,31】, with epilepsy
Autism【32】It is and fat【33】It is in close relations but unclear with the relation of tumour.The whether high expression of tumour cell
BCKDK, the regulation for whether participating in growth signals path, how to be conditioned, the effect in tumor development is necessary into one
Step research illustrates.
Bibliography：
1.Torre LA,Bray F,Siegel RL,et al.Global cancer statistics 2012.CA
Cancer J Clin, 2015,65(2):87~108.
2.Siegel RL,Miller KD,Fedewa SA,et al.Colorectal cancer statistics,
2017.CA Cancer J Clin,2017.
3.Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015.CA
Cancer J Clin, 2016,66(2):115~132.
4. national health and Family Planning Committee China Statistical Yearbooks [J] Beijing：China Concord Medical Science University publishes
Society, 2016.
5.Sakakura C,Hagiwara A,Shirahama T,et al.Infrequent activation of
mitogen-activated protein kinase in human colon
cancers.Hepatogastroenterology,1999,46 (29):2831~2834.
6.Lien GS,Lin CH,Yang YL,et al.Ghrelin induces colon cancer cell
proliferation through the GHS-R,Ras,PI3K,Akt,and mTOR signaling pathways.Eur
J Pharmacol,2016, 776:124~131.
7.Wang W,Liu H,Wang S,et al.A diterpenoid derivative 15-
oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell
tumorigenesis.Cell Res,2011,21(5):730~740.
8.Xue X,Ramakrishnan SK,Weisz K,et al.Iron Uptake via DMT1 Integrates
Cell Cycle with JAK-STAT3 Signaling to Promote Colorectal Tumorigenesis.Cell
Metab,2016,24 (3):447~461.
9.Ren SX,Cheng AS,To KF,et al.Host immune defense peptide LL-37
activates caspase-independent apoptosis and suppresses colon cancer.Cancer
Res,2012,72(24):6512~6523.
10.Van Cutsem E,Cervantes A,Nordlinger B,et al.Metastatic colorectal
cancer: ESMO Clinical Practice Guidelines for diagnosis,treatment and follow-
up.Ann Oncol,2014, 25(3):1~9.
11.Yuan Z,Shin J,Wilson A,et al.An A13 repeat within the 3'-
untranslated region of epidermal growth factor receptor(EGFR)is frequently
mutated in microsatellite instability colon cancers and is associated with
increased EGFR expression.Cancer Res,2009,69(19):7811~7818.
12.Zhang X,Nagahara H,Mimori K,et al.Mutations of epidermal growth
factor receptor in colon cancer indicate susceptibility or resistance to
gefitinib.Oncol Rep,2008,19 (6):1541~1544.
13.Rodriguez-Viciana P,Warne PH,Dhand R,et al.Phosphatidylinositol-3-
OH kinase as a direct target of Ras.Nature,1994,370(6490):527~532.
14.Ogino S,Nosho K,Kirkner GJ,et al.CpG island methylator phenotype,
microsatellite instability,BRAF mutation and clinical outcome in colon
cancer.Gut,2009,58(1):90~96.
15.Barault L,Veyrie N,Jooste V,et al.Mutations in the RAS-MAPK,PI(3)K
(phosphatidylinositol-3-OH kinase)signalling network correlate with poor
survival in a population-based series of colon cancers.Int J Cancer,2008,122
(10):2255~2259.
16.Goel A,Arnold CN,Niedzwiecki D,et al.Frequent inactivation of PTEN
by promoter hypermethylation in microsatellite instability-high sporadic
colorectal cancers.Cancer Res, 2004,64(9):3014~3021.
17.Flaherty KT,Robert C,Hersey P,et al.Improved survival with MEK
inhibition in BRAF-mutated melanoma.N Engl J Med,2012,367(2):107~114.
18.Flaherty KT,Infante JR,Daud A,et al.Combined BRAF and MEK
inhibition in melanoma with BRAF V600 mutations.N Engl J Med,2012,367(18):
1694~1703.
19.Tang Z,Dai S,He Y,et al.MEK guards proteome stability and inhibits
tumor-suppressive amyloidogenesis via HSF1.Cell,2015,160(4):729~744.
20.Gill KS,Fernandes P,O'Donovan TR,et al.Glycolysis inhibition as a
cancer treatment and its role in an anti-tumour immune response.Biochim
Biophys Acta,2016,1866 (1):87~105.
21.Pike LS,Smift AL,Croteau NJ,et al.Inhibition of fatty acid
oxidation by etomoxir impairs NADPH production and increases reactive oxygen
species resulting in ATP depletion and cell death in human glioblastoma
cells.Biochim Biophys Acta,2011,1807(6):726~734.
22.Nelson ER,Wardell SE,Jasper JS,et al.27-Hydroxycholesterol links
hypercholesterolemia and breast cancer pathophysiology.Science,2013,342
(6162):1094~1098.
23.Watanabe A,Higashi T,Sakata T,et al.Serum amino acid levels in
patients with hepatocellular carcinoma.Cancer,1984,54(9):1875~1882.
24.S Sonnet D,N O'Leary M,A Gutierrez M,et al.Metformin inhibits
Branched Chain Amino Acid(BCAA)derived ketoacidosis and promotes metabolic
homeostasis in MSUD. Sci Rep,2016,6:28775.
25.Burrage LC,Nagamani SC,Campeau PM,et al.Branched-chain amino acid
metabolism:from rare Mendelian diseases to more common disorders.Hum Mol
Genet,2014, 23(R1):R1~8.
26.Conway ME,Hull J,El Hindy M,et al.Decreased expression of the
mitochondrial bcat protein correlates with improved patient survival in idh-
wt gliomas.Brain Pathol,2016, 26(6):789~791.
27.Mitchell SM,Ross JP,Drew HR,et al.A panel of genes methylated with
high frequency in colorectal cancer.BMC Cancer,2014,14:54.
28.Billingsley KL,Park JM,Josan S,et al.The feasibility of assessing
branched-chain amino acid metabolism in cellular models of prostate cancer
with hyperpolarized[1-(13) C]-ketoisocaproate.Magn Reson Imaging,2014,32(7):
791~795.
29.Chuang JL,Wynn RM,Chuang DT.The C-terminal hinge region of lipoic
acid-bearing domain of E2b is essential for domain interaction with branched-
chain alpha-keto acid dehydrogenase kinase.J Biol Chem,2002,277(40):36905~
36908.
30.Mochel F,Charles P,Seguin F,et al.Early energy deficit in
Huntington disease: identification of a plasma biomarker traceable during
disease progression.PLoS One,2007, 2(7):e647.
31.Beaudet AL.Neuroscience.Preventable forms of autismScience 2012；
338:342-343. Science,2012,338(6105):342~343.
32.Gaia Novarino,Paul El-Fishawy,Hulya Kayserili,et al.Mutations in
BCKD-kinase Lead to a Potentially Treatable Form of Autism with
Epilepsy.Science,2012,338(6105):394~397.
33.Wubetu GY,Utsunomiya T,Ishikawa D,et al.Branched chain amino acid
suppressed insulin-initiated proliferation of human cancer cells through
induction of autophagy. Anticancer Res,2014,34(9):4789~4796.
34.Tso SC,Qi X,Gui WJ,et al.Structurebased design and mechanisms of
allosteric inhibitors for mitochondrial branched-chain alphaketoacid
dehydrogenase kinase.Proc Natl Acad Sci U S A,2013,110(24):9728~9733.
35.Regitnig P,Reiner A,Dinges HP,et al.Quality assurance for
detection of estrogen and progesterone receptors by immunohistochemistry in
Austrian pathology laboratories. Virchows Arch,2002,441(4):328~34.
The content of the invention
The purpose of the present invention is：
It was found that the crucial negative regulation kinase b CKDK in branched-chain amino acid catabolism is diagnosis colorectal cancer, glioma
Important symbol thing, can with its specific antibody by immunohistochemical method detect clinical tumor sample, help colorectal cancer,
Diagnosis, Index for diagnosis and the direction of medication usage of glioma.
What the present invention was realized in：
1. being determined that BCKDK promotes the generation of colon cancer, BCKDK is the tumor markers of colorectal cancer, and it can be applied special
Heterogenetic antibody.
1) BCKDK universal high expression in colorectal cancer cell system：We have detected first 6 plants of colorectal cancer cell systems and
The expression of BCKDK in 1 plant of people's normal intestinal epithelial cell.BCKDK expression quantity is apparently higher than just in colorectal cancer cell
Expression quantity in normal enterocyte HIEC-6.
2) silence BCKDK expression inhibitings tumour growth：We establish the colon carcinoma cell line in high expression BCKDK
BCKDK silenced cells system, observe cell growth.By being tested in cell growth assay, colony formation and nude mouse into knurl
Confirm that silence BCKDK suppresses Growth of Colon Cancer Cells.
3) it is overexpressed BCKDK and promotes JB6l41 cell transformations and low expression BCKDK Growth of Colon Cancer Cells：We are low
The cell line that stable expression BCKDK is established in BCKDK colon cancer cell and JB6l41 cells is expressed, it is real by cell growth
Test to see with colony formation and confirm that BCKDK promotes cell transformation and growth.
4) BCKDK high expression in clinical Colorectal Carcinoma
We have detected 113 colorectal cancers and 110 cancers with BCKDK specific antibodies by immunohistochemical method
BCKDK expression in the tissue of side.As a result find expression of the BCKDK in Colorectal Carcinoma apparently higher than cancer beside organism (P<
0.001)。
2. it is determined that BCKDK overexpression is related to Patients with Colorectal Cancer poor prognosis, can be by detecting BCKDK tables
Up to the clinical colorectal cancer patients prognosis of judgement.
Our the follow-ups life span of above-mentioned 110 colorectal cancer patients, and to the tumor specimen of this some patients
BCKDK expression carries out correlation statistics analysis with the survival of patients time, the results showed that height expression BCKDK colorectal cancer patients
Median survival interval be considerably shorter than low expression patient (P=0.011).
3. the mechanism of action of the generation of BCKDK promotion colorectal cancers, which is determined, is：BCKDK Direct Phosphorylation MEK, activation life
Long signal path --- MEK-ERK signal paths
1) it is overexpressed BCKDK activation MEK and ERK.
2) MEK and ERK phosphorylation that silence BCKDK suppresses.
3) silence BCKDK suppresses MEK the and ERK phosphorylations in nude mouse tumor tissue.
4) co-immunoprecipitation proves that BCKDK can interact in vivo and in vitro with MEK.
5) BCKDK Direct Phosphorylation MEK (S221) in vitro.
6) BCKDK is in dose dependent phosphorylation MEK (S221).
4. BCKDK inhibitor being determined --- benzenebutanoic acid PB suppresses MEK phosphorylations and colorectal cancer growth, and BCKDK is
Treat the potential molecular target of tumour.
Known BCKDK inhibitor benzenebutanoic acid (phenylbutyrate, PB)【34】, suppress in a dose-dependent manner
BCKDK to MEK phosphorylation modification (IP-kinase experiments) and suppress HCT116 cell BCKDK downstream targets p-MEK1/2,
P-ERK and p-BCKDHA levels and Anchorage Independent growth ability.
5. tentative confirmation BCKDK high expression in High Grade Gliomas, related to the classification of glioma
The invention has the advantages that：
1) find that BCKDK promotes colon carcinogenesis first, be the tumor markers of colorectal cancer.
2) find that BCKDK directly activates MEK1 in S221 site phosphorylations, regulates and controls growth signals path --- MEK- first
ERK signal paths.
3) colon cancer tissue sample is detected on a large scale with BCKDK antibody first, and find that BCKDK is high in colon cancer tissue
Expression and negatively correlated with patient survival, BCKDK molecules and its antibody can be applied to the early diagnosis of tumour, prognosis prediction and swollen
Knurl target therapeutic agent screens.
4) find that BCKDK expression is related to the classification of glioma first.
5) provide first using branched-chain alpha-keto acid dehydrogenase kinases (BCKDK) as biomarker identify colorectal cancer,
The method of High Grade Gliomas patient, this method can include following steps：Tissue samples are gathered from patient, detection determines
BCKDK expression in tissue samples, and identify that colorectal cancer and glioma subgroup are suffered from by BCKDK expressions
Person.Wherein, if it find that the BCKDKK in patient tissue sample is overexpressed, then the overall survival of these patients is just very poor
Cake.
Brief description of the drawings
The western blot testing results of Fig. 1 .BCKDK expressions in colorectal cancer cell system.
Fig. 2 are overexpressed influences of the BCKDK to colorectal cancer cell character mutation.
Influences of Fig. 3 silences BCKDK to colorectal cancer cell character mutation.
Influences of Fig. 4 silences BCKDK to nude mice colon carcinoma cell line one-tenth knurl ability.
Fig. 5 .t-BCKDHA overexpression is analyzed with Patients with Colorectal Cancer poor prognosis.
Fig. 6 .p-BCKDHA overexpression is analyzed with Patients with Colorectal Cancer poor prognosis.
Fig. 7 .BCKDK overexpression is analyzed with Patients with Colorectal Cancer poor prognosis.
Fig. 8 .western blot detections are overexpressed MEK the and ERK expressions of BCKDK cells.
Fig. 9 .western blot detect MEK the and ERK expressions of silence BCKDK cells.
Figure 10 silences BCKDK suppresses the testing result of MEK and ERK phosphorylation in nude mouse tumor tissue.
Phase interaction be present between external source is transferred to and expressed in Figure 11 .HEK 293T cells BCKDK albumen and MEK1 albumen
Testing result.
The detection of interaction in Figure 12 .HCT116 cells between the BCKDK albumen and MEK1 albumen of endogenous expression be present
As a result.
The testing result of Figure 13 .IP- kinase assays.
The testing result of Figure 14 cell transfection assays.
Figure 15 .MTS are tested：3200 μM of PB does not have toxic action to normal cell.
Figure 16 .IP- kinase assays：PB suppresses phosphorylation modifications of the BCKDK to MEK in a dose-dependent manner.
Figure 17 cell experiments：PB suppress in a dose-dependent manner HCT116 cell BCKDK downstream targets p-MEK1/2,
P-ERK and p-BCKDHA is horizontal.
Figure 18 Anchorage Independent growth experiments：PB suppresses HCT116 cell growths in a dose-dependent manner.
Figure 19 .BCKDK high expression in High Grade Gliomas tissue.
Embodiment
The present invention is described in detail with reference to embodiments.It should be noted that embodiments of the invention only limit
In the present invention will be described, without restriction effect.Involved relevant test method and other various experiments in embodiment
Operation, is the ordinary skill in the art, and the part being not particularly illustrated in text, one of ordinary skill in the art is referred to
The specifications of various common tool books, scientific and technical literature or correlation before the present patent application day, handbook etc. are practiced.
Embodiment one determines that BCKDK promotes the generation of colon cancer.
1) BCKDK universal high expression in colorectal cancer cell system
6 plants of colorectal cancer cell systems such as in vitro culture DLD1, HCT8, SW480, WiDr, HCT116, HCT15 and 1 plant of people are just
Normal enterocyte, extracts total protein of cell, and Western blot detects BCKDK expression.As a result show：BCKDK is being tied
High expression is generally presented in rectum cancer cell system.(see Fig. 1)
2) establish and be overexpressed BCKDK and silence BCKDK cell lines, observe Lymphocytic phenotype
Using simpleFect (being purchased from Wuhan signal dawn bio tech ltd) by pCMV-c-Flag and pCMV-
BCKDK-Flag eukaryotic expression plasmids are screened with G418, obtained into JB6Cl41 and low expression BCKDK WiDr cells
JB6-Mock, JB6-BCKDK, WiDr-Mock and WiDr-BCKDK stable cell lines.
And tested as follows：(1) cell proliferation experiment：Growth curve is drawn in cell count：By cell dissociation meter 2 ×
105Individual kind enters 10cm wares, is counted respectively in digestion in 24 hours, 48 hours, 72 hours and 96 hours, and generation growth of averaging is bent
Line)；(2) Anchorage Independent Cell transformation test：JB6l41 cells are normal epidermis cell, are easily converted after stimulation, are research
The common model of tumor transformation.Various JB6l41 stable cell lines are divided into by we to be added EGF and is not added with EGF groups, and every group sets three
Multiple holes.First the concentration containing 10%FBS is added for the BME (minimal medium, sigma companies) of 0.5% low melting-point agarose glue
Enter six orifice plates, per hole 3ml, primer is formed after to be solidified；Then cell is added into the gum concentration containing 10%FBS by packet is
In 0.33% BME, fully mix, be uniformly added dropwise on corresponding primer (8000 cells/per hole/1ml), it is placed in after gelling is solid
37 DEG C, 5%CO2Incubator culture 5-10 days, takes pictures, and counts number of cell clones, compares each group difference.As a result show：It is overexpressed
The plastidogenetic clones of BCKDK are obvious big and more (see on Fig. 2).WiDr is the colon tumor cell of BCKDK low expressions, without
EGF, which is stimulated, can also form clone, and growth and the propagation that BCKDK is obviously promoted cell are overexpressed in WiDr cells (see under Fig. 2).
As a result show that being overexpressed BCKDK promotes cell growth and cell transformation, clonality enhancing (P<0.05).
ShMock, shBCKDKs plasmid are transferred to high expression BCKDK HCT116 using the method for slow-virus infection by we
In DLD1 cells, puromycin screens to obtain the HCT116 cell lines of BCKDK silences and DLD1 cell lines.Through Western
Blot identifies that sequence shBCKDK4 and shBCKDK5 silencing efficiency are good (see the insertion figure in Fig. 3 curve maps).HCT116-
ShBCKDK cells and DLD1-shBCKDK silenced cells carry out respectively cell proliferation experiment (cell count drafting growth curve) and
Anchorage Independent Cell transformation test (method is same as above).As a result it is as shown in Figure 3：Silence BCKDK suppresses colorectal cancer cell
Propagation, conversion.Silence BCKDK suppresses growth (the picture left above of cell in HCT116 cells；*,P<0.05) and propagation (upper right
Figure), silence BCKDK suppresses growth (the lower-left figure of cell in DLD1 cells；*,P<0.05) and (bottom-right graph) is bred.
3) confirm that silence BCKDK colon carcinoma cell line one-tenth knurl ability declines by being tested in nude mouse into knurl.
4~6 week old male nude mouses are randomly divided into two groups of shMock and shBCKDK, every group 8.Digestion is got off
Two groups of cells of shMock and shBCKDK4, the side of mouse, every injection cell number 3 × 10 are subcutaneously injected into respectively6It is individual.Wait to swell
Knurl grows into about 10mm3During left and right, start to measure the length (surveying three times) of tumour every other day, and calculate its volume, calculate public
Formula is：Volume=0.52 (length × width × height).Average and generate tumor volume change curve.Reach in gross tumor volume
1000mm3When put to death mouse, peel off tumor mass, then fixed with paraffin, embedded, and cut into slices, to be ready for use on haematoxylin and Yihong (H＆
E) dye, immunohistochemical staining analysis.As a result Fig. 4 is seen：ShBCKDK groups tumor growth rate is slow compared with shMock groups, the time
Point tumor size (Fig. 4 right) (P smaller than shMock groups<0.05), finally into knurl volume again smaller than shMock groups (Fig. 4 is left).As a result
Show that silence BCKDK suppresses the nude mice one-tenth knurl ability of HCT116 cells.
High expression of the two clear and definite BCKDK of embodiment in colon cancer and related to Patients with Colorectal Cancer poor prognosis.
Strictly screened by pathologist, pick out and make a definite diagnosis colorectal cancer patients clinical tissue sample, organization chip is made, adopts
T-BCKDHA, p-BCKDHA and BCKDK expression are analyzed (according to Remmele point systems with immunohistochemical method【35】
Given a mark, score is less than 3 for feminine gender, is the positive more than or equal to 3), and investigation is tracked to each group patient, given birth to
Deposit phase association analysis.
Immunohistochemical staining (IHC) key step：
1) paraffin section prepared is positioned over 30~60min in 60 DEG C of baking boxs.Dewaxing is cut into slices successively afterwards to washing
It is positioned in dimethylbenzene I, II and dewaxes, each 15min, is then placed into each 10min in absolute ethyl alcohol I, II, then goes downwards to
95%th, each 2min in 80%, 70% ethanol, washing.
2) endogenous peroxydase is blocked：Section is placed in 3%H2O2Middle immersion 15min, washing.
3) antigen retrieval (water-bath repairing method)：Section is placed in 40min in 92~98 DEG C of water-baths, is then placed into advance
In water-bath in preheated reparation liquid, room temperature is cooled to.
4) background is closed：With 5% BSA, 37 DEG C, 30min is incubated.
5) 1 × PBS is rinsed, 5min, 3 times.
6) it is incubated primary antibody：Primary antibody (being diluted with 1%BSA) is incubated after pad, puts in wet box 4 DEG C overnight.
7) rewarming, 1 × PBS rinsings, 5min, 3 times.
8) it is incubated secondary antibody：Secondary antibody is incubated after pad, puts in wet box 37 DEG C, 40min.
9) 1 × PBS is rinsed, 5min, 3 times.
10) it is incubated SABC：Horseradish peroxidase complex is added after pad, puts in wet box 37 DEG C, 40min.
11) 1 × PBS is rinsed, 5min, 3 times.
12) develop the color (DAB-H2O2Colour developing)：Developing time is controlled under mirror, washes color development stopping.
13) redye：Haematoxylin is washed to change indigo plant after redying 2min, then is broken up with 1% acidic alcohol, and washing, which is arrived, becomes indigo plant.
14) mounting：Section is placed in 80%, 95% ethanol after each 3min, is respectively dehydrated in absolute ethyl alcohol I, II
10min；Then being placed on each 20min in dimethylbenzene I, II makes its transparent；Finally mounting is carried out with neutral gum.
1) t-BCKDHA and p-BCKDHA are overexpressed in colorectal cancer, but unrelated with prognosis.
The expression of 117 Colorectal Carcinomas and 91 cancer beside organism detection t-BCKDHA；118 Colorectal Carcinomas and
P-BCKDHA expression is detected in 97 cancer beside organisms.As a result Fig. 5, Fig. 6, BCKDHA expression quantity and the correlation of organization type are seen
Statistical analysis is as shown in intermediate table, BCKDHA expression quantity and the relation such as table below of colorectal cancer patients median survival interval
It is shown.As a result show：Expression of the t-BCKDHA in Colorectal Carcinoma is apparently higher than cancer beside organism (P<, but t- 0.001)
The median survival interval no difference of science of statistics (P=0.112) of BCKDHA height expression group patient and low expression group patient；P-BCKDHA exists
Expression quantity in Colorectal Carcinoma is higher than cancer beside organism (P<0.001), its expression quantity increases the also the median survival time with patient
Phase length is uncorrelated (P=0.368).
2) BCKDK high expression and negatively correlated with prognosis in Colorectal Carcinoma
BCKDK (being BCKDHA kinases immediately upstream) is have detected in 113 colorectal cancers and 110 cancer beside organisms
Expression, and analyze the relation of its expression quantity and organization type and the relation more afterwards with Patients with Colorectal Cancer.BCKDK is being tied
Expression in rectum cancer tissue is apparently higher than cancer beside organism (P<0.001), and height expresses the middle position of BCKDK colorectal cancer patients
Life cycle is shorter than the median survival interval of low expression patient, significant difference (P=0.011) (see Fig. 7).
Embodiment three determines that BCKDK activation MEK-ERK signal paths promote the generation of colorectal cancer
1) it is overexpressed BCKDK activation MEK and ERK.
With tumor transformation associated signal paths research common agents EGF (normal cell 20ng/mL, the ng/ of tumour cell 80
ML) stimulate BCKDK overexpressing cell 15min, extract total protein of cell immediately, with western blot detect MEK, p-MEK,
ERK and p-ERK expression.As a result see Fig. 8, be overexpressed BCKDK JB6Cl41 and WiDr cells in, in spite of with
EGF is stimulated, and p-MEK and p-ERK level are above control group, and EGF is raised after stimulating and become apparent from, and t-MEK and t-ERK
There is no difference between horizontal group.As a result show that BCKDK can activate MEK-ERK signal paths.
2) MEK and ERK phosphorylation that silence BCKDK suppresses.
P-MEK and p-ERK expression change after EGF is similarly observed in BCKDK silenced cells stimulating, method is the same as 1)
(result is shown in Fig. 9).In silence BCKDK HCT116 and DLD1 cells, p-MEK and the horizontal of p-ERK decline, t-MEK and t-
ERK expression does not change, and as a result further confirms BCKDK activation MER-ERK signal paths.
3) silence BCKDK suppresses the phosphorylation of MEK and ERK in nude mouse tumor tissue.
Gross tumor volume reaches 1000mm3After put to death mouse, peel off tumor mass, weigh, record volume size, then tumor mass is divided into
Two parts, portion send pathology department's (Xijing hospital pathology department) fixed, embeds, section, for haematoxylin and Yihong (H＆E) dyeing with
And p-MEK and p-ERK immunohistochemical stainings analysis (right see Figure 10), portion are used to extract histone Western
Blot detections p-MEK and p-ERK expression (left see Figure 10), as a result shows：P- in the tumor tissues of BCKDK silence groups
MEK and p-ERK level is less than control group.
The BCKDK Direct Phosphorylations MEK of example four S221 sites
1) allogenic immune is co-precipitated：By pCMV-Myc-MEK1 plasmids and pCMV-BCKDK-Flag plasmids difference or corotation
Contaminate in HEK293T cells, cell is collected after 48h, total protein is extracted with 1 × IP buffer cell lysis, it is first and mouse
Control IgG and Agarose-A/G beads preincubate 1h, centrifugation, take supernatant, then with the Flag antibody of mouse (or Myc
Antibody) and 4 DEG C of overnight incubations of Agarose-A/G beads, centrifugation, 1 × PBS Beads antibody protein complexs, 1
× IP buffer elute albumen, and western blot analysis (results are carried out with the Flag antibody (or Myc antibody) of rabbit source property
Such as Figure 11), with Flag antibody protein precipitations, Myc signals can be detected；With Myc antibody protein precipitations, it can also detect that Flag believes
Number.As a result show that the Flag-BCKDK and Myc-MEK of exogenous expression have interaction.
2) endogenous immune is co-precipitated：High expression BCKDK HCT116 cells are cracked with 1 × IP buffer, extract total egg
In vain, first with control IgG and Agarose-A/G the preincubate 1h of mouse, centrifuging and taking supernatant, then with same mouse
4 DEG C of overnight incubations of BCKDK antibody and Agarose-A/G, centrifugation, 1 × PBS Beads antibody protein complexs, 1 ×
IP buffer elute albumen, carry out western blot analyses with rabbit endogenous antibody, as a result see Figure 12, sunk with BCKDK antibody
Shallow lake albumen, MEK signals can be detected, showing the BCKDK and MEK of endogenous expression, there is also interaction.
3) IP- kinase assays：Kinases, prokaryotic expression are used as by the use of the BCKDK activated proteins that Flag antibody mediated immunities are co-precipitated out
His-MEK1 (62-393aa) as substrate carry out kinase reaction, with p-MEK1/2 (Ser221) antibody tests MEK1 phosphorus
Acidifying is horizontal, as a result sees Figure 13.Kinase reaction group p-MEK1/2 (Ser221) signal is significantly higher than MEK1-His groups, as a result proves
BCKDK and MEK direct interactions, are MEK1 kinases immediately upstream.
4) cell transfection assays：BCKDK is in dose dependent phosphorylation MEK.The pCMV-BCKDK-Flag of various dose
The pCMV-Myc-MEK cotransfections with same dose stimulate 20min to HEK293T cells, EGF respectively, extract albumen,
Western blot detection p-MEK1/2 (Ser221) expression change.As a result Figure 14, p-MEK1/2 (Ser221) expression are seen
Amount is as the amount of being transferred to of pCMV-BCKDK-Flag plasmids increases and raises.
The BCKDK of embodiment five inhibitor PB suppresses MEK1 phosphorylations and colorectal cancer growth
1) cytotoxicity experiment：According to cell culture step, cell dissociation is got off counting.Cell is planted in 96 orifice plates,
Density is 1000/ hole.Continue to cultivate cell, whether observe cell distribution under the microscope after 24h uniform.Old culture medium is discarded,
The fresh culture (control group not treated with medicaments) of the PB containing various concentrations is separately added into, is 200 μ l per hole cumulative volume.Point
Not by cell at 37 DEG C, 5%CO2Incubator in be incubated 24,48 and 72 hours after, culture medium is discarded, in 96 orifice plates, per hole
90 μ l fresh cultures and 10 μ l MTS one-step method cell viability detection reagents are added, cumulative volume is 100 μ l.At 37 DEG C, 5%
CO2It is incubated 3 hours in incubator.490nm reads absorbance, and statistical chart is plotted according to result, sees Figure 15, PB pairs of 3200 μM
Normal cell does not have toxic action.
2) IP- kinase assays：By the use of the BCKDK activated proteins that Flag antibody mediated immunities are co-precipitated out as kinases, first incubated with PB
30min is educated, the His-MEK1 (62-393aa) for adding prokaryotic expression carries out kinase reaction as substrate, uses p-MEK1/2
(Ser221) antibody test MEK1 phosphorylation level, as a result see that Figure 16, PB suppress BCKDK to MEK's in a dose-dependent manner
Phosphorylation modification.
3) cell experiment：We act on HCT116 cells respectively with the PB of various concentrations, and total protein of cell is extracted after 24h,
Expression horizontal western blot detection BCKDK downstream targets p-MEK1/2, p-ERK and p-BCKDHA, as a result
It is horizontal to see that Figure 17, PB suppress p-MEK1/2, p-ERK and p-BCKDHA in HCT116 cells in a dose-dependent manner.
4) Anchorage Independent growth experiment：Method is the same, as a result sees that Figure 18, PB suppress HCT116 in a dose-dependent manner
Cell growth.
The BCKDK of embodiment six high expression in High Grade Gliomas is related to the classification of glioma
The patients with gliomas surgery excision sample of different stage is collected, extracts histone, western blot detections
BCKDK is expressed.As a result Figure 19 is seen, the high expression of BCKDK in high level samples of human glioma (III level and IV levels).
Claims (2)
1. application of the branched-chain alpha-keto acid dehydrogenase kinases (BCKDK) as biomarker in tumor diagnosis kit is prepared.
2. application as claimed in claim 1, it is characterised in that：The tumour is colorectal cancer and glioma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710348306.XA CN107356461A (en) | 2017-05-17 | 2017-05-17 | Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710348306.XA CN107356461A (en) | 2017-05-17 | 2017-05-17 | Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107356461A true CN107356461A (en) | 2017-11-17 |
Family
ID=60271886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710348306.XA Pending CN107356461A (en) | 2017-05-17 | 2017-05-17 | Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107356461A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444277A (en) * | 2018-10-29 | 2019-03-08 | 郑州大学第附属医院 | Application of the metabolic markers in terms of preparing diagnosis of glioma kit |
| CN111635937A (en) * | 2020-06-29 | 2020-09-08 | 江苏省中医院 | Application of ASS1 or BCKDK inhibitor in preparation of medicine for treating ulcerative colitis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105814037A (en) * | 2013-12-17 | 2016-07-27 | 默克专利股份公司 | N1-(3,3,3-trifluoro-2-hydroxo-2-methylpropionyl)-piperidine derivatives as inhibitors of pyruvate dehydrogenase kinase |
| CN106518809A (en) * | 2015-09-15 | 2017-03-22 | 中国科学院上海药物研究所 | Pyruvate dehydrogenase kinase inhibitor and application thereof |
-
2017
- 2017-05-17 CN CN201710348306.XA patent/CN107356461A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105814037A (en) * | 2013-12-17 | 2016-07-27 | 默克专利股份公司 | N1-(3,3,3-trifluoro-2-hydroxo-2-methylpropionyl)-piperidine derivatives as inhibitors of pyruvate dehydrogenase kinase |
| CN106518809A (en) * | 2015-09-15 | 2017-03-22 | 中国科学院上海药物研究所 | Pyruvate dehydrogenase kinase inhibitor and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| PEIPEI XUE ET.AL: "BCKDK of BCAA Catabolism Cross-talking With the MAPK Pathway Promotes Tumorigenesis of Colorectal Cancer", 《EBIOMEDICINE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444277A (en) * | 2018-10-29 | 2019-03-08 | 郑州大学第附属医院 | Application of the metabolic markers in terms of preparing diagnosis of glioma kit |
| CN109444277B (en) * | 2018-10-29 | 2021-05-11 | 郑州大学第一附属医院 | Application of metabolic marker in preparation of glioma diagnostic kit |
| CN111635937A (en) * | 2020-06-29 | 2020-09-08 | 江苏省中医院 | Application of ASS1 or BCKDK inhibitor in preparation of medicine for treating ulcerative colitis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Huang et al. | Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition | |
| Nevzorova et al. | Overexpression of c-myc in hepatocytes promotes activation of hepatic stellate cells and facilitates the onset of liver fibrosis | |
| Chen et al. | Endogenous hydrogen sulfide regulates xCT stability through persulfidation of OTUB1 at cysteine 91 in colon cancer cells | |
| Liu et al. | Calpain/SHP-1 interaction by honokiol dampening peritoneal dissemination of gastric cancer in nu/nu mice | |
| Mair et al. | Metabolic imaging detects low levels of glycolytic activity that vary with levels of c-Myc expression in patient-derived xenograft models of glioblastoma | |
| Dutta et al. | Combining hyperpolarized real-time metabolic imaging and NMR spectroscopy to identify metabolic biomarkers in pancreatic cancer | |
| Guo et al. | PHLDA1 is a new therapeutic target of oxidative stress and ischemia reperfusion-induced myocardial injury | |
| Xie et al. | MARCH1 encourages tumour progression of hepatocellular carcinoma via regulation of PI3K‐AKT‐β‐catenin pathways | |
| CN109420170A (en) | Novel tumor microenvironment related target TAK1 and its application in inhibition tumour | |
| Guo et al. | Insulin gene enhancer protein 1 mediates glycolysis and tumorigenesis of gastric cancer through regulating glucose transporter 4 | |
| Tong et al. | Loss of tyrosine catabolic enzyme HPD promotes glutamine anaplerosis through mTOR signaling in liver cancer | |
| Zou et al. | Retracted: euxanthone inhibits glycolysis and triggers mitochondria‐mediated apoptosis by targeting hexokinase 2 in epithelial ovarian cancer | |
| Liu et al. | Sperm associated antigen 4 promotes SREBP1-mediated de novo lipogenesis via interaction with lamin A/C and contributes to tumor progression in hepatocellular carcinoma | |
| CN107356461A (en) | Applications of the BCKDK as biomarker in tumor diagnosis kit is prepared | |
| Wang et al. | SEMA5A‐PLXNB3 Axis Promotes PDAC Liver Metastasis Outgrowth through Enhancing the Warburg Effect | |
| Xiong et al. | AKR1C3 and β-catenin expression in non-small cell lung cancer and relationship with radiation resistance | |
| Li et al. | LASP2 is downregulated in human liver cancer and contributes to hepatoblastoma cell malignant phenotypes through MAPK/ERK pathway | |
| Jiang et al. | Ruyiping formula inhibits metastasis via the microRNA-134-SLUG axis in breast cancer | |
| Kwon et al. | Analysis of electric cigarette liquid effect on mouse brain tumor growth through EGFR and ERK activation | |
| Roh et al. | Tenofovir alafenamide alleviates nonalcoholic steatohepatitis in mice by blocking the phosphorylation of AKT in intrahepatic mononuclear phagocytes | |
| Dzhalilova et al. | Morphological and molecular-biological features of glioblastoma progression in tolerant and susceptible to hypoxia Wistar rats | |
| Madonna et al. | In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence | |
| Ren et al. | Overcoming chemoresistance to b-raf inhibitor in melanoma via targeted inhibition of phosphoenolpyruvate carboxykinase1 using 3-mercaptopropionic acid | |
| CN115282282A (en) | Application of PDK 1-targeted glucose metabolism regulation reprogramming combined with metformin in treatment of patients with endometrial cancer complicated with diabetes | |
| Suk et al. | β-HB treatment reverses sorafenib resistance by shifting glycolysis–lactate metabolism in HCC |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171117 |