CN101050473B - Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage - Google Patents
Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage Download PDFInfo
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- CN101050473B CN101050473B CN2007100637876A CN200710063787A CN101050473B CN 101050473 B CN101050473 B CN 101050473B CN 2007100637876 A CN2007100637876 A CN 2007100637876A CN 200710063787 A CN200710063787 A CN 200710063787A CN 101050473 B CN101050473 B CN 101050473B
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Abstract
This invention discloses a test kit for screening anti-tumor drugs for inhibiting tumor cell proliferation. The test kit comprises: recombinant or natural PDCD10 molecules, recombinant or natural Ste20 kinase molecules, and natural or synthetic substrate of Ste20 kinase molecules. This invention also discloses the application of recombinant or natural Ste20 kinase in screening anti-tumor drugs for inhibiting tumor cell proliferation. This invention also provides a method for screening anti-tumor drugs for inhibiting tumor cell proliferation.
Description
Technical field
The present invention relates to the screening reagent box of a kind of inhibition of cell proliferation, antitumor drug, the screening method of a kind of inhibition of cell proliferation, antitumor drug and the Ste20 family kinase purposes in screening inhibition of cell proliferation, antitumor drug.More particularly, relate to by detecting and add medicine to be measured and reorganization or the natural apoptosis factor 10 (Programmed Cell Death 10, PDCD10) after, sterile 20 (Sterile 20, and Ste20) the active variation of family kinase identifies and estimate this medicine whether be inhibition of cell proliferation, antitumor drug.The invention belongs to molecular biosciences system and biochemical field.
Background technology
The invention reside in and use PDCD10 gene and Ste20 family kinase to screen inhibition of cell proliferation, antitumor drug.
PDCD10 gene (also once called after TFAR15) clone is from the TF-1 cell of removing under the somatomedin GM-CSF culture condition, chromosomal localization is 3q26.1, mrna length 50kb, 212 amino acid of encoding, pass through database retrieval, find this gene from nematode to human high conservative, the visible following sequences table of its concrete sequence.And, analyze its aminoacid sequence and do not find signal peptide, stride film district and any known function structural domain.
Research prompting PDCD10 molecule in the past may be relevant with apoptosis and tumour.PDCD10 gene expression dose downward modulation in the atrophy skeletal muscle after the denervation, reorganization PDCD10 albumen can be suppressed to the natural death that fibrocyte ties up under some apoptosis induction condition (as staurosporine, cycloheximide, or TNF-a etc.).These results suggest PDCD10 as anti-apoptosis molecule play a role (Wang Yugang, Liu Hongtao open younger sister Ying, horse big dragon. the cDNA cloning of a human apoptosis relative new gene TFAR15 is collected life science with expressing Chinese science-C, 1999, Vol42,323-329).And then, gene chip Notes of Key Data PDCD10 gene may be transduceed relevantly with tumor signal, causes in the liver cancer Q-GY27703 cell of apoptosis and the cells such as hepatoma cell line HepG2 that IFN γ transforms that at transfer colorectal carcinoma cell line, squamous carcinoma of larynx, the antitumorigenic substance cantharidin of apoptosis of carcinoma of the pancreas, opposing cisplatin induction PDCD10mRNA expresses rise is all arranged.Kamath R.S etc. also find, close PDCD10 with the RNAi specificity and can cause 40% embryonic death at the genomic homologous gene of C.elegans; Shapshak P etc. find that the PDCD10 expression of gene all has obvious rise in this two classes patient when utilizing gene chip that relevant dementia patient's cerebral tissue of AIDS and trisomy dementia patient's the fibroblastic gene expression profile of epithelium is studied; Nearest research finds that again PDCD10 is the angiocavernous Disease-causing gene (Bergametti of brain, F., Denier, C., Labauge, P., Arnoult, M., and et al.Mutations within the Programmed Cell Death 10 Gene Cause CerebralCavernous Malformations (2005) Am J Hum Genet.76,42-51); Above result show PDCD10 with apoptosis-related disease in truly have effect.Yet the function and the mechanism of action of PDCD10 molecule also are not elaborated as yet.
Ste20 is an a kind of budding yeast serine/threonine kinase family, the conservative existence in yeast, nematode, fruit bat and mammals, existing evidence points out this family protein to play a role as the MAP4K molecule of MAPK signal transduction pathway upstream, and then the apoptosis, propagation, cellular form and the cytoskeleton that influence cell are reset (Ippeita Dan, NorinobuM.Watanabe and Akihiro Kusumi.The Ste20 group kinases as regulators of MAP kinasecascades.TRENDS in Cell Biology (2001) 11; 220-229).The MAPK path is the most classical intracellular signal transduction pathway, extensively is present in the mammalian cell, and that finds at present has topmost three kinds of MAPK paths, comprises ERK, JNK and p38 path.Various physiological processes such as wherein ERK MAPK path participation cell growth, growth, division and intercellular function are synchronous, and in pathologic processes such as malignant transformation of cells, play an important role.More and more evidences shows that ERK MAPK path is working aspect pathology generation, development and the carcinogenic behavior of human cancer.Unusual tyrosine phosphorylation acts on has all played the part of the role in the numerous disease mechanism, for example cancer, diabetes cause diseases such as retinopathy, rheumatic arthritis and Parkinson's disease.Thereby current many new drugs of developing are target with arrestin matter kinases just.
Mammals Ste20 sample protein kinase 4 (Mammalian Ste20-like Protein Kinase 4, MST4, once be named as MASK) be Ste20 kinases family member, the analysis of the Northern marking shows the MST4 wide expression, this assignment of genes gene mapping is in the Xq26 zone of disease related gene enrichment.The report prompting is arranged, and in cytoskeleton rearrangement, form generation, apoptosis and other various cell incidents, MST4 plays a role by the MAPK signal transduction pathway.Existing evidence prompting MST4 influences the growth and conversion (the Jei-Liang Lin of cell by the ERK path of regulating the non-dependence of Ras/Raf, Hua-Chien Chen, Hsin-I Fang, Dan Robinson, Hsing-Jien Kung and Hsiu-Ming Shih.MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulatingERK pathway.Oncogene (2001) 20,6559-6569).Recent research proves that by the combination of Golgi's organs stromatin GM130, (also be named as STK25, MST3) these two kinds of Ste20 kinases are positioned on the Golgi's organs for MST4 and YSK1.GM130 is a kind of turntable albumen, is the kinase whose activating molecules of MST, with combining of GM130 MST4 and YSK1 is activated.Activatory YSK1 phosphorylation 14-3-3 ζ and other may move relevant downstream target molecule with normal cell, and MST4 plays a role by unknown approach.Also have research prompting MST4 in neoplastic process, to play a role, the expression of MST4 in tumor of prostate and prostate cancer cell line takes place relevant with the AR state with tumour, crossing expression MST4 can cause non-dependence growth of cell grappling and tumour to take place, these find to support this possibility, promptly MST4 can regulate signal transduction (Victoria Sung in the prostate cancer progression, Wen Luo, Dapeng Qian, Isabelle Lee, Bahiia Jalla, and Mikhail Gishizky.The Ste20 Kinase MST4 Plays a Role in Prostate CancerProgression.CANCER RESEARCH (2001) 63,3356-3363).Although the regulation mechanism of MST4 and distribution are not illustrated as yet, kinase whose phosphorylation of Mammals Ste20 sample and binaryization can be regulated the existing report of transhipment phenomenon that shuttles back and forth of cell caryoplasm.In addition, another member MST3 of Ste20 kinases family, may comprise a two-way nuclear localization signal (amino-acid residue 278-292) at the C of its kinase domain end, this section sequence is high conservative between MST3 and MST4, and these two molecules homology in this kinases family is the highest.
The different plant species homology comparison synoptic diagram of present known PDCD10 gene opening code-reading frame sequence, PDCD10 aminoacid sequence and STE20 family kinase molecule MST4 is as follows respectively:
1.PDCD10 gene opening code-reading frame sequence
Atgaggatgacaatggaagagatgaagaatgaagctgagaccacatccatggtttctatgcccctctatgcagt
catgtatcctgtgtttaatgagctagaacgagtaaatctgtctgcagcccagacactgagagccgctttcatcaaggctg
aaaaagaaaatccaggtctcacacaagacatcattatgaaaattttagagaaaaaaagcgtggaagttaacttcacgga
gtcccttcttcgtatggcagctgatgatgtagaagagtatatgattgaacgaccagagccagaattccaagacctaaac
gaaaaggcacgagcacttaaacaaattctcagtaagatcccagatgagatcaatgacagagtgaggtttctgcagaca
atcaaggatatagctagtgcaataaaagaacttcttgatacagtgaataatgtcttcaagaaatatcaataccagaaccg
cagggcacttgaacaccaaaagaaagaatttgtaaagtactccaaaagtttcagtgatactctgaaaacgtattttaaag
atggcaaggcaataaatgtgttcgtaagtgccaaccgactaattcatcaaaccaacttaatacttcagaccttcaaaact
gtggcctga
2.PDCD10 aminoacid sequence
MRMTMEEMKNEAETTSMVSMPLYAVMYPVFNELERVNLSAAQTLRAA
FIKAEKENPGLTQDIIMKILEKKSVEVNFTESLLRMAADDVEEYMIERPEPEF
QDLNEKARALKQILSKIPDEINDRVRFLQTIKDIASAIKELLDTVNNVFKKYQ
YQNRRALEHQKKEFVKYSKSFSDTLKTYFKDGKAINVFVSANRLIHQTNLIL
QTFKTVA
3.STE20 the different plant species homology of family kinase molecule MST4 comparison synoptic diagram
Protein?Acc. Gene Organism
NP?057626.2? MST4 H.sapiens
XP?521266.2 LOC465862 P.troglodytes
XP?549261.2 LOC492140 C.familiaris
NP?598490.1 2610018G03Rik M.musculus
XP?229143.4 RGD1563568_pred R.norvegicus
icted
XP?420219.1 RCJMB04_2k24 G.gallus
NP?057626.2 --------------------------------------------
XP?521266.2 1---------MAGKSGGPPTPRLSVILPLARP-------------22
XP?549261.2 1--------MFITTKTTEKTPSWE---DDGDKMMELR--------25
NP?598490.1 --------------------------------------------
XP?229143.4 1MGRKKCKNGSIVIRSVITVVSVEIGLAEGNRLSDNTIVKSGGAK?44
XP?420219.1 1--MEKAGKLKLPESPTVTTEGVGNDGPPSAPQRSHVCKRNGSVK?42
NP?057626.2 --------------------------------------------
XP?521266.2 23 -----------GSQERLPTLPKFQLKSSPRLPVPQP-----RTP 50
XP?549261.2 26 -----------TSVRLSATWPRRGRTLTPRVTETPP-----FPP 53
NP?598490.1 --------------------------------------------
XP?229143.4 45 QTFATPDLWAATSTLASQACLQRGRVRSSGSPSYPPPHSRSRKL 88
XP?420219.1 43 LE------RNPTYRYKATDDQRAGGVASPGASHSHPQERMPCHP 80
NP?057626.2 --------------------------------------------
XP?521266.2 51 PCNVAASGTPSYPQGFPNS---SILFPGP--------------- 76
XP?549261.2 54 LAGPS---AGPPSPRLLLS--ACGGSPPP-----RRP------- 80
NP?598490.1 --------------------------------------------
XP?229143.4 89 FCSFS---RTSYVPGFSIP--AFCCSPQP-----LRAPCFANSD 122
XP?420219.1 81 LCVPSPHPEHSLAPRTARSCQGWVCSTSSSIQLSLTAPELGDYG 124
NP?057626.2 --------------------------------------------
XP?521266.2 77?--AVSQVSGSPGSPLRVPG------------------------E 94
XP?549261.2 81?--SRSPAPRGAPALLTPPG--------------------GRAEA 102
NP?598490.1 --------------------------------------------
XP?229143.4 123?YFSQPGLQLEASAPLTPPG--------------------SGVEA 146
XP?420219.1 125?CTSMGTQGQNEIGDLSPPGELEGEEITLQGFSLVAAGWLQNLLA 168
NP?057626.2 --------------------------------------------
XP?521266.2 95?SPASQTPATPAKPLR----------------------------- 109
XP?549261.2 103?GPTAEPPPTASRAQRAGGLLGR---------------------- 124
NP?598490.1 --------------------------------------------
XP?229143.4 147?GPTAEPPPAASQPRRSGGLLGR---------------------- 168
XP?420219.1 169?GPHVTARAAKAELGHGGHSPGRRASSEQGDNLAKGTNFPLRLPV 212
NP?057626.2 --------------------------------------------
XP?521266.2 110?---CLEPWLPRHRPAAGRGGEAQRSG------------------ 132
XP?549261.2 125?----LHLLFLKRRGAEERHHSSPGPG------------------ 146
NP?598490.1 --------------------------------------------
XP?229143.4 169?----LHLLFLKRRGAAEPHHWSPGPS------------------ 190
XP?420219.1 213?KAMFVTFVLWEEFGCVCTYKSSPGSGWMMTVWQGEGKEEKTDSK 256
NP?057626.2 --------------------------------------------
XP?521266.2 --------------------------------------------
XP?549261.2 --------------------------------------------
NP?598490.1 --------------------------------------------
XP?229143.4 --------------------------------------------
XP?420219.1 257 KIKDVIEKDLQALAAQENPELEKAFYSQFFWWHQAAVTVLGTRG 300
NP?057626.2 --------------------------------------------
XP?521266.2 133 ------------------------------------ITRAQVP- 139
XP?549261.2 147 --------------------------------------YRH--- 149
NP?598490.1 --------------------------------------------
XP?229143.4 191 --------------------------------------HCR--- 193
XP?420219.1 301 TSTLCPGTHLCARDTAVRALWLCLWVLRSVAQGLQGTRHCHKAP 344
NP?057626.2 --------------------------------------------
XP?521266.2 140 ---ATTTHSARRSGRGAAAEAAASAAG----------------- 163
XP?549261.2 150 ---TVRPAFGRRSGGGGGGNSGGRAPE----------------- 173
NP?598490.1 --------------------------------------------
XP?229143.4 194 ---THSSAFRKRSGTATVRKLGGRASE----------------- 217
XP?420219.1 345 EGDTALDAEARAAAAAPPHKAGAETEEAAAAQQPPPPRPPPPAR 388
NP?057626.2 --------------------------------------------
XP?521266.2 164 ----------GRQKGPVRKAWEGRRTTPGGR------------- 184
XP?549261.2 174 -----------RPRSKSLG---GPPSYPRRR------------- 190
NP?598490.1 --------------------------------------------
XP?229143.4 218 -----------RPRSRSLG---ELQSYPRRR------------- 234
XP?420219.1 389 GPTWRSEPSPGRAESRHPGSTEQPPSPLREREGGHGQGRRGEPG 432
NP?057626.2 --------------------------------------------
XP?521266.2 185 ------------SQSEPKAPPPQKRSEAAFAS------------ 204
XP?549261.2 191 ------------SQSEPKAPPPQEQCRAVFAF------------ 210
NP?598490.1 --------------------------------------------
XP?229143.4 235 ------------SQSVPQEPPPQKQREVAAAS------------ 254
XP?420219.1 433 GHGRAALVDPFPPASRSSAPAPRPQRGPAAAQQQGGREEGAAGG 476
NP?057626.2 1 -------MAHSPVAVQVPGMQNNIADPEELFTKLERIGKGSFGE 37
XP?521266.2 205 -------MAHSPVAVQVPGMQNKIXDXEELFTKLERIGKGSFGE 241
XP?549261.2 211 -------MAHSPVAVQVPGMQNNIADPEELFTKLERIGKGSFGE 247
NP?598490.1 1 -------MAHSPVAVQVPGMQNNIADPEELFTKLERIGKGSFGE 37
XP?229143.4 255 -------MAHSPVAVQVPGMQNNIADPEELFTKLERIGKGSFGE 291
XP?420219.1 477 ARPPQPAMAHSPVAVQVPGMQNHRADPEELFTKLERIGKGSFGE 520
NP?057626.2 38 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSY 81
XP?521266.2 242 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSY 285
XP?549261.2 248 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSY 291
NP?598490.1 38 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSY 81
XP?229143.4 292 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSY 335
XP?420219.1 521 VFKGIDNRTQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSPY 564
NP?057626.2 82 VTKYYGSYLKGSKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 125
XP?521266.2 286 VTKYYGSYLKGSKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 329
XP?549261.2 292 VTKYYGSYLKGSKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 335
NP?598490.1 82 VTKYYGSYLKGSKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 125
XP?229143.4 336 VTKYYGSYLKGSKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 379
XP?420219.1 565 VTKYYGSYLKGTKLWIIMEYLGGGSALDLLRAGPFDEFQIATML 608
NP?057626.2 126 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 169
XP?521266.2 330 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 373
XP?549261.2 336 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 379
NP?598490.1 126 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 169
XP?229143.4 380 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 423
XP?420219.1 609 KEILKGLDYLHSEKKIHRDIKAANVLLSEQGDVKLADFGVAGQL 652
NP?057626.2 170 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 213
XP?521266.2 374 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 417
XP?549261.2 380 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 423
NP?598490.1 170 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 213
XP?229143.4 424 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 467
XP?420219.1 653 TDTQIKRNTFVGTPFWMAPEVIQQSAYDSKADIWSLGITAIELA 696
NP?057626.2 214 KGEPPNSDMHPMRVLFLIPKNNPPTLVGDFTKSFKEFIDACLNK 257
XP?521266.2 418 KGEPPNSDMHPMRVLFLIPKNNPPTLVGDFTKSFKEFIDACLNK 461
XP?549261.2 424?KGEPPNSDMHPMRVLFLIPKNNPPTLVGDFTKSFKEFIDACLNK 467
NP?598490.1 214?KGEPPNSDMHPMRVLFLIPKNNPPTLIGDFTKSFKEFIDACLNK 257
XP?229143.4 468?KGEPPNSDMHPMRVLFLIPKNNPPTLVGDFTKSFKEFIDACLNK 511
XP?420219.1 697?KGEPPNSDMHPMRVLFLIPKNNPPTLLGEFSKPFKEFIDACLNK 740
NP?057626.2 258?DPSFRPTAKELLKHKFIVKNSKKTSYLTELIDRFKRWKAEGHSD 301
XP?521266.2 462?DPSFRPTAKELLKHKFIVKNSKKTSYLTELIDRFKRWKAEGHSD 505
XP?549261.2 468?DPSFRPTAKELLKHKFIVKNSKKTSYLTELIDRFKRWKAEGHSD 511
NP?598490.1 258?DPSFRPTAKELLKHKFIVKNSKKTSYLTELIDRFKRWKAEGHSD 301
XP?229143.4 512?DPSFRPTAKELLKHKFIVKNSKKTSYLTELIDRFKRWKAEGHSD 555
XP?420219.1 741?DPTFRPTAKELLKHKFIMKNAKKTSYLTELIDRFKRWKAEGHSS 784
NP?057626.2 302?DESDSEGSDSESTSRENNTHPEWSFTTVRKKPDPKKVQNGAEQD 345
XP?521266.2 506?DESDSEGSDSESTSRENNTHPEWSFTTVRKKPDPKKVQNG--AV 547
XP?549261.2 512?DESDSEGSDSESTSRENNTHPEWSFTTVRKKPDPKKLQNGAEQD 555
NP?598490.1 302?EESDSEGSDSESSSRESNPHPEWSFTTVRKKPDPKKLQNGEEQD 345
XP?229143.4 556?EESDSEGSDSESSSRESNTHPEWSFTTVRKKPDPKKLQNGEEQD 599
XP?420219.1 785?DESDSDGSDSESSNKENNSHPEWSFTTVRKKPDAKKLQNGTDQD 828
NP?057626.2 346?LVQTLSCLSMIITPAFAELKQQDENNASRNQAIEELEKSIAVAE 389
XP?521266.2 548?FSRRIPLRNLLLAFVSYGPREPHQTYVKINNA------------ 579
XP?549261.2 556?LVQTLSCLSMIITPAFAELKQQDENNASRNQAIEELEKSIAVAE 599
NP?598490.1 346?LVQTLSCLSMIITPAFAELKQQDENNASRNQAIEELEKSIAVAE 389
XP?229143.4 600?LVQTLSCLSMIITPAFAELKQQDENNASRNQAIEELEKSIAVAE 643
XP?420219.1 829?LVKTLSCLTMIITPVFAELKQQDTNNASRKKAIEELEKSINMAE 872
NP?057626.2 390?AACPGITDKMVKKLIEKFQKCSADESP 416
XP?521266.2 ---------------------------
XP?549261.2 600?AACPGITDKMVKKLIEKFQKCSADESP 626
NP?598490.1 390?TACPGITDKMVKKLIEKFQKCSADESP 416
XP?229143.4 644?AACPGITDKMVKKLIEKFQKCSADESP 670
XP?420219.1 873?ATCPGITDKMVKKLMEKFQK------- 892
Summary of the invention
The purpose of this invention is to provide the screening reagent box of a kind of inhibition of cell proliferation, antitumor drug, and set up the screening method of a kind of inhibition of cell proliferation, antitumor drug and the purposes of a kind of Ste20 family kinase in screening inhibition of cell proliferation, antitumor drug is provided.
At the foregoing invention purpose, the contriver with described tumour cell for example human prostata cancer PC-3 cell be target cell, make target molecule with the Ste20 family kinase, detect to add reorganization or natural PDCD10 and treating under the screening of medicaments effect, by treating screening of medicaments the Ste20 family kinase is expressed or/and suitable inhibition of cell proliferation, antitumor drug are screened in active influence.
Concrete, principle of work of the present invention is as follows: with PDCD10 molecule and Ste20 family kinase molecular mixing, with ATP is the phosphodonor of tyrosine phosphorylation reaction, after the natural substrate of Ste20 family kinase molecule or synthetic substrate are by phosphorylation, antibody with anti-this substrate phosphorylation form reacts, add two anti-and chromogenic substrates of enzyme labelling again, detect light absorption value to detect kinase activity.Utilize this test kit, if kinase whose activity changes after adding different pharmaceutical, then this medicine might be used for oncotherapy.
The present inventor tests by yeast two-hybrid, finds the interaction of MST4 and YSK1 in PDCD10 and the Ste20 family kinase, and further confirms through co-immunoprecipitation, the burnt common positioning experiment of cell inner laser copolymerization.Process is a series of to experimental results show that PDCD10 and MST4 interaction can promote the propagation and the conversion of cell by adjusting ERK MAPK path, and the expression of inhibition PDCD10 or MST4 can reduce the multiplication capacity of tumor cell line.Through extensive and deep research, the present inventor further finds, independent mistake is expressed the propagation that PDCD10 or MST4 can promote cell, and the common short proliferation function of expressing of crossing was better than far away to cross separately and expresses one of them molecule.By the siRNA that inhibition PDCD10 of transfection in tumor cell line or MST4 express, can suppress the propagation of cell.The interaction of PDCD10 and MST4 can activate ERK MAPK path, suppresses then can not activate ERK MAPK path after PDCD10 or MST4 express.The present inventor also finds simultaneously, though be endogenous or the PDCD10 that expresses can be at the kinase activity of external increase MST4, the activation of wherein crossing the PDCD10 that expresses is more than three times of contrast, and endogenous PDCD10 when being suppressed the kinase activity of MST4 slightly descend.Because the Ste20 family kinase might play a role as the MAP4K molecule of MAPK signal transduction pathway upstream, therefore can infer, can suppress the material of PDCD10, can suppress the propagation of cell, and can become the novel drugs of treatment tumour the activation of Ste20 family kinase.
Based on these results, the invention provides following technical scheme.
On the one hand, the invention provides a kind of combined prod that is used to screen inhibition cell proliferation or antitumor drug, the specific form of this combined prod can be detection kit.
Its principle is by detecting under the PDCD10 effect as previously mentioned, treat that screening of medicaments expresses or/and active influence can suppress the material of PDCD10 to the activation of Ste20 family kinase thereby filter out, promptly the Ste20 family kinase, be used to suppress cell proliferation, the medicine of treatment tumour.
This combined prod comprises following integral part: reorganization or natural PDCD10 molecule, reorganization or natural Ste20 family kinase molecule, the natural substrate of Ste20 family kinase molecule or synthetic substrate.
As claimed in claim 1 being used to screened the combined prod that suppresses cell proliferation or medicine for treating tumor thing, it is characterized in that its specific form is a detection kit.
Preferably, this combined prod also comprises: the natural substrate of ATP, anti-Ste20 family kinase molecule or the antibody of synthetic substrate phosphorylation form.Perhaps, it also comprises two anti-, the chromogenic substrate that is selected from kinase reaction damping fluid, enzyme labelling, in other reaction buffer one or more.
The described combined prod that is used to screen inhibition cell proliferation or medicine for treating tumor thing of arbitrary as described above claim is characterized in that described reorganization or natural Ste20 family kinase molecule is reorganization or natural MST4 molecule.
Preferably, the natural substrate of described Ste20 family kinase molecule or synthetic substrate are synthetic substrate Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) biotin labeling polypeptide.
On the other hand, the invention provides Ste20 family kinase a kind of reorganization or natural and wait to screen the purposes that suppresses in cell proliferation or the antitumor drug in screening.
Preferably, wherein said inhibition cell proliferation or antitumor drug comprise that albumen, polypeptide, natural drug, chemical synthetic drug are with Chinese herbal medicine extract.
The third aspect, the present invention also provides a kind of being used to screen the method that suppresses cell proliferation or antitumor drug, and described method may further comprise the steps:
Obtain PDCD10 molecule reorganization or endogenous expression;
Obtain Ste20 kinases family molecule reorganization or endogenous expression;
With the PDCD10 molecule that obtains and Ste20 kinases family molecule in external and ATP, the effect substrate of Ste20 kinases family molecule, material hybrid reaction to be checked;
Termination reaction, the expression that detects the Ste20 family kinase is or/and activity.
Provide a kind of being used to screen the detection kit that suppresses cell proliferation or antitumor drug in the embodiment preferred of the present invention, specifically comprise:
The reorganization or natural PDCD10 molecule
The reorganization or natural Ste20 family kinase molecule
3.Ste20 the natural substrate of family kinase molecule or synthetic substrate
4.ATP
5. the antibody of the natural substrate of anti-Ste20 family kinase molecule or synthetic substrate phosphorylation form
6. kinase reaction damping fluid
7. two of enzyme labelling is anti-
8. chromogenic substrate
9. other reaction buffer
Provide a kind of being used to screen the detection kit that suppresses cell proliferation or antitumor drug in the preferred embodiment of the present invention, specifically comprise:
The reorganization or natural PDCD10 molecule
The reorganization or natural MST4 molecule
3.MST4 natural substrate or synthetic substrate, as synthetic substrate Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) biotin labeling polypeptide
4.ATP
5. kinase reaction damping fluid
6. the antibody of anti-MST4 natural substrate or synthetic substrate phosphorylation form
7. two of enzyme labelling is anti-
8. chromogenic substrate
9. other reaction buffer
Provide a kind of being used to screen the method that suppresses cell proliferation or antitumor drug in the embodiment preferred of the present invention, may further comprise the steps:
1. obtain PDCD10 molecule reorganization or endogenous expression, can be by following any one approach:
1) the prokaryotic expression PDCD10 molecule of acquisition reorganization: in intestinal bacteria, culturing bacterium is extracted prokaryotic expression PDCD10 molecule from expression strain with the transfection of protokaryon PDCD10 expression plasmid.
2) the eukaryotic expression PDCD10 molecule of acquisition reorganization: in clone, culturing cell obtains eukaryotic expression PDCD10 molecule from transfectional cell with the transfection of eucaryon PDCD10 expression plasmid.
3) obtain the PDCD10 molecule of endogenous expression, from tissue that endogenous PDCD10 expresses or cell are arranged, obtain the PDCD10 molecule of endogenous expression.
2. obtain Ste20 kinases family molecule reorganization or endogenous expression, can be by following any one approach:
1) the prokaryotic expression Ste20 kinases family molecule of acquisition reorganization: in intestinal bacteria, culturing bacterium is extracted protokaryon Ste20 kinases family molecule from expression strain with the transfection of protokaryon Ste20 kinases family molecule expression plasmid.
2) the eukaryotic expression Ste20 kinases family molecule of acquisition reorganization: in clone, culturing cell obtains eukaryotic expression Ste20 kinases family molecule from transfectional cell with the transfection of eucaryon Ste20 kinases family molecule expression plasmid.
3) obtain the Ste20 kinases family molecule of endogenous expression, from tissue that endogenous Ste20 kinases family molecule expresses or cell are arranged, obtain the Ste20 kinases family molecule of endogenous expression.
3. will mix with ATP, the effect substrate of Ste20 kinases family molecule, material to be checked external by PDCD10 molecule and the Ste20 kinases family molecule that above arbitrary approach obtains, in the kinase reaction damping fluid, hatched 30 minutes for 25 ℃, with equal-volume 50mM EDTA termination reaction.
4.ELISA the reaction: reaction product be diluted at 1: 4 the bag be cushioned liquid (0.05M NaHCO3, pH 8.6) and also the bag by a pretreated elisa plate, hatched 1 hour for 37 ℃.Abandon coating buffer, wash twice with PBST (PBS/0.05%Tween20), add foetal calf serum (be diluted at 1: 100 PBST) room temperature sealing 30 minutes, the antibody (be diluted at 1: 1000 1%BSA/PBST) that adds the phosphorylation form of anti-Ste20 family kinase molecule substrate, every hole 100 μ L were hatched 1 hour for 37 ℃.Wash five times, elisa plate adds two anti-(be diluted at 1: 10000 1%BSA/PBST) of HRP mark, and every hole 100 μ l were hatched 1 hour for 37 ℃.Wash five times, every hole adds 50 μ l tmb substrates.Color development at room temperature is 450nm inspection light absorption value after 10 minutes.
Provide a kind of being used to screen the method that suppresses cell proliferation or medicine for treating tumor thing in the preferred embodiment of the present invention, may further comprise the steps:
1. obtain PDCD10 molecule recombinant eukaryon expression or endogenous expression, can be by following any one approach:
1) obtain the eukaryotic expression PDCD10 molecule of reorganization: with the transfection of eucaryon PDCD10 expression plasmid in HeLa clone, culturing cell, after 48 hours, lysing cell was also handled 1 hour with Protein G magnetic bead, hatched 2 hours with 4 ℃ of anti-PDCD10 monoclonal antibodies then, the PDCD10 in the immunocomplex of acquisition is mainly the eukaryotic expression PDCD10 molecule of reorganization.
2) the PDCD10 molecule of acquisition endogenous expression, the HeLa cell of transfection PDCD10 siRNA or not transfection siRNA, cultivate after 48 hours, lysing cell was also handled 1 hour with Protein G magnetic bead, hatched 2 hours with 4 ℃ of anti-PDCD10 monoclonal antibodies then, the PDCD10 in the immunocomplex of acquisition is the PDCD10 molecule of endogenous expression.
2. reorganization MST4 is from HTScan
TMMst4 kinases assay kit (Cell signaling, Danvers, MA)
3. will be by above arbitrary approach the PDCD10 molecule that obtains and the MST4 molecule of recombinating at external and effect substrate Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) biotin labeling polypeptide (Cell signaling ATP, MST4, Danvers, MA), material to be checked mixes, in the kinase reaction damping fluid, hatched 30 minutes for 25 ℃, with equal-volume 50mM EDTA termination reaction.
4.ELISA the reaction: reaction product be diluted at 1: 4 the bag be cushioned liquid (0.05M NaHC03, pH 8.6) and also the bag by a pretreated elisa plate, hatched 1 hour for 37 ℃.Abandon coating buffer, wash twice with PBST (PBS/0.05%Tween20), add foetal calf serum (be diluted at 1: 100 PBST) room temperature sealing 30 minutes, an anti-(Cell signaling who adds anti-phosphorylation Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) polypeptide, Danvers, MA) (be diluted at 1: 1000 1%BSA/PBST), every hole 100 μ L were hatched 1 hour for 37 ℃.Wash five times, elisa plate adds two anti-(be diluted at 1: 10000 1%BSA/PBST) of HRP mark, and every hole 100 μ l were hatched 1 hour for 37 ℃.Wash five times, every hole adds 50 μ l tmb substrates.Color development at room temperature is 450nm inspection light absorption value after 10 minutes.
The present invention is applicable to the screening of various inhibition cell proliferations or medicine for treating tumor thing, comprises that albumen, polypeptide, natural drug, chemical synthetic drug are with Chinese herbal medicine extract.Advantage of the present invention is easy and simple to handle, quick, accurate.Detect through drug screening kit provided by the invention or drug screening method, if with do not add the contrast for the treatment of screening of medicaments and compare, the material to be checked that screens can reduce the light absorption value of reaction system, illustrate that then this material can suppress the activation of PDCD10 to the Ste20 family kinase, promptly can be used as the drug candidate that suppresses cell proliferation and oncotherapy, further study and use.
Description of drawings
The present invention is further illustrated below in conjunction with accompanying drawing:
Fig. 1 is the expression of results of PDCD10 in kinds of tumor cells system;
Fig. 2 is the co-immunoprecipitation result of PDCD10 and MST4;
Fig. 3 is that PDCD10 and the laser co-focusing ubcellular of MST4 in the Hela cell are total to positioning result;
Fig. 4 is that siRNA suppresses the result that PDCD10 expresses;
Fig. 5 is that the PDCD10 adjusting is expressed MST4 excessively and crossed MTT and the H that expresses MST4 cell proliferation
3-TdR experimental result;
Fig. 6 is that PDCD10 regulates to cross to express MST4 and cross the clone who expresses MST4 cell proliferation and forms experimental result;
Fig. 7 is the result that PDCD10 suppresses the cell anoikis;
Fig. 8 is that siRNA suppresses the result that MST4 expresses;
Fig. 9 is that the siMST4 adjusting is expressed PDCD10 excessively and crossed the MTT experimental result of expressing PDCD10 cell proliferation;
Figure 10 is siMST4 suppresses phenomenon to PDCD10 inductive anoikis the result that influences;
Figure 11 is two luciferase reporter gene systems analysis PDCD10 and the MST4 result to the effect of ERK MAPK path
Figure 12 is PDCD10, MST4 and two-way interaction thereof the Western Blot result to different MAPK path tyrosine phosphorylations;
Figure 13 is that the RNAi of MST4 does not express the inhibition experimental result of PDCD10 cell ERK kinase activity to expressing PDCD10 excessively with crossing;
Figure 14 MST4 vitro kinase activity experimental result.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be not used in but these examples only limit to the present invention is described and limit the scope of the invention.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The expression study of embodiment 1 PDCD10 in kinds of tumor cells system
Culturing cell utilizes TRIZOL according to a conventional method
TMReagent extracts cell total rna, utilizes the synthesizing single-stranded cDNA of Invitrogen Corporation's Super Script II test kit, and PCR detects; Extract the albumen of culturing cell, ordinary method is carried out the SDS-PAGE and the Western marking.
PCR utilizes following PDCD10 special primer:
Upstream primer: ATGAGGATGACAATGGAAGAGATG (SEQ ID NO:1)
Downstream primer: TCAGGCCACAGTTTTGAAGGT (SEQ ID NO:2)
And confidential reference items GAPDH special primer:
Upstream primer: CCACCCATGGCAAATTCCATGGCA (SEQ ID NO:3)
Downstream primer: TCTAGACGGCAGGTCAGGTCAGGTCCACC (SEQ ID NO:4)
Figure 1A is presented on the mRNA level, and PDCD10 has expression in kinds of tumor cells system.Figure 1B is presented at protein level, and PDCD10 has expression in the system of the kinds of tumor cells except that HepG2
The experiment of embodiment 2 yeast two-hybrids
PDCD10 is implemented in the pGBK-RC bait carrier, contain the targeting vector cotransformation yeast Y190 of 1500 known gene libraries relevant with the farsighted star in Shanghai company with apoptosis, propagation and cell cycle, and on the SD/-T-L-H substratum, cultivate, plasmid in the positive yeast clone of gained is extracted, and transfection Escherichia coli, PDCD10 angle target protein comprise MST4 and YSK1.
1 crosses the co-immunoprecipitation of expressing protein
Making up N holds MST4, the YSK1 and the N end that have the c-Myc label to have the PDCD10 of FLAG label in the pCDEF carrier.It is correct that the order-checking identification of dna inserts segment.Adopt the calcium phosphate transfection method with the recon transient transfection of label to the HEK293 cell, transfection usefulness PBS washed cell three times after 48 hours scrapes culturing cell, centrifugal 500g, 5 minutes.Cell mass cracking in cell pyrolysis liquid under 4 ℃ of conditions, and blow and beat repeatedly with syringe needle.Lysate is through centrifugal (20 minutes, 14,000 * g, 4 ℃), and its supernatant liquor mixes with anti-c-Myc of 12.5 μ l or the affine agarose of anti-FLAG M2 and overnight incubation (4 ℃).Centrifugal (5 minutes, 700 * g) collect the immunosorption things, with lysate washing three times (5 minutes, 700 * g), again with 50mM Tris (pH7.5) washed twice that contains 0.1% (w/v) SDS and 150mM NaCl.With the SDS sample-loading buffer wash-out of sample with 60 μ l.Do the Western marking with anti-myc antibody and anti-Flag antibody, detect.Fig. 2 A shows that the PDCD10 of band Flag label and MST4, the YSK1 of band myc label express in the 293T cell, and proving that the MST4 of band myc label or YSK1 can be with the PDCD10 of Flag label to precipitate, the two can combine in mammalian cell in prompting.
The co-immunoprecipitation of 2 endogenous proteins
With 1 * 10
7The HeLa cell suspension is in 0.5ml lysate (50mMTris, pH8,0.4%Nonidet P-40,300mM NaCl, 10mM MgCl
2, 2.5mM CaCl
2) in, contain proteinase inhibitor (no EDTA) and DNase (10U/ul) in the lysate, hatched on ice 10 minutes.Reserve 1/10th volume lysates and be directly used in the Western marking in contrast, all the other carry out co-immunoprecipitation.Carry out co-immunoprecipitation respectively with mouse anti PDCD10 monoclonal antibody and the anti-MST4 antibody of rabbit.Immunoprecipitation was hatched 2 hours with 4 ℃ of Protein G magnetic beads, and (50mMTris, pH 8,150mM NaCl, 5mM MgCl with washings
2, 0.4%Nonidet P-40) give a baby a bath on the third day after its birth time.Bonded albumen carries out SDS-PAGE, the conventional Western marking.Fig. 2 B shows that PDCD10 and MST4 express in the Hela cell, and proof the two existence under physiological condition interacts.
1 mistake is expressed PDCD10 and is expressed the laser co-focusing ubcellular of MST4 in the Hela cell excessively and locate altogether
With pEGFP-C3-PDCD10 and pDsred-N1-MST4 transient transfection cell, cell is laid on cover glass cultivated 24 hours.Before the fluorescence microscope, cell is fixed (room temperature 30 minutes, 3% (wt/vol) PFA), cancellation (10 minutes, 50mM ammonium chloride) and infiltrationization (5 minutes, 0.1% (vol/vol) Triton X-100).All solution prepare with PBS.Cover glass is dipped in and contains 10% (wt/vol) Moviol4-88, and 1 μ g/ml DAPI is among the PBS of 10% (wt/vol) glycerine.Laser confocal microscope is observed.Fig. 3 A and Fig. 3 B are the observation image of the same visual field different multiples, and green is pEGFP-C3-PDCD10, and redness is pDsred-N1-MST4, and fused images shows that the two all is expressed in cytoplasm, and locate altogether.
2 endogenous PDCD10 and the MST4 laser co-focusing ubcellular in the Hela cell is located altogether
Cell is laid on cover glass to be cultivated 24 hours.Before the fluorescence microscope, cell is fixed, cancellation and infiltrationization processing.Successively with the anti-rabbit igg dyeing of the ox of the sheep anti-mouse igg of anti-PDCD10 antibody, FITC mark, anti-MST4 antibody and TRITC mark.Laser confocal microscope is observed.Fig. 3 C is the endogenous protein positioning image, and green is PDCD10, and redness is MST4, shows that the two all is expressed in cytoplasm, and locatees altogether.
The design of the siRNA of 1 PDCD10 below is the sequence of the siRNA that adopted
siPDCD10-1 AGCGUGGAAGUUAACUUCA(SEQ?ID?NO：5)
siPDCD10-2 GGCAAUAAAUGUGUUCGUA(SEQ?ID?NO：6)
GFPsiRNA GCAAGCUGACCCUGAAGUUC(SEQ?ID?NO：7)
Non-silencing?siRNA?UUCUCCGAACGUGUCACGU(SEQ?ID?NO：8)
2 RT-PCR detect the influence that siRNA expresses endogenous PDCD10 mRNA
Utilize TRIZOL
TMReagent extracts cell total rna, utilizes the synthesizing single-stranded cDNA of Invitrogen Corporation's Super ScriptII test kit.PCR utilizes following PDCD10 special primer:
Upstream primer: ATGAGGATGACAATGGAAGAGATG (SEQ ID NO:1)
Downstream primer: TCAGGCCACAGTTTTGAAGGT (SEQ ID NO:2)
And confidential reference items GAPDH special primer:
Upstream primer: CCACCCATGGCAAATTCCATGGCA (SEQ ID NO:3)
Downstream primer: TCTAGACGGCAGGTCAGGTCAGGTCCACC (SEQ ID NO:4)
Fig. 4 A demonstration and contrast and non-silencing group compare, and siPDCD10-1 makes endogenous PDCD10 mRNA express slightly decline, and siPDCD10-2 almost completely suppresses endogenous PDCD1D mRNA expression.
3 usefulness fluorescent microscopes and flow cytometer detect the inhibition effect that siRNA expresses GFP-PDCD10
Cell cotransfection pEGFP-PDCD10 plasmid and siRNAs detect green fluorescence intensity by fluorescent microscope and flow cytometer, with the inhibition effect of determining that siRNA expresses GFP-PDCD10.Fig. 4 B is presented under the fluorescent microscope, compare with GFP-transfected separately-PDCD10 control group (a) and non-silencing group (b), siPDCD10-1 (d), siPDCD10-2 (e) and positive control GFP siRNA (c) have the obvious suppression effect to the expression of exogenous GFP-PDCD10.Fig. 4 C demonstration flow cytometer fluorescence intensity compares with GFP-transfected separately-PDCD10 contrast and non-silencing group, and siPDCD10-1, siPDCD10-2 and GFP siRNA all obviously reduce green fluorescence intensity.
The 4 Western markings detect the influence of siRNA to endogenous PDCD10 protein expression
Extract the albumen of non-silencing, siPDCD10-1, three groups of cells of siPDCD10-2, carry out the Western marking with anti-PDCD10 antibody and detect.Fig. 4 D shows, compares with the non-silencing group, and siPDCD10-1 is to the almost not influence of endogenous PDCD10 protein level, and siPDCD10-2 significantly suppresses the proteic expression of endogenous PDCD10, and this result is consistent in two kinds of cells of HeLa and PC-3.
Embodiment 6 PDCD10 regulate not cross and express MST4 and cross MTT and the H that expresses MST4 cell proliferation
3-TdR
1 mtt assay detects cell proliferation
With transfection the cell of certain plasmid or siRNA be inoculated in 96 orifice plates with the density of 1000 cells/well, carry out MTT and analyze. add MTT solution according to specified time point, react 4-6 hour, adding first Can, 570nm detects light absorption value.Fig. 5 A shows, compares with the pCDEF group, and pCDEF-PDCD10 group and pCDEF-MST4 group can both promote cell proliferation, and cotransfection pCDEF-PDCD10 and the short cultivation effect of pCDEF-MST4 group are particularly remarkable, are higher than independent transfection group far away.Fig. 5 C shows, with transfection pGC-non-silencing (non-silencing siRNA is inserted the pGC-U6 vector construction) group relatively, pGC-PDCD10 (siPDCD10-2 siRNA is inserted the pGC-U6 vector construction) group can significantly suppress the propagation of cell.Fig. 5 D shows, compare with cotransfection pCDEF and non-silencing siRNA control group, cotransfection pCDEF-MST4 and non-silencingsiRNA group can promote cell proliferation, then can suppress to express the cell proliferation that MST4 causes by crossing because of the level that has reduced PDCD10 in cotransfection siPDCD10-2 and the pCDEF-MST4 group.
2[
3H]-the TdR method of mixing detects cell proliferation
With transfection the cell of certain plasmid or siRNA be inoculated in 96 orifice plates with the density of 1000 cells/well, by the fixed time point add [
3H]-TdR (available from Amersham Biosciences company), concentration is 1 μ Ci/ml, continue to cultivate after 6 hours, collecting cell to the Filtermat of 96-hole, MicroBeta Windows Workstation (Wallac) measure [
3H]-situation of mixing of TdR.Fig. 5 B shows and the pCDEF group compares, pCDEF-PDCD10 group and pCDEF-MST4 group [
3H]-the TdR value of mixing all has rising, and cotransfection pCDEF-PDCD10 and pCDEF-MST4 group [
3H]-the TdR value of mixing is higher than independent transfection group far away, and is consistent with the MTT analytical results, and short cultivation effect is certainly.
3 clones form experiment
With transfection the cell of certain plasmid or siRNA be inoculated in 35 millimeters culture plates with 200 or 400 density, add 800 μ g/ml Geneticin after 48 hours, changed a subculture in per three days.The 15th day, fixing (using 2%PFA/PBS) Geneticin opposing cell was counted after the violet staining.Fig. 6 shows with the pCDEF group and compares that the clone of pCDEF-PDCD10 group and pCDEF-MST4 group forms number all rising, and cotransfection pCDEF-PDCD10 and pCDEF-MST4 group clone formation number are higher than independent transfection group (A is clone's counting column diagram) far away; With transfection pGC-non-silencing group relatively, the pGC-PDCD10 group is owing to suppressed endogenous PDCD10 and expressed, the clone forms number and significantly descends (B is clone's counting column diagram), thereby has confirmed the short proliferation function of PDCD10.
Embodiment 7 PDCD10 suppress the result of cell anoikis
The anoikis analytical procedure is with reference to the report (Frisch of Frisch and Francis, S.M., and Francis, H.Disruption ofepithelial cell-matrix interactions induces apoptosis.J.Cell Biol 124,619-626,1994). culture plate, is fully embathed with PBS by twice with Poly-HEME (4mg/ml is dissolved in ethanol) bag again.Cell with various plasmids and siRNA stable transfection is resuspended in RPMI 1640 substratum, and is laid on the Poly-HEME bag by on the culture plate of crossing.Cultivate after 24 hours, use the PBS washed twice, with cell suspension in 200 μ l Annexin-V binding buffer liquid (10mMHEPES, 140mM NaCl, 2mM MgCl
2, 5mM KCl, 2.5mM CaCl
2, pH 7.4), hatch with the Annexin V of FITC mark (lucifuge, room temperature, 20 minutes).Add 400 μ l binding buffer liquid and use FACS Calibur (1 * 10 immediately
4Cells) and the CELLQuest software analysis.Fig. 7 demonstration and wild-type and pCDEF group compare, and the cell anoikis rate of pCDEF-PDCD10 group significantly descends; With transfection pGC-non-silencing group relatively, the pGC-PDCD10 group is owing to suppressed endogenous PDCD10 expression, cell anoikis rate significantly rises, and owing to anoikis rate decline showed cell malignancy ability strengthens, thereby has confirmed the short proliferation function of PDCD10.B is an anoikis percentage of cells column diagram.
The design of the siRNA of 1 MST4 below is the sequence of the siRNA that adopted
siMST4-1：GAAUAACAUAGCUGAUCCA(SEQ?ID?N0：9)
siMST4-2：CCAGAUUGCUACCAUGCUA(SEQ?ID?NO：10)
siMST4-3：AGAUUGCUACCAUGCUAAA(SEQ?ID?NO：11)
GFP?siRNA?GCAAGCUGACCCUGAAGUUC(SEQ?ID?NO：7)
Non-silencing?siRNA?UUCUCCGAACGUGUCACGU(SEQ?ID?NO：8)
2 RT-PCR detect the influence that siRNA expresses endogenous MST4 mRNA
Utilize TRIZOL
TMReagent extracts cell total rna, utilizes the synthesizing single-stranded cDNA of Invitrogen Corporation's Super Script II test kit.PCR utilizes following primer:
Upstream primer: ATGGCCCACTCGCCGGTG (SEQ ID NO:12)
Downstream primer: GGGGGATTCGTCTGCTGAACA (SEQ ID NO:13)
And confidential reference items GAPDH special primer:
Upstream primer: CCACCCATGGCAAATTCCATGGCA (SEQ ID NO:3)
Downstream primer: TCTAGACGGCAGGTCAGGTCAGGTCCACC (SEQ ID NO:4)
Fig. 8 A demonstration and contrast and non-silencing group compare, and siMST4-2 significantly suppresses the expression of endogenous MST4 mRNA, and siMST4-1, siMST4-3 express influence not quite to MST4 mRNA.
3 usefulness fluorescent microscopes and flow cytometer detect the inhibition effect that siRNA expresses GFP-MST4
Cell cotransfection pEGFP-MST4 plasmid and siRNAs detect green fluorescence intensity by fluorescent microscope and flow cytometer, with the inhibition effect of determining that siRNA expresses GFP-MST4.Fig. 8 B shows with GFP-transfected-MST4 control group (a) and non-silencing organize (b) relatively separately, siMST4-1 (d), siMST4-3 (f) do not have the obvious suppression effect to the expression of exogenous GFP-MST4, and si MST4-2 (e) and positive control GFPsiRNA (c) have the obvious suppression effect to the expression of exogenous GFP-MST4.Fig. 8 C demonstration flow cytometer fluorescence intensity compares with the non-silencing group, and si MST4-2 and GFP siRNA all obviously reduce green fluorescence intensity, and siMST4-1, siMST4-3 group does not then have noticeable change.
The 4 Western markings detect the influence of siRNA to endogenous PDCD10 protein expression
Extract the albumen of non-silencing, siMST4-1, siMST4-2, four groups of cells of siMST4-3, carry out the Western marking with anti-MST4 antibody and detect.Fig. 8 D shows, compares with the non-silencing group, and siMST4-1, siMST4-3 are to the almost not influence of endogenous MST4 protein level, and siMST4-2 significantly suppresses the proteic expression of endogenous MST4.
Embodiment 9 siMST4 regulate not cross and express PDCD10 and cross the MTT experimental result of expressing PDCD10 cell proliferation
With transfection the cell of certain plasmid or siRNA be inoculated in 96 orifice plates with the density of 1000 cells/well, carry out MTT and analyze. add MTT solution according to specified time point, react 4-6 hour, adding first Can, 570nm detects light absorption value.Fig. 9 A shows, with transfection non-silencing siRNA group relatively, the inhibition DeGrain of siMST4-1, siMST4-3 group on cell proliferation; And the siMST4-2 group significantly suppresses cell proliferation.Fig. 9 B shows, compare with cotransfection pCDEF and non-silencing siRNA control group, cotransfection pCDEF-PDCD10 and non-silencing siRNA group can promote cell proliferation, then can suppress to express the cell proliferation that PDCD10 causes by crossing because of the level that has reduced MST4 in cotransfection siMST4-2 and the pCDEF-PDCD10 group.
Embodiment 10siMST4 suppresses the influence of phenomenon to PDCD10 inductive anoikis
The anoikis analytical procedure is with embodiment 6.Figure 10 shows with the pCDEF group and compares that the cell anoikis rate of pCDEF-MST4 group significantly descends; With transfection non-silencing siRNA group relatively, suppress the tangible siMST4-2 group of expression effect cell anoikis rate and significantly rise, and the siMST4-1 and the siMST4-3 group anoikis rate that suppress weak effect do not have noticeable change; Compare with non-silencing siRNA group with cotransfection pCDEF-PDCD10, cotransfection pCDEF-PDCD10 and siMST4-2 group are because endogenous MST4 expression is suppressed, and cell anoikis rate also significantly rises.B is an anoikis percentage of cells column diagram.
Cross expression group pRL-TK (renilla luciferase carrier, as the fluorescence internal reference), reporter gene carrier (Photinus pyralis LUC carrier), Gal4-ELK1 expression vector, positive control carrier MEK1, goal gene carrier pCDEF-PDCD10, pCDEF-MST4, pCDEF is as blank, transfectional cell; PDCD10 suppresses the expression group with using pRL-TK (the renilla luciferase carrier is as the fluorescence internal reference), reporter gene carrier (Photinus pyralis LUC carrier), Gal4-ELK1 expression vector, pGC-non-silencing, pGC-PDCD10 transfectional cell; Cell cultures adds the special inhibitor PD98059 of ERK (5mM) respectively after 24 hours, p38 MAPK inhibitor SB20219 (10mM), or JNK MAPK inhibitor JNK inhibitor II (0.1uM) carry out the luciferase reporter gene analysis after 12 hours.Figure 11 A shows, compares as the blank group with pCDEF, crosses expression PDCD10 or MST4 and can improve the luciferase reporting activity, and the two is particularly remarkable to the active raising of luciferase reporting for cotransfection; And this activity can suppress by the special inhibitor PD98059 of ERK, and do not weakened by p38 MAPK inhibitor SB20219 or JNK MAPK inhibitor JNK inhibitor II, illustrates that the interaction of the two influences the ERK path; Figure 11 B shows, compares with the pGC-non-silencing group, and the pGC-PDCD10 group causes the active decline of luciferase reporting owing to suppressed the expression of endogenous PDCD10.
Embodiment 12 PDCD10, MST4 and two-way interaction thereof analyze the Western marking of different MAPK path tyrosine phosphorylations
The cell of collected transfection certain plasmid or siRNA, lysing cell extracts protein according to a conventional method, and quantitatively the experiment of the Western marking is carried out with ordinary method in the back.Figure 12 A shows, with anti-phosphorylation ERK antibody and anti-non-phosphorylating ERK antibody test, compare with the pCDEF group, pCDEF-PDCD10 group and pCDEF-MST4 group can both improve the level of phosphorylation ERK in the cell, and cotransfection pCDEF-PDCD10 and pCDEF-MST4 group raising effect are particularly remarkable, be higher than independent transfection group far away, the positive control group of MEK1.With transfection non-silencing siRNA group relatively, the ratio of siPDCD10-2 group phosphorylation ERK significantly reduces, and the ratio that suppresses the siPDCD10-1 group phosphorylation ERK of endogenous PDCD10 expression effect difference does not have considerable change.Figure 12 B shows, with anti-phosphorylation JNK antibody and anti-non-phosphorylating JNK antibody test, except that positive control MEKK group, respectively organizes the equal no significant difference of phosphorylation JNK level among the figure.Figure 12 C shows, except that positive control MEK3 organizes, respectively organizes the equal no significant difference of phosphorylation p38 level with anti-phosphorylation p38 antibody and anti-non-phosphorylating p38 antibody test among the figure.Above result confirms that the interaction of PDCD10 and MST4 realizes by regulation and control ERK MAPK path.
The RNAi of embodiment 13 MST4 is not to expressing PDCD10 excessively and crossing and express the inhibition of PDCD10 cell ERK kinase activity
Cell transfecting, proteins extraction, the experiment of the Western marking is carried out according to a conventional method.Figure 13 A shows, with anti-phosphorylation ERK antibody and anti-non-phosphorylating ERK antibody test, with transfection non-silencing siRNA group relatively, the ratio of siMST4-2 group phosphorylation ERK decreases, and the ratio of the siMST4-1 of inhibition endogenous MST4 expression effect difference and siMST4-3 group phosphorylation ERK does not have considerable change.Figure 13 B shows, with anti-phosphorylation ERK antibody and anti-non-phosphorylating ERK antibody test, compare with cotransfection pCDEF and non-silencing siRNA group, cotransfection pCDEF-PDCD10 and non-silencing siRNA group, cotransfection pCDEF-PDCD10 and siMST4-1 group, all be significantly increased with the level of phosphorylation ERK in cotransfection pCDEF-PDCD10 and the siMST4-3 group cell, and cotransfection pCDEF-PDCD10 and siMST4-2 group can reduce owing to cross the level of expressing the phosphorylation ERK that PDCD10 causes because siMST4-2 effectively suppresses endogenous MST4 and expresses.
Embodiment 14 MST4 vitro kinase activities are analyzed
Kinase activity assay HTScan
TM(Cell signaling, Danvers MA), finish according to its laboratory manual Mst4 kinases assay kit.Various expression vectors and siRNA transfection are in the HeLa cell, and after 48 hours, lysing cell was also handled 1 hour with Protein G magnetic bead, hatched 2 hours with 4 ℃ of anti-PDCD10 monoclonal antibodies then.Immunoprecipitation complex and 10U Mst4 kinases, the ATP that 200uM is cold, substrate Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) biotin labeling polypeptide was hatched 30 minutes for 25 ℃, with equal-volume 50mM EDTA termination reaction.Reaction product is diluted in bag and is cushioned liquid (0.05M NaHCO at 1: 4
3, pH8.6) and the bag by a pretreated elisa plate, hatched 1 hour for 37 ℃.Abandon coating buffer, wash twice with PBST (PBS/0.05%Tween 20), add one anti-(be diluted at 1: 1000 1%BSA/PBST) of anti-phosphorylation Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) polypeptide, every hole 100 μ L were hatched 1 hour for 37 ℃.Wash five times, elisa plate adds the anti-rabbit igg antibody (be diluted at 1: 10000 1%BSA/PBST) of HRP mark, and every hole 100 μ l were hatched 1 hour for 37 ℃.Wash five times, every hole add 50 μ l tmb substrates (TMB Substrate Kit, Pierce).Color development at room temperature is 450nm inspection light absorption value after 10 minutes.Figure 14 shows, compares with the pCDEF group, and the pCDEF-PDCD10 group can significantly strengthen the kinase activity of MST4; Compare with the non-silencingsiRNA group, the siPDCD10-2 group can reduce the kinase activity of MST4, and can not suppress the kinase activity no change of the siPDCD10-1 group MST4 of endogenous PDCD10 expression substantially.
DIC00710016.070427. sequence table
SEQUENCELISTING
＜110〉Peking University
＜120〉screening reagent box, screening method and the purposes of inhibition of cell proliferation or antitumor drug
<130>DIC07110016
<140>CN2007100637876
<141>2007-02-09
<160>2
<170>PatentInversion3.3
<210>1
<211>639
<212>DNA
＜213〉artificial sequence
<400>1
atgaggatgacaatggaagagatgaagaatgaagctgagaccacatccatggtttctatg60
cccctctatgcagtcatgtatcctgtgtttaatgagctagaacgagtaaatctgtctgca120
gcccagacactgagagccgctttcatcaaggctgaaaaagaaaatccaggtctcacacaa180
gacatcattatgaaaattttagagaaaaaaagcgtggaagttaacttcacggagtccctt240
cttcgtatggcagctgatgatgtagaagagtatatgattgaacgaccagagccagaattc300
caagacctaaacgaaaaggcacgagcacttaaacaaattctcagtaagatcccagatgag360
atcaatgacagagtgaggtttctgcagacaatcaaggatatagctagtgcaataaaagaa420
cttcttgatacagtgaataatgtcttcaagaaatatcaataccagaaccgcagggcactt480
gaacaccaaaagaaagaatttgtaaagtactccaaaagtttcagtgatactctgaaaacg540
tattttaaagatggcaaggcaataaatgtgttcgtaagtgccaaccgactaattcatcaa600
accaacttaatacttcagaccttcaaaactgtggcctga639
<210>2
<211>212
<212>PRT
＜213〉artificial sequence
<400>2
MetArgMetThrMetGluGluMetLysAsnGluAlaGluThrThrSer
151015
MetValSerMetProLeuTyrAlaValMetTyrProValPheAsnGlu
202530
LeuGluArgValAsnLeuSerAlaAlaGlnThrLeuArgAlaAlaPhe
354045
IleLysAlaGluLysGluAsnProGlyLeuThrGlnAspIleIleMet
505560
LysIleLeuGluLysLysSerValGluValAsnPheThrGluSerLeu
65707580
LeuArgMetAlaAlaAspAspValGluGluTyrMetIleGluArgPro
859095
GluProGluPheGinAspLeuAsnGluLysAlaArgAlaLeuLysGln
100105110
IleLeuSerLysIleProAspGluIleAsnAspArgValArgPheLeu
115120125
GlnThrIleLysAspIleAlaSerAlaIleLysGluLeuLeuAspThr
130135140
ValAsnAsnValPheLysLysTyrGlnTyrGlnAsnArgArgAlaLeu
145150155160
GluHisGlnLysLysGluPheValLysTyrSerLysSerPheSerAsp
165170175
ThrLeuLysThrTyrPheLysAspGlyLysAlaIleAsnValPheVal
180185190
SerAlaAsnArgLeuIleHisGlnThrAsnLeuIleLeuGlnThrPhe
195200205
LysThrValAla
210
Claims (8)
1. one kind is used to screen the combined prod that suppresses cell proliferation or anti-tumor drug, it is characterized in that, described combined prod comprises following integral part: reorganization or the natural apoptosis factor 10 molecules, the reorganization or natural Mammals STE20 sample protein kinase 4 molecules, the natural substrate or the synthetic substrate of Mammals STE20 sample protein kinase 4 molecules.
2. as claimed in claim 1 being used to screened the combined prod that suppresses cell proliferation or anti-tumor drug, it is characterized in that its specific form is a detection kit.
3. the combined prod that is used to screen inhibition cell proliferation or anti-tumor drug as claimed in claim 1 or 2, it is characterized in that it also comprises: ATP, the natural substrate of resisting mammal STE20 sample protein kinase 4 molecules or the antibody of synthetic substrate phosphorylation form.
4. as claimed in claim 3ly be used to screen the combined prod that suppresses cell proliferation or anti-tumor drug, it is characterized in that, it also comprises two anti-, the chromogenic substrate that is selected from kinase reaction damping fluid, enzyme labelling, in other reaction buffer one or more.
5. the combined prod that is used to screen inhibition cell proliferation or anti-tumor drug as claimed in claim 1, it is characterized in that the natural substrate of described Mammals STE20 sample protein kinase 4 molecules or synthetic substrate are synthetic substrate Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) biotin labeling polypeptide.
Reorganization or the natural apoptosis factor 10 molecules and Mammals STE20 sample protein kinase 4 molecules reorganization or natural in screening inhibition cell proliferation medicine to be screened or the purposes in the antitumor drug.
7. the described purposes of claim 6, wherein said inhibition cell proliferation or antitumor drug comprise albumen, polypeptide, natural drug, chemical synthetic drug and Chinese herbal medicine extract.
8. one kind is used to screen the method that suppresses cell proliferation or antitumor drug, it is characterized in that described method may further comprise the steps:
1) obtains the apoptosis factor 10 molecules reorganization or endogenous expression;
2) obtain Mammals STE20 sample protein kinase 4 molecules reorganization or endogenous expression;
3) with the apoptosis factor 10 molecules that obtain and Mammals STE20 sample protein kinase 4 molecules at the effect substrate of external and ATP, Mammals STE20 sample protein kinase 4 molecules, treat the screening of medicaments hybrid reaction;
4) termination reaction, the expression that detects Mammals STE20 sample protein kinase 4 molecules is or/and activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2007100637876A CN101050473B (en) | 2007-02-09 | 2007-02-09 | Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007100637876A CN101050473B (en) | 2007-02-09 | 2007-02-09 | Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage |
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| Publication Number | Publication Date |
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| CN101050473A CN101050473A (en) | 2007-10-10 |
| CN101050473B true CN101050473B (en) | 2010-08-18 |
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| CN2007100637876A Expired - Fee Related CN101050473B (en) | 2007-02-09 | 2007-02-09 | Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105695562B (en) * | 2014-11-27 | 2020-01-07 | 中国科学院上海生命科学研究院 | Use of MST4 gene diagnosis and cell treatment for infectious diseases and related medicine thereof |
| CN106226531B (en) * | 2016-07-25 | 2018-05-22 | 广州道瑞医药科技有限公司 | ANGPT2 is screening or is preparing to diagnose or treat the application in angiomatous drug |
| CN109112106B (en) * | 2018-09-07 | 2022-01-04 | 广州长峰生物技术有限公司 | Method for establishing in vitro model of human primary liver cancer tissue |
| CN113577288A (en) * | 2021-09-15 | 2021-11-02 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Application of MST4 related substance in preparing medicine for treating neuroinflammation reaction after cerebral hemorrhage |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1072722C (en) * | 1997-12-30 | 2001-10-10 | 中国科学院上海细胞生物学研究所 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
| EP1334659A1 (en) * | 2000-11-15 | 2003-08-13 | Ono Pharmaceutical Co., Ltd. | Pd-1-lacking mouse and use thereof |
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2007
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1072722C (en) * | 1997-12-30 | 2001-10-10 | 中国科学院上海细胞生物学研究所 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
| EP1334659A1 (en) * | 2000-11-15 | 2003-08-13 | Ono Pharmaceutical Co., Ltd. | Pd-1-lacking mouse and use thereof |
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