CN108329387A - Relevant tomour specific transcript LIN28B-TST of cancer and application thereof - Google Patents

Relevant tomour specific transcript LIN28B-TST of cancer and application thereof Download PDF

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CN108329387A
CN108329387A CN201710035081.2A CN201710035081A CN108329387A CN 108329387 A CN108329387 A CN 108329387A CN 201710035081 A CN201710035081 A CN 201710035081A CN 108329387 A CN108329387 A CN 108329387A
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lin28b
tst
cell
polypeptide
expression
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黄胜林
何祥火
李焱
郭维杰
胡治祥
包怡超
郑秋鹏
吕洞宾
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Fudan University Shanghai Cancer Center
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Abstract

The present invention provides relevant tomour specific transcript LIN28B TST of cancer and application thereof, specifically, LIN28B TST provided by the invention can be expressed in kinds of tumors tissue, the high expression in certain tumor tissues, but it is not expressed in normal differentiated tissue and embryonic tissue, and can be obviously promoted growth and proliferation, the generation for promoting tumour of tumour cell in vitro, in vivo.

Description

Relevant tomour specific transcript LIN28B-TST of cancer and application thereof
Technical field
The present invention relates to therapeutic field of tumor.In particular it relates to the relevant tomour specific transcript LIN28B-TST of cancer And application thereof.
Background technology
Depth RNA sequencing from a brand-new visual angle present human transcription group characteristic and cut mode it is various Property and complexity.Cancer is a kind of complex disease with heterogeneity, contains the change of science of heredity and epigenetics level, So that it forms more complicated transcript profile.Therefore, the transcript profile of cancer has prodigious potential to generate tomour specific transcript (Tumor-Specific Transcripts,TST).The identification of tomour specific transcript has very important clinical effect, The classification of tomour specific transcript, such as fusion transcript (fusion transcripts) is to understanding the generating process of tumour Very great meaning (fusion transcript is one kind of tomour specific transcript, derives from the transposition of DNA).Lose normal regulation Transcription and epigenetics change may cause between a large amount of tomour specific transcript, including special mRNA and gene Completely new transcript.However, the identification of these specific transcription sheets and function are not yet fully elucidated.
The protein family of LIN28, including LIN28A and LIN28B, which are that one kind is high in embryo occurs, expresses, but adult The rna binding protein that do not expressed in tissue.LIN28 albumen can prevent tumor suppressor let-7 family RNA (a kind of small RNA maturation), and regulate and control the self-renewing of mammalian stem cell.However, cause the mechanism of LIN28B gene activations herein it It is preceding not to be elucidated with.
Therefore, the new mechanism activated in cancer there is an urgent need in the art to study LIN28B genes, and exploitation and tumour Grow and be proliferated relevant albumen, and the target drug for the treatment of tumour.
Invention content
It is an object of the invention to provide a kind of novel targets LIN28B-TST related with tumor susceptibility and/or prognosis and The application of its diagnosis, treatment etc..
The first aspect of the present invention provides a kind of LIN28B-TST polypeptides of separation, and the polypeptide is selected from the group：
(a) there is SEQ ID NO:The polypeptide of 1 amino acid sequence；
(b) by SEQ ID NO:1 amino acid sequence is by one or more amino acid residues (preferably, 1-30 is a, more preferably Ground, 1-15, more preferably, 1-6) replace, miss or add and formed, and there is SEQ ID NO:1-22 in 1 Shown in 22 amino acid amino acid sequence, have tumor cell specific expression, and with promote tumour cell life Long and proliferation promotes the tumorigenic active polypeptide derived from (a)；
(c) with SEQ ID NO:1 amino acid sequence has >=80% homology (homology preferably, >=90%；Preferably >=95% homology；Most preferably, >=97% homology, such as 98% or more, 99% or more), and there is SEQ ID NO:1 In 22 amino acid shown in 1-22 amino acid sequence, express with tumor cell specific and with promoting to swell The growth of oncocyte and proliferation promote the tumorigenic active polypeptide derived from (a).
In another preferred example, the amino acid sequence of the polypeptide such as SEQ ID NO.:Shown in 1.
In another preferred example, the polypeptide comes from mammal, preferably, people, rat, mouse, it is preferable that people.
In another preferred example, the polypeptide is by LIN28B-WT (SEQ ID NO.:3) one or more of N-terminal is (preferable Ground, 3) amino acid substitution is SEQ ID NO:The amino acid sequence of 22 Amino acid profiles shown in 1-22 in 1.
Second aspect of the present invention provides a kind of polynucleotides of separation, the polynucleotide encoding first aspect present invention The polypeptide.
In another preferred example, the polynucleotides are selected from the group：DNA sequence dna, RNA sequence.
In another preferred example, the DNA sequence dna is selected from the group：Genome sequence, cDNA sequence.
In another preferred example, the polynucleotides are mRNA or cDNA.
In another preferred example, the polynucleotides include a nucleotide sequence, the nucleotide sequence with it is selected from the group below A kind of nucleotides sequence shows at least 80% (preferably at least 90%, more preferably at least 95%) the phase same sex：
(i) polynucleotides of the polypeptide of coding as described in the first aspect of the invention；With
The polynucleotides of (i i) and polynucleotides (i) complementation.
In another preferred example, the polynucleotide encoding amino acid sequence such as SEQ ID NO.:Polypeptide shown in 1.
In another preferred example, the sequence of the polynucleotides has such as SEQ ID NO.:Sequence shown in 2.
Third aspect present invention provides a kind of carrier, and the carrier contains the multinuclear glycosides described in second aspect of the present invention Acid.
Fourth aspect present invention provides a kind of host cell, and the host cell contains described in third aspect present invention The polynucleotides described in second aspect of the present invention are integrated in carrier or genome.
Fifth aspect present invention provides a kind of preparation method of polypeptide, including：
Under conditions suitable for the expression, the host cell described in fourth aspect present invention is cultivated, to give expression to the present invention Polypeptide described in first aspect；The polypeptide with separation.
Sixth aspect present invention provides a kind of specific inhibitor, the Inhibitor specificity with first party of the present invention Polypeptide described in face combines or inhibits LIN28B-TST expression.
In another preferred example, the inhibitor include antisense nucleic acid, antibody, micromolecular compound, CrispR reagents, Or combinations thereof.
In another preferred example, the antisense nucleic acid includes siRNA, shRNA, miRNA.
In another preferred example, the inhibitor does not inhibit or does not inhibit substantially wild type LIN28B.
In another preferred example, the inhibitor preferentially inhibits LIN28B-TST (compared with wild type LIN28B).
In another preferred example, the Inhibitor specificity is directed to the specific epitopes of LIN28B-TST.
In another preferred example, the specific epitopes refer to what LIN28B-TST has but wild type LIN288 did not had Epitope.
In another preferred example, the specific epitopes are selected from the group：
(a)SEQ ID NO.:The epitope that 1-22 amino acid sequences are constituted in 1；
(b)SEQ ID NO.:1 1-22 amino acid sequence and LIN28B-TST polypeptides other amino acid sequence institutes The epitope of composition.
In another preferred example, the specific inhibitor is incorporated into the specific epitopes of the LIN28B-TST polypeptides, Or it is incorporated into the multi-epitope of the specific epitopes and other sequences composition of the LIN28B-TST polypeptides, the specific epitopes are selected from The following group：SEQ ID NO.:1-22 amino acid sequences in 1：MSHRRQVLQKRMRSFNQVSSAP(SEQ ID NO.:8).
In another preferred example, the specific inhibitor specific can inhibit the activity of the promoter of LIN28B-TST.
In another preferred example, the promoter of the LIN28B-TST such as SEQ ID NO.:Shown in 9.
Seventh aspect present invention provides a kind of purposes of the specific inhibitor described in sixth aspect present invention, the spy Specific inhibitor is used to prepare drug or preparation, the diagnosis or Index for diagnosis of the drug or preparation for (i) tumour；(ii) press down Growth of tumour cell processed and proliferation；And/or (iii) inhibits tumorigenic ability.
In another preferred example, the tumour cell is selected from the group the cell of tumour：Liver cancer, cervical carcinoma, carcinoma of endometrium, Glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or combinations thereof.
In another preferred example, the tumour is selected from the group：Liver cancer, cervical carcinoma, carcinoma of endometrium, glioma, breast cancer, Melanoma, lung cancer, colon cancer, gastric cancer, or combinations thereof.
Eighth aspect present invention provides a kind of pharmaceutical composition, including：
Specific inhibitor described in sixth aspect present invention；And pharmaceutically acceptable carrier.
In another preferred example, described pharmaceutical composition further includes lowering the table of the LIN28B-TST polypeptides or nucleotide Up to amount or active micromolecular compound, and/or nucleic acid.
In another preferred example, the nucleic acid includes siRNA, shRNA, miRNA.
Ninth aspect present invention provides whether there is in a kind of detection (vitro detection especially non-diagnosticly) sample The method of LIN28B-TST, including：Sample is contacted with the specific antibody of LIN28B-TST, sees whether to form antibody compound Object forms antibody complex and means that there are LIN28B-TST in sample.
Tenth aspect present invention provides a kind of detect and the relevant disease of LIN28B-TST unconventionality expressions or disease-susceptible humans The method of property, including：It detects in the nucleic acid sequence of coding said polypeptide with the presence or absence of mutation.
Tenth one side of the invention provides described in a kind of polypeptide described in first aspect present invention, second aspect of the present invention Polynucleotides purposes, for screen inhibit the active inhibitor of LIN28B-TST or for the identification of peptide fingerprinting spectrum or Reference substance as detection of nucleic acids.
The twelfth aspect of the present invention provide a kind of determining tester whether be LIN28B-TST inhibitor method, packet Include step：
(a) in test group, in cultivating system, in the presence of tester, the cell of culture expression LIN28B-TST；And And in there is no the tester and the identical control group of other conditions, cultivate identical cell；
(b) the expression quantity E1 or its activity A1 of LIN28B-TST described in cell described in test group are detected；And with compare The expression quantity E2 or activity A2 of LIN28B-TST described in cell described in group is compared；
Wherein, if the expression quantity E1 is substantially less than the expression quantity E2 or described activity A1 and is substantially less than the activity A2, then it represents that the object to be tested is the inhibitor of LIN28B-TST.
In another preferred example, the detection includes the detection of nucleic acid level, and/or the detection of protein level.
In another preferred example, " being substantially less than " refers to ratio≤1/2 of E1/E2 or A1/A2, preferably≤1/3, More preferably≤1/4.
In another preferred example, the cell includes tumour cell.
In another preferred example, the tumour cell comes from people.
In another preferred example, the method further includes step (c)：The LIN28B-TST polypeptides that step (b) is determined Inhibitor further test its to the inhibiting effect of growth of tumour cell and proliferation (including In vitro cell experiment or animal it is real It tests).
In another preferred example, the tester includes：Antibody, compound, nucleic acid.
In another preferred example, the tumour cell is selected from the group the cell of tumour：Liver cancer, cervical carcinoma, carcinoma of endometrium, Glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or combinations thereof.
In another preferred example, the method is non-diagnostic and non-therapeutic.
The present invention the 13rd aspect provide a kind of LIN28B-TST polypeptides or its transcript or its coded sequence or its The purposes of detection reagent, is used to prepare a detection kit, and the kit is used for the diagnosis or prognosis of tumour.
In another preferred example, the tumour is selected from the group：Liver cancer, cervical carcinoma, carcinoma of endometrium, glioma, mammary gland Cancer, melanoma, lung cancer, colon cancer, gastric cancer, or combinations thereof.
In another preferred example, the LIN28B-TST polypeptides or its transcript or its coded sequence are used as reference Or standard items.
In another preferred example, the detection reagent is selected from the group：Primer, probe, antibody, protein chip, nucleic acid core Piece, or combinations thereof.
In another preferred example, the detection reagent is the detection reagent of specific detection LIN28B-TST.
In another preferred example, the detection reagent is to discriminate between the detection reagent of LIN28B-TST and LIN28B-WT.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows that transcript profile sequencing analysis is found that new LIN28B tomour specific transcripts in kinds cancer LIN28B-TST.Wherein,
(A) in different transcript profile sequencing libraries, the reads sequences of LIN28B gene locations are distributed.
(B) in TCGA databases, the expression of LIN28B-TST and LIN28B-WT in various cancers type. HCC, i.e. hepatocellular carcinoma (sample number=348)；Cancer (sample number=296) in CESC, i.e. uterine neck and uterine neck；LGG, i.e. brain are rudimentary Other glioma (sample number=529)；DLBC, i.e. diffusivity large B cell lymphoid tumor (sample number=45)；BRCA, i.e. mammary gland infiltration Property cancer (sample number=1093)；SKCM, i.e. skin epidermis melanoma (sample number=294)；UCEC, i.e. corpus uteri inner membrance sample cancer (sample number=175)；BLCA, i.e. vesicourethral epithelioma (sample number=388)；HNSC, i.e. squamous cell lung carcinoma (sample number= 494)；KIRP, i.e. renal papilla cell cancer (sample number=290)；LUAD, i.e. adenocarcinoma of lung (sample number=504)；GBM, i.e. pleomorphism Glioblastoma (sample number=158)；UCS, i.e. sarcoma of uterus (sample number=42)；ESCA, the i.e. cancer of the esophagus (sample number= 179)；STAD, i.e. sdenocarcinoma of stomach (sample number=384), COAD, i.e. adenocarcinoma of colon (sample number=288).
Fig. 2 shows the identification of LIN28B-TST transcripts.Wherein,
(a) in Huh7 cells LIN28B-TST Northern blot results.
(b) in HuH-7 cells LIN28B-TST 5 ' RACE experiment cDNA segments agarose gel electrophoresis and Sanger Sequencing result.
(c) in HuH-7 cells LIN28B-TST 3 ' RACE experiment cDNA segments agarose gel electrophoresis and Sanger Sequencing result.
(d) compared with LIN28B-WT transcripts, LIN28B-TST transcripts have 2 special exons at 5 ' ends. The red mark of this use of LIN28-TST specific transcriptions.First base-pair (hg19chr6 of transcription initiation probability highest:104,937, 026) it is considered as transcription initiation site.There are two (ATGs for translation initiation site；It is marked with blue overstriking underscore)：First ATG,hg19chr6:104,937,099-104,937,101；Second ATG, chr6:104,950,476-104,950,478. Terminator codon (TAA) is marked with blue underscore.3 ' UTR Green Markers.
(e) amino acid sequence of LIN28B-TST.Translation initiation is in 1 or No. 2 initiation codon.It is corresponding that there are two eggs Propylhomoserin (being marked with blue underscore) leads to that there are two types of albumen, respectively 29.38kDa (269 amino acid) and 27.96kDa (258 amino acid).Amino acid sequence red-label special LIN28B-TST.
Fig. 3 shows expression of the LIN28B in various kinds of cell system.Detection LIN28B-WT and LIN28B- is tested with qPCR TST transcripts expression in various kinds of cell system.
Fig. 4 shows LIN28B-TST and LIN28B-WT in the expression of tissue and its relationship with tumor prognosis.Wherein,
(A) LIN28B-TST and LIN28B-WT is in fetus liver, normal liver, nonneoplastic tissue and hepatocellular carcinoma (HCC) expression in tissue.
(B) there is the total survivorship curve of patient of the HCC of LIN28B-TST expression with (-) with (+) or not.P values use logarithm order (Log-rank test) is examined to be calculated.
Fig. 5 shows the expression regulation of LIN28B-TST.Wherein,
(A) LIN28B-TST is in different transcription initiation sites, and in HuH-7 cell lines, progress chromosome is immunized coprecipitated Form sediment sequencing (ChIP-seq) and the 5 ' ends section cDNA rapid amplifyings (Rapid Amplification of cDNA Ends).
(B) in HEK-293T cell lines, LIN28B-TST promoters carry out the fluorescein after a series of knockouts of 5 ' sections Enzyme reporter assay.
(C) position view on 2 islands CpG of LIN28B-TST promoter regions.The state of promoter methylation uses Sodium hydrogensulfite is sequenced.In the specified sites CpG, what solid circles represented is methylated cytosine, and what empty circles represented It is unmethylated cytimidine.
(D) after being handled 6 days with or without the use of 10 μM of 5- aza deoxycytidines (5-AZA), LIN28B is in cell line The immunoblot results of expression.
(E) after the siRNA for using 1 μM of JQ1 or transfection targeting c-Myc, what LIN28B and cMyc were expressed in cell line exempts from Epidemic disease Blot results.β actins (β-actin) are used as internal reference.
Fig. 6 shows that LIN28B-TST encodes one section of long isomer protein with additional N-terminal amino acid.Wherein,
(A) LIN28B transcript variants thereofs and corresponding isomers schematic diagram.Initiation codon (ATG) and terminator codon (TAA) it is indicated with black arrow.
(B) immunoblot results of the cell of expression LIN28B-WT and LIN28B-TST.Two predictions of diagram LIN28B-TST initiation codons are from ATG to AGG by respectively and common mutation.
(C) expression of the LIN28B-WT and LIN28B-TST in various kinds of cell system.
Fig. 7 shows expression of the LIN28B-TST in tumour patient tissue.Wherein,
(A) it is carried out in HEK-293T cell lines, HuH-7 cell lines and HCC tissue of patient using LIN28B-TST antibody Immunoblot experiment.T, i.e. HCC are organized；N, i.e., corresponding nonneoplastic tissue.
(B) immunohistochemical staining is carried out in HCC and normal liver tissue using LIN28B-TST antibody.
Fig. 8 shows influences of the LIN28B-TST to tumor cell proliferation.Wherein,
(A) in NCI-H1299 cell lines, siRNA is lowered and is carried out colony formation and thin after LIN28B-TST expression Born of the same parents' proliferation experiment.Use two siRNA, i.e. si-LIN28B-TST-1 and si-LIN28B-TST-2 of targeting LIN28B-TST. There is no the siRNA of target by as negative control (Ctrl).
(B) in Huh-7 cell lines, siRNA carries out colony formation after lowering LIN28B-TST expression and cell increases Grow experiment.
Fig. 9 shows the influence lowered using CRISPR/Cas9 technologies after LIN28B-TST is expressed to tumor cell proliferation. Wherein,
(A) with the site of red solid triangular marker sgRNA targetings in figure.With immune in Huh-7 cells Blot experiment demonstrates knockout efficiency.The sgRNA of no target site is as internal reference (Ctrl).
(B) LIN28B-TST is lowered to Clone formation and ability of cell proliferation using CRISPR/Cas9 in Huh-7 cells Influence.***,p<0.001.
Figure 10 shows that the influence of cell proliferation in vivo after LIN28B-TST is lowered in zoopery detection.It uses CRISPR/Cas9 lowers the expression of LIN28B-TST.Wherein,
(A) volume of the mice-transplanted tumor for the HuH-7 that LIN28B-TST is knocked out.*p<0.05,**p<0.01,***p< 0.001.
(B) quality of the mice-transplanted tumor for the HuH-7 that LIN28B-TST is knocked out.*p<0.05,**p<0.01,***p< 0.001
Specific implementation mode
The present inventor passes through long-term and in-depth study unexpectedly finds by largely screening and detached one for the first time The new transcript LIN28B-TST (variant) of kind, it shows high expression level and high shear Index Score, it has following several A feature：1) 22 new amino acid sequences are increased in the N-terminal of wild type LIN28B-WT albumen；2) it is in kinds of tumors group Middle expression is knitted, the high expression in certain tumor tissues, but do not express in the normal tissue.3) variant in vitro can be bright The aobvious growth for promoting tumour cell and proliferation, the generation for promoting tumour.4) it is thin can be obviously promoted tumour in vivo for the variant The growth of born of the same parents and proliferation, the generation for promoting tumour.The present invention is completed on this basis.
Term
As used herein, term " LIN28B-TST polypeptides ", " LIN28B-TST albumen ", " variant ", " LIN28B-TST Variant " is used interchangeably, and is referred both to LIN28B-TST variant amino acid sequences (SEQ ID NO.:1) albumen or Polypeptide, it can be obviously promoted growth and proliferation, the generation for promoting tumour of tumour cell in vivo, in vitro.
As used herein, term " LIN28B-WT polypeptides ", " the LIN28B albumen of wild type ", " LIN28B-WT albumen " can It is used interchangeably, refers both to natural LIN28B albumen, sequence such as SEQ ID NO.:Shown in 3, nucleotide sequence such as SEQ ID NO.:Shown in 4.
LIN28B-TST
The present inventor is found that the completely new transcript of a LIN28B gene, its table by transcript profile sequencing (RNA-seq) Reveal high expression level and high shear Index Score, is named as LIN28B-TST.LIN28B-TST is in hepatocellular carcinoma and very much Specifically expressing in other human cancer cells, is not expressed in the normal tissue.LIN28B-TST expresses high patient's HCC prognosis It is poor.Different from wild type LIN28B transcripts, LIN28B-TST is generated from a new transcription initiation site, and is contained One strong promoter not regulated and controled by c-Myc.It is swollen that the tumour of expression LIN28B-TST may be classified as a kind of new invasion Tumor hypotype.Experiment and experiment in vitro show that tumor cell proliferation can be significantly inhibited by lowering the expression of LIN28B-TST in vivo. These discoveries demonstrate the new mechanism that LIN28B genes activate in cancer, and prompt LIN28B-TST as diagnosing tumor and The potential using value for the treatment of.
In the present invention, representative LIN28B-TST polypeptides refer to LIN28B-TST variant amino acid sequences (SEQ ID NO.:1) albumen or polypeptide.
In the present invention, representative LIN28B-TST polypeptides refer to by LIN28B-WT (SEQ ID NO.:3) the one of N-terminal A or multiple (preferably, 3) amino acid substitution is SEQ ID NO:The ammonia of 22 Amino acid profiles shown in 1-22 in 1 Base acid sequence.
In the present invention, representative wild type LIN28B polypeptides (LIN28B-WT polypeptides) refer to SEQ ID NO.:3 The albumen or polypeptide of shown sequence.
As used herein, " separation " refers to that substance is separated from its primal environment (if it is crude, original Beginning environment is natural surroundings).As under the native state in active somatic cell polynucleotide and polypeptide be not isolate and purify , but same polynucleotide or polypeptide such as from native state with separated in other existing substances, then to isolate and purify 's.
As used herein, " the LIN28B-TST albumen or polypeptide of separation " refers to LIN28B-TST polypeptides substantially free of day Right relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use the protein purification of standard Technology purifies LIN28B-TST albumen.Substantially pure polypeptide can generate single master in non-reducing polyacrylamide gel Band.The purity of LIN28B-TST polypeptides can use amino acid sequence analysis.
The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The present invention's is more Peptide can be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (example Such as, bacterium, yeast, higher plant, insect and mammalian cell) in generate.According to the host used in recombinant production scheme, originally The polypeptide of invention can be glycosylated, or can be nonglycosylated.The polypeptide of the present invention may also include or not include starting Methionine residues.
The invention also includes the segment of people's LIN28B-TST albumen, derivative and analogue.As used herein, term " piece Section ", " derivative " and " analog " refer to the identical biology of natural human LIN28B-TST albumen for being kept substantially the present invention Function or active polypeptide.Polypeptide fragment, the derivative or the like of the present invention can be (i) there are one or it is multiple conservative or non- Conservative amino acid (preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can May not be by genetic code encoding, or (ii) more with substituent group in one or more amino acid residues Peptide, or (iii) mature polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion Be formed by polypeptide, or (iv) additional amino acid sequence be fused to this polypeptide sequence and formed polypeptide (such as targeting sequencing or Secretion sequence or for purifying the sequence or proprotein sequence of this polypeptide, or the fusion protein with the formation of antigen I gG segments). According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.
In the present invention, term " people LIN28B-TST polypeptides " refers to the SEQ ID with people's LIN28B-TST protein actives NO:The polypeptide of 1 sequence.The term further includes having and people's LIN28B-TST albumen identical functions, SEQ ID NO:1 sequence Variant form.These variant forms include (but being not limited to)：One or more (it is usually 1-50, preferably 1-30, more Good ground 1-20, most preferably 1-10) amino acid missing, insertion and/or substitution, and added in C-terminal and/or N-terminal One or several (being usually within 20, be more preferably within 5 within preferably 10) amino acid.For example, in ability In domain, when being substituted with similar nature or similar amino acid, the function of protein is not usually changed.For another example, at the ends C End and/or N-terminal add one or several amino acid generally also and will not change the function of protein.The term further includes people The active fragment and reactive derivative of LIN28B-TST albumen.
The variant form of the polypeptide includes：Homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, can be with the encoded albumen of DNA, the Yi Jili of people's LIN28B-TST DNA hybridizations under the conditions of high or low stringency The more peptide or proteins obtained with the antiserum of anti-human LIN28B-TST polypeptides.The present invention also provides other polypeptides, such as comprising people LIN28B-TST polypeptides or its segment are formed by fusion protein with other polypeptides.Other than the almost polypeptide of overall length, the present invention Further comprise the soluble fragments of people's LIN28B-TST polypeptides.In general, the segment has people LIN28B-TST polypeptide sequences extremely Few about 10 continuous amino acids, typically at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably At least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.
Invention also provides the analog of people LIN28B-TST albumen or polypeptide.These analogs and natural human LIN28B-TST The difference of polypeptide can be the difference on amino acid sequence, can also be the difference on the modified forms for do not influence sequence, or Have both at the same time.These polypeptides include natural or induction genetic variant.Induction variant can be obtained by various technologies, such as Random mutagenesis is generated by radiating or being exposed to mutagens, can also pass through site-directed mutagenesis or other known molecular biology Technology.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and with non- The analog of natural or synthetic amino acid (such as β, gamma-amino acid).It should be understood that the polypeptide of the present invention is not limited to State the representative polypeptide enumerated.
Modification (not changing primary structure usually) form include：The chemical derivative form of in vivo or in vitro polypeptide such as acetyl Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.Further include being modified to improve its anti-proteolytic properties or optimization The polypeptide of solubility property.
In the present invention, " people's LIN28B-TST albumen conservative variation's polypeptides " refer to and SEQ ID NO:1 amino acid sequence Row are compared, and have at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid by property it is similar or Similar amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and produce It is raw.
Table 1
The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide Coding region sequence can be with SEQ ID NO:Coding region sequence shown in 2 is identical or the variant of degeneracy.Such as this paper institutes With " variant of degeneracy ", which refers to coding in the present invention, has SEQ ID NO:1 protein, but with SEQ ID NO:2 institutes The differentiated nucleic acid sequence of coding region sequence shown.
Encode SEQ ID NO:The polynucleotides of 1 mature polypeptide include：The coded sequence of encoding mature polypeptide；It is ripe The coded sequence of polypeptide and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and Non-coding sequence.
In an example of the present invention, a kind of polynucleotides of separation are provided, it, which is encoded, has SEQ ID NO:1 institute Show the polypeptide of amino acid sequence.Its nucleotide sequence such as SEQ ID NO:Shown in 2, the polynucleotide sequence overall length that it includes is 5428 bases, open reading frame are located at 74-880, and encoding full leng is the LIN28B-TST albumen (SEQ of 269 amino acid ID NO:1)。
The preparation of albumen of the present invention
Polypeptide and polynucleotides in the present invention preferably provide in a separate form, are more preferably purified to homogeneous.
The people LIN28B-TST nucleotide full length sequences or its segment of the present invention can usually use PCR amplification method, recombination method Or artificial synthesized method obtains.It, can be according to related nucleotide sequence disclosed in this invention, especially for PCR amplification method Open reading frame sequence carrys out design primer, and the commercially available libraries cDNA or made by conventional method well known by persons skilled in the art are used in combination The standby libraries cDNA expand as template and obtain related sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR expands Increase, then the segment that each time amplifies is stitched together by proper order again.
Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
At present, it is already possible to completely by chemical synthesis come obtain encoding albumen of the present invention (its segment or its derivative Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
Using method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA；230：1350-1354) It is optimized for obtaining the gene of the present invention.Especially it is difficult to when obtaining the cDNA of overall length from library, RACE is preferably used Method (ends RACE-cDNA rapid amplification), the primer for PCR can be suitable according to the sequence information of invention disclosed herein Locality selection, and available conventional method synthesis.The DNA/RNA of amplification such as can be detached and purified by gel electrophoresis with conventional method Segment.
The present invention also relates to the carriers for the polynucleotides for including the present invention, and with of the invention carrier or TRA2B- The genetically engineered host cell of DNAH5 albumen coded sequences, and generate polypeptide of the present invention through recombinant technique Method.
By the recombinant dna technology of routine (Science, 1984；224：1431), using the polynucleotide of the present invention Sequence can be used to express or produce the TRA2B-DNAH5 polypeptides of recombination.In general there are following steps：
(1) polynucleotides (or variant) of the encoding human LIN28B-TST polypeptides of the present invention, or with contain the multinuclear The recombinant expression carrier of thuja acid converts or suitable host cell of transduceing；
(2) host cell that is cultivated in suitable culture medium；
(3) be separated from culture medium or cell, protein purification.
The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
Specific antibody
On the other hand, the invention also includes the polypeptides to LIN28B-TSTDNA of the present invention or its fragment coding to have spy Anisotropic polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity ", which refers to antibody, can be incorporated into this Invent LIN28B-TST products or segment.Preferably, referring to those can be combined with LIN28B-TST gene outcomes of the present invention or segment But nonrecognition and the antibody for being incorporated into other non related antigen molecules, especially specific antibody nonrecognition LIN28B-WT days Right albumen.The present invention also provides a kind of particularly preferred specific antibodies, it identifies and combine SEQ ID NO.:1- in 1 22 amino acid sequences, preferably, recognizable and combination SEQ ID NO.:3-22 amino acid sequences in 1.
Antibody includes the molecule that those can combine and inhibit LIN28B-TST of the present invention in the present invention, also simultaneously including those The antibody of LIN28B-TST functions of the present invention is not influenced.The invention also includes those can be with modification or this hair of unmodified form The antibody that bright LIN28B-TST gene outcomes combine.
The present invention includes not only complete monoclonal or polyclonal antibody, but also includes having immunocompetent antibody piece Section, such as Fab ' or (Fab)2Segment；Heavy chain of antibody；Antibody light chain；Genetically engineered Single Chain Fv Molecule A (Ladner et al., United States Patent (USP) No.4,946,778)；Or chimeric antibody, such as there is mouse antibody binding specificity but still retain the antibody portion from people The antibody divided.
The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.For example, purifying LIN28B-TST gene outcomes of the present invention or its with antigenic segment, animal can be applied to induce Anti-TNF-α The generation of body.Similar, it expresses LIN28B-TST albumen of the present invention or its cell with antigenic segment can be used to Immune animal produces antibody.The antibody of the present invention can also be monoclonal antibody.Such monoclonal antibody can utilize hybridization Tumor technology come prepare (see Kohler et al.,Nature256；495,1975；Kohler et al.,Eur.J.Immunol.6：511, 1976；Kohler et al.,Eur.J.Immunol.6：292,1976；Hammerl ing et al.,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981).The antibody of the present invention includes that can block this It invents the antibody of LIN28B-TST functions and does not influence the antibody of LIN28B-TST functions of the present invention.All kinds of antibody of the present invention Segment or the functional areas that LIN28B-TST gene outcomes of the present invention can be utilized, are obtained by common immunological techniques.These segments Or functional areas can utilize recombination method to prepare or synthesized using Peptide synthesizer.With LIN28B-TST gene outcomes of the present invention Unmodified form in conjunction with antibody can be produced with the gene outcome produced in prokaryotic cell (such as E.Coli) animal is immunized It is raw；The antibody (such as the albumen or polypeptide of glycosylation or phosphorylation) combined with posttranslational modification form, can use eukaryocyte (example Such as yeast or insect cell) in the gene outcome that generates obtain animal is immunized.Anti- LIN28B-TST albumen of the present invention resists Body can be used in immunohistochemistry technology, detect the LIN28B-TST albumen of the present invention in biopsy specimen.
Antibody in the present invention can be used to treat or prevent and the relevant disease of LIN28B-TST albumen of the present invention.It gives suitable When the antibody of dosage can stimulate or block generation or the activity of LIN28B-TST albumen of the present invention.
Antibody can also be used for being designed to the immunotoxin for internal a certain privileged sites.Such as LIN28B-TST eggs of the present invention The monoclonal antibody of white high-affinity can covalently be tied with bacterium or phytotoxin (such as diphtheria toxin, ricin, abrine etc.) It closes.A kind of usual way is the amino of antibody to be attacked, by the exchange of disulfide bond, by toxin with thiol crosslinkers such as SPDP It is incorporated on antibody, this hybrid antibody can be used for killing the cell of LIN28B-TST protein positives of the present invention.
The production of polyclonal antibody can use LIN28B-TST albumen of the present invention or polypeptide immune animal, such as rabbit, mouse, greatly Mouse etc..A variety of adjuvants can be used for enhancing immune response, including but not limited to Freund's adjuvant etc..
Inhibitor of LIN28B-TST and application thereof
Using LIN28B-TST of the present invention, by various conventional screening assays, it can filter out and inhibit LIN28B-TST or work Property expression substance, such as receptor, inhibitor, agonist or antagonist.
Typically, the example of inhibitor of the invention includes (but being not limited to)：Micromolecular compound, antibody, antisense core Sour (including siRNA, shRNA, miRNA), Crispr reagents.Wherein, the inhibitor of the present invention is the inhibitor of specificity, It specifically binds to or targets the special polypeptides of the LIN28B-TST or nucleic acid sequence specific epitopes, or is incorporated into described The multi-epitope of the specific epitopes of LIN28B-TST polypeptides and other sequences composition, or the specificity of inhibition LIN28B-TST are strong Promoter.
Also, in a preferred embodiment, inhibitor of the present invention preferentially inhibits LIN28B-TST (with wild type LIN28B is compared).
Protein antibodies, inhibitor, antagonist or receptor of the present invention etc. can be used when being administered (administration) in the treatment In the growth for inhibiting tumour cell.In general, these substances can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous In mounting medium, wherein pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH value can with the property for being formulated substance and Illness to be treated and be varied from.Prepared pharmaceutical composition can be administered by conventional route, including (but It is not limited to)：Tumor is interior, intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or local administration.
The inhibitor of the present invention can be directly used for disease treatment, for example, for the treatment in terms of cancer (especially liver cancer). When using the inhibitor of LIN28B-TST of the present invention, other tumor therapeutic agents, such as cis-platinum are also can be used simultaneously.
The present invention also provides a kind of pharmaceutical compositions, it contains the inhibition of the LIN28B-TST of the present invention of safe and effective amount Agent (such as antibody or micromolecular compound) and pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but and unlimited In)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Pharmaceutical preparation should match with administering mode.
The pharmaceutical composition of the present invention can be made into injection form, such as with physiological saline or contain glucose and other The aqueous solution of adjuvant is prepared by conventional method.The pharmaceutical composition of such as tablet and capsule etc can pass through conventional side It is prepared by method.Pharmaceutical composition such as injection, solution, tablet and capsule preferably aseptically manufacture.The administration of active constituent Amount is therapeutically effective amount, such as about 100 mg/kg weight of about 1 microgram/kg body weight-daily.In addition, the inhibition of the present invention Agent can be also used together with other therapeutic agents.
It is that the inhibitor of the LIN28B-TST of safe and effective amount is applied to mammal when using pharmaceutical composition, In the safe and effective amount typically at least about 10 micrograms/kg body weight, and in most cases be no more than about 100 milligrams/thousand Gram weight, preferably the dosage is about 10 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also taken an examination Administration route, the factors such as patient health situation are considered, within the scope of these are all skilled practitioners technical ability.
The polynucleotide of people's LIN28B-TST albumen can also be used for a variety of therapeutic purposes.Gene therapy technology can be used for controlling The cell caused by the expression of the LIN28B-TST albumen without expression or exception/inactive of LIN28B-TST albumen is treated to increase It grows, develop or metabolic disorder.The gene therapy vector (such as viral vectors) of recombination may be designed to the LIN28B-TST of expression variance Albumen, to inhibit endogenic LIN28B-TST protein actives.From the expression vector such as retrovirus, adenopathy of virus Poison, adeno-associated virus (AAV), herpes simplex virus, parvovirus etc. can be used for LIN28B-TST gene transfers to intracellular. The method that structure carries the recombinant viral vector of LIN28B-TST genes is found in existing document (Sambrook, et al.).Separately Outer recombined human LIN28B-TST genes can be packaged into liposome, then be transferred to intracellular.
The oligonucleotide (including antisense RNA and DNA) and ribozyme of inhibition LIN28B-TST mRNA is also the present invention's Within the scope of.Ribozyme be it is a kind of can specific cleavage specific RNA enzyme sample RNA molecule, mechanism of action be ribozyme molecule with mutually Endonuclease effect is carried out after the target RNA-specific hybridization of benefit.The RNA and DNA and ribozyme of antisense can with existing any RNA or DNA synthetic technologys obtain, as the technology of solid phase phosphoamide chemical synthesis synthetic oligonucleotide has been widely used.Antisense RNA Molecule can transcribe acquisition in vitro or in vivo by encoding the DNA sequence dna of the RNA.This DNA sequence dna has been integrated into the RNA of carrier The downstream of polymerase promoter.In order to increase the stability of nucleic acid molecules, it can be modified with a variety of methods, such as increase by two The sequence length of side, the connection application phosphorothioates key or peptide bond between ribonucleotide rather than phosphodiester bond.
Polynucleotide imports tissue or intracellular method includes：Polynucleotide is directly injected into in-vivo tissue In；Or in vitro first imported polynucleotide in cell by carrier (such as virus, bacteriophage or plasmid), then cell is moved It plants internal etc..
Inhibit the inhibitor of LIN28B-TST promoter activities also within the scope of the invention.LIN28B-TST and LIN28B-WT promoters are different, and the small-molecule drug or traditional Chinese medicinal components for inhibiting LIN28B-TST can be selected by drug sieve, right LIN28B-WT does not influence.
Diagnostic method
The invention further relates to quantitative and detection and localization people LIN28B-TST nucleic acid or protein level diagnostic testing process.This A little experiments are known in the art, and include FISH measurement and radiommunoassay.The people LIN28B- detected in experiment TST protein levels may be used as explaining importance of the people LIN28B-TST albumen in various diseases and for diagnosing LIN28B- The disease that TST albumen works.
In a kind of detection detection sample LIN28B-TST albumen is utilized with the presence or absence of the method for LIN28B-TST albumen Specific antibody is detected, it includes：Sample is contacted with LIN28B-TST protein specific antibodies；It sees whether to be formed anti- Nanocrystal composition forms antibody complex and means that there are LIN28B-TST albumen in sample.
The polynucleotide of LIN28B-TST albumen can be used for the diagnosing and treating of LIN28B-TST protein related diseases. In terms of diagnosis, whether the polynucleotide of LIN28B-TST albumen can be used for detecting the expression of LIN28B-TST albumen or in disease The unconventionality expression of LIN28B-TST albumen under state.As LIN28B-TST DNA sequence dnas can be used for the hybridization to biopsy specimen to sentence The abnormal expression of disconnected LIN28B-TST albumen.Hybridization technique includes Southern blottings, Northern blottings, in situ hybridization Deng.These technical methods are all disclosed mature technologies, and relevant kit all commercially obtains.The multinuclear of the present invention Part or all of thuja acid can be used as probe and be fixed on microarray (microarray) or DNA chip (also known as " gene core Piece ") on, for analyzing the Differential expression analysis of gene and gene diagnosis in tissue.With the special primer of LIN28B-TST albumen Carry out the transcription product that RNA- polymerase chain reactions (RT-PCR) amplification in vitro also can detect LIN28B-TST albumen.
Main advantages of the present invention include：
(1) present invention firstly discovers that new transcript LIN28B-TST, it is in hepatocellular carcinoma and a lot of other human carcinomas Specifically expressing in disease cell, is not expressed in the normal tissue, and there are one new transcription initiation site, expression to be opened by LIN28B-TST The regulation and control of mover zone methylation.
(2) present invention firstly discovers that comparing egg of albumen LIN28B-WT, the LIN28B-TST albumen in wild type of wild type The more one section of new sequence of the N-terminal of white LIN28B-WT, which can be identified by special antibody, and lower LIN28B- The expression of TST can significantly inhibit the proliferation of tumour cell in vitro and in vivo.
(3) present invention firstly discovers that LIN28B-TST can be as the target molecule of new tumor diagnosis and therapy.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.
Unless stated otherwise, otherwise reagent used in description of the invention and material are commercial product.
Universal method
I. MATERIALS METHODS
1, cell culture and chemical reagent
Human embryonic kidney cells HEK-293T, HuH-7 and SK-HEP-1 cell (purchased from ATCC and Japanese cell bank) exist It is cultivated in DMEM (Gibco, New York, USA) culture medium；NCI-H1299 cells (being purchased from ATCC) are in RPMI-1640 (Gibco) it is cultivated in culture medium；HCT-116 cells (being purchased from ATCC) are cultivated in McCoy ' s 5A (Gibco) culture medium；AGS It is purchased from ATCC with A549 cells) it is cultivated in F-12K (Gibco) culture medium.10% N of tire serum is added in each culture medium (Gibco), 100IU/mL Pen .- Streps (Gibco).Cell is placed in 37 DEG C, contains 5%CO2Incubator in cultivated. JQ1 is purchased from Selleck Chemicals (Houston, USA) company.Decitabine (5-Aza-2 '- Deoxycytidine, 5AZA) it is purchased from Sigma-Aldrich (St.Louis, USA) company.
2, RNA is extracted, and reverse transcription and real-time quantitative PCR react (Quantitative real-time polymerase chain reaction)
Total serum IgE is extracted with TRIzol reagents from cell or tissue according to the operating guidance of manufacturer.CDNA is used PrimeScriptTMRT reagent Kit (TaKaRa, Tokyo, Japan) are synthesized.Real-time quantitative PCR reacts (qPCR) and uses SYBR Premix Ex Taq II (TaKaRa) β-actin are used as internal reference.The primer sequence used is as shown in table 2.Ripe MiRNAs is usedIt is special in MicroRNA Assays (Applied Biosystems, Foster City, USA) Primer and probe is quantified.U6small nuclear (snRNA) are used as internal reference.
2 primer table of table
3、Northern blot
Digoxigenin labeled is prepared using DIG Northern Starter Kit (Roche, Indianapolis, IN, USA) Rna probe, the template that corresponding PCR product is transcribed as T7.PCR primer is listed in supplementary tables 2.It uses NorthernMax Kit (Thermo Fisher Scientific, Waltham, USA) are carried out.10 μ g total serum IgEs are 2% It is detached, is then transferred on nylon membrane (GE Healthcare, Uppsala, Sweden) in agarose glue.After film drying Carry out UV crosslinking (265nm, 200mJ/cm2)1min.Prehybridization 1h is carried out at 62 DEG C, then 62 DEG C of progress hybridized overnights.Room Under temperature, film is washed using the 2x SSC containing 0.1%SDS, then film 30min is washed for 62 DEG C, washes twice.Next with containing 0.1%SDS's 0.2x SSC wash film, and 62 DEG C are washed film 30min, are washed twice.Developed (Typhoon, Molecular after film is washed Devices)。
4、RACE(Rapid Amplification of cDNA Ends)
5 '-RACE and 3 '-RACE experiments are according to SMARTerTM RACE cDNA Amplification Kit The specification of (Clontech, Mountain View, USA) is operated.The primer that 5 '-RACE and 3 '-RACE are used is listed in benefit Fill table 2.PCR product is cloned into pMD20-T or pMD18-T vector (TaKaRa) and carries out sanger sequencings.
5, antibody
Used antibody includes：H3K27ac(39133；ChIP-qPCR and ChIP-seq) and RNApol II (61667；ChIP-qPCR and ChIP-seq) it is bought from Active Motif (Carlsbad, USA) company；H3K4me3 (9751；ChIP-qPCR and ChIP-seq),c-Myc(13987；) and LIN28B (11965 WB；WB) from CST (Danvers, USA) company buys；IGFBP1(22803-1-AP；) and IGFBP3 (14642-1-AP WB；WB) from Proteintech (Rosemont, USA) company buys；ACTB(A2228；WB) bought from Sigma-Aldrich companies；IGFBP2 (AP10127b；WB) bought from Abgent (San Diego, USA) company；LIN28B-TST is by GenScript The synthesis of (Nanjing, China) company, there is special N-terminal De-immunized sequences (HRRQVLQKRMRSFNQVSSAP (SEQ ID NO.:3-22 in 1)
6、Western blot
Sodium dodecyl sulfate-polypropylene amide (sodium dodecyl sulfate- of the protein 10% or 14% Polyacrylamide, SDS) detached in gel, be then transferred into nitrocellulose filter (Bio-Rad, Hercules, USA on).Film is closed with 5% skimmed milk power and is incubated with primary antibody.Protein band chemiluminescence detection reagent (Thermo Fisher Scientific) it is detected in 6100 chemiluminescence imaging instrument of Tanon.
7, oligonucleotides transfects
SiRNA (Small interfering RNA, siRNA) is by RiboBio (Guangzhou, China) company Synthesis, LIN28B-TST interference sequences are：5′-GGAAAGCACAUUAGACCAU-3′(SEQ ID NO.:And 5 ' -5) AGGUUCUUCAGAAGAGGAU-3’(SEQ ID NO.:6)；The interference sequence of c-Myc is 5 '-GGUCAGAGUCUGGAUCACC- 3′(SEQ ID NO.:7).Cell usesThe transfection of RNAiMAXreagent (Invitrogen) reagent is few Nucleotide, ultimate density 50nM, 48h is tested after transfection.
8, cell Proliferation and colony formation
It is detected using CCK8 (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) The proliferation of cell.In colony formation, 1 × 103A cell kind is in each hole of 6 orifice plates, 37 DEG C of 1-2 weeks of culture.Carefully Born of the same parents are fixed using 4% paraformaldehyde and 1% crystal violet (Sigma-Aldrich) are used to dye.Finally carry out cell clone count and Analysis.
9, immunohistochemical staining
Tissue samples are fixed using neutral formalin, parafilm wrap, and are cut into 5 μm of slabs.Histotomy dewaxes Distilled water flushing is used in combination in rehydration afterwards.Slice is placed in 10mM sodium citrate buffer solutions (pH 6.0) and uses microwave stove heat 20min Carry out antigen retrieval.3%H is used at room temperature2O230min is acted on to remove endogenous peroxydase.Slice uses confining liquid (Thermo Fisher Scientific) room temperature closes 1h, then adds LIN28B-TST primary antibodies (1:500), the room temperature in wet box It is incubated 1h.Washing slice after incubation is incubated the anti-rabbit secondary antibody of biotin labeling, is then incubated the Streptavidin of HRP labels (streptavidin).Diaminobenzidine (DAB) (DAKO, Glostrup, Denmark) Liquid Substrate is as color developing agent, Soviet Union Lignin dye liquor (Mayer ' s hematoxylin) (DAKO) is used as counterstain.All slice uses Axioskop 2microscope microscopes (Carl Zeiss, Oberkochen, Germany) observation is taken pictures.
10, nude inoculation
The HuH-7 cells and control cell for collecting the LIN28B knocked out with CRISPR/Cas9 technologies carry out weight with DMEM It is outstanding.At the back of every nude mice (male BALB/c-nu/nu, 6 week old) 2 × 10 are inoculated by lower portion6A cell (is resuspended in 200μL DMEM).Every the major diameter (L) and minor axis (W) that 3-4 days use vernier caliper measurement tumour.Tumour is calculated with following equation Volume (V):V=(L x W2Mouse is put to death behind)/2.4 week, take out the tumour of formation and is weighed.The raising of all nude mices and reality Operation is tested to carry out all in accordance with the regulation of the Shanghai medical experiment animal welfare committee.
Embodiment 1 obtains the sequence of LIN28B-TST
Pass through the transcript profile of different normal structures and cancerous tissue and several cell lines sequencing (RNA-seq) analysis and research Potential specific transcription sheet.By largely screening, it was found that a large amount of new tomour specific transcript, including between 430 genes Completely new transcript and 1385 covering known transcripts.In these tomour specific transcripts, it is found surprisingly that The completely new transcript of one LIN28B gene, it shows high expression level and high shear Index Score, is named as LIN28B- TST.And it was found that LIN28B-TST is in Hepatocellular Carcinoma (HCC), HuH-7 hepatocellular carcinomas cell line and NCI- It is expressed in H1299 lung cancer cell lines, and wild type LIN28 transcripts (LIN28B-WT) are then expressed in HEK-293T human embryos In kidney cell line (Figure 1A).All variant transcriptions originally express (Figure 1A) not in normal liver tissue.LIN28-TST is in 5 ' end tools There are 2 new exons, in addition then exon 2-4 contained by the transcript with wild type is consistent (Figure 1A) for 3 exons.
Pass through northern blot method (Northern Blot) and the ends cDNA rapid amplification (Rapid Amplification of cDNA Ends) it confirmed the presence (Fig. 2) of LIN28B-TST transcripts.
Embodiment 2 detects the mRNA expressions of LIN28B-TST in tumour cell and tissue
In order to measure the expression of LIN28B-TST, 22 different tumours have been screened from TCGA transcript sequencing data storehouses It is more than 7000 samples.LIN28B is expressed in 10.2% tumour, in the tumor sample of all expression LIN28B, LIN28B-TST is specifically expressed again in HCC (91.5%, 54/59) and a lot of other such as cervical carcinomas (CESC, 86.2%, 25/ And endocervix cancer, brain Low grade glioma (LGG, 85.2%, 23/27), breast invasive carcinoma (BRCA, 69.8%, 30/ 29) 43), skin epidermis melanoma (SKCM, 68.1%, 32/47), the cancer of squamous cell lung carcinoma (LUSC, 50%, 59/118) In type (Figure 1B).In order to distinguish the expression with quantitative analysis LIN28B-WT and LIN28B-TST, special draw is devised Object (table 2), and real-time polymerase chain reaction (RT-PCR) (Fig. 3) has been carried out in various kinds of cell system.Later further at 120 pairs LIN28B-TST wide expressions in HCC tissues, but the not table in normal liver tissue or fetus hepatic tissue are confirmed in liver cancer sample It reaches；By contrast, LIN28B-WT can detect expression in fetus hepatic tissue, and express LIN28B-WT with being only dispersed in liver cancer (Fig. 4 A).Survival analysis shows that patient's prognosis with HCC and expression LIN28B-TST is worse (Fig. 4 B).It is sent out by RT-PCR Expression is not detected in tissues such as the normal heart, lung, brain, stomach, intestines, lung, liver, spleen, pancreas, skeletal muscle in existing LIN28B-TST.
The Regulation Mechanism and generation Identification of Fusion Protein of embodiment 3LIN28B-TST
The transcript sequencing result of LIN28B has prompted an alternative transcription in the upstreams LIN28B-WT distance 20kb or so Starting point (ATI).5 ' RACE amplifications have been carried out later, and ATI above-mentioned is navigated to the chr6 of No. 6 chromosome:104,937, 026 region.Chromosome co-immunoprecipitation sequencing (ChIP-seq) is found that histone H 3 K4me3, H3K27Ac and RNA polymerase For II (RNApol II) in the significant enrichment (Fig. 5 A) of ATI location proximates, this is the movable typical performance of promoter.These data Display LIN28B-TST occurs in making a variation the newly generated sites ATI from a typical chromatin.In order to detect the area in the sites ATI Domain could function as a promoter, and by this section, there may be the DNA fragmentations of LIN28B-TST promoters to be cloned into In luciferase reporter gene.Luciferase reporting analysis shows that, the promoter activity of LIN28B-TST is HEK-293T cells The promoter activity of LIN28B-WT or SV40 is more than 20 times (Fig. 5 B) in system.
Series of genes knocks out the core promoter area that experiment is found that one section of about 200 bp length close to the sites ATI Domain (Fig. 5 B).The islands CpG in order to study LIN28B-TST promoter regions methylate it is whether related to the expression of LIN28B-TST, The region of ATI and its both sides has carried out sulphite sequencing.In the promoter region of LIN28B-TST, the islands Liang Ge CpG are found. The sample that do not express with LIN28B-TST compares, express LIN28B-TST sample be demonstrated by second island CpG it is lower CpG methylation levels (Fig. 5 C).
In order to confirm that the demethylation in the sites CpG leads to the expression of LIN28B-TST enough, LIN28B or table are not being expressed Up in the cell of LIN28B-WT LIN28B- is had studied with dnmt rna inhibitor 5- aza deoxycytidines (5-AZA) TST.In this does not express the SK-HEP1 and ags cell system of LIN28B, the processing of 5-AZA consumingly has activated LIN28B-TST Expression；And in HEK-293T the and A549 cell lines of expression LIN28B-WT, the processing of 5-AZA can be activated significantly LIN28B-TST and the expression (Fig. 5 D) for inhibiting LIN28B-WT.
These are the results show that the demethylation of LIN28B-TST promoters may be the premise of LIN28B-TST transcriptions.Hair A person of good sense is investigated whether LIN28B-TST can be regulated and controled by c-Myc oncogenes, the results showed that, c-Myc can be with direct regulation and control The expression of LIN28B-WT.Inhibition by siRNA (siRNA) or BET inhibitor JQ1 to c-Myc, significantly reduces LIN28B is in the expression of the HepG2 cell lines of expression LIN28B-WT, and LIN28B-TST is not as c-Myc is being expressed Expression silencing in the HuH-7 cell lines of LIN28B-TST is changed (Fig. 5 E).This shows that the activation of LIN28B-TST is independent In the expression of c-Myc.
LIN28B-TST transcripts include that the in-frame start codon (ATGs) of 2 predictions is held 5 ', result in its coding Albumen has additional Amino-terminal amino acid (Fig. 6 A and 6B).Various cancers cell line to expressing LIN28B-TST is immunized Engram analysis finds a main band, shows that LIN28B-TST is mainly translated (Fig. 6 C) from first initiation codon. Inventor has further made a variation two initiation codons separately or together.Immunoblotting shows the shape of each LIN28B-TST variations Formula all no longer generates corresponding protein band (Fig. 6 B).
Embodiment 4 prepares LIN28B-TST antibody
It is prepared for a kind of rabbit source antibody of the anti-additional Amino-terminal amino acid of LIN28B-TST albumen.Synthesize LIN28B-TST Specific polypeptides HRRQVLQKRMRSFNQVSSAP, i.e. SEQ ID NO.:It is even to carry out KLH for 3-22 amino acid sequences in 1 Connection, 5mg be injected into rabbit carry out 3 times it is immune, take rabbit anteserum, and carry out affinity chromatography, acquisition specifically resists for LIN28B-TST Body.
Immunoblotting the results show that this antibody can specifically identify main LIN28B-TST isomers, and simultaneously It is unable to the smaller LIN28B-TST isomers of specific recognition or LIN28B-WT (Fig. 6 B).Exempted from using above-mentioned specific antibody Epidemic disease trace and immunohistochemistry technique can also detect the expression of LIN28B-TST in HCC tissues, and cannot be in normal hepatocytes It is detected in dirty tissue (Fig. 7 A and 7B).
5 cell growth of embodiment and proliferation experiment
Inventor further uses the expression of siRNA and CRISPR/Cas9 gene editing technology silences LIN28B-TST, and Observe that significant cell growth inhibition and cell colony form inhibiting effect (Fig. 8 and Fig. 9).Pass through nude mice by subcutaneous tumor formation Animal experiment finds that silence LIN28B-TST can significantly inhibit the one-tenth knurl ability of Huh-7 liver cancer cells, and has lowered tumour Quality and volume (Figure 10).
These results indicate that LIN28B-TST is that very crucial transcript occurs for cancer cell proliferation and tumour, And have to a certain extent as treatment of cancer specific target target potential.
Embodiment 6 screens the inhibitor of LIN28B-TST
By the method described in the present invention, following two groups of substances are applied to Huh-7 cell lines, then the table of LIN28B-TST Up to activity and the growth of following two groups of materials onto cells and the influence of proliferation detection：(a) candidate substances；(b) blank control.
If the expression activity of the LIN28B-TST of the cell of group (a) and following two groups of substances are to the journey that grows and be proliferated Degree statistically be substantially less than group (b) LIN28B-TST expression activity and following two groups of materials onto cells growth and Proliferation, then show that the candidate substances are the inhibitor of LIN28B-TST.
Similarly, by the method described in the present invention, following two groups of substances are applied to the (expression of NCI-H1299 cell lines LIN28B-TST), the influence to the LIN28B-TST activity expressed and to cell growth and proliferation is then observed：(a) candidate Substance；(b) blank control.
If the growth of expression activity and cell of the LIN28B-TST of group (a) and the degree of proliferation are statistically shown Growth and the proliferation for writing the expression activity and cell of the LIN28B-TST less than group (b), then show that the candidate substances are The inhibitor of LIN28B-TST.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Tumor Hispital Attached to Fudan Univ
<120>Relevant tomour specific transcript LIN28B-TST of cancer and application thereof
<130> P2016-1869
<160> 45
<170> PatentIn version 3.5
<210> 1
<211> 269
<212> PRT
<213>Homo sapiens (Homo sapiens)
<400> 1
Met Ser His Arg Arg Gln Val Leu Gln Lys Arg Met Arg Ser Phe Asn
1 5 10 15
Gln Val Ser Ser Ala Pro Gly Gly Ala Ser Lys Gly Gly Gly Glu Glu
20 25 30
Pro Gly Lys Leu Pro Glu Pro Ala Glu Glu Glu Ser Gln Val Leu Arg
35 40 45
Gly Thr Gly His Cys Lys Trp Phe Asn Val Arg Met Gly Phe Gly Phe
50 55 60
Ile Ser Met Ile Asn Arg Glu Gly Ser Pro Leu Asp Ile Pro Val Asp
65 70 75 80
Val Phe Val His Gln Ser Lys Leu Phe Met Glu Gly Phe Arg Ser Leu
85 90 95
Lys Glu Gly Glu Pro Val Glu Phe Thr Phe Lys Lys Ser Ser Lys Gly
100 105 110
Leu Glu Ser Ile Arg Val Thr Gly Pro Gly Gly Ser Pro Cys Leu Gly
115 120 125
Ser Glu Arg Arg Pro Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro Lys
130 135 140
Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys Glu
145 150 155 160
Cys Ser Leu Pro Pro Gln Pro Lys Lys Cys His Tyr Cys Gln Ser Ile
165 170 175
Met His Met Val Ala Asn Cys Pro His Lys Asn Val Ala Gln Pro Pro
180 185 190
Ala Ser Ser Gln Gly Arg Gln Glu Ala Glu Ser Gln Pro Cys Thr Ser
195 200 205
Thr Leu Pro Arg Glu Val Gly Gly Gly His Gly Cys Thr Ser Pro Pro
210 215 220
Phe Pro Gln Glu Ala Arg Ala Glu Ile Ser Glu Arg Ser Gly Arg Ser
225 230 235 240
Pro Gln Glu Ala Ser Ser Thr Lys Ser Ser Ile Ala Pro Glu Glu Gln
245 250 255
Ser Lys Lys Gly Pro Ser Val Gln Lys Arg Lys Lys Thr
260 265
<210> 2
<211> 5444
<212> DNA
<213>Homo sapiens (Homo sapiens)
<400> 2
aggctggtgt tgaactcctg ggctcaagcg attcgccacg tcaacctctg aagaaggtgc 60
tgggattaca agcatgagcc accgccgcca ggttcttcag aagaggatga ggtcattcaa 120
ccaggtttca tcagccccag gcggggctag caaaggtggt ggagaagagc ccgggaagct 180
gccggagccg gcagaggagg aatcccaggt tttgcgcgga actggccact gtaagtggtt 240
caatgtgcgc atgggatttg gattcatctc catgataaac cgagagggaa gccccttgga 300
tattccagtc gatgtatttg tacaccaaag caaactattc atggaaggat ttagaagcct 360
aaaagaagga gaaccagtgg aattcacatt taaaaaatct tccaaaggcc ttgagtcaat 420
acgggtaaca ggacctggtg ggagcccctg tttaggaagt gaaagaagac ccaaagggaa 480
gacactacag aaaagaaaac caaagggaga tagatgctac aactgtggtg gccttgatca 540
tcatgctaag gaatgtagtc tacctcctca gccaaagaag tgccattact gtcagagcat 600
catgcacatg gtggcaaact gcccacataa aaatgttgca cagccacccg cgagttctca 660
gggaagacag gaagcagaat cccagccatg cacttcaact ctccctcgag aagtgggagg 720
cgggcatggc tgtacatcac caccgtttcc tcaggaggct agggcagaga tctcagaacg 780
gtcaggcagg tcacctcaag aagcttcctc cacgaagtca tctatagcac cagaagagca 840
aagcaaaaag gggccttcag ttcaaaaaag gaaaaagaca taacaggtct tcttcatatg 900
ttctttcctt tacccggttg caaagtctac ctcatgcaag tataggggaa cagtatttca 960
caagcagtag ctgacctggg attttaacta ctattgggga actgtgaatt ttttaaacag 1020
acaaatcact ctaagcaaat tacatttgag cagggtgtca tgttttatgt taattcagag 1080
aataagatac tatgtctgtc aatatgtgca tgtgtgagag ggagagagcc tgagtctgtg 1140
tgtgtacatg aggattttta tataggaatg tagacacata tataaagagg ctttgtcttt 1200
atatatttgt gtatagatca aagcacacac cctctctcat ataattggat atttccaaga 1260
attgaaaacc catgtgaagc attatagata gttttaaatt taacccactg gagttttctt 1320
gaaataccac ttcttttata ttatataaaa ctaaaaacac gactgttacc ttttgtgtga 1380
accaaaggat acttcagatc tcagagctgc caattatggg gtactaaagg tttttaagac 1440
atccagttct cccgaatttg ggattgcctc tttttcttga aatctctgga gtagtaattt 1500
ttttccccct tttttgaagg cagtacctta acttcatatg cctctgactg ccataagctt 1560
ttttgattct gggataacat aactccagaa aagacaatga atgtgtaatt tgggccgata 1620
tttcactgtt ttaaattctg tgtttaattg taaaattaga tgcctattaa gagaaatgaa 1680
ggggaggatc atcttagtgg cttgttttca gtagtatttt aatatcagct tcttgtaacc 1740
ttttccatgt tgtgagggtt gtaagggatt gtgtggcaac agcagcttcc cttggctaac 1800
tcaatcttct acccattgct tagagcaggg agccctcctt atttactact gaagacctta 1860
gagaactcca attgtttggc atatattttt ggtggtggtt tttattcctc ctggagagtt 1920
atctaatttg tttctaaaac aaacaagcag caaagaaatg aattaaatac tggggttgag 1980
aattaaaatt aagtggatgt tcacagttgc ccaatatata tgacctgcaa atgatacgaa 2040
aaagtgcagc atttagtggc agttaacaag agtgacaagc ctggggcaga ggtaccaaac 2100
ctctcccacc agagagctag aagtatttta tacagtaact ttgatcttat ggaagtgacc 2160
ttcaatgctt attctgaagt aacctatatg gtggatacag gatgaacatt cagtgccagg 2220
gagaatcttc tcaggttggt tctcgttaga gtgataaact ggctaggggc catagtattg 2280
gtcctgttag gtttcggtca tggaaaaaaa aattattttg gggtcatcct ggctctagat 2340
gttatgggca aatttctgaa acatctgcaa gaaggtacca gttaattata gtgcttaata 2400
ttgggaataa gattaagcat tataattata atgtatgggc ctgttggtgt aagctcagat 2460
aattaaataa aaatagcatg actcaaatga gacatattct gctgaacagt ttctacttcc 2520
tctcccgcct gtcctgtcat gggagacgtg tatagttgct gctgtttcag caaaccacca 2580
taagacgaaa atgcctcagg ttgggttgcc agtcctttac aactcagctt gaatttcaca 2640
acagtgattg tgagaatctg cgtggtatac actgaaatat cggtgtgctg tgatgcaaag 2700
cttacctttg acgatattga atgtgatata gctgtagaga agtacttcct tgccttatgt 2760
gaggatttca aacttattta aattatgtag acaaatcaaa gtggcattgc ttaattttta 2820
gcaggcataa taagcaagtt aacagtaaaa tgcaaaacat gataagcgtt gctcaatttt 2880
tagcaggtat aataagcagg ttaacagtaa aaatgcaaaa catgatagat aagtcacttt 2940
gaaaattcaa accaaagttc cttcacctta tggaaatagg aaattatgga cttcaaaatt 3000
ggacacttcc tgtttacaaa aagaaattca gagctaaaat catggtaaaa aaaaatagaa 3060
acacttgaga actatggtct ttatgggtgc aatttgaaat ccttttcatc atcttaccag 3120
actaaactaa gagcacatac caaacctatc ttatggttga aagttggggt ttatttttta 3180
tatgagaata ttatcactat tacataacat actcaggaca aagaactttg ctcagggaac 3240
ataccatgta atatttttgt tgtttcttta cagactagtc tacagtcctg cttactcaaa 3300
acaaaccaaa taacttatac ctttatataa gtattatgta ctgatgatag taactacctc 3360
tgagtttgac acagatcaaa atttttgaat atcagatatc agttatccta tttttatttc 3420
atgtgaaaac tcctctaaag cagattccct caactctgtg catatgtgaa tatcactgat 3480
gtgaacacat tgttcattta cataggtaaa atattactct gtttacagca aaaggctacc 3540
tcatagttga tacatagcac acctgtatgt atgctgttcc agccttacag gtggctgata 3600
attctctggt acagaacctt tttatctgta ttataaatag caattcacaa ctgcatgttt 3660
ctgacaaaca cttgtgaata atgaagcatc tcgttttagt tagcaaagtc tccaaacatt 3720
tccttaaaat aatcatgtat ttagtttaaa gaattatggg cactgttcaa cttaagcaaa 3780
acagaacacg gaagcagtct tagaagcacc actttgccca gaggtggagg ttggaagggg 3840
tagcagggag aggggttggt gtatgcaggt attcatgcta ggcaaagagt ttaaaagacg 3900
ccaatgtcct tcatttactg tctgtgctgc cctgaagcca agcgtattgc agcattatag 3960
ccccaggcac ataactaact agcactggct tgccaaggaa tgaacatgca atgccattac 4020
tagctattga gggaaaaggg tctgtgtgaa gcatcacttt gcagggatta ctaatggtgg 4080
ggcagcaggt ctgtgaatta agttatctct tgacctcacc ctcatgtcaa cacaaatgta 4140
attcctaaac aagatgcatt gccagtctct tagccctgta agctgatctt ttgctacatg 4200
gcagactata atgaaaacat ttttatactt gggtttctag tcttcactag aaggccttgg 4260
atgtattttt gcagttgaaa gatttagaaa gatttttacc tgcttataac ttggaagttt 4320
agagtgcaat gtaagaaaaa agatcaagaa atgtcatgtt attagcatca gtccacctcc 4380
aatattgccg atactttttt tattctggct cagttttatt ttgcaccagt gcggccccaa 4440
gttactgctg gttgtattta gtttgtgaat aggagcccat aagtgttaat agactttgta 4500
acattcacta taagatgaat tatacaggac atgggaaatc tcattaagtc ttaaagttaa 4560
tttaaattaa tttatctgtt ttctctaaga aatgtttatc ataaaatata tatgtgtatt 4620
tcccctttgg ttataaaatt tgggaaagta tgtacaagtg cagctgcact gactttaatt 4680
ttctagatgt cttaatgaga tttatttgtt ttagagaaga acatcttgtt aaaagcatca 4740
aactctgtct tacatagctg tcaacagcct ctttaagatg tggtggttgt atgatctgtg 4800
tcttaattgt tcagttagag tgagaagttg acctatgatt catttttaaa ttttatattt 4860
ggaacaaagc tgcaagttat ggtaaagtac tgtactgtga gaagtattat gatatttaat 4920
gcatctgtgg cttaacactt gtgagagtta ccagcttgaa aatgatggtg ttgactacct 4980
cttgaatcac atctatcaac cactggcacc taccaccaag ctggcttcaa ttagtatgtg 5040
ttgctttttg gtattaacaa ctaaccgtac tagagaccaa agtgaaccct gatttttata 5100
tgtctttaat aatggtgttt tatctagtgt ttttaaatta tcctgtgtag tatttagatt 5160
acctcattgt ccattttgac tcatgttgtt tacaagtgaa aataaaaaca cttgaactgt 5220
atgtttttaa aagacaaaaa aggggtagat gtttggaatg cgtttcactc gcatgcagtc 5280
atctggaggg actgaagcac tgtttgcctt tctgtacact ctgggtttta tattctcatt 5340
tcatgcctaa tgtcttattc tgtcaattat ggatatgttg aggtttaaaa aaattacttg 5400
attaaaaata aaacatataa cgttggcatt taaaaaaaaa aaaa 5444
<210> 3
<211> 250
<212> PRT
<213>Homo sapiens (Homo sapiens)
<400> 3
Met Ala Glu Gly Gly Ala Ser Lys Gly Gly Gly Glu Glu Pro Gly Lys
1 5 10 15
Leu Pro Glu Pro Ala Glu Glu Glu Ser Gln Val Leu Arg Gly Thr Gly
20 25 30
His Cys Lys Trp Phe Asn Val Arg Met Gly Phe Gly Phe Ile Ser Met
35 40 45
Ile Asn Arg Glu Gly Ser Pro Leu Asp Ile Pro Val Asp Val Phe Val
50 55 60
His Gln Ser Lys Leu Phe Met Glu Gly Phe Arg Ser Leu Lys Glu Gly
65 70 75 80
Glu Pro Val Glu Phe Thr Phe Lys Lys Ser Ser Lys Gly Leu Glu Ser
85 90 95
Ile Arg Val Thr Gly Pro Gly Gly Ser Pro Cys Leu Gly Ser Glu Arg
100 105 110
Arg Pro Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro Lys Gly Asp Arg
115 120 125
Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys Glu Cys Ser Leu
130 135 140
Pro Pro Gln Pro Lys Lys Cys His Tyr Cys Gln Ser Ile Met His Met
145 150 155 160
Val Ala Asn Cys Pro His Lys Asn Val Ala Gln Pro Pro Ala Ser Ser
165 170 175
Gln Gly Arg Gln Glu Ala Glu Ser Gln Pro Cys Thr Ser Thr Leu Pro
180 185 190
Arg Glu Val Gly Gly Gly His Gly Cys Thr Ser Pro Pro Phe Pro Gln
195 200 205
Glu Ala Arg Ala Glu Ile Ser Glu Arg Ser Gly Arg Ser Pro Gln Glu
210 215 220
Ala Ser Ser Thr Lys Ser Ser Ile Ala Pro Glu Glu Gln Ser Lys Lys
225 230 235 240
Gly Pro Ser Val Gln Lys Arg Lys Lys Thr
245 250
<210> 4
<211> 5517
<212> DNA
<213>Homo sapiens (Homo sapiens)
<400> 4
aattgacaaa gtcacgtgtg ctcagggggc cagaaactgg agagaggaga gaaaaaaatc 60
aaaagaagga aagcacatta gaccatgcga gctaaatttg tgatcgcaca aaatcaagat 120
gttagattga tgcagaagat cactccgttc caaagggaaa gttttcatct cacgagtttg 180
gagctgaggg cccgtggggc aacatggccg aaggcggggc tagcaaaggt ggtggagaag 240
agcccgggaa gctgccggag ccggcagagg aggaatccca ggttttgcgc ggaactggcc 300
actgtaagtg gttcaatgtg cgcatgggat ttggattcat ctccatgata aaccgagagg 360
gaagcccctt ggatattcca gtcgatgtat ttgtacacca aagcaaacta ttcatggaag 420
gatttagaag cctaaaagaa ggagaaccag tggaattcac atttaaaaaa tcttccaaag 480
gccttgagtc aatacgggta acaggacctg gtgggagccc ctgtttagga agtgaaagaa 540
gacccaaagg gaagacacta cagaaaagaa aaccaaaggg agatagatgc tacaactgtg 600
gtggccttga tcatcatgct aaggaatgta gtctacctcc tcagccaaag aagtgccatt 660
actgtcagag catcatgcac atggtggcaa actgcccaca taaaaatgtt gcacagccac 720
ccgcgagttc tcagggaaga caggaagcag aatcccagcc atgcacttca actctccctc 780
gagaagtggg aggcgggcat ggctgtacat caccaccgtt tcctcaggag gctagggcag 840
agatctcaga acggtcaggc aggtcacctc aagaagcttc ctccacgaag tcatctatag 900
caccagaaga gcaaagcaaa aaggggcctt cagttcaaaa aaggaaaaag acataacagg 960
tcttcttcat atgttctttc ctttacccgg ttgcaaagtc tacctcatgc aagtataggg 1020
gaacagtatt tcacaagcag tagctgacct gggattttaa ctactattgg ggaactgtga 1080
attttttaaa cagacaaatc actctaagca aattacattt gagcagggtg tcatgtttta 1140
tgttaattca gagaataaga tactatgtct gtcaatatgt gcatgtgtga gagggagaga 1200
gcctgagtct gtgtgtgtac atgaggattt ttatatagga atgtagacac atatataaag 1260
aggctttgtc tttatatatt tgtgtataga tcaaagcaca caccctctct catataattg 1320
gatatttcca agaattgaaa acccatgtga agcattatag atagttttaa atttaaccca 1380
ctggagtttt cttgaaatac cacttctttt atattatata aaactaaaaa cacgactgtt 1440
accttttgtg tgaaccaaag gatacttcag atctcagagc tgccaattat ggggtactaa 1500
aggtttttaa gacatccagt tctcccgaat ttgggattgc ctctttttct tgaaatctct 1560
ggagtagtaa tttttttccc ccttttttga aggcagtacc ttaacttcat atgcctctga 1620
ctgccataag cttttttgat tctgggataa cataactcca gaaaagacaa tgaatgtgta 1680
atttgggccg atatttcact gttttaaatt ctgtgtttaa ttgtaaaatt agatgcctat 1740
taagagaaat gaaggggagg atcatcttag tggcttgttt tcagtagtat tttaatatca 1800
gcttcttgta accttttcca tgttgtgagg gttgtaaggg attgtgtggc aacagcagct 1860
tcccttggct aactcaatct tctacccatt gcttagagca gggagccctc cttatttact 1920
actgaagacc ttagagaact ccaattgttt ggcatatatt tttggtggtg gtttttattc 1980
ctcctggaga gttatctaat ttgtttctaa aacaaacaag cagcaaagaa atgaattaaa 2040
tactggggtt gagaattaaa attaagtgga tgttcacagt tgcccaatat atatgacctg 2100
caaatgatac gaaaaagtgc agcatttagt ggcagttaac aagagtgaca agcctggggc 2160
agaggtacca aacctctccc accagagagc tagaagtatt ttatacagta actttgatct 2220
tatggaagtg accttcaatg cttattctga agtaacctat atggtggata caggatgaac 2280
attcagtgcc agggagaatc ttctcaggtt ggttctcgtt agagtgataa actggctagg 2340
ggccatagta ttggtcctgt taggtttcgg tcatggaaaa aaaaattatt ttggggtcat 2400
cctggctcta gatgttatgg gcaaatttct gaaacatctg caagaaggta ccagttaatt 2460
atagtgctta atattgggaa taagattaag cattataatt ataatgtatg ggcctgttgg 2520
tgtaagctca gataattaaa taaaaatagc atgactcaaa tgagacatat tctgctgaac 2580
agtttctact tcctctcccg cctgtcctgt catgggagac gtgtatagtt gctgctgttt 2640
cagcaaacca ccataagacg aaaatgcctc aggttgggtt gccagtcctt tacaactcag 2700
cttgaatttc acaacagtga ttgtgagaat ctgcgtggta tacactgaaa tatcggtgtg 2760
ctgtgatgca aagcttacct ttgacgatat tgaatgtgat atagctgtag agaagtactt 2820
ccttgcctta tgtgaggatt tcaaacttat ttaaattatg tagacaaatc aaagtggcat 2880
tgcttaattt ttagcaggca taataagcaa gttaacagta aaatgcaaaa catgataagc 2940
gttgctcaat ttttagcagg tataataagc aggttaacag taaaaatgca aaacatgata 3000
gataagtcac tttgaaaatt caaaccaaag ttccttcacc ttatggaaat aggaaattat 3060
ggacttcaaa attggacact tcctgtttac aaaaagaaat tcagagctaa aatcatggta 3120
aaaaaaaata gaaacacttg agaactatgg tctttatggg tgcaatttga aatccttttc 3180
atcatcttac cagactaaac taagagcaca taccaaacct atcttatggt tgaaagttgg 3240
ggtttatttt ttatatgaga atattatcac tattacataa catactcagg acaaagaact 3300
ttgctcaggg aacataccat gtaatatttt tgttgtttct ttacagacta gtctacagtc 3360
ctgcttactc aaaacaaacc aaataactta tacctttata taagtattat gtactgatga 3420
tagtaactac ctctgagttt gacacagatc aaaatttttg aatatcagat atcagttatc 3480
ctatttttat ttcatgtgaa aactcctcta aagcagattc cctcaactct gtgcatatgt 3540
gaatatcact gatgtgaaca cattgttcat ttacataggt aaaatattac tctgtttaca 3600
gcaaaaggct acctcatagt tgatacatag cacacctgta tgtatgctgt tccagcctta 3660
caggtggctg ataattctct ggtacagaac ctttttatct gtattataaa tagcaattca 3720
caactgcatg tttctgacaa acacttgtga ataatgaagc atctcgtttt agttagcaaa 3780
gtctccaaac atttccttaa aataatcatg tatttagttt aaagaattat gggcactgtt 3840
caacttaagc aaaacagaac acggaagcag tcttagaagc accactttgc ccagaggtgg 3900
aggttggaag gggtagcagg gagaggggtt ggtgtatgca ggtattcatg ctaggcaaag 3960
agtttaaaag acgccaatgt ccttcattta ctgtctgtgc tgccctgaag ccaagcgtat 4020
tgcagcatta tagccccagg cacataacta actagcactg gcttgccaag gaatgaacat 4080
gcaatgccat tactagctat tgagggaaaa gggtctgtgt gaagcatcac tttgcaggga 4140
ttactaatgg tggggcagca ggtctgtgaa ttaagttatc tcttgacctc accctcatgt 4200
caacacaaat gtaattccta aacaagatgc attgccagtc tcttagccct gtaagctgat 4260
cttttgctac atggcagact ataatgaaaa catttttata cttgggtttc tagtcttcac 4320
tagaaggcct tggatgtatt tttgcagttg aaagatttag aaagattttt acctgcttat 4380
aacttggaag tttagagtgc aatgtaagaa aaaagatcaa gaaatgtcat gttattagca 4440
tcagtccacc tccaatattg ccgatacttt ttttattctg gctcagtttt attttgcacc 4500
agtgcggccc caagttactg ctggttgtat ttagtttgtg aataggagcc cataagtgtt 4560
aatagacttt gtaacattca ctataagatg aattatacag gacatgggaa atctcattaa 4620
gtcttaaagt taatttaaat taatttatct gttttctcta agaaatgttt atcataaaat 4680
atatatgtgt atttcccctt tggttataaa atttgggaaa gtatgtacaa gtgcagctgc 4740
actgacttta attttctaga tgtcttaatg agatttattt gttttagaga agaacatctt 4800
gttaaaagca tcaaactctg tcttacatag ctgtcaacag cctctttaag atgtggtggt 4860
tgtatgatct gtgtcttaat tgttcagtta gagtgagaag ttgacctatg attcattttt 4920
aaattttata tttggaacaa agctgcaagt tatggtaaag tactgtactg tgagaagtat 4980
tatgatattt aatgcatctg tggcttaaca cttgtgagag ttaccagctt gaaaatgatg 5040
gtgttgacta cctcttgaat cacatctatc aaccactggc acctaccacc aagctggctt 5100
caattagtat gtgttgcttt ttggtattaa caactaaccg tactagagac caaagtgaac 5160
cctgattttt atatgtcttt aataatggtg ttttatctag tgtttttaaa ttatcctgtg 5220
tagtatttag attacctcat tgtccatttt gactcatgtt gtttacaagt gaaaataaaa 5280
acacttgaac tgtatgtttt taaaagacaa aaaaggggta gatgtttgga atgcgtttca 5340
ctcgcatgca gtcatctgga gggactgaag cactgtttgc ctttctgtac actctgggtt 5400
ttatattctc atttcatgcc taatgtctta ttctgtcaat tatggatatg ttgaggttta 5460
aaaaaattac ttgattaaaa ataaaacata taacgttggc atttaaaaaa aaaaaaa 5517
<210> 5
<211> 19
<212> RNA
<213>Artificial sequence
<400> 5
ggaaagcaca uuagaccau 19
<210> 6
<211> 19
<212> RNA
<213>Artificial sequence
<400> 6
agguucuuca gaagaggau 19
<210> 7
<211> 19
<212> RNA
<213>Artificial sequence
<400> 7
ggucagaguc uggaucacc 19
<210> 8
<211> 22
<212> PRT
<213>Homo sapiens (Homo sapiens)
<400> 8
Met Ser His Arg Arg Gln Val Leu Gln Lys Arg Met Arg Ser Phe Asn
1 5 10 15
Gln Val Ser Ser Ala Pro
20
<210> 9
<211> 1708
<212> DNA
<213>Homo sapiens (Homo sapiens)
<400> 9
acgtaggcta tttccattct tttgctatta taaaaatcaa tccaggtata cacatcattt 60
gctgtataag atgcacttat atctctggga taatttccca ggaaagggat tgctaagtca 120
tagagtatat tttaaatttt gagagatttc accaaatttc cctctctaaa tgttgtgccc 180
ccttggtacc cccaccagga atgtatcaaa gtatgggttt atctatagcc tcaccaaaaa 240
attattttaa aactttaaag ctgtatttta acagtgtgtt gtcaaacgtt aggatttttg 300
ccagtttaat agacaaacac aagtttgtca caatttttat gaattattat tatttttttg 360
agatggagtc ttgctctgtc gcccaggctg gaatgcagtg gcgcaatctc ggctcattgc 420
aatctccccc tcccgggttc aagcgatttt cccgcctcag cccccggagt agctgggact 480
acaggcgccc gccaccacgc ccggctaatt tttgtatttt cagtagagac gcggtgtcac 540
catgttggcc agctggtatt gaactcctga tatcaggtga tccacccacc tcggtctccc 600
aaagtgctgg gattacagga gtgacccacc gcgcccagcc aacagttttt atgaatgtta 660
aacttcctgt tcatttctca ttttcctctt ctgtacataa gggagtgctc aatagataca 720
atataatttg ggtgacagtg cttgctttat taacttttga tgttggaaat aatttgtatt 780
tgtgataggt atgtagcagt ctaaaacaaa cttttgtgac tgatttcttt tactatgata 840
aatgcaaaaa atctgtgcca tgtgcagcaa gtcagttctc tgcagaatga tattaatttt 900
tcagtgaaga agcaaaatca tttctaacaa tgattcttgt tatgggttga actgcatccc 960
tccaaaaaga tgttgaagtc ctacccccca gtctctgtaa atgtgtactt aaaaataggg 1020
tccttgtagt taatcaagtt aagatgagat cattagggtg ggtcctaatc tgactggtgt 1080
cctgaaaagg aaaaattggg gcatggggac agataagtag aaagggaaga caatgtgatg 1140
acacaagcag cagccattta caagctaagg aatgcccgag tctaccagaa gccaggagag 1200
aggcttggca cagattctca tcacagtcca gaatcgaccc ggaagccttc agatctgtaa 1260
gacgttacat ttttgttgtt tcagccaccc tagaaagcaa atacaatgct attcaccctg 1320
ttgcccacgc tggcctggaa cttctgtact caagcgatcg gcccatctcg gtctcctaat 1380
gtgccgggat tacaggcatg agccactgca cccggccagc tagttttaat ctttttcttt 1440
ctttcttcca ttttaggaca ggatcgcact gttgcccagg gtggagtgca gtggcgcaat 1500
cttggctcac tgcaacctcc gcccccctgc tggagagatc ctcccacctc agcctcccca 1560
atagctggga ccactggcgc gcagaccacg cccagcgaat tttttgtaga gagccaggtc 1620
gccattttcc acaggctggt gttgaactcc tgggctcaag cgattcgcca cgtcaacctc 1680
tgaagaaggt gctgggatta caagcatg 1708
<210> 10
<211> 21
<212> DNA
<213>Artificial sequence
<400> 10
tgatgcagaa gatcactccg t 21
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
atatccaagg ggcttccctc t 21
<210> 12
<211> 19
<212> DNA
<213>Artificial sequence
<400> 12
ttacaagcat gagccaccg 19
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
gctcttctcc accacctttg 20
<210> 14
<211> 22
<212> DNA
<213>Artificial sequence
<400> 14
ttgttacagg aagtcccttg cc 22
<210> 15
<211> 22
<212> DNA
<213>Artificial sequence
<400> 15
atgctatcac ctcccctgtg tg 22
<210> 16
<211> 19
<212> DNA
<213>Artificial sequence
<400> 16
ggagccctcc ttatttact 19
<210> 17
<211> 45
<212> DNA
<213>Artificial sequence
<400> 17
gatcactaat acgactcact ataggggaaa cctaacagga ccaat 45
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
ttctttggct gaggaggtag 20
<210> 19
<211> 33
<212> DNA
<213>Artificial sequence
<400> 19
gactacctct tgaatcacat ctatcaacca ctg 33
<210> 20
<211> 53
<212> DNA
<213>Artificial sequence
<400> 20
agaagattct agagctagcg aattcatggc cgaaggcggg gctagcaaag gtg 53
<210> 21
<211> 52
<212> DNA
<213>Artificial sequence
<400> 21
agaagattct agagctagcg aattcatgag ccaccgccgc caggttcttc ag 52
<210> 22
<211> 56
<212> DNA
<213>Artificial sequence
<400> 22
cgcagatcct tcgcggccgc ggatcctact tgcatgaggt agactttgca accggg 56
<210> 23
<211> 45
<212> DNA
<213>Artificial sequence
<400> 23
cgataggtac cgagctctta tgagaaagta gccctttttc ctgag 45
<210> 24
<211> 44
<212> DNA
<213>Artificial sequence
<400> 24
gcttacttag atcgcagatc cctcagctcc aaactcgtga gatg 44
<210> 25
<211> 44
<212> DNA
<213>Artificial sequence
<400> 25
cgataggtac cgagctctta cgtaggctat ttccattctt ttgc 44
<210> 26
<211> 44
<212> DNA
<213>Artificial sequence
<400> 26
gcttacttag atcgcagatc gcttgtaatc ccagcacctt cttc 44
<210> 27
<211> 49
<212> DNA
<213>Artificial sequence
<400> 27
cgataggtac cgagctctta caaagtatgg gtttatctat agcctcacc 49
<210> 28
<211> 49
<212> DNA
<213>Artificial sequence
<400> 28
cgataggtac cgagctctta gagtagctgg gactacaggc gcccgccac 49
<210> 29
<211> 49
<212> DNA
<213>Artificial sequence
<400> 29
cgataggtac cgagctctta tttgtatttg tgataggtat gtagcagtc 49
<210> 30
<211> 49
<212> DNA
<213>Artificial sequence
<400> 30
cgataggtac cgagctctta gggtgggtcc taatctgact ggtgtcctg 49
<210> 31
<211> 49
<212> DNA
<213>Artificial sequence
<400> 31
cgataggtac cgagctctta agaaagcaaa tacaatgcta ttcaccctg 49
<210> 32
<211> 49
<212> DNA
<213>Artificial sequence
<400> 32
cgataggtac cgagctctta tggagtgcag tggcgcaatc ttggctcac 49
<210> 33
<211> 49
<212> DNA
<213>Artificial sequence
<400> 33
cgataggtac cgagctctta ggagagatcc tcccacctca gcctcccca 49
<210> 34
<211> 49
<212> DNA
<213>Artificial sequence
<400> 34
cgataggtac cgagctctta cagaccacgc ccagcgaatt ttttgtaga 49
<210> 35
<211> 49
<212> DNA
<213>Artificial sequence
<400> 35
cgataggtac cgagctctta caggctggtg ttgaactcct gggctcaag 49
<210> 36
<211> 24
<212> DNA
<213>Artificial sequence
<400> 36
caccgaaata aactgacctg gcgg 24
<210> 37
<211> 24
<212> DNA
<213>Artificial sequence
<400> 37
aaacccgcca ggtcagttta tttc 24
<210> 38
<211> 25
<212> DNA
<213>Artificial sequence
<400> 38
caccgaacca ggtttcatca gcccc 25
<210> 39
<211> 25
<212> DNA
<213>Artificial sequence
<400> 39
aaacggggct gatgaaacct ggttc 25
<210> 40
<211> 25
<212> DNA
<213>Artificial sequence
<400> 40
caccgtgcta tagatgactt cgtgg 25
<210> 41
<211> 25
<212> DNA
<213>Artificial sequence
<400> 41
aaacccacga agtcatctat agcac 25
<210> 42
<211> 25
<212> DNA
<213>Artificial sequence
<400> 42
ttttgggata attttttagg aaagg 25
<210> 43
<211> 25
<212> DNA
<213>Artificial sequence
<400> 43
accaactaac caacataata acacc 25
<210> 44
<211> 25
<212> DNA
<213>Artificial sequence
<400> 44
tattagggtg ggttttaatt tgatt 25
<210> 45
<211> 25
<212> DNA
<213>Artificial sequence
<400> 45
aaaaaaatct ctccaacaaa aaaac 25

Claims (10)

1. a kind of LIN28B-TST polypeptides of separation, which is characterized in that the polypeptide is selected from the group：
(a) there is SEQ ID NO:The polypeptide of 1 amino acid sequence；
(b) by SEQ ID NO:The one or more amino acid residues of 1 amino acid sequence process (preferably, 1-30, more preferably, 1-15, more preferably, 1-6) replace, miss or add and formed, and there is SEQ ID NO:In 1 shown in 1-22 22 amino acid amino acid sequence, have tumor cell specific expression, and with promote tumour cell growth and It is proliferated, promotes the tumorigenic active polypeptide derived from (a)；
(c) with SEQ ID NO:1 amino acid sequence has >=80% homology (homology preferably, >=90%；Preferably >= 95% homology；Most preferably, >=97% homology, such as 98% or more, 99% or more), and there is SEQ ID NO:In 1 The amino acid sequence of 22 amino acid shown in 1-22 is expressed with tumor cell specific and with promotion tumour The growth of cell and proliferation promote the tumorigenic active polypeptide derived from (a).
2. a kind of polynucleotides of separation, which is characterized in that the polypeptide described in the polynucleotide encoding claim 1.
3. a kind of carrier, which is characterized in that the carrier contains the polynucleotides described in claim 2.
4. a kind of host cell, which is characterized in that the host cell contains whole in carrier or genome described in claim 3 Close the polynucleotides having the right described in requirement 2.
5. a kind of preparation method of polypeptide, which is characterized in that including：
Under conditions suitable for the expression, the host cell described in claim 4 is cultivated, it is described in claim 1 to give expression to Polypeptide；The polypeptide with separation.
6. a kind of specific inhibitor, which is characterized in that combined with polypeptide described in claim 1 to the Inhibitor specificity Or inhibit LIN28B-TST expression.
7. a kind of purposes of the specific inhibitor described in claim 6, which is characterized in that the specific inhibitor is for making Standby drug or preparation, the diagnosis or Index for diagnosis of the drug or preparation for (i) tumour；(ii) inhibit growth of tumour cell and Proliferation；And/or (iii) inhibits tumorigenic ability.
8. a kind of pharmaceutical composition, which is characterized in that including：
Specific inhibitor described in claim 6；And pharmaceutically acceptable carrier.
9. a kind of determining tester whether be LIN28B-TST inhibitor method, which is characterized in that including step：
(a) in test group, in cultivating system, in the presence of tester, the cell of culture expression LIN28B-TST；And There is no in the tester and the identical control group of other conditions, identical cell is cultivated；
(b) the expression quantity E1 or its activity A1 of LIN28B-TST described in cell described in test group are detected；And in control group The expression quantity E2 or activity A2 of LIN28B-TST described in the cell is compared；
Wherein, if the expression quantity E1 is substantially less than the expression quantity E2 or described activity A1 and is substantially less than the activity A2, Then indicate that the object to be tested is the inhibitor of LIN28B-TST.
10. the purposes of a kind of LIN28B-TST polypeptides or its transcript or its coded sequence or its detection reagent, feature exist In being used to prepare a detection kit, the kit is used for the diagnosis or prognosis of tumour.
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