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 PDFInfo
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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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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
Technical Field
The present invention relates to the field of tumor therapy. In particular, it relates to the cancer-associated tumor-specific transcript LIN28B-TST and its uses.
Background
Deep RNA sequencing has revealed the diversity and complexity of the properties and splicing patterns of the human transcriptome from a completely new perspective. Cancer is a complex disease with heterogeneity, involving genetic and epigenetic level changes, making it a more complex transcriptome. Thus, the cancer transcriptome has a great potential to generate Tumor-Specific Transcripts (TST). The identification of tumor-specific transcripts has a very important clinical role, and the classification of tumor-specific transcripts, such as fusion transcripts, is also of great importance for understanding the process of tumor development (fusion transcripts are one of the tumor-specific transcripts, are derived from DNA translocations). Loss of normal regulatory transcriptional processes and epigenetic changes may lead to a large number of tumor-specific transcripts, including specific mrnas and novel transcripts between genes. However, the identification and function of these specific transcripts has not yet been fully elucidated.
The protein family of LIN28, including LIN28A and LIN28B, is an RNA-binding protein that is highly expressed in embryogenesis, but not in adult tissues. The LIN28 protein can prevent the maturation of tumor suppressor let-7 family RNA (a micro RNA) and regulate the self-renewal of mammalian stem cells. However, the mechanism that leads to the activation of LIN28B gene has not been elucidated before.
Therefore, there is an urgent need in the art to study the completely new mechanism of LIN28B gene activation in cancer, and to develop proteins associated with tumor growth and proliferation, and target drugs for treating tumors.
Disclosure of Invention
The invention aims to provide a novel target LIN28B-TST related to tumor susceptibility and/or prognosis and application thereof in diagnosis, treatment and the like.
In a first aspect, the invention provides an isolated LIN28B-TST polypeptide selected from the group consisting of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO 1;
(b) a polypeptide derived from (a) which is formed by substituting, deleting or adding one or more amino acid residues (preferably, 1-30, more preferably, 1-15, more preferably, 1-6) in the amino acid sequence of SEQ ID NO. 1, has an amino acid sequence of 22 amino acids shown in positions 1-22 of SEQ ID NO. 1, has tumor cell-specific expression, and has the activity of promoting the growth and proliferation of tumor cells and the tumorigenesis;
(c) the polypeptide which has more than or equal to 80 percent of homology (preferably more than or equal to 90 percent of homology; preferably more than or equal to 95 percent of homology; most preferably more than or equal to 97 percent of homology, such as more than 98 percent and more than 99 percent) with the amino acid sequence of SEQ ID NO. 1, has the amino acid sequence of 22 amino acids shown in the 1 st to 22 th positions of SEQ ID NO. 1, has tumor cell specific expression, and has the activity of promoting the growth and the proliferation of tumor cells and the tumorigenesis.
In another preferred embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID No. 1.
In another preferred embodiment, the polypeptide is derived from a mammal, preferably a human, rat, mouse, preferably, human.
In another preferred example, the polypeptide replaces one or more (preferably, 3) amino acids from the N-terminus of LIN28B-WT (SEQ ID NO.:3) with an amino acid sequence consisting of 22 amino acids shown in positions 1-22 of SEQ ID NO. 1.
In a second aspect, the present invention provides an isolated polynucleotide encoding a polypeptide according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide is selected from the group consisting of: DNA sequence, RNA sequence.
In another preferred embodiment, the DNA sequence is selected from the group consisting of: genome sequence and cDNA sequence.
In another preferred embodiment, the polynucleotide is mRNA or cDNA.
In another preferred embodiment, the polynucleotide comprises a nucleotide sequence that is at least 80% (preferably at least 90%, more preferably at least 95%) identical to a nucleotide sequence selected from the group consisting of:
(i) a polynucleotide encoding a polypeptide according to the first aspect of the invention; and
(i i) a polynucleotide complementary to polynucleotide (i).
In another preferred embodiment, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 1.
In another preferred embodiment, the sequence of the polynucleotide has the sequence shown in SEQ ID No. 2.
In a third aspect, the present invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
In a fourth aspect, the invention provides a host cell comprising a vector according to the third aspect of the invention or a genome into which has been integrated a polynucleotide according to the second aspect of the invention.
In a fifth aspect, the present invention provides a method for preparing a polypeptide, comprising:
culturing a host cell according to the fourth aspect of the invention under conditions suitable for expression, thereby expressing a polypeptide according to the first aspect of the invention; and isolating said polypeptide.
In a sixth aspect, the invention provides a specific inhibitor that specifically binds to a polypeptide according to the first aspect of the invention or inhibits LIN28B-TST expression.
In another preferred embodiment, the inhibitor comprises an antisense nucleic acid, an antibody, a small molecule compound, a CrispR agent, or a combination thereof.
In another preferred example, the antisense nucleic acid comprises siRNA, shRNA, miRNA.
In another preferred embodiment, the inhibitor does not inhibit or does not substantially inhibit wild-type LIN 28B.
In another preferred example, the inhibitor preferentially inhibits LIN28B-TST (as compared to wild-type LIN 28B).
In another preferred embodiment, the inhibitor is specific for a specific epitope of LIN 28B-TST.
In another preferred embodiment, the specific epitope refers to an epitope which LIN28B-TST has but not wild-type LIN 288.
In another preferred embodiment, said specific epitope is selected from the group consisting of:
(a) 1, an epitope consisting of the amino acid sequence from position 1 to position 22 in SEQ ID NO;
(b) 1 amino acid sequence from 1 st to 22 nd of the epitope consisting of the other amino acid sequences of the LIN28B-TST polypeptide.
In another preferred embodiment, said specific inhibitor binds to a specific epitope of said LIN28B-TST polypeptide, or to a complex epitope of a specific epitope of said LIN28B-TST polypeptide with other sequences, said specific epitope being selected from the group consisting of: 1, amino acid sequence from position 1 to position 22 in SEQ ID NO: MSHRRQVLQKRMRSFNQVSSAP (SEQ ID NO: 8).
In another preferred example, the specific inhibitor can specifically inhibit the activity of the promoter of LIN 28B-TST.
In another preferred embodiment, the promoter of LIN28B-TST is shown in SEQ ID No. 9.
The seventh aspect of the present invention provides a use of a specific inhibitor according to the sixth aspect of the present invention for the preparation of a medicament or formulation for (i) diagnosis or prognosis of a tumour; (ii) inhibiting tumor cell growth and proliferation; and/or (iii) the ability to inhibit tumorigenesis.
In another preferred embodiment, the tumor cell is selected from the group consisting of cells of tumors: liver cancer, cervical cancer, endometrial cancer, glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: liver cancer, cervical cancer, endometrial cancer, glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or a combination thereof.
In an eighth aspect, the present invention provides a pharmaceutical composition, comprising:
a specific inhibitor according to the sixth aspect of the invention; and a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises a small molecule compound, and/or a nucleic acid, which down-regulates the expression level or activity of the LIN28B-TST polypeptide or nucleotide.
In another preferred embodiment, the nucleic acid comprises siRNA, shRNA, miRNA.
In a ninth aspect, the invention provides a method of detecting (particularly non-diagnostic in vitro detection) the presence or absence of LIN28B-TST in a sample, comprising: the sample was contacted with an antibody specific for LIN28B-TST and observed for the formation of an antibody complex, which indicates the presence of LIN28B-TST in the sample.
In a tenth aspect, the present invention provides a method for detecting a disease or a susceptibility to a disease associated with aberrant expression of LIN28B-TST, comprising: detecting the presence or absence of a mutation in a nucleic acid sequence encoding said polypeptide.
In an eleventh aspect, the invention provides a polypeptide of the first aspect of the invention, or a polynucleotide of the second aspect of the invention, for use in screening for an inhibitor of LIN28B-TST activity, or for use in peptide fingerprinting, or as a control for nucleic acid detection.
In a twelfth aspect, the invention provides a method of determining whether a test agent is an inhibitor of LIN28B-TST, comprising the steps of:
(a) culturing LIN28B-TST expressing cells in the presence of the test substance in a culture system in a test group; and culturing the same cells in a control group otherwise identical in the absence of the test agent;
(b) detecting the expression level of LIN28B-TST E1 or activity A1 of the cells in the test group; and comparing the expression level E2 or activity A2 of LIN28B-TST in the cells of the control group;
wherein, if the expression level E1 is significantly lower than the expression level E2 or the activity A1 is significantly lower than the activity A2, it indicates that the test substance is an inhibitor of LIN 28B-TST.
In another preferred embodiment, the detection comprises detection at the nucleic acid level, and/or detection at the protein level.
In another preferred embodiment, said "significantly lower" means that the ratio E1/E2 or A1/A2 is 1/2, preferably 1/3, more preferably 1/4.
In another preferred embodiment, the cells comprise tumor cells.
In another preferred embodiment, the tumor cell is from a human.
In another preferred embodiment, the method further comprises the step (c): the inhibitor of LIN28B-TST polypeptide determined in step (b) was further tested for its inhibitory effect on tumor cell growth and proliferation (including in vitro cell experiments, or animal experiments).
In another preferred embodiment, the test article comprises: antibodies, compounds, nucleic acids.
In another preferred embodiment, the tumor cell is selected from the group consisting of cells of tumors: liver cancer, cervical cancer, endometrial cancer, glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or a combination thereof.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
The thirteenth aspect of the invention provides a use of a LIN28B-TST polypeptide, or a transcript thereof, or a coding sequence thereof, or a detection reagent thereof, for preparing a detection kit, wherein the kit is used for diagnosis or prognosis of tumors.
In another preferred embodiment, the tumor is selected from the group consisting of: liver cancer, cervical cancer, endometrial cancer, glioma, breast cancer, melanoma, lung cancer, colon cancer, gastric cancer, or a combination thereof.
In another preferred embodiment, the LIN28B-TST polypeptide, or a transcript or coding sequence thereof, is used as a reference or standard.
In another preferred embodiment, the detection reagent is selected from the group consisting of: primers, probes, antibodies, protein chips, nucleic acid chips, or combinations thereof.
In another preferred embodiment, the detection reagent is a detection reagent that specifically detects LIN 28B-TST.
In another preferred embodiment, the detection reagent is a detection reagent for distinguishing LIN28B-TST from LIN 28B-WT.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Fig. 1 shows that transcriptome sequencing analysis found a novel LIN28B tumor-specific transcript LIN28B-TST in various cancers. Wherein,
(A) distribution of the reads sequence at the position of LIN28B gene among the different transcriptome sequencing libraries.
(B) The expression levels of LIN28B-TST and LIN28B-WT in different cancer species in the TCGA database. HCC, hepatocellular carcinoma (sample number 348); CESC, i.e., cervical and endocervical cancer (sample number 296); LGG, brain low-grade glioma (sample number 529); DLBC, diffuse large B-cell lymphoma (sample number 45); BRCA, i.e., breast invasive carcinoma (sample number 1093); SKCM, i.e. cutaneous epithelial melanoma (sample number 294); UCEC, endometrioid carcinoma (sample number 175); BLCA, bladder urothelial carcinoma (sample number 388); HNSC, i.e., lung squamous cell carcinoma (sample number 494); KIRP, renal papillary cell carcinoma (sample number 290); LUAD, lung adenocarcinoma (sample number 504); GBM, glioblastoma multiforme (sample number 158); UCS, uterine sarcoma (sample number 42); ESCA, instant ductal carcinoma (sample number 179); STAD, gastric adenocarcinoma (sample number 384), COAD, colon adenocarcinoma (sample number 288).
FIG. 2 shows the identification of LIN28B-TST transcript. Wherein,
(a) northern blot results of LIN28B-TST in Huh7 cells.
(b) Agarose gel electrophoresis and Sanger sequencing of 5' RACE experimental cDNA fragments of LIN28B-TST in HuH-7 cells.
(c) Agarose gel electrophoresis and Sanger sequencing of the 3' RACE experimental cDNA fragment of LIN28B-TST in HuH-7 cells.
(d) The LIN28B-TST transcript had 2 specific exons at the 5' end compared to the LIN28B-WT transcript. LIN28-TST specific transcripts were marked in red. The first base pair with the highest probability of transcription initiation (hg19chr6:104,937,026) was considered as the transcription initiation site. Two translation initiation sites (ATGs; marked with blue bold underline): the first ATG, hg19chr6:104,937,099-104,937, 101; the second ATG, chr6:104,950,476-104,950,478. stop codon (TAA) is marked with blue underlining. The 3' UTR is marked with green.
(e) The amino acid sequence of LIN 28B-TST. Translation begins with start codon 1 or 2. The corresponding two methionine residues (marked with blue underlining) resulted in two proteins, 29.38kDa (269 amino acids) and 27.96kDa (258 amino acids), respectively. The amino acid sequence specific for LIN28B-TST was labeled with red.
Fig. 3 shows the expression of LIN28B in various cell lines. qPCR experiments were used to test the expression of LIN28B-WT and LIN28B-TST transcripts in various cell lines.
FIG. 4 shows the expression of LIN28B-TST and LIN28B-WT in tissues and their relationship to tumor prognosis. Wherein,
(A) expression of LIN28B-TST and LIN28B-WT in fetal liver, normal liver, non-tumor tissue and hepatocellular carcinoma (HCC) tissue.
(B) Total survival curves of patients with (+) or without (-) HCC expressing LIN 28B-TST. P-values were calculated using the Log-rank test (Log-rank test).
FIG. 5 shows the expression regulation of LIN 28B-TST. Wherein,
(A) LIN28B-TST was subjected to chromosomal co-immunoprecipitation sequencing (ChIP-seq) and Rapid Amplification of 5' segment cDNA Ends (Rapid Amplification of cDNA Ends) at different transcription initiation sites in the HuH-7 cell line.
(B) In HEK-293T cell line, LIN28B-TST promoter was subjected to a series of knockout luciferase reporter experiments following 5' segment.
(C) Schematic location of 2 CpG islands of LIN28B-TST promoter region. The status of promoter methylation was sequenced using sodium bisulfite. At the indicated CpG sites, filled circles represent methylated cytosines, while open circles represent unmethylated cytosines.
(D) Immunoblot results of the expression of LIN28B in cell lines 6 days after treatment with or without 10. mu.M 5-azadeoxycytidine (5-AZA).
(E) Immunoblot results of LIN28B and cMyc expression in cell lines after using 1. mu.M JQ1 or transfection of siRNA targeting c-Myc β actin (β -actin) was used as an internal reference.
Fig. 6 shows that LIN28B-TST encodes a long protein isoform with an extra N-terminal amino acid. Wherein,
(A) schematic representation of LIN28B transcript variants and the corresponding isoforms. The initiation codon (ATG) and the termination codon (TAA) are indicated by black arrows.
(B) Immunoblot results of cells expressing LIN28B-WT and LIN 28B-TST. Two predicted LIN28B-TST start codons are shown mutated separately and together from ATG to AGG.
(C) Expression of LIN28B-WT and LIN28B-TST in a variety of cell lines.
Fig. 7 shows the expression of LIN28B-TST in the tissues of tumor patients. Wherein,
(A) immunoblot experiments were performed using the LIN28B-TST antibody in HEK-293T cell line, HuH-7 cell line and HCC patient tissues. T, HCC tissue; n, i.e. the corresponding non-tumor tissue.
(B) Immunohistochemical staining was performed in HCC and normal liver tissues using LIN28B-TST antibody.
FIG. 8 shows the effect of LIN28B-TST on tumor cell proliferation. Wherein,
(A) in NCI-H1299 cell line, the siRNA down-regulated LIN28B-TST expression and clone formation experiment and cell proliferation experiment. Two siRNAs targeting LIN28B-TST were used, namely si-LIN28B-TST-1 and si-LIN 28B-TST-2. siRNA without target was taken as negative control (Ctrl).
(B) In the Huh-7 cell line, the siRNA down-regulates LIN28B-TST expression and then carries out clone formation experiments and cell proliferation experiments.
Fig. 9 shows the effect on tumor cell proliferation after downregulation of LIN28B-TST expression using CRISPR/Cas9 technology. Wherein,
(A) the sites targeted by the sgrnas are marked in the figure with red filled triangles. Knockout efficiency was verified by immunoblotting experiments in Huh-7 cells. sgRNA without targeting site served as internal reference (Ctrl).
(B) The effect of LIN28B-TST down-regulation on clonogenic and cell proliferative capacities using CRISPR/Cas9 in Huh-7 cells. P <0.001.
FIG. 10 shows an animal experiment examining the effect on cell proliferation in vivo following downregulation of LIN 28B-TST. Expression of LIN28B-TST was downregulated using CRISPR/Cas 9. Wherein,
(A) volume of LIN28B-TST knockout HuH-7 mice transplanted tumors. P <0.05, p <0.01, p <0.001.
(B) Quality of LIN28B-TST knockout HuH-7 mice transplantable tumors. P <0.05, p <0.01, p <0.001
Detailed Description
The present inventors have conducted extensive and intensive studies and extensive screening, and have unexpectedly discovered and isolated a novel transcript LIN28B-TST (variant) which exhibits high expression levels and high shear index scores for the first time, and which has several characteristics: 1) 22 new amino acid sequences are added at the N terminal of the wild-type LIN28B-WT protein; 2) it is expressed in various tumor tissues, is highly expressed in some tumor tissues, but is not expressed in normal tissues. 3) The variant can obviously promote the growth and proliferation of tumor cells in vitro and promote the generation of tumors. 4) The variant can obviously promote the growth and proliferation of tumor cells and the generation of tumors in vivo. The present invention has been completed based on this finding.
Term(s) for
As used herein, the terms "LIN 28B-TST polypeptide", "LIN 28B-TST protein", "variant", "LIN 28B-TST variant" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of LIN28B-TST variant (SEQ ID No.:1) that significantly promotes the growth and proliferation of tumor cells, as well as the development of tumors, in vitro and in vivo.
As used herein, the terms "LIN 28B-WT polypeptide", "wild-type LIN28B protein", "LIN 28B-WT protein" are used interchangeably and refer to the native LIN28B protein, the sequence of which is set forth in SEQ ID No. 3 and the nucleotide sequence of which is set forth in SEQ ID No. 4.
LIN28B-TST
The present inventors found a novel transcript of LIN28B gene by transcriptome sequencing (RNA-seq), which exhibited high expression level and high shear index score and was named LIN 28B-TST. LIN28B-TST is specifically expressed in hepatocellular carcinoma and many other human cancer cells, and is not expressed in normal tissues. HCC patients with high LIN28B-TST expression had a poorer prognosis. Unlike the wild-type LIN28B transcript, LIN28B-TST was generated from a new transcription initiation site and contained a strong promoter that was not regulated by c-Myc. Tumors expressing LIN28B-TST may be classified as a new aggressive tumor subtype. In vivo experiments and in vitro experiments show that the tumor cell proliferation can be remarkably inhibited by down-regulating the expression of LIN 28B-TST. These findings demonstrate a completely new mechanism for the activation of the LIN28B gene in cancer and suggest a potential application value of LIN28B-TST as a tumor diagnosis and treatment.
In the present invention, a representative LIN28B-TST polypeptide refers to a protein or polypeptide having the amino acid sequence of LIN28B-TST variant (SEQ ID No.: 1).
In the present invention, a representative LIN28B-TST polypeptide refers to an amino acid sequence consisting of one or more (preferably, 3) amino acids from the N-terminus of LIN28B-WT (SEQ ID NO.:3) replaced with 22 amino acids shown in positions 1-22 of SEQ ID NO. 1.
In the present invention, a representative wild-type LIN28B polypeptide (LIN28B-WT polypeptide) refers to a protein or polypeptide having the sequence shown in SEQ ID No. 3.
As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.
As used herein, "isolated LIN28B-TST protein or polypeptide" means that LIN28B-TST polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. One skilled in the art can purify the LIN28B-TST protein using standard protein purification techniques. Substantially pure polypeptides are capable of producing a single major band on a non-reducing polyacrylamide gel. The purity of LIN28B-TST polypeptide can be analyzed by amino acid sequence analysis.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.
The invention also includes fragments, derivatives and analogs of the human LIN28B-TST protein. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the native human LIN28B-TST protein of the present invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an antigenic IgG fragment). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
In the present invention, the term "human LIN28B-TST polypeptide" refers to the polypeptide of SEQ ID NO:1 having the activity of human LIN28B-TST protein. The term also includes variants of the sequence of SEQ ID NO. 1 that have the same function as the human LIN28B-TST protein. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the human LIN28B-TST protein.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to human LIN28B-TST DNA under high or low stringency conditions, and polypeptides or proteins obtained using antisera to human LIN28B-TST polypeptides. The invention also provides other polypeptides, such as fusion proteins comprising human LIN28B-TST polypeptide or its fragment and other polypeptides. In addition to the nearly full-length polypeptide, the invention also includes soluble fragments of the human LIN28B-TST polypeptide. Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the human LIN28B-TST polypeptide sequence.
The invention also provides human LIN28B-TST protein or polypeptide analogues, these analogues and natural human LIN28B-TST polypeptide differences can be amino acid sequence differences, can also not affect the sequence of the modified form of the difference, or both, these polypeptides include natural or induced genetic variants.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.
In the present invention, the "human LIN28B-TST protein conservative variant polypeptide" refers to the polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties, compared with the amino acid sequence of SEQ ID NO. 1. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 2 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 1, but differ from the sequence of the coding region shown in SEQ ID NO. 2.
The polynucleotide encoding the mature polypeptide of SEQ ID NO. 1 comprises: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
In one embodiment of the present invention, an isolated polynucleotide is provided that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO. 1. The nucleotide sequence is shown in SEQ ID NO. 2, and the full length of the polynucleotide sequence contained in the polynucleotide sequence is 5428 bases, the open reading frame of the polynucleotide sequence is positioned at 74-880, and the polynucleotide sequence encodes LIN28B-TST protein (SEQ ID NO:1) with 269 amino acids in full length.
Preparation of the proteins of the invention
The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
The full-length nucleotide sequence of human LIN28B-TST or its fragment can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
A method of amplifying DNA/RNA using PCR technology (Saiki, et al science 1985; 230: 1350-. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells engineered with the vectors of the invention or the coding sequence of the TRA2B-DNAH5 protein, and methods for producing the polypeptides of the invention by recombinant techniques.
The polynucleotide sequence of the present invention may be used to express or produce a recombinant TRA2B-DNAH5 polypeptide by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human LIN28B-TST polypeptide, or with a recombinant expression vector comprising the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Specific antibodies
In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptide encoded by LIN28B-TSTDNA of the invention or a fragment thereof. Herein, "specific" means that the antibody binds to the LIN28B-TST product or fragment of the present invention. Preferably, it refers to those antibodies which bind to the LIN28B-TST gene product or fragment of the invention but do not recognize and bind to other non-related antigenic molecules, in particular the specific antibodies do not recognize the LIN28B-WT native protein. The invention also provides a particularly preferred specific antibody which recognizes and binds to the amino acid sequence from positions 1 to 22 of SEQ ID No. 1, preferably to the amino acid sequence from positions 3 to 22 of SEQ ID No. 1.
The antibodies of the invention include those molecules that bind to and inhibit LIN28B-TST of the invention, as well as those antibodies that do not affect the function of LIN28B-TST of the invention. The invention also includes those antibodies which bind to the LIN28B-TST gene product of the invention in modified or unmodified form.
The invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2A fragment; an antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but retain portions of the antibody from a human.
The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified LIN28B-TST gene product of the present invention, or an antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing the LIN28B-TST protein of the present invention or an antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibody of the present invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al,Nature256 of; 495, 1975; the result of Kohler et al,Eur.J.Immunol.6: 511,1976, respectively; the result of Kohler et al,Eur.J.Immunol.6: 292,1976, respectively; hammerl's et al,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). The antibody of the invention includes an antibody capable of blocking the function of LIN28B-TST of the invention and an antibody which does not affect the function of LIN28B-TST of the invention. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the LIN28B-TST gene product of the invention. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the LIN28B-TST gene product of the invention can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell). Antibodies against the LIN28B-TST protein of the inventionThe body can be used in immunohistochemical technique to detect LIN28B-TST protein in biopsy specimens.
The antibodies of the invention can be used for treating or preventing diseases related to the LIN28B-TST protein of the invention. The production or activity of the LIN28B-TST protein can be stimulated or blocked by administration of appropriate doses of the antibody.
Antibodies can also be used to design immunotoxins directed to a particular site in the body. For example, the monoclonal antibody with high affinity for LIN28B-TST protein of the present invention can be covalently bound to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). One common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody by exchange of disulfide bonds, and this hybrid antibody can be used to kill the cells positive for LIN28B-TST protein of the present invention.
Production of polyclonal antibodies animals, such as rabbits, mice, rats, etc., can be immunized with the LIN28B-TST protein or polypeptide of the present invention. Various adjuvants may be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.
Inhibitor of LIN28B-TST and application thereof
By utilizing the LIN28B-TST of the invention, substances which inhibit LIN28B-TST or activity expression, such as receptors, inhibitors, agonists or antagonists and the like, can be screened out by various conventional screening methods.
Typically, examples of inhibitors of the invention include (but are not limited to): small molecule compounds, antibodies, antisense nucleic acids (including siRNA, shRNA, miRNA), Crispr reagents. Wherein, the inhibitor of the invention is a specific inhibitor, which is specifically bound or targeted to a specific polypeptide or nucleic acid sequence epitope of the LIN28B-TST, or bound to a composite epitope consisting of the specific epitope of the LIN28B-TST polypeptide and other sequences, or inhibits a specific strong promoter of the LIN 28B-TST.
Also, in a preferred embodiment, the inhibitors of the invention preferentially inhibit LIN28B-TST (as compared to wild-type LIN 28B).
The protein antibody, inhibitor, antagonist or receptor of the present invention, etc., when administered (dosed) therapeutically, can be used to inhibit the growth of tumor cells. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
The inhibitor of the invention can be directly used for disease treatment, for example, for the treatment of cancer (especially liver cancer). When the inhibitor of LIN28B-TST of the invention is used, other tumor therapeutic agents, such as cisplatin, etc., can also be used simultaneously.
The invention also provides a pharmaceutical composition which contains a safe and effective amount of an inhibitor (such as an antibody or a small molecule compound) of LIN28B-TST of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration.
The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 100 milligrams per kilogram of body weight per day. In addition, the inhibitors of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of an inhibitor of LIN28B-TST is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 100 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Polynucleotides of the human LIN28B-TST protein can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat cell proliferation, development or metabolic abnormalities that result from the non-expression or abnormal/inactive expression of LIN28B-TST protein, LIN28B-TST protein. Recombinant gene therapy vectors (e.g., viral vectors) can be designed to express a variant LIN28B-TST protein to inhibit the activity of the endogenous LIN28B-TST protein. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, and the like can be used to transfer the LIN28B-TST gene into cells. Methods for constructing recombinant viral vectors carrying the LIN28B-TST gene can be found in the literature (Sambrook, et al.). In addition, the recombinant human LIN28B-TST gene can be packaged into liposome and then transferred into cells.
Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit LIN28B-TST mRNA are also within the scope of the invention. Ribozymes are enzyme-like RNA molecules that specifically cleave specific RNAs and act by specifically hybridizing the ribozyme molecules with complementary target RNAs followed by endonucleolytic action. Antisense RNA and DNA and ribozymes can be obtained by any existing RNA or DNA synthesis techniques, such as solid phase phosphoramidite chemical synthesis method for oligonucleotide synthesis. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in various ways, such as increasing the length of the flanking sequences, using phosphothioester or peptide bonds instead of phosphodiester linkages for the linkages between ribonucleosides.
The method for introducing a polynucleotide into a tissue or cell comprises: injecting the polynucleotide directly into the in vivo tissue; or introducing the polynucleotide into cells in vitro via a vector (e.g., a virus, a phage, or a plasmid), and then transplanting the cells into the body.
Inhibitors that inhibit the activity of the LIN28B-TST promoter are also within the scope of the present invention. The LIN28B-TST and LIN28B-WT promoters are different, and small molecule drugs or traditional Chinese medicine components for inhibiting LIN28B-TST can be screened out through drugs, and do not affect LIN 28B-WT.
Diagnostic method
The invention also relates to a diagnostic test method for quantitative and local detection of human LIN28B-TST nucleic acid or protein level. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of human LIN28B-TST protein detected in the assay can be used to explain the importance of human LIN28B-TST protein in various diseases and to diagnose the diseases in which LIN28B-TST protein plays a role.
A method for detecting whether LIN28B-TST protein exists in a sample is to detect by using specific antibody of LIN28B-TST protein, which comprises the following steps: contacting the sample with an antibody specific for LIN28B-TST protein; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of LIN28B-TST protein in the sample.
The polynucleotide of LIN28B-TST protein can be used for diagnosing and treating LIN28B-TST protein related diseases. In diagnostic terms, a polynucleotide of the LIN28B-TST protein can be used to detect the presence or absence of expression of the LIN28B-TST protein or abnormal expression of the LIN28B-TST protein under disease conditions. For example, the LIN28B-TST DNA sequence can be used for hybridization of biopsy specimens to determine abnormal expression of LIN28B-TST protein. The hybridization techniques include Southern blotting, Northern blotting, in situ blotting, etc. The technical methods are all published mature technologies, and related kits are all available from commercial sources. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes in tissues and gene diagnosis. The transcript of LIN28B-TST protein was also detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for LIN28B-TST protein.
The main advantages of the invention include:
(1) the invention discovers for the first time that a new transcript LIN28B-TST is specifically expressed in liver cell carcinoma and many other human cancer cells and is not expressed in normal tissues, and LIN28B-TST has a new transcription initiation site, and the expression is regulated and controlled by the methylation of a promoter region.
(2) Compared with the wild-type protein LIN28B-WT, the LIN28B-TST protein has a new sequence at the N-terminal of the wild-type protein LIN28B-WT for the first time, the new sequence can be recognized by a specific antibody, and the proliferation of tumor cells can be remarkably inhibited in vitro and in vivo by down regulating the expression of LIN 28B-TST.
(3) The invention discovers for the first time that LIN28B-TST can be used as a novel target molecule for tumor diagnosis and treatment.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Unless otherwise indicated, all reagents and materials used in the present specification are commercially available products.
General procedure
I. Material method
1. Cell culture and chemical reagents
Human embryonic kidney cells HEK-293T, HuH-7 and SK-HEP-1 cells (purchased from ATCC and Japanese cell Bank) were cultured in DMEM (Gibco, New York, USA) medium; NCI-H1299 cells(purchased from ATCC) in RPMI-1640(Gibco) medium; HCT-116 cells (purchased from ATCC) were cultured in McCoy's 5A (Gibco) medium; AGS and A549 cells were purchased from ATCC) and cultured in F-12K (Gibco) medium. To each medium was added 10% fetal bovine serum (Gibco), 100IU/mL penicillin-streptomycin (Gibco). The cells were incubated at 37 ℃ with 5% CO2The incubator of (2) for cultivation. JQ1 was purchased from Selleck Chemicals (Houston, USA). 5-Aza-2 '-deoxycytidine (5-Aza-2' -deoxycytidine, 5AZA) was purchased from Sigma-Aldrich (St. Louis, USA).
2. RNA extraction, reverse transcription and real-time Quantitative PCR reaction (Quantitative real-time polymerase chain reaction)
Total RNA was extracted from cells or tissues using TRIzol reagent according to the manufacturer's instructions. PrimeScript for cDNATMRT reagent Kit (TaKaRa, Tokyo, Japan) Synthesis real-time quantitative PCR reaction (qPCR) Using SYBR Premix Ex Taq II (TaKaRa), β -actin as an internal reference the primer sequences used are shown in Table 2 mature miRNAs were usedSpecific primers and probes in MicroRNA Assays (Applied Biosystems, Foster City, USA) were quantified. U6small nuclear (snRNA) serves as an internal reference.
TABLE 2 primer Table
3、Northern blot
DIG Northern Starter Kit (Roche, Indianapolis, IN, USA) was used to prepare digoxigenin-labeled RNA probes, the corresponding PCR product serving as a template for transcription of T7. The PCR primers are listed in supplementary Table 2. Northern Max Kit (Thermo) was usedFisher Scientific, Waltham, USA). Mu.g of total RNA was separated in 2% agarose gel and transferred to a nylon membrane (GE Healthcare, Uppsala, Sweden). UV crosslinking (265nm,200 mJ/cm) after drying of the membrane2) For 1 min. Prehybridization was performed at 62 ℃ for 1h, and then hybridization was performed at 62 ℃ overnight. The membrane was washed twice at room temperature using 2x SSC containing 0.1% SDS, then 30min at 62 ℃. The membrane was then washed twice with 0.2 XSSC containing 0.1% SDS at 62 ℃ for 30 min. After washing the membrane, development was carried out (Typhoon, molecular devices).
4、RACE(Rapid Amplification of cDNA Ends)
5 '-RACE and 3' -RACE experiments according to SMARTerTMThe RACE cDNA Amplification Kit (Clontech, Mountain View, USA) was described in the specification. The primers used for 5 '-RACE and 3' -RACE are listed in supplementary Table 2. The PCR products were cloned into pMD20-T or pMD18-T vector (TaKaRa) for sanger sequencing.
5. Antibodies
The antibodies used included: H3K27ac (39133; ChIP-qPCR and ChIP-seq) and RNApol II (61667; ChIP-qPCR and ChIP-seq) were purchased from Active Motif (Carlsbad, USA) Inc.; H3K4me3 (9751; ChIP-qPCR and ChIP-seq), c-Myc (13987; WB) and LIN28B (11965; WB) were purchased from CST (Danvers, USA) corporation; IGFBP1 (22803-1-AP; WB) and IGFBP3 (14642-1-AP; WB) were purchased from Proteitech (Rosemont, USA) Inc.; ACTB (A2228; WB) was purchased from Sigma-Aldrich; IGFBP2(AP10127 b; WB) was purchased from Abgent (San Diego, USA); LIN28B-TST was synthesized by GenScript (Nanjing, China) and has a specific N-terminal immune sequence (HRRQVLQKRMRSFNQVSSAP (positions 3-22 in SEQ ID NO.: 1).
6、Western blot
Proteins were separated on a 10% or 14% sodium dodecyl sulfate-polyacrylamide (SDS) gel and transferred to nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked with 5% nonfat dry milk and incubated with primary antibody. Protein bands were detected with chemiluminescent detection reagent (ThermoFisher Scientific) on a Tanon 6100 chemiluminescent imager.
7. Transfection with oligonucleotides
Small interfering RNA (siRNA) was synthesized by RiboBio (Guingzhou, China) and the LIN28B-TST interference sequence was: 5'-GGAAAGCACAUUAGACCAU-3' (SEQ ID No.:5) and 5'-AGGUUCUUCAGAAGAGGAU-3' (SEQ ID No.: 6); the interference sequence of c-Myc is 5'-GGUCAGAGUCUGGAUCACC-3' (SEQ ID NO.: 7). Cell useRNAiMAXreagent (Invitrogen) reagent transfection oligonucleotide, final concentration of 50nM, 48h after transfection experiment.
8. Cell proliferation and colony formation experiments
Cell proliferation was detected using CCK8(Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). In the clone formation experiment, 1X 103Individual cell species were cultured in each well of 6-well plates at 37 ℃ for 1-2 weeks. Cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet (Sigma-Aldrich). Finally, cell clone counting and analysis are carried out.
9. Immunohistochemical staining
Tissue samples were fixed using neutral formalin, paraffin-wrapped, and cut into 5 μm thick sections. Tissue sections were dewaxed, rehydrated, and rinsed with distilled water. The sections were placed in 10mM sodium citrate buffer (pH 6.0) and heated for 20min using a microwave oven for antigen retrieval. At room temperature with 3% H2O2The endogenous peroxidase is removed by action for 30 min. Sections were blocked with blocking solution (Thermo Fisher Scientific) for 1h at room temperature, then LIN28B-TST primary antibody (1:500) was added and incubated in a wet box for 1h at room temperature. After incubation, sections were washed, biotin-labeled anti-rabbit secondary antibody was incubated, and then HRP-labeled streptavidin (streptavidin) was incubated. Diaminobenzidine (DAB) (DAKO, Glostrup, Denmark) liquid substrate as a color developer, and Mayer's hematoxylin (DAKO) as a counterstain. All areSections were photographed using an Axioskop2microscope (Carl Zeiss, Oberkochen, Germany).
10. Inoculation of nude mice
HuH-7 cells of LIN28B knocked out with CRISPR/Cas9 technology, as well as control cells, were collected and resuspended with DMEM. Each nude mouse (male BALB/c-nu/nu,6 weeks old) was inoculated subcutaneously at the lower back of 2X 10 mice6Individual cells (resuspended in 200 μ L DMEM). The long (L) and short (W) diameters of the tumors were measured with a vernier caliper every 3-4 days. The tumor volume (V) was calculated by the following formula, V ═ L x W2) Mice were sacrificed after 2.4 weeks and the formed tumors were removed and weighed. All the nude mice were bred and subjected to experimental procedures according to the rules of the Shanghai medical laboratory animal ethics Committee.
Example 1 sequence for obtaining LIN28B-TST
Potential specific transcripts were investigated by transcriptome sequencing (RNA-seq) analysis of different normal and cancer tissues and several cell lines. Through extensive screening, a large number of new tumor-specific transcripts were found, including a whole new transcript among 430 genes and 1385 transcripts covering known genes. Among these tumor-specific transcripts, a novel transcript of the LIN28B gene was unexpectedly found, which exhibited high expression levels and a high shear index score, designated LIN 28B-TST. And LIN28B-TST was found to be expressed in hepatocellular carcinoma tissue (HCC), HuH-7 hepatocellular carcinoma cell line, and NCI-H1299 lung cancer cell line, while the wild-type LIN28 transcript (LIN28B-WT) was expressed in HEK-293T human embryonic kidney cell line (FIG. 1A). None of the variant transcripts was expressed in normal liver tissue (FIG. 1A). LIN28-TST has 2 new exons at the 5' end, and the other 3 exons are identical to exons 2-4 contained in the wild type transcript (FIG. 1A).
The presence of LIN28B-TST transcript was confirmed by Northern Blot (Northern Blot) and by rapid amplification of cDNA Ends (RapidAmplification of cDNA Ends) (FIG. 2).
Example 2 detection of the mRNA expression level of LIN28B-TST in tumor cells and tissues
To determine the expression of LIN28B-TST, more than 7000 samples of 22 different tumors were screened from the TCGA transcript sequencing database. LIN28B was expressed in 10.2% of tumors, and LIN28B-TST was in turn specifically expressed in HCC (91.5%, 54/59) and many other cancer species such as cervical cancer (CESC, 86.2%, 25/29) and endocervical carcinoma, brain low-grade glioma (LGG, 85.2%, 23/27), breast invasive carcinoma (BRCA, 69.8%, 30/43), cutaneous epithelial melanoma (SKCM, 68.1%, 32/47), lung squamous cell carcinoma (lus, 50%, 59/118) in all tumor samples expressing LIN28B (fig. 1B). In order to distinguish and quantify the expression levels of LIN28B-WT and LIN28B-TST, specific primers were designed (table 2) and real-time polymerase chain reaction (RT-PCR) was performed in various cell lines (fig. 3). Then, LIN28B-TST is further confirmed to be widely expressed in HCC tissues but not expressed in normal liver tissues or fetal liver tissues in 120 pairs of liver cancer samples; in contrast, LIN28B-WT was able to detect expression in fetal liver tissue, and only LIN28B-WT was sporadically expressed in liver cancer (fig. 4A). Survival analysis indicated that patients with HCC and expressing LIN28B-TST had a poorer prognosis (FIG. 4B). No expression of LIN28B-TST was detected in normal heart, lung, brain, stomach, intestine, lung, liver, spleen, pancreas, skeletal muscle and other tissues by RT-PCR.
Example 3 Regulation mechanism of LIN28B-TST and identification of the protein produced
The transcript sequencing result of LIN28B suggested a variable transcription start point (ATI) at a distance of about 20kb upstream of LIN 28B-WT. 5' RACE amplification was then performed and the previously described ATI was mapped to the chr6:104,937,026 region of chromosome 6. Chromosomal co-immunoprecipitation sequencing (ChIP-seq) found a significant enrichment of histones H3K4me3, H3K27Ac, and RNA polymerase ii (rnasol ii) near the ATI site (fig. 5A), which is typical of promoter activity. These data show that LIN28B-TST appears from a typical newly generated ATI site of chromatin variation. To determine whether the region of the ATI site could function as a promoter, the DNA fragment in which the LIN28B-TST promoter was present was cloned into the luciferase reporter gene. Luciferase reporter assays showed that the promoter activity of LIN28B-TST was more than 20-fold higher than that of LIN28B-WT or SV40 in HEK-293T cell line (FIG. 5B).
A series of gene knock-out experiments found a core promoter region of approximately 200 bp in length near the ATI site (FIG. 5B). To investigate whether CpG island methylation in the promoter region of LIN28B-TST correlates with the expression of LIN28B-TST, sulfite sequencing was performed in the ATI and regions flanking it. In the promoter region of LIN28B-TST, two CpG islands were found. Samples expressing LIN28B-TST showed lower CpG methylation levels in the second CpG island compared to samples not expressing LIN28B-TST (FIG. 5C).
To confirm that demethylation of CpG sites was sufficient to result in the expression of LIN28B-TST, LIN28B-TST was studied with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-AZA) in cells that did not express LIN28B or that expressed LIN 28B-WT. In SK-HEP1 and AGS cell lines that do not express LIN28B, treatment with 5-AZA strongly activated expression of LIN 28B-TST; also in HEK-293T and a549 cell lines expressing LIN28B-WT, treatment with 5-AZA significantly activated LIN28B-TST and inhibited expression of LIN28B-WT (fig. 5D).
These results show that demethylation of the LIN28B-TST promoter may be a prerequisite for LIN28B-TST transcription. The inventors also investigated whether LIN28B-TST can be regulated by c-Myc oncogene, and the results showed that c-Myc can directly regulate the expression of LIN 28B-WT. The inhibition of c-Myc by small interfering rna (sirna) or BET inhibitor JQ1 significantly reduced the expression of LIN28B in the HepG2 cell line expressing LIN28B-WT, while LIN28B-TST was not altered with the silencing of c-Myc expression in the HuH-7 cell line expressing LIN28B-TST (fig. 5E). This indicates that the activation of LIN28B-TST is independent of the expression of c-Myc.
The LIN28B-TST transcript contained 2 predicted in-frame start codons (ATGs) at the 5' end, resulting in its encoded protein with additional amino-terminal amino acids (fig. 6A and 6B). Immunoblot analysis of different cancer cell lines expressing LIN28B-TST revealed a major band, indicating that LIN28B-TST was translated predominantly from the first initiation codon (fig. 6C). The inventors further mutated the two start codons separately or together. Immunoblots showed that each of the forms of LIN28B-TST variation no longer produced the corresponding protein band (fig. 6B).
EXAMPLE 4 preparation of LIN28B-TST antibody
A rabbit antibody was prepared against the additional amino-terminal amino acid of the LIN28B-TST protein. Synthesizing LIN28B-TST specific polypeptide HRRQVLQKRMRSFNQVSSAP, namely the amino acid sequence of 3-22 th position in SEQ ID NO. 1, carrying out KLH coupling, injecting 5mg into a rabbit for 3 times of immunization, taking rabbit serum, and carrying out affinity chromatography to obtain the specific antibody against LIN 28B-TST.
The immunoblot results showed that this antibody specifically recognized the major LIN28B-TST isoform, but not the smaller LIN28B-TST isoform or LIN28B-WT (fig. 6B). Using immunoblotting and immunohistochemistry techniques with the specific antibodies described above, it was also possible to detect the expression of LIN28B-TST in HCC tissues, but not in normal liver tissues (FIGS. 7A and 7B).
Example 5 cell growth and proliferation experiments
The inventors further used siRNA and CRISPR/Cas9 gene editing techniques to silence the expression of LIN28B-TST and observed significant inhibition of cell growth and inhibition of cell colony formation (fig. 8 and 9). Through a nude mouse subcutaneous tumorigenic animal experiment, the silencing LIN28B-TST is found to remarkably inhibit the tumorigenic capacity of Huh-7 hepatoma cells, and the mass and the volume of a tumor are down-regulated (figure 10).
These results indicate that LIN28B-TST is a transcript critical for cancer cell proliferation and tumorigenesis and has potential as a specific target for cancer therapy to some extent.
EXAMPLE 6 screening for inhibitors of LIN28B-TST
According to the method of the present invention, the following two groups of substances were administered to the Huh-7 cell line, and then the expression activity of LIN28B-TST and the effects of the following two groups of substances on cell growth and proliferation were examined: (a) a candidate substance; (b) blank control.
If the expression activity of LIN28B-TST of the cells of group (a) and the extent of growth and proliferation of the following two groups of substances are statistically significantly lower than the expression activity of LIN28B-TST of group (b) and the growth and proliferation of the cells of the following two groups of substances, it is indicated that the candidate substance is an inhibitor of LIN 28B-TST.
Similarly, the following two groups of substances were administered to the NCI-H1299 cell line (expressing LIN28B-TST) and then observed for activity on LIN28B-TST expression and effect on cell growth and proliferation according to the method of the present invention: (a) a candidate substance; (b) blank control.
If the degree of expression activity of LIN28B-TST and growth and proliferation of cells of group (a) was statistically significantly lower than the degree of expression activity of LIN28B-TST and growth and proliferation of cells of group (b), it was suggested that the candidate substance was an inhibitor of LIN 28B-TST.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> affiliated tumor hospital of double-denier university
<120> cancer-associated tumor-specific transcript LIN28B-TST and uses thereof
<130>P2016-1869
<160>45
<170>PatentIn version 3.5
<210>1
<211>269
<212>PRT
<213> Intelligent (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 GlnGlu 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> Intelligent (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> Intelligent (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
5055 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> Intelligent (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> Intelligent (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> Intelligent (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. An isolated LIN28B-TST polypeptide, wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO 1;
(b) a polypeptide derived from (a) which is formed by substituting, deleting or adding one or more amino acid residues (preferably, 1-30, more preferably, 1-15, more preferably, 1-6) in the amino acid sequence of SEQ ID NO. 1, has an amino acid sequence of 22 amino acids shown in positions 1-22 of SEQ ID NO. 1, has tumor cell-specific expression, and has the activity of promoting the growth and proliferation of tumor cells and the tumorigenesis;
(c) the polypeptide which has more than or equal to 80 percent of homology (preferably more than or equal to 90 percent of homology; preferably more than or equal to 95 percent of homology; most preferably more than or equal to 97 percent of homology, such as more than 98 percent and more than 99 percent) with the amino acid sequence of SEQ ID NO. 1, has the amino acid sequence of 22 amino acids shown in the 1 st to 22 th positions of SEQ ID NO. 1, has tumor cell specific expression, and has the activity of promoting the growth and the proliferation of tumor cells and the tumorigenesis.
2. An isolated polynucleotide encoding the polypeptide of claim 1.
3. A vector comprising the polynucleotide of claim 2.
4. A host cell comprising the vector or genome of claim 3 and having the polynucleotide of claim 2 integrated therein.
5. A method for producing a polypeptide, comprising:
culturing the host cell of claim 4 under conditions suitable for expression, thereby expressing the polypeptide of claim 1; and isolating said polypeptide.
6. A specific inhibitor that specifically binds to the polypeptide of claim 1 or inhibits LIN28B-TST expression.
7. Use of a specific inhibitor according to claim 6 for the preparation of a medicament or formulation for (i) diagnosis or prognosis of a tumour; (ii) inhibiting tumor cell growth and proliferation; and/or (iii) the ability to inhibit tumorigenesis.
8. A pharmaceutical composition, comprising:
a specific inhibitor according to claim 6; and a pharmaceutically acceptable carrier.
9. A method of determining whether a test agent is an inhibitor of LIN28B-TST, comprising the steps of:
(a) culturing LIN28B-TST expressing cells in the presence of the test substance in a culture system in a test group; and culturing the same cells in a control group otherwise identical in the absence of the test agent;
(b) detecting the expression level of LIN28B-TST E1 or activity A1 of the cells in the test group; and comparing the expression level E2 or activity A2 of LIN28B-TST in the cells of the control group;
wherein, if the expression level E1 is significantly lower than the expression level E2 or the activity A1 is significantly lower than the activity A2, it indicates that the test substance is an inhibitor of LIN 28B-TST.
10. Use of a LIN28B-TST polypeptide, or a transcript or a coding sequence thereof, or a detection reagent thereof, for preparing a detection kit for the diagnosis or prognosis of a tumor.
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WO2024152945A1 (en) * | 2023-01-17 | 2024-07-25 | 复旦大学附属肿瘤医院 | Use of brca2-thr10 site phosphorylation as tumor biomarker |
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