WO2009030086A1

WO2009030086A1 - Functions and uses of human bone marrow stromal cell-derived growth inhibitor

Info

Publication number: WO2009030086A1
Application number: PCT/CN2007/070626
Authority: WO
Inventors: Xuetao Cao; Nan Li; Dajing Xia; Tao Wang
Original assignee: Zhejiang University
Priority date: 2007-09-05
Filing date: 2007-09-05
Publication date: 2009-03-12

Abstract

The present invention provides uses of human bone marrow stromal cell-derived growth inhibitor (BDGI) or its coding sequence for manufacturing the compositions which inhibit migration of tumors and promote apoptosis of tumor cells. The protein in the present invention is a growth-inhibiting protein which inhibits growth and migration of tumors and induces apoptosis of tumor cells.

Description

The function and use of novel human growth inhibitory protein BDGI

Technical field

The present invention belongs to the fields of biotechnology and medicine, and in particular, the present invention relates to a novel human growth inhibitory protein, Bone Marrow Stromal Cell-derived Growth Inhibitor (BDGI), and a polypeptide encoding the same. The use of a polynucleotide for the preparation of a composition that inhibits migration of tumor cells. Background technique

Tumors are diseases that seriously endanger human health. According to WHO statistics, the average number of people who die of malignant tumors per year among the more than 6 billion people in the world is 7.2 million, and the number of new cases is 8.9 million, and the number is increasing year by year. This is also the case in the domestic situation. The composition ratio of malignant tumors in the cause of death has increased from 12.6% to 17.9% in the past 20 years. As a result, governments, research institutions, and pharmaceutical companies have long placed a high priority on cancer research and anti-cancer drugs.

The disorder of cell growth regulation is the main cause of tumorigenesis, which is caused by the imbalance of positive regulatory signals that promote cell growth and proliferation, prevent terminal differentiation, and negative regulation signals that promote cell maturation and apoptosis. A slow process in which multiple factors work together and multi-step tumorigenesis. The mechanism involves activation of proto-oncogenes, inactivation of anti-oncogenes, persistence of growth and proliferation signals, and abnormal cell cycle. Therefore, studying the molecular mechanism of tumor formation, occurrence and development at multiple levels is conducive to re-establishing the dynamic balance between positive and negative regulatory signals at the molecular level, in order to achieve the desired therapeutic effect. ]

Apoptosis is a gradual death of genes that are actively regulated by specific genes in specific time and space. It plays an important role in tumorigenesis, development, tumor therapy, embryonic development, immune response, nervous system development, and tissue cell metabolism. effect. Apoptosis is an important component of the cell monitoring system. If it is abnormal, it will cause the cells that should be apoptotic to "illegally" survive, resulting in uncontrolled proliferation of cells, resulting in highly degraded tumor cells. In the process of cell carcinogenesis, many apoptotic activating genes are blocked, and the function of apoptosis-inhibiting genes is enhanced. It has been found that many tumors are caused by obstruction of apoptosis, such as lymphoma, breast cancer, prostate cancer, ovarian cancer, and chronic leukemia.

Since apoptosis abnormalities play an important role in the occurrence and development of many human malignancies, the theory and technology for selectively inducing tumor cell apoptosis has become one of the main strategies for the treatment of malignant tumors.

The discovery of new candidate tumor drugs is the hope of treating tumors. In modern drug research, the establishment of new targets is often the premise and guarantee for the creation of new drugs. The international community has invested a lot of manpower, material resources and the latest biotechnology in this field. So far, there are about 400 molecular-level drug screening targets in the world. With the research of the human genome, it is estimated that by 2005, about 30,000 people will be found from about 100,000 human genes. A new drug target. The discovery of novel drug targets at the gene level is conducive to elucidating the etiology and mechanism of tumors, finding new methods for tumor treatment, and providing favorable conditions for the diagnosis, prevention and treatment of cancer patients.

In 2000, the world market for anticancer drugs reached $14.8 billion. The average annual growth rate is 12.5%, and by 2005 the market is expected to exceed $26.7 billion. Therefore, finding new malignant tumor target genes and new treatment methods has important social and economic value.

Growth inhibitory proteins having a cell growth inhibiting action are generally low molecular weight polypeptides or glycoprotein molecules which are capable of inhibiting cell growth in vivo by binding to corresponding receptors on the cell surface. Growth inhibitors such as transforming growth factor bl (TGF-bl), mammastatin (mammastatin neuregulin (NDF), mammary gland growth inhibitory factor (MDGI), and MDGI-related genes have been reported in the literature in the last decade. MRG) and the like have potential anti-mammary tumors and other tumor formation effects.

Growth inhibitors such as TGF-bl can significantly inhibit tumor formation and growth; mammastatin is a peptide derived from the conditional medium of normal mammary epithelial cells, which inhibits normal and transformed cultures in vitro. DNA synthesis of mammary epithelial cells; MDGI and MRG can stimulate the differentiation of breast cancer epithelial cells in vitro, cause local cell proliferation inhibition, and inhibit tumor growth in nude mice. These findings suggest that abnormal expression of growth inhibitory factors is closely related to tumorigenesis. Therefore, increasing the production of growth inhibitory factors or fully exerting its growth inhibition may be a new strategy for tumor therapy.

BDGI is a novel growth inhibitory factor obtained from large-scale random sequencing of BMSC cDNA gene library. It has a total length of 1766 bp and contains a complete open reading frame of 1434 bp, which encodes 477 amino acids. The gene is located at 16q24. 1. This gene has been shown to inhibit the growth of breast cancer cell line MCF-7.

The use of new growth inhibitory factors to inhibit the migration of tumor cells to treat tumors is a hot research topic in the field of cancer therapy. There is an urgent need in the art to develop effective growth inhibitory factors to meet the needs of tumor treatment. Summary of the invention

It is an object of the present invention to provide a human growth inhibitory protein BDGI protein and its coding sequence for inhibiting migration of tumor cells. In a first aspect of the invention, there is provided a use of a human bone marrow stromal cell-derived growth inhibitory factor, BDGI polypeptide or a coding sequence thereof, for use in the preparation of a composition for inhibiting tumor cell migration.

In one embodiment of the invention, the BDGI polypeptide is selected from the group consisting of

(a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;

(b) a polypeptide derived from (a) having the amino acid sequence of SEQ ID NO: 2 formed by substitution, deletion or addition of one or more amino acid residues, and having a function of inhibiting soft agar colony formation of MCF-7 cells; with

(c) a fusion protein of a BDGI polypeptide having a soft agar clone inhibiting MCF-7 cells The function formed.

In another embodiment of the invention, the composition is also for promoting tumor cell apoptosis. In another embodiment of the invention, the composition is a pharmaceutical composition.

In another embodiment of the present invention, the composition contains a pharmaceutically acceptable carrier, diluent or excipient, and 0.001 to 99.99% by weight of a human BDGI polypeptide or a coding sequence thereof, to which the composition The total weight is based on the basis.

In a preferred embodiment, the human BDGI polypeptide or a coding sequence thereof is contained in an amount of from 0.01 to 95% by weight, preferably from 0.1 to 90% by weight, more preferably from 0.5 to 80% by weight.

In another embodiment of the present invention, the tumor cells are selected from the group consisting of breast cancer cells and lung cancer cells.

In another embodiment of the invention, the pharmaceutical composition further comprises an additional anti-cancer drug. In another embodiment of the invention, the coding sequence is selected from the group consisting of:

(i) the sequence shown in positions 147-1577 of SEQ ID NO: 1; or

(ii) the sequence shown in 1-1766 of SEQ ID NO: 1.

In a second aspect of the invention, there is provided a method of screening a human bone marrow stromal cell-derived growth inhibitory factor, an agonist or inhibitor of a BDGI polypeptide, in vitro, the method comprising:

(a) providing a test group and a control group, wherein the control group is a culture system of a tumor cell expressing a BDGI polypeptide or a culture system of a tumor cell to which a BDGI polypeptide is added, and the test group is added with a test substance a culture system of a tumor cell expressing a BDGI polypeptide, or a culture system of a tumor cell to which a test substance and a BDGI polypeptide are added;

(b) Observing the migration of the tumor cells in the test group and comparing with the tumor cell migration of the control group;

Wherein, the migration ability of the tumor cells in the test group is increased compared with the control group, indicating that the test substance is an inhibitor of a potential BDGI polypeptide; wherein, in comparison with the control group, the migration of the tumor cells in the test group A decrease in capacity indicates that the test substance is an agonist of a potential BDGI polypeptide.

In one embodiment of the present invention, the culture system of the tumor cell expressing the BDGI polypeptide or the culture system of the tumor cell to which the BDGI polypeptide is added is a culture system of MCF-7 cells overexpressing the BDGI polypeptide.

In another preferred embodiment, the method is for screening for an inhibitor, and in step (b), further comprising comparing migration of the tumor cells in the test group to migration of a positive control group, The positive control group was a culture system of tumor cells expressing a BDGI polypeptide to which an anti-BDGI antibody was added, or a culture system to which tumor cells to which an anti-BDGI antibody and a BDGI polypeptide were added.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

The following drawings are used to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

Figure 1 shows the Western blot analysis of human BDGI eukaryotic recombinant expression vector transfected human tumor cells expressing BDGI protein. Figure 1A: Human MCF-7 breast cancer cells; Figure 1B: Human A549 lung cancer cells.

Figure 2 shows the inhibition of tumor cell proliferation by overexpression of human BDGI protein of the present invention. Figure 2A: AMCF-7 breast cancer cells; Figure 2B: Human A549 lung cancer cells.

Figure 3 shows the inhibition of tumor cell colony formation by human BDGI protein overexpression.

Figure 4 shows the inhibition of tumor cell migration by human BDGI protein overexpression. Figure 4A: Average number of cells migrating per field of view; Figure 4B: Average distance of cell migration per field of view.

Figure 5 is the induction of tumor cell apoptosis by overexpression of human BDGI protein. Figure 5A: Annexin V/PI staining results; Figure 5B: Scanning electron micrographs. detailed description

The present inventors conducted extensive and in-depth research on the anticancer effect of the growth inhibitory factor-Bone marrow stromal cell-derived growth inhibitory factor BDGI obtained from large-scale random sequencing of the BMSC cDNA gene library, and confirmed in various aspects through various experiments. The BDGI polypeptide has a function of inhibiting tumor cell migration and promoting apoptosis of tumor cells, and is effective for inhibiting and treating tumors, thereby completing the present invention.

Specifically, overexpression of the BDGI protein of the present invention can significantly inhibit the migration of human tumor cells (e.g., MCF-7 breast cancer cells and A549 lung cancer cells), and reduce the colony formation rate of tumor cells in soft agar. At the same time, overexpression of BDGI protein can also induce tumor cell apoptosis. The inhibition of tumor cell migration and induction of tumor cell apoptosis by BDGI protein suggests that BDGI protein is an important growth inhibitory factor in malignant transformation of normal cells, and it is involved in the occurrence, development and metastasis of tumors. All of the above results indicate that BDGI is involved in tumorigenesis and biological behavior, and can regulate a variety of physiological and pathological activities, and thus has important development and application value in immunodiagnosis and immunotherapy in various fields such as anti-tumor and immune function regulation.

The invention also provides the use of a coding sequence for a BDGI polypeptide encoding a polypeptide having the BDGI amino acid sequence set forth in SEQ ID NO: 2, for use in inhibiting and treating a tumor. The polynucleotide of the present invention is isolated from a human bone marrow stromal cell cDNA library. Its sequence is shown in SEQ ID NO: 1: It contains a polynucleotide sequence of 1766 bases in full length and its open reading frame is located at 147-14580, encoding a human BDGI protein of 477 amino acids in length (SEQ ID). NO: 2). The isolated polynucleotide produces a BDGI polypeptide by encoding to achieve an effect of inhibiting and treating a tumor.

BDGI protein or peptide In the present invention, the terms "BDGI protein", "BDGI polypeptide" or "bone marrow stromal cell-derived growth inhibitory factor BDGI" are used interchangeably and refer to a growth inhibitory factor BDGI amino acid sequence derived from human bone marrow stromal cells (SEQ ID N0 : 2) A protein or polypeptide, a conservative variant polypeptide thereof, an active fragment thereof, or an active derivative thereof.

Preferably, the "BDGI polypeptide" is selected from the group consisting of:

(a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;

(; b) Derived from (a) the amino acid sequence of SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acid residues, and having the function of inhibiting soft agar colony formation of MCF-7 cells Peptide.

As can be seen from the above definition, the term "BDGI protein" includes a variant form of the sequence of SEQ ID NO: 2 which has the same function as the human BDGI protein. These variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions And/or substitution, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein. The term also encompasses active fragments and active derivatives of human BDGI proteins.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to human BDGI DNA under high or low stringency conditions And a polypeptide or protein obtained using an antiserum against an anti-human BDGI polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising a human BDGI polypeptide or a fragment thereof. In addition to nearly full length polypeptides, the invention also includes soluble fragments of human BDGI polypeptides. Typically, the fragment has at least about 10 contiguous amino acids of the human BDGI polypeptide sequence, 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, optimally at least about 100 consecutive amino acids.

As used herein, the terms "fragment" and "derivative" refer to a polypeptide that substantially retains the same biological function or activity of the native human BDGI protein of the invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It 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 mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state of living cells It is not isolated and purified, but the same polynucleotide or polypeptide, if separated from other substances present in the natural state, is isolated and purified.

As used herein, "isolated BDGI protein or polypeptide" means that the BDGI polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art will be able to purify the BDGI protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.

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 may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). The polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.

The polypeptides of the invention may also be used in conjunction with proteins of molecular weight such as BSA to form polypeptide conjugates. Typically, the conjugate consists of a polypeptide, a crosslinker, and BSA, wherein the crosslinker is preferably glutaraldehyde, EDAC. Polynucleotide encoding a BDGI protein or polypeptide

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. For human BDGI, the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1, or may be a degenerate variant. As used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.

The term "polynucleotide encoding a BDGI protein or polypeptide" and "BDGI polypeptide coding sequence" are used interchangeably and may include a polynucleotide encoding BDGI or a polynucleoside further comprising additional coding and/or non-coding sequences. acid.

The present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity. The full-length human BDGI nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be used with commercially available cDNA libraries or as known to those skilled in the art. The cDNA library prepared by the method was used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

Once the relevant sequences have been obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.

At present, it has been possible to obtain a DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or BDGI protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention can be used to express or produce recombinant BDGI polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:

(1) using a polynucleotide (or variant) encoding a BDGI polypeptide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) Separating and purifying proteins from the culture medium or cells.

An agonist or inhibitor of a BDGI polypeptide

The invention also relates to agonists or inhibitors of BDGI polypeptides.

As used herein, an inhibitor of a BDGI polypeptide refers to a substance that is capable of combating, inhibiting or reducing the activity or expression of a BDGI polypeptide. Common inhibitors include antibodies, antisense nucleotides, interfering RNA (RNAi), certain natural extracts or compounds.

As used herein, an agonist of a BDGI polypeptide refers to a substance that is capable of increasing, or promoting, the activity or expression of a BDGI polypeptide. Common accelerators include BDGI fusion proteins, certain natural extracts or compounds, and the like.

In the present invention, an agonist or inhibitor of a BDGI polypeptide can be screened by an in vitro screening method. In one embodiment, the method includes:

(a) providing a test group and a control group, wherein the control group is a culture system of a tumor cell expressing a BDGI polypeptide or a culture system of a tumor cell to which a BDGI polypeptide is added, the test group is added with a test substance a culture system of a tumor cell expressing a BDGI polypeptide, or a culture system of a tumor cell to which a test substance and a BDGI polypeptide are added;

(b) observing the migration of the tumor cells in the test group and the migration of the tumor cells with the control group Compare the situation;

Among them, the increased migration ability of the tumor cells in the test group compared with the control group indicates that the test substance is an inhibitor of a potential BDGI polypeptide; and the decreased ability to migrate indicates that the test substance is a potential BDGI polypeptide. Agent. Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising a safe therapeutically effective amount (e.g., 0.001 to 99% by weight) of a BDGI polypeptide of the present invention or a polynucleotide encoding the same (or an agonist thereof), and a pharmaceutically acceptable An acceptable carrier or excipient. In a preferred embodiment of the invention, the human BDGI polypeptide is present in an amount of from 0.001 to 99.9% by weight, preferably from 0.01 to 95% by weight, more preferably from 0.1 to 90% by weight, most preferably from 0.5 to 80% by weight.

The term "effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given condition, routine experimentation can be used to determine the effective amount that the clinician can determine.

For the purposes of the present invention, an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the invention. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991). Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

The pharmaceutically acceptable carrier in the compositions of the present invention may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

The composition of the present invention may contain an additional anticancer drug in addition to the BDGI polypeptide as an active ingredient. Preferably, the additional anticancer drug is selected from the group consisting of: TNF-oc, TGF-β, IFN-oc, glycophosphoryl mustard, hematoporphyrin, lycopene betaine, Brucea javanica, and B.甙 (ie etoposide), dehydrated dulcitol, doxorubicin, tamoxifen, 5-fluorouracil, norcantharidin, difuran fluorouracil, cucurbitacin, harringtonine, oridonin, scorpion , Yunzhi glycopeptide, cytarabine, carbopol, paclitaxel, lentinan, flutamide, ifosfamide, umbrel, leuprolide acetate, deoxyfluorouridine, lopoplatin, Elinoxone, letrozole, teniposide, or a combination thereof. Mode of administration

The pharmaceutical preparation should be matched to 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 body weight to about 5 milligrams per kilogram body weight per day.

The pharmaceutical composition of the present invention can be administered orally, subcutaneously, intradermally, intravenously or the like. The therapeutic dosage regimen can be a single dose regimen or a multiple dose regimen.

When a pharmaceutical composition is used, a safe and effective amount of the BDGI polypeptide is administered to a mammal, especially a human. Specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

Furthermore, the BDGI polypeptide of the present invention can be used alone to inhibit tumor cell migration, and can also be used in combination with other substances. Advantages of the invention

The main advantages of the invention are:

(a) A novel use of a BDGI polypeptide and a polynucleotide encoding the same, which is effective for inhibiting migration of tumor cells and promoting apoptosis of tumor cells, thereby being used for cancer treatment;

(b) The BDGI polypeptide and its encoding polynucleotide as a pharmaceutical composition may be used alone or in combination with other polypeptides, polynucleotides, or in combination with other drugs and therapeutic means for the treatment of malignant tumors. Example

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or The conditions recommended by the manufacturer.

Percentages and parts are by weight unless otherwise stated. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. Example 1 : Cloning of human BDGI cDNA

Total RNA from human bone marrow stromal cells was extracted using Trizol reagent (Life Technologies). Then, poly(A) mRNA is isolated from total RNA. After reverse transcription of poly (A) mRNA to form cDNA, the cDNA fragment was inserted into the vector by SuperScriptl l cloning kit (Life Technologies). At the cloning site, DH5a bacteria were transformed to form a cDNA plasmid library. The sequence of the 5' end of the randomly selected clone was determined by the dideoxy method. The determined cDNA sequence was compared with an existing public DNA sequence database, and it was found that the DNA sequence of one cDNA clone was a new full-length cDNA. The DNA sequence contained in the new clone was bidirectionally determined by synthesizing a series of primers. Computer analysis indicated that the full-length cDNA contained in the clone was a new cDNA sequence (shown as SEQ ID NO: 1) encoding a new protein (as shown in SEQ ID NO: 2). This protein is the human growth inhibitory protein BDGI, and its coding gene is the human growth inhibitory protein BDGI gene.

Sequence SEQ ID NO: 1 is 1766 bp in length and includes a 146 bp 5' non-coding region and a 186 bp 3' non-coding region encoding a polypeptide of 477 amino acids. Theoretically, the molecular weight of the unglycosylated mature molecule is about 52 kD. Example 2: Cloning of the coding sequence of human BDGI protein by RT-PCR

Total RNA in human bone marrow stromal cells was extracted with Trizol reagent, and 5 mg of total RNA was mixed with 1 mg of 01 igo-dT12-18 for reverse transcription. The reverse transcription system was 20 ml, and 80 ml dd3⁄40 was added for dilution after the reaction.

The primers used for PCR amplification of BDGI are as follows:

Sense Primer 5, - GC GAA TTC ATG AGC TCC TCC AGA AAG GAC-3, (SEQ ID NO: 3), Antisense Primer 5, - G CM GCT TTG TTA GGG TGG CTT CCT GGT-3, (SEQ ID NO : 4), with β-ac tin as a positive control.

The PCR reaction volume was 50 ml, which contained 10 ml of reverse transcription template, 0.5 mM primer, 0.2 mM dNTP, and 1 U rTaq DNA polymerase (Takara). The amplification parameters were: 95 ° C, 15 sec, 58 ° C, 30 sec, 72 ° C, 90 sec. After 35 cycles, the PCR product was subjected to 1.0% agarose gel electrophoresis. The result of DNA sequence analysis revealed that the DNA sequence of the PCR product was identical to that of 147-1582 shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human BDGI

Northern blotting was performed as follows: The filter to be tested was placed in 10 ml of a pre-warmed hybridization solution at 68 ° C, pre-hybridized in a hybridization oven (Bellco) at 68 ° C for 30 minutes; the labeled cDNA probe was Denature at 95~100 °C for 2~5 minutes, add rapidly to the ice and add the hybridization solution (final concentration of cDNA probe is 2~10ng/ml or 1~2 X 106cpm/ml), mix well, at 68 °C Hybridize for 2 hours. After the hybridization, the filter was rinsed several times with 2 'SSC, 0.05% SDS at room temperature, followed by shaking for 30 to 40 minutes, during which the lotion was changed several times. Subsequently, it was rinsed with 0. l 'SSC, 0.1% SDS at 50 ° C for 20 to 40 minutes. Finally, the filter was wrapped in a plastic wrap and exposed to -7 CTC for 24 to 48 hours.

Northern shows that human BDGI is highly expressed in tissues such as normal human testis, liver and skeletal muscle. Example 4: Prokaryotic expression of human BDGI protein In this example, the full-length plasmid DNA of Example 1 was used as a template, and amplification was carried out using PCR oligonucleotide primers of the 5' and 3' ends of the sequence below to obtain human BDGI DNA as an insert.

The 5' oligonucleotide primer (upstream primer 2) sequence used in the PCR reaction is:

5,-GC CAT ATG AGC TCC TCC AGA AAG GAC-3' (SEQ ID NO: 5).

The 3' primer (upstream primer 2) sequence is:

5,- TC CTC GAG GGG TGG CTT CCT GGT-3, (SEQ ID NO: 6).

The obtained PCR product was purified, digested with Nde I-Xhol, and then recombined with the expression vector plasmid pET-24a (+) (Novagen) according to a conventional method and transformed into competent E. coli DH5a, and cloned. The positive clone was Nde. l-Xhol was identified by enzyme digestion, and the product was analyzed by 0.8% agarose gel electrophoresis. After purification, it was purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE). It was confirmed by the sequence that the complete BDGI coding sequence has been inserted.

The positive DH5a clone expressing BDGI was inoculated into 100 ml of 2' YTA medium, cultured at 37 ° C, shaking at 300 rpm for 12 to 15 hours, and diluted 1:1 in pre-warmed 2 'YTA medium. Continue to shake culture to 0D ₆ . . =0. 5~0. 8 , after adding 1M IPTG to 0. ImM, induced at 30 ° C for 5 hours, centrifuged at 4 ° C, 8, OOO rpm for 10 minutes, collecting the cells; adding 1 / 20 bacterial growth volume GuNTA- 0 buffer (20 mM Tris-HCl pH 7. 9, 0. 5 M NaCl, 10% Glycerol, 6M Guanidium HC1) and PMSF; suspended bacteria, blasting bacteria on ice (Β· Braun Labsonic U); placed at room temperature for 30 minutes, Mix or mix with magnetic force; 15, OOOrpm, 4 ° C, centrifuge for 20 minutes, remove the supernatant with a 0.8 mm filter, after 1 ml Ni-NTA column, the flow rate is controlled at 15ml / hour, with 5 times NTA volume of GuNTA-O Buffer wash, the flow rate is controlled at about 30ml / hour; respectively with 5 times NTA volume of GuNTA-40, GuNTA-60, GuNTA-100, GuNTA-500 (40, 60, 100, 500 The mM imidazole concentration was eluted, the flow rate was controlled at about 15 ml/hour, and the eluate was collected to obtain a human BDGI-His fusion protein.

The N-amino acid sequence analysis by Edams hydrolysis confirmed that the N-terminal sequence was identical to the N-terminal sequence shown in SEQ ID NO: 2. Example 5: Production of anti-human BDGI antibody

The human BDGI recombinant protein obtained in Example 4 was used to immunize an animal to produce an antibody, and the specific method is as follows. The recombinant molecules are separated by chromatography and used. Separation can also be performed by SDS-PAGE gel electrophoresis, and the electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were intraperitoneally injected with 50-100 μg/0.2 ml of the emulsified protein. After 14 days, mice were intraperitoneally injected with a dose of 50-100 μg/0.2 ml with the same antigen emulsified with incomplete Freimd's adjuvant to boost the immunization. A booster immunization was performed every 14 days, at least three times. The specific reactivity of the obtained antiserum was evaluated by its ability to precipitate a human BDGI gene translation product in vitro. As a result, it was found that the antibody specifically binds to the protein of the present invention. Example 6: Construction of human BDGI eukaryotic expression vector and eukaryotic gene transfection

In this example, using the full-length plasmid DNA of Example 1 as a template, amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human BDGI DNA as an insert.

The 5'-end oligonucleotide primer sequence (upstream primer 1) and the 3'-end primer (downstream primer 1) sequences used in the PCR reaction are the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The obtained PCR product was purified, digested with EcoR I-Hind III, and then recombined with the eukaryotic expression vector plasmid pcDNA3.1/myc-His (-) B (Invitrogen) according to a conventional method and transformed into competent Escherichia coli DH5a. Positive clones were picked and identified, purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE). It was confirmed by sequencing that the complete BDGI coding sequence has been inserted.

The BDGI eukaryotic expression plasmid DNA was transfected into human breast cancer MCF-7 cells and human lung cancer A549 cells by liposome LipofectAMINE reagent (Invitrogen), and the pcDNA3.1 plasmid vector was used as an unrelated control. Follow the instructions. The main steps are: the plasmid DNA to be transfected is mixed with the liposome LipofectAMINE in a certain ratio, and allowed to react at room temperature for 45 minutes; 60~80% confluent cells grown in a 6-well cell culture plate are used, and serum is used in 0PTI-MEM. After washing the medium (Invitrogen) twice, add the plasmid DNA-liposome mixture, incubate at 37 ° C, 5% CO ₂ , culture for 6-8 hours, add an equal volume of normal medium containing 20% serum, continue to culture Replace the fresh medium after 6 hours. Transient expression was performed, and cells were collected for Western blot analysis 48 hours after transfection to detect the transfection effect. Example 7: Western blot detection

MCF-7 cells and A549 cells transiently transfected with BDGI-expressing proteins in Example 6 were lysed with a cell lysate (Cell Signaling). Centrifuge at 4 ° C, 13, OOO rpm for 10 minutes. The supernatant was taken and protein quantification was performed using the BCA Protein Assay Kit (PIERCE). The protein sample was subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 100 V at 4 °C. Dye with Ponceau and mark the size and orientation. Block at room temperature for 2 hours (5% skim milk powder in TBST solution) and dilute the primary antibody with blocking solution and incubate for 1 hour at room temperature. TBST (0. 05% Tween 20 in TBS solution) was washed for 15 minutes, 3 times, and the secondary antibody was diluted with blocking solution and incubated for 2 hours at room temperature. The TBST was washed for 15 minutes, 3 times, and washed with TBS (10 mM Tri s-HC1, pH 8.0, 150 mM NaCl) for 15 minutes, then added with a chemiluminescent substrate (Pierce) for 1 minute, and rapidly sealed and auto-developed. The primary antibody used for Western blot detection was the anti-BDGI antibody obtained in Example 5. The secondary antibody is HRP-labeled anti-rabbit IgG (Cell Signaling).

The results showed that humans were detectable in cells transfected with human BDGI eukaryotic expression vector 48 hours after transfection.

The expression product of BDGI has a molecular weight of 52 kD (Fig. 1). Example 8: [3⁄4] Thymine Deoxynucleoside Incorporation Method for Cell Proliferation

MCF-7 cells overexpressing BDGI protein in Example 6 in logarithmic growth phase at 2 X 10 ⁴ cells/ The wells were inoculated into 96-well plates, and after 56 hours of culture, ³ H-TdR (1 Ci/well, Amersham) was added for further 16 hours, and then the cells were collected on a glass fiber filter paper using a multi-head cell harvester, followed by PBS. Trichloroacetic acid and ethanol were rinsed to remove the free ³ H-TdR, the filter paper was dried, and finally radioactively detected by a LS 6500 liquid scintillation counter, and the cpm value was counted.

The results showed that the amount of ³ H-TdR in MCF-7 cells overexpressing BDGI was significantly lower than that in untransfected MCF-7 cells and mock-transfected MCF-7 cells (P < 0.01). Overexpression can reduce the rate of DNA synthesis in MCF-7 cells and inhibit cell proliferation in vitro (Fig. 2A). Example 9: MTT assay for cell proliferation

The A549 cells overexpressing the BDGI protein in Example 6 in the logarithmic growth phase were seeded in a 96-well flat-bottomed plate, and each of the three wells was set and cultured at 37 ° C, 5% CO ₂ . On the day of the test, 100 ml of medium containing 10% MTT (5 mg/ml, Sigma) was added, and incubation was continued for 4 hours. The supernatant was carefully aspirated and 150 ml/well of DMS0 was added thereto, and incubated at 37 ° C for 10 minutes to dissolve the cells. Inside the formazan, the 0D value of the 570 nm wavelength was measured by an enzyme-linked instrument.

The results showed that the 0D value of A549 cells overexpressing BDGI was significantly lower than that of untransfected A549 cells and mock-transfected A549 cells (P<0.01). The results showed that overexpression of BDGI could inhibit cell proliferation in vitro (see figure 2B). Example 10: Soft agar colony formation experiment

Take 2 X DMEM medium (containing 20% FCS) and 1.2% low melting point agar each, wait for agar to heat and melt slightly, then mix well, add 6-well plate, set at room temperature for use; then take logarithmic growth phase The BDGI protein-expressing MCF-7 cells were prepared in a single cell suspension and counted under a microscope while taking 2 X DMEM medium (containing 20% FCS) and 0.6% low melting point agar. Serve, agar is heated and melted, cooled slightly, and then mixed. When cooled to about 37 °C, mix well with the prepared single cell suspension and add 6-well plate to 500 cells/well. Chromosomes with a diameter above μ ηι were counted at 37 ° C, 5% CO ₂ , for three weeks.

The results showed that the cloning rate of MCF-7 cells overexpressing BDGI was significantly lower than that of MCF-7 cells and mock-transfected MCF-7 cells (P<0.01), indicating that stable expression of BDGI can decrease MCF-7 cells. The rate of clone formation (see Figure 3). Example 11: Migration experiment

The MCF-7 cells overexpressing the BDGI protein in Example 6 in the logarithmic growth phase were inoculated into a 60 mm diameter petri dish at ⁵ ×10 ⁵ cells/well, and when they reached 80% confluence, the blade was used. Draw a groove in the Petri dish as a marker, hold the blade, wipe the cells on one side of the blade with a sterile cotton ball, and immediately observe under the microscope to ensure that there is no cell residue on the side of the marked scratch line, and then use PBS. Wash 2 times, add fresh medium The culture was continued, and after 36 hours and 48 hours, the following two results were observed under the microscope: 1 number of cells migrating per field of view 2 maximum distance of cell migration per field.

The results showed that MCF-7 cells overexpressing BDGI were smaller than mock-transfected MCF-7 cells and untransfected MCF-7 cells, both in the number of migrating cells (Fig. 4A) and in cell migration distance (Fig. 4B). This indicates that overexpression of BDGI can inhibit the migration ability of MCF-7 cells. Example 12: Detection of apoptosis

Apoptosis was detected using the Annexin V/FITC kit (Bender), and the procedure was carried out according to the instructions, which are briefly described as follows: MCF-7 cells overexpressing BDGI protein in Example 6 were washed with PBS and then resuspended in Binding to the buffer. Dilute with Annexin V/FITC for 10 minutes at room temperature, PBS wash, resuspend in binding buffer, add PI, load immediately, analyze by flow cytometry, and analyze software as ModFit

3. 0 LTTM Software.

The sufficiency of the 5% glutaraldehyde in the 5% glutaraldehyde is 0. 1M pity. In the acid salt buffer (pH 7.4), the cells were fixed at 4 ° C for 2 hours, and the cell pellet was washed twice with PBS in 0.1 M phosphate buffer (pH 7. 4) containing 1% citric acid. ), fixed at 4 ° C for 1 hour, washed twice with PBS, using gradient ethanol (50%, 70%,

80%, 90%, 100%) and acetone (100%) dehydrated, each time 15 minutes, embedded with Epon 812, ultra-thin section, double staining with uranium acetate and lead citrate, PHILIPS TECNAI 10 transmission electron microscope Observe, the acceleration voltage is 80KV.

The results showed that MCF-7 cells overexpressing BDGI were significantly higher than those of mock-transfected MCF-7 cells and untransfected MCF-7 cells, regardless of the proportion of early apoptosis or late apoptosis (Fig. 5A). (Fig. 5B) It is shown that, 48 hours after overexpression of BDGI, MCF-7 cells shrink in size, cytoplasm concentrates, chromatin condensation occurs in the nucleus, and edge collection shows increased electron density and nuclear fragmentation in some cells. Multiple electron density-enhanced nuclear fragments are visible in the cytoplasm. These results indicate that BDGI can induce apoptosis in MCF-7 cells. Example 13: Preparation of BDGI polypeptide injection and BDGI-His fusion protein injection

The lmg BDGI polypeptide and the BDGI-His fusion protein prepared in Example 4 were separately subjected to a conventional mixing method.

200 ml of saline was injected into an injection solution and dispensed in a 10 ml vial.

Using the method of testing the mobility in Example 11, the effects of the test group administered with the two injections and the control group administered with the same volume of physiological saline on the cell migration were compared. The results showed that both injections inhibited the migration of MCF-7 tumor cells in vitro compared to the control. Example 14: In vitro screening of BDGI inhibitors and agonists

The MCF-7 cells of Example 11 were treated with the test substance and the control at a concentration of 0.1 mg/ml, respectively. Their effects on cell mobility were tested separately according to the method described in Example 11. Wherein: Test substance 1 is the anti-BDGI antibody prepared in Example 5; Test substance 2 is the antisense sequence of the sequence of positions 147-1582 of SEQ ID NO: 1 (obtained according to a conventional molecular biological method); The BDGI-His fusion protein prepared in Example 4; the control group was an equal volume of physiological saline.

The results showed that Test 1 and Test 2 increased the migration ability of MCF-7 cells compared to the control group, indicating that the two test substances were inhibitors of potential BDGI polypeptides; and Test 3 made MCF-7 The ability of the cells to migrate decreased, indicating that Test 3 is an agonist of the potential BDGI polypeptide. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

Rights request

A use of a human bone marrow stromal cell-derived growth inhibitory factor, i.e., a BDGI polypeptide or a coding sequence thereof, for use in the preparation of a composition for inhibiting migration of tumor cells.

2. The use according to claim 1, wherein the BDGI polypeptide is selected from the group consisting of:

(a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;

(c) A fusion protein of a BDGI polypeptide having a function of inhibiting the formation of a soft agar clone of MCF-7 cells.

3. The use according to claim 1, wherein the composition is further for promoting apoptosis of tumor cells.

4. Use according to claim 1 wherein the composition is a pharmaceutical composition.

5. The use according to claim 1, wherein the composition comprises a pharmaceutically acceptable carrier, diluent or excipient, and 0.001 to 99.99% by weight of a human BDGI polypeptide or a coding sequence thereof. Based on the total weight of the composition.

6. The use according to claim 1, wherein the tumor cells are selected from the group consisting of breast cancer cells and lung cancer cells.

7. The use according to claim 1, wherein the pharmaceutical composition further contains an additional anticancer drug.

8. The use according to claim 1, wherein the coding sequence is selected from the group consisting of:

(i) the sequence shown in positions 147-1577 of SEQ ID NO: 1; or

(ii) the sequence shown in 1-1766 of SEQ ID NO: 1.

A method for in vitro screening of a human bone marrow stromal cell-derived growth inhibitory factor, an agonist or inhibitor of a BDGI polypeptide, characterized in that the method comprises:

(b) observing the migration of the tumor cells in the test group and comparing with the tumor cell migration of the control group; Wherein, the migration ability of the tumor cells in the test group is increased compared with the control group, indicating that the test substance is an inhibitor of a potential BDGI polypeptide;

Among them, the decreased migration ability of the tumor cells in the test group compared with the control group indicates that the test substance is an agonist of a potential BDGI polypeptide.

10. The method according to claim 9, wherein the culture system of the tumor cell expressing the BDGI polypeptide or the culture system of the tumor cell to which the BDGI polypeptide is added is the culture of the MCF-7 cell overexpressing the BDGI polypeptide. system.