WO2009030084A1 - NEW FUNCTIONS AND USES OF HUMAN ONCOGENE-LIKE SMALL G PROTEIN RabJ - Google Patents

Abstract

Human oncogene-like small G protein RabJ (Rab protein with J domain) is provided. The uses of said oncogene-like small G protein RabJ molecule and its coding sequence are also provided. RabJ molecule is a small G protein having GTPase activity, which is related to the control of cell proliferation and differentiation, and possesses an oncogene-like action.

Description

 The function and use of new human oncogene-like small G protein RabJ

 Technical field

 The present invention is in the field of biotechnology and medicine, and in particular, the present invention relates to human oncogene-like small G protein RabJ and its coding sequence. The polypeptide of the present invention is a novel molecule having GTPase activity, which is involved in the regulation of cell proliferation and cell differentiation, and has an oncogene-like effect. Background technique

 Small G proteins refer to single subunit G proteins with a molecular weight of about 20-30 kDa. At present, with the completion of the Human Genome Project, more than one hundred members of this class of proteins have been discovered, and their common features are that they can bind guanosine triphosphate (GTP) and guanosine diphosphate (GDP), and can hydrolyze GTP γ. Phosphoric acid becomes GDP (ie GTPase activity;). According to the homology of its primary structure, it can be divided into six families, including six families including Ras, Rab, Rho, Arf, Sari and Ran.

 The molecules of the Ras and Rho families are extremely important small G proteins because they are involved in the regulation of signaling mechanisms, so that cells can respond correctly to external stimuli and play an important role in basic life activities such as cell survival and apoptosis. Members of the Ras family include Ras, Ral, and Rap, and the most widely known of them are the Ras subfamily, including Ha-Ras, Ki-Ras, and N-Ras. Both knockout mouse models and defective cell models have demonstrated that Ras family molecules play an important role in embryogenesis and ontogeny. Members of the Rho family mainly include Rho, Rac, Cdc42, etc., by regulating the functions and activities of Rho-associated kinase (ROK), myosin binding subunit (MBS) phospholipase and other target proteins. Regulate cellular activities such as cytoskeleton, cell proliferation, and stress response.

 Ras and tumorigenesis and their role in the regulation of cell proliferation have attracted attention. In almost all types of tumor cells or tumor tissues, there are mutations in the H-Ras, K-Ras or N-Ras genes, and Ras mutations are the most likely in tumor-mutated genes. Molecules associated with the regulation of Ras protein activity are also involved in tumorigenesis. Molecules of the Rho family also regulate the formation of intracellular actin-stressing fibers, cell membrane processes, and depressions by regulating the cellular actin system. In the fibroblast cell line NIH3T3, a mutant protein molecule that regulates the activity of the Rho family can cause cell transformation. These oncogene-like molecules that regulate the GTP/GDP binding activity and GTPase activity of the Rho family include Dbl, Vav, Lbc, Tiaml, Tim, Ost, Ect2, Lsc, and FGD1, which can cause abnormalities in the function of the cell actin system, causing cells. Changes in migration infiltration capacity.

Rab (Ras-like protein in rat brain) is a small molecular weight GTPase belonging to the Ras superfamily. The basic biochemical feature is that it can bind GTP/GDP and hydrolyze GTP. So far, the family has grown to more than 100 members, including in many species including nematodes, yeast, fruit fly, zebrafish, mice, rats, humans, etc., mainly involved in intracellular proteins. The transport process regulates the fusion of membranous organelles and the process of budding, anchoring and fusion of microvesicles, regulating cell activities such as protein synthesis, endocytosis, degradation and secretion. Different Rab proteins often have different subcellular localizations and thus can locally regulate the fine protein transport process. E.g Rabla, Rablb, Rab2 localize to the endoplasmic reticulum and Golgi and mediate the transport of substances between the two; Rab6, Rab8, Rabl2 are associated with the Golgi apparatus and mediate the transport of substances between different Golgi sites.

 Abnormal function of the Rab protein causes some pathological phenomena and diseases. Since Rab family proteins play important regulatory roles in certain physiological processes, there is an urgent need in the art to develop new human Rab proteins. Summary of the invention

 It is an object of the present invention to provide a novel human oncogene-like small G protein RabJ protein and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide the use of the polypeptide and the coding sequence. In a first aspect of the invention, there is provided the use of an antagonist of a human RabJ polypeptide for use in the preparation of a composition for inhibiting tumor formation or tumor growth.

 In another preferred embodiment, the antagonist is an antibody, an antisense nucleotide or an interfering RNA.

 In another preferred embodiment, the RabJ polypeptide is selected from the group consisting of:

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

 (b) The amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of one or more amino acid residues, and has the function of promoting the formation of clones of Hela cells or MCF-7 cells on soft agar in vitro. (a) a derived polypeptide.

 In another preferred embodiment, the composition is a pharmaceutical composition.

 In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

 In another preferred embodiment, the sequence of the interfering RNA is SEQ ID NO: 9.

 In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, or gastric cancer.

 In another preferred embodiment, the composition is also useful for reducing tumorigenicity of tumor cells in vivo.

 In a second aspect of the invention, there is provided the use of a human RabJ polypeptide or a coding sequence thereof for screening for a therapeutic agent which inhibits tumor formation or tumor growth by interaction with a human RabJ polypeptide or a coding sequence thereof .

 In a third aspect of the invention, a method of screening for a therapeutic agent that inhibits tumor cell growth is provided, comprising the steps of:

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

 (b) observing the growth of the tumor cells in the test group and comparing with the growth of the tumor cells of the control group;

Among them, the growth rate of the tumor cells in the test group was smaller than that of the control group, indicating that the test substance is a potential therapeutic agent for inhibiting the growth of tumor cells. In another preferred embodiment, the test substance is an interfering RNA.

 In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a human

An antagonist of the RabJ polypeptide.

 In another preferred embodiment, the antagonist is an interfering RNA having the sequence SEQ ID NO: 9. 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 defined by the claims.

 Figure 1 is a Northern blot analysis of human RabJ protein human RabJ expression in normal human tissues of the present invention. The knot prompts that the RabJ protein is selectively expressed in testicular tissue.

 Fig. 2 is a diagram showing the expression and purification of the human RabJ protein of the present invention and the biochemical characteristics of the RabJ protein. The results showed that the RabJ protein has binding GTP/GDP activity and GTPase activity.

 Among them, (A) expression and purification of RabJ and mutant GST fusion proteins. (B) Specific binding of RabJ GTP/GDP activity analysis. (C) Analysis of Rab J GTPase activity.

 Figure 3 is a Western blot analysis of RabJ protein expressed by NIH3T3 cells transfected with eukaryotic recombinant expression vector of human RabJ of the present invention.

 Figure 4 is an analysis of RNA interference of human RabJ protein expression in HeLa and B MCF-7 cells.

 Figure 5 is a proliferative effect of human RabJ protein overexpression on NIH3T3 cells of the present invention. The results suggest that overexpression of RabJ protein significantly promoted the proliferation of NIH3T3 cells.

 Fig. 6 is a graph showing the inhibitory effect on the proliferation of HeLa and MCF-7 cells after down-regulation of human RabJ protein expression of the present invention. The results suggest that down-regulation of RabJ protein expression can inhibit the proliferation of HeLa and MCF-7 cells.

 Figure 7 is a graph showing the in vitro transformation of NIH3T3 cells induced by high expression of the human RabJ protein of the present invention. Among them, the results are expressed as the number of cells forming the focus or the number of clones/plating χ100%.

 Figure 8 is a graph showing that the down-regulation of human RabJ protein expression of the present invention inhibits the clonality of HeLa and MCF-7 cells. Figure 9 is a graph showing that the high expression of the human RabJ protein of the present invention causes NIH3T3 cells to form tumorigenicity in nude mice (n = 10).

 Figure 10 is a graph showing that down-regulation of human RabJ protein inhibits in vivo tumorigenicity (A) and tumor growth rate (B) of HeLa cells. detailed description

Through extensive and intensive research, the present invention cloned a new Rab family member, RabJ. The coding sequence of RabJ is isolated from a human dendritic cell cDNA library, and its sequence is shown in SEQ ID NO: 1, which comprises a polynucleotide sequence of 1787 bases in length and an open reading frame at 25- 846 digits, the full length of the code is 273 Amino acid human RabJ protein (SEQ ID NO: 2). Experiments have shown that RabJ protein is highly expressed in a variety of tumor cells. Normal fibroblasts NIH3T3 are transformed after high expression of RabJ, which is expressed as colonies on semi-solid agar plates, and RabJ-transformed NIH3T3 cells can be used in nude mice. Formation of fibroblastic sarcoma. RabJ can promote cell proliferation. Blocking the expression of RabJ using RNA interference technology can inhibit the proliferation of tumor cells, resulting in decreased tumorigenicity and decreased tumor growth rate in vivo. Therefore, RabJ can be used as an effective target for tumor therapy.

RabJ polypeptide

 In the present invention, the terms "RabJ protein", "RabJ polypeptide" or "testicular function-related protein RabJ" are used interchangeably and refer to a protein having the testis function-related protein RabJ amino acid sequence (SEQ ID NO: 2 or 4) or Peptide. They include the testicular function-related protein RabJ with or without the initial methionine. In the present invention, hRabJ refers to a human RabJ molecule, and mRabJ refers to a mouse-derived homolog of hRabJ. Studies suggest that RabJ may be involved in the thermal protection of testes and other protection against adverse factors; it may be used for the diagnosis and treatment of reproductive dysfunction and testicular diseases such as tumors, trauma, dysplasia, etc.; The diagnosis and treatment of abnormal diseases is also extremely meaningful.

 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, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .

 As used herein, "isolated RabJ protein or polypeptide" means that the RabJ 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 RabJ protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the RabJ polypeptide can be analyzed by amino acid sequence.

 The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced by recombinant techniques 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 invention also includes fragments, derivatives and analogs of RabJ polypeptides. As used herein, the terms "fragment,""derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of a native RabJ polypeptide of the invention. The polypeptide fragment, derivative or analog of the present invention may be a polypeptide in which ω has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues;), and such substituted amino acid residues may Is not also encoded by the genetic code, or (H) a polypeptide having a substituent group in one or more amino acid residues, or o a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene-2) An alcohol; a polypeptide formed by fusion of the polypeptide, or (IV) an additional amino acid sequence fused to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or with an antigen IgG) Fragment Formation of the fusion protein ;). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

 In the present invention, the term "RabJ polypeptide" means a polypeptide having the sequence of SEQ ID NO. 2 or 4 having RabJ polypeptide activity. The term also encompasses variant forms of the sequence of SEQ ID NO. 2 or 4 that have the same function as the RabJ polypeptide. These variants include (but are not limited to;): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10;) amino acid deletions Insertion and/or substitution, and addition of one or several (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 usually 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 RabJ polypeptides.

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

 The invention also provides analogs of RabJ polypeptides or polypeptides. The difference between these analogs and the native RabJ polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.

 In the present invention, "RabJ polypeptide conservative variant polypeptide" means up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of SEQ ID NO: 2 or 4. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.

RabJ's coding sequence

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be either a coding strand or a non-coding strand. For human RabJ, the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: l or 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.

 Polynucleotides encoding mature polypeptides of RabJ include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences;) and non-coding sequences .

 The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising an additional coding and/or non-coding sequence.

The present invention also relates to a variant of the above polynucleotide which encodes the same amino acid sequence as the present invention. Fragments, analogs and derivatives of peptides or polypeptides. 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 which 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 invention also relates to hybridization to the sequences described above and having at least 50% between the two sequences, preferably at least

70%, more preferably at least 80% identical polynucleotide. The invention particularly relates to polynucleotides which hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) denaturation at the time of hybridization Agent, such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) the identity between only two sequences is at least 90% or more, better Hybridization occurs when it is more than 95%. Further, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.

 The full-length human RabJ 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 prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is 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.

 The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or RabJ 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 utilized to express or produce recombinant RabJ polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:

 (1) using a polynucleotide (or variant) encoding a RabJ 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.

RabJ polypeptide antagonist

The invention also relates to antagonists of RabJ polypeptides. As used herein, an antagonist of a RabJ polypeptide refers to a substance that is capable of combating, or reducing, the activity or expression of a RabJ polypeptide. Common antagonists are antibodies, antisense nucleotides, and interfering RNA (RNAi). Pharmaceutical composition and method of administration

 An antagonist or the like of the protein of the present invention, when administered therapeutically (administered;), can be used for inhibiting tumor formation or for inhibiting growth of tumor cells. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

 In the present invention, an antagonist of RabJ polypeptide can be directly used for the treatment of diseases, for example, for the treatment of diseases such as tumors (such as lung cancer, breast cancer). When an antagonist of the RabJ protein of the present invention is used, other therapeutic agents can also be used at the same time.

 The invention also provides a pharmaceutical composition comprising a safe and effective amount of a RabJ polypeptide antagonist of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to;: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. 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 prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day.

 When a pharmaceutical composition is used, a safe and effective amount of a RabJ protein antagonist is administered to the mammal, wherein the safe and effective amount is usually at least about 1 microgram per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician. The main advantages of the invention are:

 (a) Based on the correlation between RabJ polypeptide and its encoding polynucleotide and tumor formation and tumorigenicity, the corresponding antagonist can be used to inhibit tumor formation and reduce tumorigenicity for cancer treatment;

 (b) Based on the correlation of RabJ with cell proliferation, RabJ can be used as an effective tumor therapeutic target for screening therapeutic agents for treating tumors by interacting with RabJ. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not 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 according to the manufacturer. The suggested conditions. Example 1: Cloning of human RabJ cDNA

Total RNA from human dendritic cells was extracted with Tnzol reagent (Invitrogen). Then, poly(A) mRNA is isolated from total RNA. After the reverse transcription of poly(A) mRNA to form cDNA, SuperScnptll cloning reagent was used. The cassette (Invitrogen) was inserted into the cloning site of the vector, and the DH5oc 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. When the determined cDNA sequence was compared with an existing public DNA sequence database, 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 was named Rab family protein RabJ with human J domain, and its coding gene was named Rab family protein RabJ gene with human J domain.

 SEQ ID NO: 1 is 1787 bp in length and comprises a 24 bp 5' non-coding region and a 941 bp 3' non-coding region encoding a polypeptide of 273 amino acids. Theoretically, the unglycosylated mature molecule has a molecular weight of about 30 kD and belongs to the Rab family of molecules. Example 2: Cloning of the coding sequence of human RabJ protein by RT-PCR

Tnzol reagent is extracted with human dendritic cells total RNA, total RNA from cells taken 5μ _{§ 12} and 1 μ _§ 01igo-dT - ₁₈ were mixed and reverse transcription. The reverse transcription system was 20 μl, and after the reaction was completed, 80 μl dd 3⁄40 was added for dilution. The primers used for PCR amplification of RabJ were as follows: sense primer 5'-AATGGAGGCCAACATGCCG-3' (SEQ ID NO: 3), antisense primer 5'- TACTTTCTACTTGATGTT-3' (SEQ ID NO: 4), and β-actin As a positive control. The PCR reaction volume was 50 μl, including the reverse transcription template 10 μl, 0.5 mM primer, 0.2 mM dNTP and B 1U rTaq DNA polymerase (Takara), and the amplification parameters were 95 ° C for 15 seconds, 57 ° C for 30 seconds, 72. After 30 seconds at °C, the PCR products were confirmed by 1.5% agarose gel electrophoresis after 28 cycles. The result of DNA sequence analysis indicated that the coding DNA sequence of the PCR product was identical to that of position 24-852 shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human RabJ

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 95 Denature at ~100 °C for 2 to 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 10 ⁶ cpm/ml), mix well, at 68° C hybridization for 2 hours. After the end of the hybridization, the filter was rinsed several times with 2xSSC, 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 washed with 0.1 x SSC, 0.1% SDS at 50 ° C for 20 to 40 minutes. Finally, the filter was wrapped in a plastic wrap and exposed to X-ray film at -70 ° C for 24 to 48 hours.

 Northern shows that human RabJ is highly expressed in normal human testis tissue, and is weakly expressed in ovarian and brain tissues, and is not expressed in other tissues examined (Fig. 1). Example 4: Prokaryotic expression of human RabJ protein

In this example, the human PCR amplification product of Example 2 was used as a template, and amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human RabJ DNA as an insert. The 5'-end oligonucleotide primer sequence used in the PCR reaction was: 5'-GCGGATCC ATGGAGGCC AAC ATGC-3 '(SEQ ID NO: 5). The 3' end primer sequence was: 5'-TCGGATCC CTACTTGATGTTTTTC -3' (SEQ ID NO: 6).

 The obtained PCR product was purified, digested with BamH I and then recombined with the expression vector plasmid pGEX-2T (Pharmacia) according to a conventional method and transformed into competent E. coli DH5a, and the clone was picked, and the positive clone was identified by BamH I digestion. The forward clones were identified by EcoR I digestion, and the products were 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 sequencing that the complete RabJ coding sequence has been inserted.

The cloning of the positive DH5a clone expressing RabJ was inoculated in 100 ml of 2xYTA medium, and cultured for 12-15 hrs at 37 °C, 300 rpm, 1 : 10 diluted in pre-warmed 2xYTA medium, and cultured for 1.5 hr, with 100 mM IPTG to O. lmM After induction at 30 ° C for 2-6 hr, 5,000 g 4 ° C centrifugation 10 mm to remove the supernatant, placed on ice with 50 ml lxPBS (0.14 M NaCl, 2.7 mM KC1, l O. l mM Na ₂ HP0 ₄ , 1.8 mM KH ₂ P0 ₄ , pH 7.3) Resuspend, ultrasonic (B. Braun Labsonic U) was crushed and then added with 20% Triton X-100 to 1% for 30 min, then centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was 0.8 μηι filter. After filtration, after washing with 1 ml of 50% glutathione Sepharose 4B column, lxPBS, add 500 ul of glutathione elution buffer (10 mM glutathione, 50 mM Tris-HCl, pH 8.0) at room temperature. After standing for 30 minutes, the eluate was collected and eluted 2-3 times to obtain a human RabJ-GST fusion protein. (Figure 2A)

 After removal of GST by thrombin (thrombin XSigma), the RabJ protein was obtained with a molecular weight of approximately 30 kD, which was consistent with the predicted value.

 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 RabJ antibody

 The human RabJ 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 carried out 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 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 non-complete Freund's adjuvant to boost the immunization. Enhance immunization every 14 days, at least three times. The specific reactivity of the obtained antiserum was evaluated by its ability to precipitate the human RabJ gene translation product in vitro.

 As a result, it was found that the prepared antibody specifically binds to the protein of the present invention. Example 6: GTP/GDP binding activity and GTPase activity analysis of human RabJ protein

10 μ _§ human RabJ-GST fusion protein, GST protein or BSA as control, 50 mM Tris-HCl, 2 mM EDTA, 1 mM containing 25 nM a-[ ³² P]-GTP (Amersham-Phamacia) DTT, 5 mM MgCl ₂ , pH 8.0 in a 50 μΐ reaction system at 30 ° C for 30 min. Or add it separately in the buffer 100 μΜ of GTP, GDP, GTP YS or ATP was applied at 30 ° C for 30 min in the above buffer. After termination of the reaction with 1 M EDTA, 10 μM was dissolved in 10 μΐ of non-denatured protein loading buffer and separated by 15% SDS-PAGE. The gel was then wrapped in a packaging film and subjected to autoradiography at -80 °C. After the end of the binding reaction, the concentration of MgCl _{2 was} increased to 10 mM, and incubation was continued at 30 ° C. After stopping the reaction with 1 M EDTA at different time points, Sepharose G50 column (Amersham-Phamacia) was centrifuged to remove free oc- [ ³² P]-GTP, take 1 μΐ of eluate, spot it on polyethyleneimine cellulose filter (Sigma), perform thin layer chromatography in 1 M UC1/1 M formic acid solution, then -80 °C Autoradiography was performed.

 The results showed that RabJ can bind to p32-tagged GTP, which can be blocked by excess GDP, unlabeled GTP and GTP analog GTPyS, but not by ATP, suggesting that RabJ is a specific binding GTP/GDP. Small G protein. Removal of the amino terminal 209 amino acid protein RabJ(209-273)-GST could not bind to GTP/GDP, suggesting that this GTP/GDP binding activity is mediated by the Rab domain. In addition, RabJ showed significant GTP hydrolysis activity, and this activity was also mediated by the Rab domain. Therefore, RabJ is a small G protein with GTPase activity. (Figs. 2B and 2C) Example 7: Construction of human RabJ eukaryotic expression vector and eukaryotic gene transfection

 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 at the 5' and 3' ends of the sequence below to obtain human RabJ DNA as an insert.

 The 5' oligonucleotide primer sequence used in the PCR reaction is:

 5'- CGGGATCC AATGGAGGCCAACATGCCGA -3' (SEQ ID NO: 7)

 The 3' primer sequence is:

 5'- GCGGTACC GACTACTTGATGTTTTTCAG -3' (SEQ ID NO: 8)

The obtained PCR product was purified, digested with BamH I-Kpn I and recombined with the eukaryotic expression vector plasmid pcDNA3.1/myc-His (-) B (Invitrogen;) by conventional methods and transformed into competent Escherichia coli DH5a. Positive clones were picked and identified, purified and sequenced (ABI's Model 377 Sequencer, B _lg Dye Terminator Kit, PE). It was confirmed by sequencing that the complete RabJ coding sequence has been inserted.

The RabJ eukaryotic expression plasmid DNA was transfected into NIH3T3 (mouse normal fibroblast, ATCC: CRL-1658) with liposome LipofectAMINE reagent (Invitrogen) to pcDNA3.1/myc-His(-)B plasmid vector. As an irrelevant control. Follow the instructions. The main steps are as follows: 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 is grown in MCF-7 cells of 6-well cell culture plate, using OPTI- After washing twice with MEM serum-free medium (Invitrogen), add the plasmid DNA-liposome mixture, incubate at 37 ° C 5% CO _{2 for} 6-8 hours, add an equal volume of normal medium containing 20% serum, continue Fresh medium was replaced after 6 hours of culture. Transient expression was collected 48 hours after transfection and subjected to Western blot analysis to detect transfection effects. Example 8: Western blot detection

Cellular lysate (Cell) of NIH3T3 cells transiently transfected with RabJ protein in Example 7 Signaling Inc. lyses cells. The supernatant was taken by centrifugation at 13,000 rpm x l Omin at 4 ° C, and protein quantification was performed using a BCA protein detection kit (PIERCE). The protein samples were subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 100 V at 4 ° C, stained with Ponceau and labeled with size and orientation. Block at room temperature for 2 hours (5% skim milk powder in TBST solution), 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, washed with TBS (10 mM Tns-HCl, pH 8.0, 150 mM NaCl) for 15 minutes, then added with a chemiluminescent substrate (Pierce) for 1 mm, and rapidly sealed and auto-developed. The primary antibody used for Western blot detection was the anti-RabJ antibody obtained in Example 5. The secondary antibody was HRP-labeled anti-rabbit IgG (Cell Signaling).

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

The expression product of RabJ has a molecular weight of ³⁰ KD (Fig. 3). Example 9: RNA interference analysis of human RabJ

 In vitro synthesis of 19-nt long RabJ oligonucleotide fragment (Shanghai Biotech Co., Ltd.;), insertion of eukaryotic RNAi vector pSuppressorNeo (IMGENEX): sense strand 5'- U CAU CUC CAU GGG CAA CGC -3' (SEQ ID NO: 9) and the antisense strand 5'-GCG UUG CCC AUG GAG AUG A (SEQ ID NO: 10). A 21-nt long randomized control oligonucleotide was also inserted into this vector as a control: sense strand 5'-UCA GUC ACG UUA AUG GUC GUU (SEQ ID NO: 1 1) and antisense strand 5'-AAC GAC CAU UAA CGU GAC UGA (SEQ ID NO: 12).

 Transfection of HeLa (human cervical cancer cells) with liposome Lipofect AMINE reagent (Invitrogen)

ATCC: CCL-2) and MCF-7 (human breast cancer cells, ATCC: HTB-22) are used to interfere with the expression of RabJ protein in cells. The main steps are the same as in Example 7. 48 hours after transfection, cells were harvested for RT-PCR (main steps as in Example 2) and Western blot analysis (main steps as in Example 8) for RabJ protein expression.

 RT-PCR and Western blotting showed that RabJ RNA interference successfully inhibited the expression of human RabJ mRNA and protein in HeLa and MCF-7 cells, while 21nt randomized control had no effect on RabJ mRNA and protein expression. (Figure 4). Example 10: Cell proliferation assay

The [ ³ H]-thymidine incorporation method was employed. NIH3T3 cells overexpressing RabJ in Example He, HeLa or MCF-7 cells inhibiting RabJ expression in Example 9 were plated at ⁵ ×10 ⁴ /well in 24-well culture plates (Falcon), three cells per cell. hole. After 6 h of culture, the culture was continued for 24 to 48 h in serum-free medium, and 0.5 μL was added to each well for the last 4 h. ι (1 Ci = 37 GBq) of [ ³ H]-thymidine (Amersham-Phamacia). Then, it was washed three times with PBS, and the cells were dissolved in PBS containing 1% Triton X-100, collected with a glass fiber filter, and radioactivity was measured with a liquid scintillation meter.

The results showed that untransfected wild-type NIH3T3 cells grew slowly under serum-free conditions, but cells transfected with RabJ were able to grow rapidly and were control cells (untransfected wild-type and blank vector-transfected controls). 25-35 fold, suggesting that NIH3T3 cells have acquired autonomous growth ability after RabJ overexpression (Fig. 5). After the down-regulation of RabJ protein expression, the growth rate of HeLa and MCF-7 cells was significantly decreased (Fig. 6). Example 11: Analysis of in vitro conversion activity

A clone formation assay was used. In order to examine the ability of cells to grow independently of growth, the NIH3T3 cells stably overexpressing RabJ in Example 7, and the HeLa or MCF-7 cells (5 x 10 ⁴ ) inhibiting RabJ expression in Example 9 were suspended in 0.5% soft agar. Place in a 6-well plate containing 1% soft agar. After 3 weeks of culture, clones larger than 50 cells were recorded as positive by light microscopy.

 The results showed that ΝΙΗ3Τ3 cells stably expressing RabJ could form cell clones on soft agar (Fig. 7), whereas untransfected normal wild-type cells and empty vector control cells did not. After down-regulation of RabJ protein expression, the ability of HeLa and MCF-7 cells to form clones on soft agar was inhibited (Fig. 8). Example 12: In vivo tumorigenicity test

The NIH3T3 cells stably expressing RabJ in Example 7, and the HeLa cells (1 x 10 ⁶ ) inhibiting RabJ expression in Example 9 were subcutaneously injected into the right lower abdomen of Balb/C nude mice, and then the growth rate of the tumor was observed or The incidence of tumors (number of mice in which tumors appeared / number of nude mice injected with cells). Tumor size was measured with a vernier caliper (unit: mm) and the result was length X width X width (tumor volume; unit: mm ³ ). Observe for six weeks.

 The results showed that NIH3T3 cells expressing full-length RabJ could form tumor nodules (100%) in vivo, whereas wild-type and control vector-transfected NIH3T3 cells could not form tumors (0% Figure 9), while RabJ protein expression was down-regulated. Post-HeLa cells showed a decrease in tumorigenicity in vivo (Fig. 10A), and the rate of tumor growth that had formed was also reduced (Fig. 10B). Discussion

 Rab (Ras-like protein in rat brain) is a small molecular weight GTPase belonging to the Ras superfamily. The basic biochemical feature is that it can bind GTP/GDP and hydrolyze GTP.

Different Rab proteins have different functions. In cells, Rab3a is mainly distributed in neural tissue and secretory cell lines, and is mainly involved in the regulation of neurotransmitter secretion. Rab3a knockout mice develop normally, but show short- and long-term synaptic plasticity defects in the mossy fiber pathway, and abnormal circadian activity cycles. Rab5 is a widely distributed Rab protein that is primarily localized in early endocytosis and is involved in the regulation of endocytic pathways, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF) receptor EGFR, and PDGFR endocytosis, thereby participating in signals. Regulation of conduction. Abnormalities in Rab proteins and their regulatory and effector proteins can also cause disease, such as certain bleeding and hyperpigmented diseases (GnscelU syndrome;), depression, Charcot-Mane-Tooth neurosis, kidney disease (kidney) Tumor sclerosis;), blindness (no choroidal disease;) and other diseases are associated with dysfunction of Rab-related proteins; high expression of Rab protein is often associated with certain pathological processes of vascular, pulmonary, and thyroid diseases. GnscelU syndrome is a disease caused by abnormal pigmentation and pigmentation and T lymphocyte killing dysfunction. It is found that the mutation of Rab27a is the occurrence of the disease. Genetic cause. Abnormalities in the Rab7 protein can cause abnormalities in the metabolism of lipoproteins, causing hyperlipidemia and vascular diseases; some intracellular bacteria, such as Mycobacterium tuberculosis, can inhibit the function of Rab7 to infect the host and cause disease. Abnormalities in Rab regulatory proteins are also the cause of certain diseases, such as choroidal disorders, X-linked neurodegenerative disorders; abnormalities in post-translational modification of Rab proteins can cause Hermansky-Pudlak syndrome.

 Some studies have also suggested that certain Rab proteins may be involved in tumorigenesis. First, abnormal expression of Rab protein is found in certain tumor cells or tumor types. Rab25 is highly expressed in prostate cancer, and its expression level is closely related to the differentiation and clinical stage of prostate cancer. Rab38 expression is also significantly increased in melanoma cell lines; Rab3 is also highly expressed in pituitary adenocarcinoma. Rab2 expression was significantly increased in peripheral blood mononuclear cells of hematopoietic tumors and solid tumor patients; Rab2 expression was also abnormal in mouse adrenal and mouse lung cancers. Second, a deletion mutation in the Rab gene was found in the tumor. A deletion of the chromosome 22ql l.2 region was found in malignant rhabdomyosarcoma, which contains the coding region of Rab36. In addition, abnormalities in Rab regulatory proteins have also been found in tumors. In neuroblastoma and hematopoietic tumors, the expression of the Rab protein regulatory protein RabGDI is significantly increased. In prostate cancer, the expression of Rab GTPase-activating protein, PRC17, which regulates GTP hydrolysis of Rab5 protein, can affect tumor growth characteristics.

 The RabJ protein of the present invention is highly expressed in various tumor cells, and the normal fibroblast NIH3T3 is transformed after high expression of RabJ, which is expressed as a colony formed on a semi-solid agar plate, and RabJ-transformed NIH3T3 cells can be used in nude mice. Formation of fibroblastic sarcoma. RabJ can promote cell proliferation. Blocking the expression of RabJ using RNA interference technology can inhibit the proliferation of tumor cells, resulting in decreased tumorigenicity and decreased tumor growth rate in vivo. The above experimental results of the present invention suggest that RabJ is involved in cell proliferation and cell cycle progression, and thus RabJ can be used as an effective tumor therapeutic target.

 In addition, antagonists of human RabJ protein (such as antibodies or interfering RNA) can be used to inhibit the growth of tumor cells, and thus have great application prospects. 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 invention.

Claims

Rights request Use of an antagonist of a human RabJ polypeptide, characterized in that the antagonist is used for the preparation of a composition for inhibiting tumor formation or tumor growth.  The use according to claim 1, wherein the antagonist is an antibody, an antisense nucleotide or an interfering RNA.  3. The use according to claim 1, wherein the RabJ polypeptide is selected from the group consisting of:  (a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;  (b) The amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of one or more amino acid residues, and has the function of promoting the formation of clones of Hela cells or MCF-7 cells on soft agar in vitro. (a) a derived polypeptide.  The use according to claim 1, wherein the composition is a pharmaceutical composition.  The use according to claim 2, wherein the sequence of the interfering RNA is SEQ ID NO: 9.  The use according to claim 1, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, or gastric cancer.  7. The use according to claim 1, wherein the composition is further for reducing the tumorigenicity of tumor cells in vivo.  8. Use of a human RabJ polypeptide or a coding sequence thereof for screening for a therapeutic agent which inhibits tumor formation or tumor growth by interaction with a human RabJ polypeptide or a coding sequence thereof.  9. A method of screening for a therapeutic agent that inhibits tumor cell growth, comprising the steps of:  (a) providing a test group and a control group, wherein the control group is a culture system of a tumor cell expressing a RabJ polypeptide or a culture system of a tumor cell to which a RabJ polypeptide is added, and the test group is added with a test substance a culture system of a tumor cell expressing a RabJ polypeptide, or a culture system of a tumor cell to which a test substance and a RabJ polypeptide are added;  (b) observing the growth of the tumor cells in the test group and comparing with the growth of the tumor cells of the control group;  Among them, the growth rate of the tumor cells in the test group was lower than that of the control group, indicating that the test substance was a potential therapeutic agent for inhibiting the growth of tumor cells.  A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antagonist of a human RabJ polypeptide.