CN1948482B - Antisensedigonucleotides sequence for inhibiting human Rabj gene expression and its application - Google Patents

Abstract

The present invention provides an antisense oligonucleotide for oncogene-like molecule RabJ. Specifically, the present invention relates to an antisense oligonucleotide sequence aimed at the expression and function of the oncogene-like molecule RabJ, the antisense oligonucleotide sequence has targeted anti-tumor cell RabJ gene expression and function, inhibition The function of tumor cell growth and inhibition of tumorigenicity of tumor cells, thus playing a role in the treatment of tumors. The invention also relates to a pharmaceutical composition containing the antisense oligonucleotide, and discloses a method for using the antisense oligonucleotide drug in disease treatment, especially its use in the treatment of malignant tumors.

Description

Antisense oligonucleotide sequence for inhibiting human RabJ gene expression and its application

technical field

The invention belongs to the fields of biotechnology and medicine. The present invention relates to antisense oligonucleotides directed against the novel oncogene-like molecule RabJ. Specifically, the present invention relates to a group of antisense oligonucleotide sequences aimed at the expression and function of the oncogene-like molecule RabJ, the antisense oligonucleotide sequences have targeted anti-tumor cell RabJ gene expression and function, Inhibit the growth of tumor cells and inhibit the tumorigenicity of tumor cells, thus playing a role in the treatment of tumors. The invention also relates to a pharmaceutical composition containing the antisense oligonucleotide, and discloses a method for treating diseases with the antisense oligonucleotide drug, especially the use in the treatment of malignant tumors.

Background technique

Small G proteins refer to single-subunit G proteins 1-5 with a molecular weight of about 20-30 kDa. At present, with the completion of the Human Genome Project, more than 100 members of this class of proteins have been found, and their common characteristics are that they can bind guanosine triphosphate (GTP) and guanosine diphosphate (GDP), and can hydrolyze the GTPγ position. Phosphoric acid becomes GDP (ie GTPase activity, GTPase activity) (Wittinghofer and Pai, Trends Biochem Sci.1991, 16:382-387; Polakis and McCormick, J Biol Chem.1993, 268:9157-9160; Moodie et al., Oncogene. 1995, 11:447-454; Marshall, Trends Biochem Sci. 1993, 18:250-254). Small G proteins play a very important role in regulating cell growth, proliferation, differentiation, migration, and intracellular protein transport and other basic life activities.

The abnormal function of small G protein can cause many pathological phenomena and diseases, among which Ras and tumorigenesis and its role in the regulation of cell proliferation have received special attention. In almost all types of tumor cells or tumor tissues, there are mutations of H-Ras, K-Ras or N-Ras genes, and among the genes mutated in tumors, the probability of Ras mutations is the highest (Bos, Cancer Res.1989 ; 49:4682-4689).

Rab (rat brain Ras-like protein, Ras-like protein in rat brain) is a small molecular weight GTPase belonging to the Ras superfamily, and its basic biochemical characteristics are that it can bind GTP/GDP and hydrolyze GTP (Simons and Zerial, Neuron.1993 , 11:789-799; Takai et al., Physiol Rev.2001, 81:153-208; Martinez and Goud, Biochim Biophys Acta.1998; 1404:101-112; Stenmark and Olkkonen, Genome Biol.2001, 2: REVIEWS3007) . Abnormalities of Rab protein and its regulatory proteins and effector proteins can also cause diseases, such as certain bleeding and abnormal pigmentation diseases (Griscelli syndrome), depression, Charcot-Marie-Tooth neurosis, kidney disease (tubular Diseases such as sclerosis), blindness (choroideremia) and other diseases are all related to the abnormal function of Rab-related proteins; in some pathological processes of blood vessel, lung and thyroid diseases, the high expression of Rab protein is often accompanied (Seabra et al., Trends Mol Med.2002 , 8:23-30).

In the study of Griscelli syndrome (a disease accompanied by pigmentation and abnormal killing function of T lymphocytes caused by abnormal pigment transport), it was found that the mutation of Rab27a is the genetic cause of the disease (Menasche et al., Blood.2003, 101:2736-2742; Bahadoran et al., J Biol Chem. 2003; 278:11386-11392; Barral et al., J Clin Inyest. 2002, 110:247-257). The abnormality of Rab7 protein can cause abnormal metabolism of lipoprotein, causing hyperlipidemia and vascular diseases; some intracellular bacteria, such as Mycobacterium tuberculosis, can infect the host and cause disease by inhibiting the function of Rab7 (Runz et al., J Neurosci. 2002, 22: 1679-1689; Kim et al., Biochem Biophys Res Commun. 2000, 293: 375-382; Choudhury et al., J Clin Invest. 2002, 109: 1541-1550; Via et al., J Biol Chem. 1997, 272 : 13326-13331; Clemens et al., Infect Immun. 2000, 68: 5154-5166; Rupper et al., J Cell Sci. 2001, 114: 2449-2460).

Earlier studies believed that the relationship between Rab protein and tumorigenesis is not very close, but the current research suggests that Rab protein may be involved in tumorigenesis. First, abnormal expression of Rab protein was 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 (He et al., 6ene Expr. 2002, 10: 231-242); the expression of Rab38 in melanoma cell lines is also Significantly increased (Jager et al., CancerRes.2000, 60:3584-3591; Loftus et al., Proc.Natl.Acad.Sci.USA.2000, 99:4471-4476); the expression of Rab3 is also high in pituitary adenocarcinoma (Culine et al., Cancer.1992, 70:2552-2556); in the peripheral blood mononuclear cells of patients with hematopoietic tumors and solid tumors, the expression of Rab2 was significantly increased; in mouse adrenal carcinoma and mouse lung cancer, the expression of Rab2 Also appear abnormal (Culine et al., Cancer Res. 1992, 52:3083-3088). Second, a deletion mutation of the Rab gene was found in the tumor. A deletion in the region of chromosome 1622q11.2, which contains the coding region of Rab36, was found in malignant rhabdomyosarcoma (Mori et al., Biochem Biophys Res Commun. 1999, 254: 594-600; Zhou et al., Gene. 2000, 241: 133- 141). In addition, there are also abnormalities of Rab regulatory proteins in tumors. In neuroblastoma and hematopoietic tumors, the expression of RabGDI, a regulatory protein of Rab protein, is significantly increased. PRC17 (prostate cancer gene 17, prostate cancer gene 17) is a RabGTPase activating protein (GTPase activating protein, GAP), which can regulate the GTP hydrolysis of Rab5 protein, and its expression is increased in prostate cancer, thereby affecting the growth characteristics of tumors (Pei et al., Cancer Res. 2002, 62:5420-5424).

Human RabJ is a new member of the small G protein family derived from dendritic cells derived from peripheral blood mononuclear cells of healthy adults, characterized by the inclusion of three classes of functional domains in its protein composition: N-terminal (1-18 amino acids) And the nuclear localization signal (nuclear localization signal, NLS) in the middle (210-216 amino acids), the middle Rab-like functional domain (19-209 amino acids) and the J functional domain (217-273 amino acids) at the C-terminal, and with the Ras family molecules have high homology. The three types of functional domains mediate the nuclear localization of RabJ respectively; Phosphatidylinositol 3-kinase (phosphatidylinositol 3-kinase, PI3K) P85 subunit, P53 subunit interaction; interaction with HSC70 and Raf (Ras-associated factor, Ras-associated factor) and other functions. The nucleotide sequence, amino acid sequence and preparation method of RabJ are disclosed in Chinese patent application CN01126826.3.

Antisense drugs, also known as antisense oligonucleotide drugs, refer to artificially synthesized DNA molecules and their analogs with a length of 10-30 bases used as drugs. According to the principle of nucleotide hybridization, antisense drugs can hybridize with specific genes and interfere with the production of disease-causing proteins at the genetic level. Protein plays an important role in the human body's metabolic process. Most diseases are caused by protein abnormalities. Regardless of tumors, cardiovascular diseases or infectious diseases, traditional drugs mainly act directly on the pathogenic protein itself, while antisense drugs act on Genes that produce proteins can be widely used in the treatment of various diseases. They are more selective than traditional drugs, and have the characteristics of high efficiency, low toxicity, and less dosage. A few years ago, antisense drugs were difficult to carry out extensive clinical trials due to the high cost of synthesis. In recent years, due to the improvement of synthesis technology and the successful development of synthesis equipment, the cost of antisense drugs has been greatly reduced, thus accelerating the research and development of antisense drugs.

From the perspective of pharmacokinetics, due to the presence of nucleases in various organs and tissues in the body, natural oligonucleotides are easily decomposed and inactivated when entering the body, so antisense drugs are mostly modified oligonucleotides to enhance their Resistance to nuclease degradation. There are many ways to modify oligonucleotides. Thio-oligonucleotides are commonly used because these antisense drugs have good water solubility and are easy to synthesize in large quantities, which can meet clinical needs. Administered by subcutaneous, intramuscular and intravenous injection, except for brain tissue, thiooligonucleotides can be distributed in various organs and tissues, which can bind to plasma proteins, but with low affinity, and are mainly excreted in urine.

Based on the principle of base complementarity, antisense gene therapy uses nucleic acids that are complementary to the target gene or its mRNA to induce RNase H (RNase H) activity or form a triple helix with the target DNA through steric repression. ), at the level of gene replication, transcription, splicing, mRNA transport and translation, inhibits the properties of protein synthesis, inhibits the expression of oncogenes, inhibits the secretion of growth factors or blocks its receptors, thereby blocking abnormal signal transduction in cancer cells And autocrine and paracrine loops, make cancer cells enter the normalization track or cause cancer cell apoptosis.

Antisense nucleic acids include antisense ribonucleic acid (antisence RNA), ribozyme (ribozyme) and antisense oligonucleotides (antisense oligodeoxynucleotides, AONs). Through the development and research of antisense oligonucleotide drugs, 17 antisense oligonucleotide drugs have entered clinical trials. Among them, Vitravene is an antisense drug approved by the FDA to enter the market in 1998. There is a modified cap at both ends of its molecular structure, thereby enhancing its stability. The drug has strong antiviral effect and is used for treating cytomegalovirus retinitis and AIDS complicated with giant cell retinitis. Adverse reactions include iritis and vitritis, with an incidence rate of 25%. Treatment with glucocorticoids can alleviate or eliminate the inflammatory reaction.

In order for AON to enter the target cells smoothly and play a therapeutic role, targeted transport is very important. There are many problems in direct injection, such as: AON is easily degraded, the cell uptake rate is not high, non-specific binding reduces the efficacy of AON, and non-specific blocking of mRNA. To this end, the following methods have been tried to improve targeted transport: (1) linking AON to transferrin/poly-L-lysine complex to enhance its biological effect; (2) linking AON to positively charged Lipids form complexes to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (3) AON is mediated into cells after being wrapped with liposomes, which is conducive to the smooth entry of macromolecules and is free from various extracellular substances. Enzyme hydrolysis; (4) Combine AON with cholesterol to increase its cytoplasmic retention time by 10 times; (5) Transport AON with immunoliposomes to specifically transport it to target tissues and cells; (6) In vitro transfection of AON to transfected cells (such as fibroblasts) can also better load AON into target cells; (7) electroporation, that is, to introduce AON into target cells by means of electric current.

In recent years, although antisense drugs have made some progress in the treatment of certain tumors and viral infections in clinical trials or applications, the results obtained are not yet very satisfactory. Therefore, there is an urgent need in this field to further develop antisense drugs that can effectively inhibit tumors.

Contents of the invention

The object of the present invention is to provide a kind of antisense oligonucleotide that can effectively suppress tumor, and described antisense specifically targets the oncogene-like molecule RabJ gene at the mRNA level, and has the effect on the biological behavior of tumor cells expressing RabJ. Intervention can effectively inhibit the expression of RabJ and achieve anti-tumor effect.

In the first aspect of the present invention, an antisense oligonucleotide is provided, the antisense oligonucleotide is reverse complementary to human RabJ gene mRNA, its length is 18-25bp, and the antisense oligonucleotide Sense oligonucleotides can reduce the growth of HeLa cells by more than 50%.

In another preferred example, the antisense oligonucleotide is bound to the corresponding nucleotide sequence of the following regions of RabJ: nuclear localization signal, Rab functional domain, J functional domain, or a combination thereof.

In another preferred example, the antisense oligonucleotide contains nucleotide sequences selected from SEQ ID NO: 5-8.

More preferably, the antisense oligonucleotide is selected from the following group:

AON-RabJ1: 5'-CTTCCGCTTCGGCATGTTGG-3';

AON-RabJ2: 5'-CTGCCAGCCACGCATCAAGG-3';

AON-RabJ3: 5'-CTCCCAGCATGTCCCAACTG-3';

AON-RabJ4: 5'-CAGGAGGGCTGTCCGAGCAT-3'.

In another preferred example, the antisense oligonucleotide has modified nucleotides, and the modification method is selected from the group consisting of sulfur modification, methylation modification, phosphorothioation modification, or its combination.

In another preferred example, the modified nucleotides are full-chain phosphate-thio-modified nucleotides.

The second aspect of the present invention also relates to a composition, which contains 0.001-99.99wt% of the aforementioned antisense oligonucleotides and acceptable carriers, diluents or excipients.

Preferably, the composition is a pharmaceutical composition, and the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.

The third aspect of the present invention provides the use of the antisense oligonucleotide as described above, which is used to prepare a composition for inhibiting tumor cell growth or a pharmaceutical composition for treating tumor. More preferably, said tumor cells or tumors express RabJ protein and are selected from the group consisting of cervical cancer, breast cancer, and colon cancer.

Description of drawings

Figure 1: Analysis of the expression of RabJ at the mRNA level after the transient expression of RabJ antisense oligonucleotides;

Figure 2: Analysis of the expression of RabJ at the protein level after the transient expression of RabJ antisense oligonucleotides;

Figure 3: RabJ antisense oligonucleotides inhibited the growth of tumor cells after transient expression;

Figure 4: After transient expression of RabJ antisense oligonucleotides, its inhibition of tumorigenic activity of tumor cells in vitro;

Figure 5: Intratumoral injection of RabJ antisense oligonucleotides inhibits the growth of tumor cells in vivo;

Figure 6: RabJ antisense oligonucleotides promote tumor cell differentiation after intratumoral injection. Figure 6A is a wild-type HeLa tumor; Figure 6B is SON-RabJ1; Figure 6C is SON-RabJ4; Figures 6D-6F are AON-RabJ1; Figures 6G-6I are AON-RabJ4. Arrows indicate fibrous connective tissue insertions; small asterisks indicate squamous differentiation; large asterisks indicate necrotic areas; flower patterns indicate vacuolar structures.

Detailed ways

After extensive and in-depth research, the present inventors found that the expression of RabJ in tumor cells tends to increase, and inhibiting the expression of RabJ can inhibit the proliferation of tumor cells and the tumorigenic activity in vivo and in vitro. The inventors further synthesized and tested a variety of RabJ antisense oligonucleotides, and screened out antisense sequences that can strongly inhibit the expression of RabJ, thereby inhibiting tumor cell proliferation and tumorigenicity in vivo and in vitro. The present invention has been accomplished on this basis.

Specifically, the inventor's research shows that RabJ is mainly distributed in primary and secondary spermatocytes (spermatocytes) and early spermatids (spermatids) in testicular tissue, and has no expression in interstitial cells and Sertoli cells. The expression patterns of cyclins cyclin A and cyclin D1 were similar in mouse testes, suggesting that RabJ may be involved in cell cycle regulation.

In the NIH3T3 cell line, the overexpression of RabJ can promote the expression of cell cycle proteins, promote cell proliferation, and promote cell cycle progression, and can promote the formation of colonies on soft agar, and can also form fibroblastosarcoma in nude mice , suggesting that it may be an oncogene-like small G protein.

The study of the present invention also found that the expression of RabJ in tumor cells is often increased, and inhibiting the expression of RabJ can inhibit the proliferation of tumor cells and the tumorigenic activity in vivo and in vitro. Since RabJ can form complexes with a variety of proteins, kinase inhibitors were used to inhibit the activity of each kinase, and it was found that MEK/ERK inhibitors could completely inhibit the tumorigenic ability of RabJ in vitro, suggesting that the ERK1/2-dependent signal transduction mechanism may be RabJ tumorigenic mechanism.

The results of confocal microscopy also suggested that RabJ is a nuclear anchoring protein of MEK1/2. By increasing the level of phosphorylated MEK1/2 in the nucleus, the sustained activation of ERK1/2 is formed locally in the nucleus, causing tumorigenesis.

Therefore, the inventor's research shows that RabJ, as a member of the small G protein family, plays an important role in cell cycle regulation, cell proliferation, tumorigenesis and other processes, and its mechanism may be promoted by nuclear anchor phosphorylated MEK1/2 ERK1/2-mediated effects that promote cell proliferation and tumorigenesis. Therefore, RabJ is an oncogene-like small G protein, which can be used as a potential candidate target in the diagnosis and treatment of clinical tumors.

active ingredient

In the present invention, the active ingredient is RabJ antisense oligonucleotide. As used herein, the term "active ingredient" refers to such RabJ antisense oligonucleotide: said oligonucleotide is reverse complementary to human RabJ gene mRNA, its length is preferably 18-25bp, and said antisense Oligonucleotides can reduce the growth of HeLa cells by more than 50% (eg, 50%-90% or more).

The cDNA sequence of RabJ is shown in SEQ ID NO: 19, which encodes RabJ protein (shown in SEQ ID NO: 20). The full length of SEQ ID NO: 19 is 1787bp, including a 24bp 5' non-coding region and a 316bp 3' non-coding region, encoding a polypeptide containing 273 amino acids.

As used herein, "antisense oligonucleotide" refers to a nucleotide oligomer that is antisense. Antisense oligonucleotides form triple strands (antigene) with double-stranded DNA through complementary base (A-T, A-U, and G-C) pairing, or form hybrid double strands (antisense) with single-stranded RNA, thereby blocking gene replication , transcriptional or post-transcriptional processing and translation of mRNA. At the same time, double-stranded RNA can be degraded by intracellular ribonuclease H (RNase H), thereby more effectively blocking the expression of target genes. Since the antisense nucleotide can only be combined with the reverse complementary target sequence, it has the characteristics of high specificity and small side effects.

Preferred antisense oligonucleotides are bound to the corresponding nucleotide sequences of the following regions of RabJ: nuclear localization signal (NLS, 1-18 amino acids), Rab functional domain (19-200 amino acids), J functional domain ( 201-273 amino acids), or a combination thereof.

In one embodiment of the present invention, the antisense oligonucleotide is selected from the following group:

AON-RabJ1: 5'-CTTCCGCTTCGGCATGTTGG-3';

AON-RabJ2: 5'-CTGCCAGCCACGCATCAAGG-3';

AON-RabJ3: 5'-CTCCCAGCATGTCCCAACTG-3';

AON-RabJ4: 5'-CAGGAGGGCTGTCCGAGCAT-3'.

Among them, AON-RabJ1 and AON-RabJ2 correspond to the RabJ nuclear localization signal (NLS, 1-18 amino acids); AON-RabJ3 corresponds to the Rab functional domain of RabJ (19-200 amino acids); AON-RabJ4 corresponds to the RabJ J functional domain (amino acids 201-273).

Various modifications of nucleotides are known to those skilled in the art. The stability of the antisense oligonucleotide and/or the efficiency of the antisense oligonucleotide drug can be further improved by modifying the nucleotide. Therefore, the present invention also includes various nucleotide modification forms, and representative examples of modification forms include, but are not limited to: thio-modification, methylation modification, phosphothio-modification, or combinations thereof.

The RabJ antisense oligonucleotide of the invention can effectively inhibit the expression of RabJ, thereby inhibiting the proliferation of tumor cells. Specifically, the RabJ antisense oligonucleotide of the present invention targets the RabJ gene at the mRNA level, and reverses the biological behavior of tumor cells positively expressing RabJ. Experiments have proved that: (1) inhibiting the expression of RabJ can inhibit the proliferation of tumor cells; (2) inhibiting the expression of RabJ in vivo can inhibit the tumorigenicity of tumor cells in vitro; (3) inhibiting the expression of RabJ can inhibit the growth of tumor cells in vivo; (4) Inhibiting the expression of RabJ can promote the differentiation of tumor cells.

Pharmaceutical composition and method of administration

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount (such as 0.001-99wt%) of the RabJ antisense oligonucleotide of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or 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 active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day.

When using the pharmaceutical composition, a safe and effective amount of RabJ antisense oligonucleotide is administered to the mammal, wherein the safe and effective amount is usually at least about 1 microgram/kg body weight, and in most cases no more than about 5 mg/kg body weight. kg body weight, preferably the dosage is about 10 μg/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

When preparing a pharmaceutical composition, usually, these antisense oligonucleotides of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, relatively Preferably the pH is about 6-8, although pH may vary with the nature of the substance being formulated and the condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

Preferred routes of administration of the antisense oligonucleotide drug of the present invention include but are not limited to: (1) direct naked DNA injection; (2) linking AON to transferrin/poly-L-lysine complex ; (3) AON is formed into a complex with positively charged lipids; (4) AON is mediated into cells after being wrapped with liposomes; (5) AON is combined with cholesterol; (6) transported by immunoliposomes AON; (7) in vitro transfection of AON to transfected cells (such as fibroblasts); (8) electroporation, that is, introducing AON into target cells by means of electric current.

In addition, the antisense oligonucleotides of the present invention can be directly used for disease treatment alone, and can also be used in combination with other therapeutic agents, such as TNF-α, TGF-β, IFN-α, Angiostatin, Endostatin, glycyphosphoryl mustard, Hematoporphyrin, lycorine betaine, Brucea javanica milk, etoposide (i.e. etoposide), anhydrodulcitol, doxorubicin, tamoxifen, 5-fluorouracil, norcanthin, difuran fluorouracil , cucurbitacin, harringtonine, oridonin, irinazolin, versicolor, cytarabine, carboplatin, paclitaxel, lentinan, flutamide, ifosfamide, ubene Mesi, leuprolide acetate, deoxyfluridine, loboplatin, Elinnotken, letrozole and teniposide, etc.

The main advantages of the present invention are:

(a) The four antisense oligonucleotide sequences designed for different target sites of the RabJ gene in the present invention can effectively inhibit the expression of the oncogene-like molecule RabJ, thereby being used for cancer treatment;

(b) The antisense oligonucleotide of the present invention can be used alone or in combination with several antisense oligonucleotides, and can also be used in combination with other drugs and treatment means for the treatment of malignant tumors.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as the people such as Sambrook, Molecular Cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacture conditions recommended by the manufacturer.

Example

General Methods and Materials

(a) Oligonucleotides

The sequence of the RabJ antisense oligonucleotide is shown below:

AON-RabJ1: 5'-CTTCCGCTTCGGCATGTTGG-3'(+7-+26) (SEQ ID NO: 5);

AON-RabJ2: 5'-CTGCCAGCCACGCATCAAGG-3'(+318-+337) (SEQ 1D No.6);

AON-RabJ3: 5'-CTCCCAGCATGTCCCAACTG-3'(+651-+670) (SEQ ID NO: 7);

AON-RabJ4: 5'-CAGGAGGGCTGTCCGAGCAT-3' (+788-+807) (SEQ ID NO: 8).

The oligonucleotide sequence used as a negative control is as follows:

AON-RabJ1neg:

(+7-+26 AON sequence negative control) (SEQ ID NO: 9)

AON-RabJ2neg:

(+318-+337 AON sequence negative control) (SEQ ID No.10)

AON-RabJ3neg:

(+651-+670 AON sequence negative control) (SEQ ID NO: 11)

AON-RabJ4neg:

(+788-+807 AON sequence negative control) (SEQ ID NO: 12)

(b) cell lines

HeLa cell line: ATCC: CCL-2 (purchased from ATCC), which is a cervical cancer tumor cell line positively expressing RabJ.

MCF-7 cell line: ATCC: HTB-22 (purchased from ATCC), which is a breast cancer tumor cell line positively expressing RabJ.

SW480 cell line: ATCC: CCL-28 (purchased from ATCC), which is a colon cancer tumor cell line positively expressing RabJ.

The cultivation and treatment methods of the cells are as follows: the cells are inoculated in the RPMI1640 (Invitrogen Company) culture medium containing 10% calf serum, and placed in a CO _incubator with a volume fraction of 5% at 37 degrees Celsius for routine culture, without drugs before the experiment. Grow for two weeks. Phosphothio-modified SON-RabJ1 (SEQ ID NO: 1), SON-RabJ2 (SEQ ID NO: 2), SON-RabJ3 (SEQ ID NO: 3), SON-RabJ4 (SEQ ID NO: 4), AON -RabJ1 (SEQ ID NO: 5), AON-RabJ2 (SEQ ID NO: 6), AON-RabJ3 (SEQ ID NO: 7), AON-RabJ4 (SEQ ID NO: 8) and the corresponding negative control AON-RabJ1neg ( SEQ ID NO: 9), AON-RabJ1neg (SEQ ID NO: 10), AON-RabJ1neg (SEQ ID NO: 11) and AON-RabJneg (SEQ ID NO: 12) were added with LipofectAMINE (InvitroGen Company) at a final concentration of 1 μmol Cell culture medium, cells were harvested for detection after 24-48 hours of incubation.

Example 1: Analysis of the expression of RabJ at the mRNA and protein levels after the transient expression of RabJ antisense oligonucleotides

The forward sequence SON-RabJ1 (SEQ ID NO: 1), SON-RabJ2 (SEQ ID NO: 2), SON-RabJ3 (SEQ ID NO: 3), SON-RabJ4 (SEQ ID NO: 4) modified by phosphorothioation ), antisense oligonucleotide AON-RabJ1 (SEQ ID NO: 5), AON-RabJ2 (SEQ ID NO: 6), AON-RabJ3 (SEQ ID NO: 7), AON-RabJ4 (SEQ ID NO: 8 ) and corresponding negative controls AON-RabJ1neg (SEQ ID NO: 9), AON-RabJ1neg (SEQ ID NO: 10), AON-RabJ1neg (SEQ ID NO: 11) and AON-RabJneg (SEQ ID NO: 12) ( Synthesized by Shanghai Sangon Bioengineering Co., Ltd.), with a final concentration of 1 μmol and the transfection reagent LipofectAMINE (Invitrogen Company) were added to HeLa cervical cancer cell culture medium, and the cells were harvested 24-48 hours after incubation for detection.

In this example, the inhibition of antisense oligonucleotides on the expression of RabJ gene, an oncogene-like molecule, was detected from two levels of mRNA and protein levels.

(a) TaqMan quantitative RT-PCR method to detect mRNA

Methods as below:

(1) Reverse transcription: extract total RNA according to TRIzol Total RNA Extraction Kit. The quality of RNA was identified by electrophoresis and quantified by UV spectrophotometer. Take 2.5 μg of total RNA, add 50 μl of reverse transcription reaction system, use SuperScript™ II reverse transcription kit (purchased from Invitrogen), and perform reverse transcription according to the instructions.

(2) PCR amplification: The PCR reaction system was conventional (reagents were purchased from Invitrogen).

The RabJ specific primers used were: 5'-GGA GTC AAA CCT GGG GCC TCA AG-3' (upstream, SEQ ID NO: 13) and 5'-CAG GAG GGC TGT CCG AGC ATT C-3' (downstream, SEQ ID NO: 14);

The RabJ specific probe used is: 5'FAM-GTA GCA CCT GGC AGT GAA GATG-3'TAMRA (SEQ ID NO: 15);

The β-actin specific primers used were: 5'-TCA CCC ACA CTG TGC CCA TCT AC6A-3' (upstream, SEQ ID NO: 16) and 5'-CAG CGG AAC CGC TCA TTG CCA ATG G-3' (downstream, SEQ ID NO: 17);

The β-actin-specific probe used was: 5'FAM-ATG CCC TCC CCC ATG CCA TCCTGC GT-3'TAMRA (SEQ ID NO: 18).

Cycling conditions: denaturation at 94°C for 45 seconds, annealing at 58°C for 1 minute, extension at 72°C for 1 minute, and final extension for 10 minutes.

(3) Quantification of PCR products: dynamically measure the fluorescence intensity, and use the ratio of the fluorescence intensity of RabJ and β-actin to express the level of RabJ mRNA.

(b) Western blot technique to detect the expression level of RabJ protein

Cells in each group were collected for 48 hours, and cell lysate was prepared, and Western blot detection was performed with RabJ polyclonal antibody (prepared according to the method in Example 6 in CN01126826.3).

result

RabJ antisense oligonucleotides (SEQ ID NO: 5-8) can significantly inhibit RabJ mRNA (see Figure 1) and protein expression (see Figure 2) in HeLa tumor cells. Its corresponding forward sequence (SEQ ID NO: 1-4) and negative control (SEQ ID NO: 9-12) had no effect on the mRNA (see Figure 1 ) and protein expression of RabJ.

Example 2: Growth inhibition of tumor cells after transient expression of RabJ antisense oligonucleotides

In this example, the [ ³ H]-thymidine incorporation method was adopted.

After 48 hours of transient expression of RabJ antisense oligonucleotides, HeLa, MCF-7 and SW480 cells were seeded in a 24-well culture plate (Falcon) at a cell density of 5× ¹⁰⁴ /well, and each cell was seeded in three wells . After culturing for 6 hours, continue culturing in serum-free medium for 24-48 hours. In the last 4 hours, 0.5 μCi (1Ci=37 GBq) of [ ³ H]-thymidine was added to each well. Then, wash three times with PBS, suspend cells in PBS containing 1% Triton X-100, collect with glass fiber filter membrane, and measure radioactivity with liquid scintillation apparatus. Alternatively, after 24 hours of serum starvation, normal medium containing 10% FCS was added, the culture was continued for 24 hours, and [ ³ H]-thymidine was added for the last 4 hours, and then radioactivity was measured.

The results showed that RabJ antisense oligonucleotides (SEQ ID NO: 5-8) could significantly inhibit the growth ability of HeLa, MCF-7 and SW480 tumor cells in vitro. The inhibitory effect on HeLa is shown in Figure 3.

Example 3: Inhibition of tumorigenic activity of tumor cells in vitro after transient expression of RabJ antisense oligonucleotides

In this example, the soft agar colony formation method was used. HeLa cells transiently expressed with 5×10 ⁴ RabJ antisense oligonucleotides suspended in 0.5% soft agar were inoculated in a 6-well plate with 1% soft agar at the bottom to detect the ability of the cells to grow contact-independently. After culturing for 3 weeks, the clones with more than 50 cells were recorded as positive under the light microscope.

The results showed that RabJ antisense oligonucleotides (SEQ ID NO: 5-8) could significantly inhibit the colony formation ability of HeLa tumor cells on soft agar in vitro (see FIG. 4 ).

Example 4: Inhibition of tumor cell growth rate by intratumoral injection of RabJ antisense oligonucleotides

Subcutaneously inject 1×10 ⁶ HeLa cells into the right lower abdomen of Balb/C nude mice (purchased from Shanghai Bikai Experimental Animal Co., Ltd.) Nude mice, combined test group is 2), after 4 weeks, inject RabJ antisense oligonucleotide (20μg/20μl 0.9%NaCl/mouse) (SEQ ID NO:5,6,7,8 Or use SEQ ID NO: 5 and 6) in combination to observe the tumor growth rate.

The results showed that when HeLa wild-type cells formed tumors in nude mice, continuous injection of RabJ antisense oligonucleotides in the tumors could significantly inhibit the growth rate of HeLa tumors, suggesting that RabJ antisense oligonucleotides (SEQ ID NO: 5 -8) It has a therapeutic effect on tumors in vivo, and the test results of the AON-RabJ1 antisense oligonucleotide are shown in Figure 5 .

In addition, the combined use of AON-RabJ1 and AON-RabJ2 (each 20 μg/20 μl of 0.9% NaCl/mouse) resulted in smaller tumor volumes than the single use of AON-RabJ1 or AON-RabJ2. This indicated that combined use of RabJ antisense oligonucleotides could obtain better inhibitory effect.

Example 5: Intratumoral injection of RabJ antisense oligonucleotides promotes differentiation of HeLa cells in vivo

1×10 ⁶ HeLa cells were subcutaneously injected into the right lower flank of Balb/C nude mice, and 4 weeks later, RabJ antisense oligonucleotides (20 μg/20 μl 0.9% NaCl/mouse) were injected into the tumor, 4 One week later, tumor samples were taken for HE staining.

The results showed that the HeLa cells treated with RabJ antisense oligonucleotides differentiated to form cancer nests composed of squamous epithelial cells, and the insertion of fibrous tissue-like connective tissue, the generation of vacuolar cells and the occurrence of extensive cell death phenomenon, suggesting the mature differentiation of HeLa tumor tissue (see Figure 6). This indicates that RabJ antisense oligonucleotides have a differentiation-promoting effect on tumors in vivo, thereby producing tumor therapeutic effects.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> The Second Military Medical University of the Chinese People's Liberation Army

<120> antisense oligonucleotide sequence for inhibiting human RabJ gene expression and application thereof

<130>057872

<160>20

<170>PatentIn version 3.1

<210>1

<211>20

<212>DNA

<213> oligonucleotide

<400>1

ccaacatgcc gaagcggaag 20

<210>2

<211>20

<212>DNA

<213> oligonucleotide

<400>2

ccttgatgcg tggctggcag 20

<210>3

<211>20

<212>DNA

<213> oligonucleotide

<400>3

cagttgggac atgctggggag 20

<210>4

<211>20

<212>DNA

<213> oligonucleotide

<400>4

atgctcggac agccctcctg 20

<210>5

<211>20

<212>DNA

<213> oligonucleotide

<400>5

cttccgcttc ggcatgttgg 20

<210>6

<211>20

<212>DNA

<213> oligonucleotide

<400>6

ctgccagcca cgcatcaagg 20

<210>7

<211>20

<212>DNA

<213> oligonucleotide

<400>7

ctcccagcat gtcccaactg 20

<210>8

<211>20

<212>DNA

<213> oligonucleotide

<400>8

caggagggct gtccgagcat 20

<210>9

<211>20

<212>DNA

<213> oligonucleotide

<400>9

ctagcgcttc cgcatgatgg 20

<210>10

<211>20

<212>DNA

<213> oligonucleotide

<400>10

ctggcagcca ggcatctacg 20

<210>11

<211>20

<212>DNA

<213> oligonucleotide

<400>11

ctcgcagcat gtcgcatgtg 20

<210>12

<211>20

<212>DNA

<213> oligonucleotide

<400>12

cacgacgcct gtgcgagcat 20

<210>13

<211>23

<212>DNA

<213> oligonucleotide

<400>13

ggagtcaaac ctggggcctc aag 23

<210>14

<211>22

<212>DNA

<213> oligonucleotide

<400>14

caggagggct gtccgagcat tc 22

<210>15

<211>22

<212>DNA

<213> oligonucleotide

<400>15

gtagcacctg gcagtgaaga tg 22

<210>16

<211>25

<212>DNA

<213> oligonucleotide

<400>16

tcacccacac tgtgcccatc tacga 25

<210>17

<211>25

<212>DNA

<213> oligonucleotide

<400>17

cagcggaacc gctcattgcc aatgg 25

<210>18

<211>26

<212>DNA

<213> oligonucleotide

<400>18

atgccctccc ccatgccatc ctgcgt 26

<210>19

<211>1787

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(25)..(843)

<223>

<400>19

caagcttatg catgcggccg ctga atg gag gcc aac atg ccg aag cgg aag 51

Met Glu Ala Asn Met Pro Lys Arg Lys

1 5

gag ccc ggc agg tct ctc cgc atc aaa gtc atc tcc atg ggc aac gcc 99

Glu Pro Gly Arg Ser Leu Arg Ile Lys Val Ile Ser Met Gly Asn Ala

10 15 20 25

gaa gtg ggg aaa agc tgt att ata aag cga tac tgt gag aaa aga ttc 147

Glu Val Gly Lys Ser Cys Ile Ile Lys Arg Tyr Cys Glu Lys Arg Phe

30 35 40

gtg tct aaa tac ctg gca aca att gga att gac tat gga gtc aca aag 195

Val Ser Lys Tyr Leu Ala Thr Ile Gly Ile Asp Tyr Gly Val Thr Lys

45 50 55

gta cac gtc aga gac aga gaa atc aaa gtt aac atc ttt gat atg gct 243

Val His Val Arg Asp Arg Glu Ile Lys Val Asn Ile Phe Asp Met Ala

60 65 70

gga cat ccc ttc ttc tat gag gtt cga aat gag ttt tac aag gac aca 291

Gly His Pro Phe Phe Tyr Glu Val Arg Asn Glu Phe Tyr Lys Asp Thr

75 80 85

cag ggt gtg ata ctg gtc tat gat gtt ggg cag aaa gac tcc ttt gac 339

Gln Gly Val Ile Leu Val Tyr Asp Val Gly Gln Lys Asp Ser Phe Asp

90 95 100 105

gcc ctt gat gcg tgg ctg gca gaa atg aag caa gag ctt gga cct cat 387

Ala Leu Asp Ala Trp Leu Ala Glu Met Lys Gln Glu Leu Gly Pro His

110 115 120

gga aac atg gaa aat att ata ttt gta gtt tgt gcc aac aag att gat 435

Gly Asn Met Glu Asn Ile Ile Phe Val Val Cys Ala Asn Lys Ile Asp

125 130 135

tgt acc aaa cat cgc tgt gta gat gaa agt gaa gga cgt ctt tgg gct 483

Cys Thr Lys His Arg Cys Val Asp Glu Ser Glu Gly Arg Leu Trp Ala

140 145 150

gaa agc aaa ggg ttc ctg tac ttt gaa act tca gca caa act gga gaa 531

Glu Ser Lys Gly Phe Leu Tyr Phe Glu Thr Ser Ala Gln Thr Gly Glu

155 160 165

ggc att aat gag atg ttc cag acc ttt tat ata tcc ata gtt gat tta 579

Gly Ile Asn Glu Met Phe Gln Thr Phe Tyr Ile Ser Ile Val Asp Leu

170 175 180 185

tgt gaa aat ggc ggg aaa cgc cct acc acc aat agc agt gct agt ttc 627

Cys Glu Asn Gly Gly Lys Arg Pro Thr Thr Asn Ser Ser Ala Ser Phe

190 195 200

acc aaa gaa caa gca gat gcc att cgc aga att cga aat agt aaa gac 675

Thr Lys Glu Gln Ala Asp Ala Ile Arg Arg Ile Arg Asn Ser Lys Asp

205 210 215

agt tgg gac atg ctg gga gtc aaa cct ggg gcc tca agg gat gaa gtc 723

Ser Trp Asp Met Leu Gly Val Lys Pro Gly Ala Ser Arg Asp Glu Val

220 225 230

aat aaa gcg tat cgg aaa ctt gct gtg ctt ctt cac cct gac aaa tgt 771

Asn Lys Ala Tyr Arg Lys Leu Ala Val Leu Leu His Pro Asp Lys Cys

235 240 245

gta gca cct ggc agt gaa gat gcc ttc aaa gca gtt gtg aat gct cgg 819

Val Ala Pro Gly Ser Glu Asp Ala Phe Lys Ala Val Val Asn Ala Arg

250 255 260 265

aca gcc ctc ctg aaa aac atc aag tagaaagtac agaaaaaagc cacatgtggg 873

Thr Ala Leu Leu Lys Asn Ile Lys

270

actcaaatgc aaacagactt tccctagagg tgaaataacc aacgtggagt tttccttccc 933

agaatctcac tgctcttttc attcatgtgt tgtcatttgt atatcagtaa ttcaggtacc 993

catttcatag acatttact gagaaatgac ctgcatttgt atgaagtgaa ctgagcgtca 1053

caccctgtac ttcatttcat atttctagat aattctgaat ttttttctca ttcgtcagct 1113

ctgtaattat agtatcactt agacatttca cttggggaaa tccacaaggt tcctggaggg 1173

agggaagaga ggacaagagg accctttcac tttttctttt ttacggaatt catcatcaga 1233

gaagaaaata acaaaaatgg aagcaaacaa catcagaacc cctgtaagtt tggtgtgacc 1293

ttacagacaa gttgctgctt ttacaatgag ttccttaggt ggtattttaa cccatcgatc 1353

tataatgatg actcttggca gccctttggg agtttgtaaa atgaggtgat acagttctga 1413

attgagcatt cctttatgat attcactctg ttcctcttct gcagccacca gtgggagaga 1473

caagccagtc ctaagagaaa aggtggtggc agccacaaat tctaggtaca ctggctgctg 1533

cctatcctgt ccctggatct gaggcctttc ccttgccata gaaatggttg ctggtagcag 1593

tagagagcac tgtgcacctg ggaatgagga atcaggcccc aagacagaag tacttggagg 1653

agccagctgc agtagtatcc gcctgtagtc ccagctactc aggaggctga gacaggagga 1713

ttgcttaagc ccaggagctc aagtcccacc tgggcaacat agtaagatct tgtctcttaa 1773

aaaaaaaaaaaaaa 1787

<210>20

<211>273

<212>PRT

<213> Homo sapiens

<400>20

Met Glu Ala Asn Met Pro Lys Arg Lys Glu Pro Gly Arg Ser Leu Arg

1 5 10 15

Ile Lys Val Ile Ser Met Gly Asn Ala Glu Val Gly Lys Ser Cys Ile

20 25 30

Ile Lys Arg Tyr Cys Glu Lys Arg Phe Val Ser Lys Tyr Leu Ala Thr

35 40 45

Ile Gly Ile Asp Tyr Gly Val Thr Lys Val His Val Arg Asp Arg Glu

50 55 60

Ile Lys Val Asn Ile Phe Asp Met Ala Gly His Pro Phe Phe Tyr Glu

65 70 75 80

Val Arg Asn Glu Phe Tyr Lys Asp Thr Gln Gly Val Ile Leu Val Tyr

85 90 95

Asp Val Gly Gln Lys Asp Ser Phe Asp Ala Leu Asp Ala Trp Leu Ala

100 105 110

Glu Met Lys Gln Glu Leu Gly Pro His Gly Asn Met Glu Asn Ile Ile

115 120 125

Phe Val Val Cys Ala Asn Lys Ile Asp Cys Thr Lys His Arg Cys Val

130 135 140

Asp Glu Ser Glu Gly Arg Leu Trp Ala Glu Ser Lys Gly Phe Leu Tyr

145 150 155 160

Phe Glu Thr Ser Ala Gln Thr Gly Glu Gly Ile Asn Glu Met Phe Gln

165 170 175

Thr Phe Tyr Ile Ser Ile Val Asp Leu Cys Glu Asn Gly Gly Lys Arg

180 185 190

Pro Thr Thr Asn Ser Ser Ala Ser Phe Thr Lys Glu Gln Ala Asp Ala

195 200 205

Ile Arg Arg Ile Arg Asn Ser Lys Asp Ser Trp Asp Met Leu Gly Val

210 215 220

Lys Pro Gly Ala Ser Arg Asp Glu Val Asn Lys Ala Tyr Arg Lys Leu

225 230 235 240

Ala Val Leu Leu His Pro Asp Lys Cys Val Ala Pro Gly Ser Glu Asp

245 250 255

Ala Phe Lys Ala Val Val Asn Ala Arg Thr Ala Leu Leu Lys Asn Ile

260 265 270

Lys

Claims (7)

1. an antisense oligonucleotide is characterized in that, described antisense oligonucleotide is selected from down group:

AON-RabJ1：5’-CTTCCGCTTCGGCATGTTGG-3’；

AON-RabJ2：5’-CTGCCAGCCACGCATCAAGG-3’；

AON-RabJ3:5 '-CTCCCAGCATGTCCCAACTG-3 '; With

AON-RabJ4：5’-CAGGAGGGCTGTCCGAGCAT-3’。

2. antisense oligonucleotide as claimed in claim 1 is characterized in that, has the nucleotide through modifying in the described antisense oligonucleotide, and described modification mode is selected from down group: thio-modification, the modification that methylates, phosphoric acid thio-modification or its combination.

3. antisense oligonucleotide as claimed in claim 2 is characterized in that, it is characterized in that, described nucleotide through modification is the nucleotide of full chain warp phosphoric acid thio-modification.

4. a compositions is characterized in that, it contains each described antisense oligonucleotide and acceptable carrier, diluent or excipient among the 0.001-99.99wt% claim 1-3.

5. compositions as claimed in claim 4 is characterized in that described compositions is a pharmaceutical composition, and described carrier, diluent or excipient are pharmaceutically acceptable carrier, diluent or excipient.

6. the purposes of the described antisense oligonucleotide of claim 1 is characterized in that, is used to prepare the pharmaceutical composition that suppresses growth of tumour cell compositions or treatment tumor.

7. purposes as claimed in claim 6 is characterized in that, described tumor cell or tumor are expressed RabJ albumen, and are selected from down group: cervical cancer, breast carcinoma, colon cancer.

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