CN101328475A - Nucleic acid molecule NRN1SR1 and its application in the preparation of anticancer drugs - Google Patents

Abstract

The invention belongs to the biomedical field and relates to NRN1SR1 and an application of the NRN1SR1 in the preparation of an antitumor drug. The tumor threatens the health of people. The invention provides the nucleotide molecule NRN1SR1 used for preparing the antitumor drug, and the sequence comprises 5'-GGCAGCUUAUUCGAACUCU-3' or 5'- GGCAGCTTATTCGAACTCT-3'. Compared with nude mice without the NRN1SR1 in the same culture condition, the nude mice containing the NRN1SR1 has a tumor body prevented from growing evidently; moreover, the phenomenon is more evident as time goes on. The product provides a novel way and method for treating and relieving the tumor.

Description

Nucleic acid molecule NRN1SR1 and its application in the preparation of anticancer drugs

technical field

The invention belongs to the field of biomedicine and relates to a nucleotide molecule and its application in the preparation of antitumor drugs.

Background technique

Tumor disease has now risen to be the No. 2 "killer" in the world, and its death toll is second only to cardiovascular disease. Over the past few years, the foreign medical community has gained a new understanding of the pathogenesis of tumor diseases on the basis of cells. Based on the further understanding of the pathogenesis of tumors, people use various approaches to research and develop drugs that can specifically and effectively kill tumor cells without toxicity to normal cells. At present, chemotherapy and radiotherapy are still the first choice for the treatment of cancer. Although the two have achieved considerable curative effect on the treatment of tumors, they have relatively large toxic and side effects due to the lack of specificity to tumor cells, and some tumor cells have a certain effect on chemotherapy. And radiotherapy treatment insensitivity, thus limiting their clinical application to a large extent. In recent years, in order to develop drugs that can specifically kill cancer cells and have no toxic and negative effects on normal cells, people have paid great attention to and invested heavily in research on the pathogenesis of cancer from the cellular and molecular levels.

Contents of the invention

The object of the present invention is to provide a nucleotide molecule for preparing antitumor drugs.

Another object of the present invention is to provide applications of the above-mentioned nucleotide molecules.

The present invention provides a nucleotide molecule for the preparation of antitumor drugs, which has the activity of inhibiting tumor growth and its sequence includes 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCU-3', which is referred to in the present invention as Called NRN1SR1.

Wherein, A, U, G and C are adenine nucleotides, uracil nucleotides, guanine nucleotides and cytosine nucleotides, respectively.

In the present invention, the term "nucleotide molecule NRN1SR1" refers to a nucleic acid sequence that has the activity of inhibiting NRN1 protein and contains a high degree of homology to the 5'-GGCAGCUUAUUCGAACUCU-3' sequence. The term also includes nucleotide sequences that are at least 70%, preferably at least 80%, more preferably at least 90% homologous to 5'-GGCAGCUUAUUCGAACUCU-3'.

The term also includes nucleotide sequence variants of 5'-GGCAGCUUAUUCGAACUCU-3'. These variations include (but are not limited to): deletions, insertions and/or substitutions of several (usually 1-15, preferably 1-10, more preferably 1-5) nucleotides, and Several (usually within 10, preferably within 5) nucleotides are added at the 5' and/or 3' end. For example, the sequence formed by adding some dT (deoxythymidine) after 5'-GGCAGCUUAUUCGAACUCU-3' (3' end).

The sequence of the nucleotide molecule in the present invention may include 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCUdTdT-3'.

The sequence of the nucleotide molecules in the present invention may include 5'-GGCAGCUUAUUCGAACUCU-3'.

The sequence of the nucleotide molecule in the present invention may include 5'-GGCAGCUUAUUCGAACUCUdTdT-3'.

The sequence of the nucleotide molecule in the present invention may include 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCUdTdT-3'.

The sequence of the nucleotide molecule in the present invention may be 5'-GGCAGCUUAUUCGAACUCUdTdT-3'.

The present invention also provides a vector, which contains a nucleotide molecule whose sequence includes 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCUdTdT-3'.

The above vector of the present invention may include 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCU-3' in its sequence.

In the present invention, various vectors known in the art, such as commercially available vectors, can be used. For example, a commercially available vector is selected, and then 5'-GGCAGCUUAUUCGAACUCU-3' containing the NRN1SR1 nucleotide sequence of the present invention is operably linked to the expression control sequence to form a recombinant vector. For example, lentivirus, adenovirus or Forest Encephalitis virus expression system (Semliki ForestVirus) can be used. Lentivirus belongs to the subgenus of retrovirus, represented by human immunodeficiency virus (human immunodefficiency virus, HIV). Lentivirus not only has the ability to infect and divide target cells and integrate their genomes, especially to infect neuronal cells , Macrophages, liver cells, cardiomyocytes and stem cells, etc., a variety of non-dividing cell capabilities. Therefore, lentiviral vectors have been used as effective tools for gene transfer and have been widely used in the study of gene function, especially gene therapy.

As used herein, "operably linked to" refers to a situation where some part of a linear RNA sequence is capable of affecting the activity of other parts of the same linear RNA sequence, for example, if a signal peptide RNA is expressed as a precursor and participates in the expression of a polypeptide Secretion, then the signal peptide (secretion leader sequence) RNA is operably linked to the polypeptide RNA; if the promoter controls the transcription of the sequence, it is operably linked to the coding sequence; if the ribosome binding site is placed so that When its translational position is present, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for nucleic acid molecules of the invention it means adjacent without interfering with function.

The present invention also provides a host cell, which contains a nucleotide molecule whose sequence includes 5'-GGCAGCTTATTCGAACTCT-3' or 5'-GGCAGCUUAUUCGAACUCU-3'.

In the present invention, the term "host cell" is mainly a eukaryotic cell. Commonly used are: CHO cells, COS-7, 293 cells, and so on.

On the other hand, the present invention also provides the preparation method of the above-mentioned nucleotide molecule NRN1SR1, that is, according to the sequence of NRN1SR1, each ribonucleic acid molecule is sequentially dehydrated and condensed.

The nucleotide molecule NRN1SR1 of the present invention can be prepared by various conventional preparation methods. The nucleotide molecule NRN1SR1 sequence of the present invention can usually be obtained by enzymatic hydrolysis or artificial synthesis.

The present invention also provides the application of the above-mentioned nucleotide molecule NRN1SR1 in the preparation of antitumor drugs.

Both in vivo and in vitro results have verified that the NRN1SR1 of the present invention can significantly reduce the expression of NRN1.

Experiments have proved that NRN1 (NCBI accession number NM_016588) can promote tumor growth, therefore, reducing the expression of NRN1 means inhibiting tumor growth.

Injecting cells containing NRN1SR1 into tumors of nude mice, compared with nude mice not injected with NRN1SR1 under the same culture conditions, the growth of tumors was significantly inhibited, and this phenomenon became more and more obvious over time. This shows that NRN1SR1 of the present invention can be used as a tumor suppressor.

Said tumor can be benign or malignant tumor in liver, lung or breast.

The nucleotide molecule NRN1SR1 and its analogues of the present invention can provide various effects when administered (administered) therapeutically. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the nucleotide molecule NRN1SR1 of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound 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 nucleotide molecule NRN1SR1 of the present invention can be prepared in the form of an injection, for example, by using normal saline or an aqueous solution containing glucose and other auxiliary agents through conventional methods. 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 10 mg/kg body weight per day. In addition, NRN1SR1 of the present invention can also be used with other therapeutic agents.

When the nucleotide molecule NRN1SR1 of the present invention is used as a drug, a therapeutically effective dose of the polypeptide can be administered to mammals, wherein the therapeutically effective dose is usually at least about 10 μg/kg body weight, and in most cases no more than About 8 mg/kg body weight, preferably the dose is about 10 microgram/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.

In the present invention, the antitumor drug is a pharmaceutical composition comprising an effective therapeutic amount of the nucleotide molecule NRN1SR1 and a pharmaceutical carrier or excipient. The pharmaceutical composition can be injection or tablet. Its therapeutically effective amount may range from 1 microgram/kg to 10 mg/kg body weight per day.

In the present invention, the medicine may be injection, powder or tablet.

When the nucleotide molecule NRN1SR1 of the present invention is injected into the tumor of nude mice, compared with nude mice not injected with NRN1SR1 under the same culture conditions, the growth of the tumor is obviously inhibited, and this phenomenon becomes more and more obvious as time goes by. The invention provides a new approach and means for the treatment and remission of tumors.

Detailed ways

Example 1 NRN1 promotes tumor growth experiment in nude mice

1) The cell line b6 stably transfected with NRN1 and the cell line stably transfected with an empty vector were expanded for culture

2) Digest the cells with trypsin and centrifuge at 1000rpm×5min

3) Wash twice with PBS and count with a cell counting plate

4) Dilute the cell suspension with PBS to 2x10 ⁶ cells/200 microliters

5) Inoculate subcutaneously in the armpit of nude mice (Animal Center, Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, BALB/c), and inject 200 μl of cell suspension once. The same nude mouse was injected with stable cell line V1 stably transfected with empty vector in the left armpit, and injected with cell line N1 stably expressing exogenous NRN1 in the right side.

6) About 2 weeks later, the inoculated nude mice grew cell tumors, and the tumors were taken out after being sacrificed through the cervical spine, and the length, diameter and weight of the tumors were measured.

The results showed that the tumor growth in the NRN1 group was significantly faster than that in the empty vector group. Two weeks after injection into nude mice, the tumor volume in the NRN1 group was larger than that in the control group, and there was a significant difference after three weeks. This suggests that overexpression of NRN1 can promote tumor growth.

Example 2 Endogenous screening of NRN1 inhibitors

According to the sequence of NRN1, the nucleotide molecule with the sequence of 5'-GGCAGCUUAUUCGAACUCUdTdT-3' was designed as a candidate inhibitor of NRN1. Called NRN1SR1.

1. Seed 293T cells in a 24-well plate with a seeding density of 2*10 ⁴ /well

2. 24h (hour), 72h, and 120h after seeding the plate, the NRN1SR1 fragment was repeatedly transfected, and dissolved in Opti-MEM (Opti-MEM I Reduced Serum Medium, Invitrogen Company, 31985-070) as a solvent to make the final concentration reach 40nM. The transfection reagent used lipofectamin2000 (11668-027, Invitrogene Company).

3. Digest the cells at 24h, 72h, and 120h after the first transfection, and collect cell samples at 72h and 120h (can you give an order of magnitude 5*10 ⁴ ). The expression level of NRN1 endogenous protein was detected by Western detection using Anti-Neuritin (FL-142) (Santa Cruz Company) for the antibody.

Conclusion: Compared with the siRNA-negative fragment NS (5'-UUCUCCGAACGUCACGUdTdT-3'), the expression level of NRN1 protein can be down-regulated by about 60% 72 hours after transfection of NRN1SR1. This shows that the NRN1SR1 of the present invention can obviously inhibit NRN1 and can be used as a tumor suppressor.

Example 3 The cellular level screening experiment of the reduction effect of NRN1 gene expression

Experimental steps:

1. 293T cells (purchased from the Chinese Academy of Sciences Cell Bank) in 96-well plates, the seeding density is 0.8*10 ⁴ /well

2. After 24 hours, the lentivirus (lentivirus, Jikai Company) carrying NRN1SR1 was transfected into the cells. The titer of Lentivirus was 10 ⁶ TU/ul, and the MOI was taken as 100, that is, 80ul of virus liquid was added to each well. The specific transduction method is as follows:

Replace each well of cells with 50ul of fresh medium (pH7.0), add an appropriate amount of virus solution, and add 50ul of medium to each well after 7-8 hours. Change the medium after 24h. Observe the GFP (green fluorescence) fluorescence at 48-72h to detect the transduction efficiency.

3. The cells were expanded and cultured, and the cells were sorted by GFP green fluorescence by flow cytometry, and the positive cells for virus transduction were screened out, and the positive rate was detected.

4.Western detection of the expression level of NRN1 endogenous protein in positive cells and negative cells.

in conclusion:

A. The GFP fluorescence was observed at 48 hours, and the positive rate was 46%, which indicated that the experimental method of virus transduction was correct.

B. Sorting transduced cells by flow cytometry, the transduction rate of the positive group: 90%. Transduction rate of negative group (cells containing negative fragment NS): 96% (data displayed by flow cytometry).

C. The expression level of NRN1 protein in positive cells by Western assay was 89% lower than that in negative cells.

Example 4 NRN1 Inhibitor Inhibits Tumor Growth Experiment in Nude Mice

1. Experimental materials

1. Nude mice: Shanghai Slack Experimental Animal Co., Ltd.

2. Lentivirus vector (Lentivirus vector): Shanghai Jikai Gene Technology Co., Ltd.

2. Experimental steps

1. Put the nude mice into the SPF grade animal room (specific pathogen-free laboratory animal room), and make them adapt to the culture environment for about a week.

2. Culture SMMC-7721 cells with DMEM (Dulbecco's Mediified Eagle Medium, invitrogene company, 12800-82)+10% FBS.

3. Digest SMMC-7721 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) with trypsin and centrifuge, remove the supernatant, resuspend with serum-free DMEM, remove the supernatant, add an appropriate amount of PBS, and make about 4× per ml Suspension of ¹⁰⁷ cells.

4. Inject 0.2 ml of PBS suspension containing approximately 8×10 ⁶ units of cells into the underarm of 4-6 week-old nude mice.

5. When the diameter of the tumor body reaches 3-5mm (millimeters), the same 2 mice are classified into the experimental group and the control group according to the tumor volume size, and each mouse in the 2 groups is given an ear number, and the record is recorded. The long and short diameters and body weight of each mouse tumor at that time.

6. Directly inject 0.1 ml (milliliter) of virus and virus empty vector into the tumors of mice in the two groups respectively, and take the injection of virus empty vector as a control. Injections were repeated every 4 days for a total of three injections.

7. From the day of virus injection for the first time, measure and record the major and minor diameters and body weight of the tumor body of each mouse every 3 days, the volume v=ab of the tumor body ^2/2 (a major diameter length, b short diameter length). The tumor was removed about 4 weeks later.

The results showed that, compared with the control group, the tumor growth of the nude mice injected with the virus (containing NRN1SR1) was significantly slowed down, and the tumor body of the NRN1SR1 group was about 40% of the tumor body of the control group after 25 days of virus injection. It shows that NRN1SR1 of the present invention can obviously inhibit tumor growth.

SEQUENCE LISTING

<110> Fudan University

<120>Nucleic acid molecule NRN1SR1 and its application in the preparation of anticancer drugs

<130>41

<140>200810037442.8

<141>2008-02-05

<160>3

<170>PatentIn version 3.1

<210>1

<211>19

<212>DNA

<213>Artificial

<400>1

ggcagcttat tcgaactct 19

<210>2

<211>19

<212> RNA

<213>Artificial

<400>2

ggcagcuuau ucgaacucu 19

<210>3

<211>17

<212> RNA

<213>Artificial

<400>3

uucuccgaac gucacgu 17

Claims (8)

1. A nucleotide molecule, characterized in that it has the activity of inhibiting tumor growth and its sequence comprises 5'-GGCAGCUUAUUCGAACUCU-3' or 5'-GGCAGCTTATTCGAACTCT-3'. 2. A nucleotide molecule as claimed in claim 1, wherein its sequence comprises 5'-GGCAGCUUAUUCGAACUCUdTdT-3'. 3. A nucleotide molecule as claimed in claim 1, characterized in that its sequence comprises 5'-GGCAGCUUAUUCGAACUCU-3' or 5'-GGCAGCTTATTCGAACTCT-3'. 4. A nucleotide molecule as claimed in claim 1, characterized in that its sequence is 5'-GGCAGCUUAUUCGAACUCUdTdT-3'. 5. a kind of preparation method as any nucleotide molecule in claim 1-4, it is characterized in that, by the sequence of any nucleotide molecule in claim 1-4, each ribonucleic acid group is successively Dehydration and shrinkage. 6. The use of any one of the nucleotide molecules as claimed in claims 1-4 in the preparation of antitumor drugs. 7. The application according to claim 6, characterized in that the antitumor drug is composed of any one of the nucleotide molecules in claims 1-4 containing an effective therapeutic amount and a pharmaceutical carrier or excipient pharmaceutical composition. 8. A kind of application as claimed in claim 6, is characterized in that, described medicine is injection, powder or tablet.