CN101328475B - Nucleic acid molecule NRN1SR11 and use thereof in anti-cancer medicine preparation - 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 NRN 1 SR 11 and the application in the preparation cancer therapy drug thereof

Technical field

The invention belongs to biomedicine field, relate to a kind of nucleic acid molecule and the application in the preparation antitumor drug thereof.

Background technology

Tumor disease has now risen to No. the 2nd, the world " killer ", and its death toll is only second to cardiovascular diseases.In the past few years, the foreign medical science bound pair has had new understanding again in the pathogeny of tumor disease on cell base.Based on the further understanding to tumor invasion mechanism, people utilize various approach to develop and develop can be special, effectively killing tumor cell and to the avirulent medicine of normal cell.At present, for treatment for cancer is first-selection with chemotherapy and radiotherapy still, though both have obtained suitable curative effect to tumor treatment, but since lack to the specificity of tumour cell thus have bigger toxic side effect and some tumour cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.In recent years, can to kill and wound cancer cells specifically and normal cell is not had the medicine of toxic side effect in order to develop, from cell, paid much attention to and huge investment by the research on the molecular level to the pathogenesis of cancer for people.

Summary of the invention

The purpose of this invention is to provide a kind of nucleic acid molecule that is used to prepare antitumor drug.

Another object of the present invention provides the application of above-mentioned nucleic acid molecule.

The invention provides a kind of nucleic acid molecule that is used to prepare antitumor drug, it has active and its sequence that suppresses tumor growth and comprises 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCU-3 ', is called as NRN1SR1 in the present invention.

Wherein, A, U, G and C are respectively adenine nucleotide, uridylate, guanylic acid and cytidylic acid(CMP).

In the present invention, term " nucleic acid molecule NRN1SR1 " refers to have inhibition NRN1 protein-active and contains and 5 '-GGCAGCUUAUUCGAACUCU-3 ' sequence height homologous nucleotide sequence.This term also comprises the homology at least 70% with 5 '-GGCAGCUUAUUCGAACUCU-3 ', preferably at least 80%, and at least 90% nucleotide sequence more preferably.

This term also comprises the nucleotide sequence variation form of 5 '-GGCAGCUUAUUCGAACUCU-3 '.These variant forms comprise (but being not limited to): several (are generally 1-15, preferably 1-10,1-5 more preferably) disappearance, insertion and/or the replacement of Nucleotide, and add several (being generally in 10, preferably is in 5) Nucleotide at 5 ' and/or 3 ' end.For example, (3 ' end) add the sequence that some dT (deoxythymidine) back forms in 5 '-GGCAGCUUAUUCGAACUCU-3 ' back.

Nucleic acid molecule in the present invention, its sequence can comprise 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

Nucleic acid molecule among the present invention, its sequence can comprise 5 '-GGCAGCUUAUUCGAACUCU-3 '.

Nucleic acid molecule among the present invention, its sequence can comprise 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

Nucleic acid molecule among the present invention, its sequence can comprise 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

Nucleic acid molecule among the present invention, its sequence can be 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

The present invention also provides a kind of carrier, and it contains the nucleic acid molecule that sequence comprises 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

Above-mentioned carrier of the present invention can comprise 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCU-3 ' in its sequence.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, will contain the 5 '-GGCAGCUUAUUCGAACUCU-3 ' of NRN1SR1 nucleotide sequence of the present invention then, operationally be connected in expression regulation sequence, can form recombinant vectors.For example, can adopt slow virus, adenovirus or fores encephalitis virus expression system (Semliki ForestVirus).Slow virus (Lentivirus) belongs to the retrovirus subgenus, with human immunodeficiency virus (human immunod efficiency virus, HIV) being representative. slow virus not only has the division of infection target cell and integrates in its genome, especially has the multiple Unseparated Cell ability that comprises neuronal cell, scavenger cell, liver cell, myocardial cell and stem cell etc. that infects, thereby, lentiviral vectors is widely used in the especially research of gene therapy of gene function as the effective tool of transgenosis.

As used herein, " operationally be connected in " and refer to such a case, be that some part of linear rna sequence ground can influence same linear rna sequence other are partly active, for example, if signal peptide RNA is as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) RNA operationally is connected in polypeptide RNA so; If transcribe to the promotor control sequence, it is operationally to be connected in encoding sequence so; If ribosome bind site is placed in the time of making its position, translation ground, it is can operate to be connected in encoding sequence so.Generally, " can operate and be connected in " mean mutually and close on, and then means adjacent and do not influence function for nucleic acid molecule of the present invention.

A kind of host cell also is provided among the present invention, and it contains the nucleic acid molecule that sequence comprises 5 '-GGCAGCTTATTCGAACTCT-3 ' or 5 '-GGCAGCUUAUUCGAACUCU-3 '.

In the present invention, term " host cell " mainly is an eukaryotic cell.Commonly used has: Chinese hamster ovary celI, COS-7,293 cells, or the like.

On the other hand, the present invention also provides the preparation method of above-mentioned nucleic acid molecule NRN1SR1, and the sequence of promptly pressing NRN1SR1 is with each ribonucleic acid molecule dehydrating condensation successively.

Nucleic acid molecule NRN1SR1 of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule NRN1SR1 sequence of the present invention can obtain with the method for enzymolysis process or synthetic usually.

The present invention also provides the above-mentioned application of nucleic acid molecule NRN1SR1 in the preparation antitumor drug.

Verify all that with in vitro results NRN1SR1 of the present invention can obviously cut down the expression of NRN1 in the body.

Experiment showed, that NRN1 (the NCBI number of landing NM_016588) can promote tumor growth, therefore, the expression of cutting down NRN1 just means the inhibition tumor growth.

To contain in the tumour of injection cell nude mice of NRN1SR1, and compare with the nude mice of not injecting NRN1SR1 under the same culture conditions, the growth of knurl body obviously is suppressed, and this phenomenon is As time goes on obvious day by day.This explanation, NRN1SR1 of the present invention can be used as tumor inhibitor.

Described tumour can be the optimum or malignant tumour at liver, lung or mammary gland position.

Nucleic acid molecule NRN1SR1 of the present invention and analogue thereof when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

With nucleic acid molecule NRN1SR1 of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains compound and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Nucleic acid molecule NRN1SR1 of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, NRN1SR1 of the present invention also can use with the other treatment agent.

When nucleic acid molecule NRN1SR1 of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Among the present invention, the pharmaceutical composition that described antitumor drug is made up of the nucleic acid molecule NRN1SR1 that contains effective therapeutic dose and carrier pharmaceutically or vehicle.Described pharmaceutical composition can be injection or tablet.Its effective therapeutic dose can for every day 1 microgram/kilogram to 10 mg/kg body weight.

Among the present invention, described medicine can be injection, pulvis or tablet.

Nucleic acid molecule NRN1SR1 of the present invention is injected in the tumour of nude mice, compares with the nude mice of not injecting NRN1SR1 under the same culture conditions, and the growth of knurl body obviously is suppressed, and this phenomenon is As time goes on obvious day by day.The present invention is for tumor treatment and a kind of new approach and the means of providing are provided.

Embodiment

The short nude mice tumor growth experiment of embodiment 1 NRN1

1) the cell strain enlarged culturing of the cell strain b6 of stable transfection NRN1 and stable transfection empty carrier

2) use trypsin digestion cell, centrifugal 1000rpm * 5min

3) PBS washes twice, counts with cell counting count board

4) with PBS diluting cells suspension to 2 * 10 ⁶Cell/200 microlitres

5) (medicine institute of Shanghai Chinese Academy of Sciences animal center, BALB/c) subcutaneous vaccination is carried out in the oxter, a shot 200 μ l cell suspensions nude mice.The stable cell line V1 of oxter, same nude mice left side injection stable transfection empty carrier, the cell strain N1 of the right injection stable expression of exogenous NRN1.

6) after about 2 weeks, the nude mice of inoculation grows glucagonoma, takes out the knurl body after cervical vertebra is put to death, and measures the knurl body line of apsides and weight.

The result shows that NRN1 group tumor growth is obviously faster than the empty carrier group, and after two weeks, NRN1 group knurl body volume occurs significant difference greater than control group after three weeks the injection nude mice.This showed that expression NRN1 can promote tumor growth.

Embodiment 2 endogenous screening NRN1 inhibitor

According to the sequence of NRN1, implementation sequence is that the nucleic acid molecule of 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 ' is as the NRN1 candidate inhibitor.Be called NRN1SR1.

1. plant the 293T cell in 24 orifice plates, plant plate density 2*10 ⁴/ hole

Behind kind of plate 24h (hour), 72h, 120h repeat transfection NRN1SR1 fragment, (Opti-MEM I Reduced Serum Medium, invitrogen company 31985-070) is dissolution with solvents, makes final concentration reach 40nM with Opti-MEM.Transfection reagent uses lipofectamin2000 (11668-027, invitrogene company).

3. 24h, 72h, 120h peptic cell after the transfection first time are collected 72h and 120h part and (can probably be given an order of magnitude 5*10 ⁴) cell sample.Western detects, antibody Anti-Neuritin (FL-142) (santa cruz company) NRN1 intrinsic protein changes of expression level.

Conclusion: relatively, 72h is that visible NRN1 protein expression level reduces about 60% after the NRN1SR1 transfection with the negative fragment NS of siRNA (5 '-UUCUCCGAACGUCACGUdTdT-3 ').This explanation, NRN1SR1 of the present invention can obviously suppress NRN1, can be used as tumor inhibitor.

The cell levels screening experiment of the consumption of 3 pairs of NRN1 genetic expressions of embodiment

Experimental procedure:

1. plant 293T cell (available from Chinese Academy of Sciences's cell bank) in 96 orifice plates, plant plate density 0.8*10 ⁴/ hole

2.24h after will carry NRN1SR1 lentivirus (slow virus, Ji Kai company) transduction go into cell.The Lentivirus titre is 10 ⁶TU/ul, getting MOI is 100, promptly every hole adds 80ul virus liquid.Concrete transduction method is as follows:

Change 50ul fresh culture (PH7.0) for every porocyte, add an amount of viral liquid, the 50ul substratum is added in every hole behind the 7-8h.Change liquid behind the 24h.48-72h observes GFP (green fluorescence) fluorescence, detects transduction efficiency.

3. with the cell enlarged culturing, utilize the flow cytometry pair cell to carry out the sorting of GFP green fluorescence, filter out virus transduction positive cell, and detect its positive rate.

4.Western detect the NRN1 intrinsic protein changes of expression level of positive cell and negative cells.

Conclusion:

A.48h observe GFP fluorescence, positive rate is 46%, illustrates that virus transduction experimental technique is correct.

B. flow cytometry sorting transducer cell, the transduction rate of positive group: 90%.(cell that contains negative fragment NS) transduction rate of negative group: 96% (FCM results show data).

C.Western detects positive cell NRN1 protein expression level and reduces by 89% than negative cells.

Embodiment 4 NRN1 inhibitor suppress the experiment of nude mice tumor growth

One, experiment material

1. nude mice: Shanghai Slac Experimental Animal Co., Ltd.

2.Lentivirus vector (lentiviral vectors): the Shanghai triumphant gene engineering of Ji company limited

Two, experimental procedure

1. nude mice is put into SPF level Animal House (no-special pathogen level experimental animal room), make it adapt to an about week of culture environment.

With DMEM (Dulbecco ' s Medified Eagle Medium, invitrogene company, 12800-82)+10%FBS cultivates the SMMC-7721 cell.

3. SMMC-7721 cell (available from Chinese Academy of Sciences's cell bank) with centrifugal after the trysinization, is removed supernatant, resuspended with the DMEM of serum-free, remove supernatant then, add an amount of PBS, make every milliliter about 4 * 10 ⁷The suspension of individual cell.

4. the oxter injection 0.2ml to the nude mice in age in 4-6 week contains 8 * 10 approximately ⁶The PBS suspension of the cell of individual unit.

5. when treating that knurl body diameter reaches 3-5mm (millimeter), be classified as experimental group and control group respectively, and all cut the ear numbering for this every mouse of 2 groups, the major diameter minor axis and the body weight of every mouse tumor body of record this moment by identical 2 of the big young pathbreaker of knurl volume.

6. virus and the viral empty carrier of difference direct injection 0.1ml (milliliter) in the knurl body of 2 groups of mouse are contrast with the injecting virus empty carrier.Duplicate injection in per 4 days is once injected three times altogether.

7. since the day of the injecting virus first time, measured and write down the major diameter minor axis and the body weight of the knurl body of every mouse, the volume v=ab of knurl body in per 3 days ²/ 2 (a major diameter length, b minor axis length).Get knurl after about 4 week.

The result shows that compare with control group, the nude mice knurl bulk-growth of injecting virus (containing NRN1SR1) obviously slows down, and is about 40% of control group knurl body at NRN1SR1 group knurl body after behind the virus injection 25 days.Illustrate that NRN1SR1 of the present invention can obviously suppress tumor growth.SEQUENCE?LISTING

＜110〉Fudan University

＜120〉nucleic acid molecule NRN 1 SR 11 and the application in the preparation cancer therapy drug thereof

<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 (6)

1. a nucleic acid molecule is characterized in that, it has active and its sequence that suppresses tumor growth is 5 '-GGCAGCUUAUUCGAACUCU-3 ' or 5 '-GGCAGCTTATTCGAACTCT-3 '.

2. a nucleic acid molecule is characterized in that, its sequence is 5 '-GGCAGCUUAUUCGAACUCUdTdT-3 '.

3. the preparation method of claim 1 or 2 described nucleic acid molecules is characterized in that, by the sequence of claim 1 or 2 described nucleic acid molecules, obtains with artificial synthetic method.

4. as the application of nucleic acid molecule as described in claim 1 or 2 in the preparation antitumor drug.

5. application as claimed in claim 4 is characterized in that the pharmaceutical composition that described antitumor drug is made up of the claim 1 that contains effective therapeutic dose or 2 described nucleic acid molecules and carrier pharmaceutically or vehicle.

6. an application as claimed in claim 5 is characterized in that, described medicine is injection, pulvis or tablet.