CN101016324A - Nucleic acid molecule RTN4BSR62 and application in preparation of anti-cancer drugs - Google Patents

Abstract

The invention discloses a making method of RTN4BSR32 and application to prepare tumour-proof drug, whose sequence contains 5'-AATGTCTCTCCAGTACAGG-3' or 5'-AAUGUCUCUCCAGUACAGG-3'. The molecular can inhibit tumour from growing obviously, which provides a new path and method to treat and release tumour.

Description

Nucleic acid molecule RTN4BSR62 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, its sequence comprises 5 '-ATAATGATCTATCTGTGCC-3 ' or 5 '-GGCACAGAUAGAUCAUUAU-3 ', is called as RTN4BSR62 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 RTN4BSR62 " refers to have inhibition RTN4B protein-active and contains and 5 '-GGCACAGAUAGAUCAUUAU-3 ' sequence height homologous nucleotide sequence.This term also comprises the homology at least 70% with 5 '-GGCACAGAUAGAUCAUUAU-3 ', preferably at least 80%, and at least 90% nucleotide sequence more preferably.

This term also comprises the nucleotide sequence variation form of 5 '-GGCACAGAUAGAUCAUUAU-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 '-GGCACAGAUAGAUCAUUAU-3 ' back.

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

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

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

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

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

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

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, selecting commercially available carrier for use, the 5 '-GGCACAGAUAGAUCAUUAU-3 ' that will contain RTN4BSR62 nucleotide sequence of the present invention then operationally is connected in expression regulation sequence, can form recombinant vectors., for example, can adopt slow virus, adenovirus or fores encephalitis virus expression system (Semliki Forest Virus).Slow virus (Lentivirus) belongs to the retrovirus subgenus, and (human immunod efficiency virus HIV) is representative with human immunodeficiency virus.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 being connected in " refers to a kind of like this situation, and promptly some part of linear rna sequence can influence the activity of same other parts of linear rna sequence.For example, if signal peptide RNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) RNA operationally is connected in polypeptide RNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, 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 '-ATAATGATCTATCTGTGCC-3 ' or 5 '-GGCACAGAUAGAUCAUUAU-3 '.

In the present invention, term " host cell " is an eukaryotic cell, as Chinese hamster ovary celI, COS-7,293 cells etc.

On the other hand, the present invention also provides the preparation method of above-mentioned nucleic acid molecule RTN4BSR62, and the sequence of promptly pressing RTN4BSR62 is with each ribonucleic acid molecule dehydrating condensation successively.

Nucleic acid molecule RTN4BSR62 of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule RTN4BSR62 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 RTN4BSR62 in the preparation antitumor drug.

Verify all that with in vitro results RTN4BSR62 of the present invention can obviously cut down the expression of RTN4B in the body.

Experiment showed, that RTN4B (the NCBI number of landing AF148538) can promote tumor growth, therefore, the expression of cutting down RTN4B just means the inhibition tumor growth.

The injection cell that will contain RTN4BSR62 is gone in the tumour of nude mice, compares with the nude mice of not injecting RTN4BSR62 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.

Nucleic acid molecule RTN4BSR62 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 RTN4BSR62 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 RTN4BSR62 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, BRSK2 of the present invention also can use with the other treatment agent.

When nucleic acid molecule RTN4BSR62 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 RTN4BSR62 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 RTN4BSR62 of the present invention is injected in the tumour of nude mice, compares with the nude mice of not injecting RTN4BSR62 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.

Description of drawings

After Fig. 1 is injection RTN4B, the long knurl situation map in oxter, nude mice left side.The negative contrast of pcDNA3.1Myc-His empty carrier cell strain inoculation nude mice.The inoculating cell number is 3 * 10 ⁶Individual.After inoculating about 2 weeks, the nude mice of inoculation grows glucagonoma.

After Fig. 2 is injection RTN4B, the long knurl situation map in oxter, nude mice left side.The negative contrast of pcDNA3.1Myc-His empty carrier cell strain inoculation nude mice.The inoculating cell number is 3 * 10 ⁶Individual.After inoculating about 2 weeks, the nude mice of inoculation grows glucagonoma.The same Fig. 1 of nude mice order.

Fig. 3 is Fig. 1 and Fig. 2 and the knurl body figure as a result after peeling off.Wherein, a6 is a left oxter knurl situation; B6 is left armpit knurl body situation.The knurl body that forms behind stable transfection RTN4B and the pcDNA3.1Myc-His empty carrier cell strain inoculation nude mice.Last row is the knurl body of left side a6 cell strain, and following row is the knurl body of same nude mice right side b6 cell strain.The knurl body puts in order and puts in order consistent with Fig. 1 and Fig. 2 nude mice.

Fig. 4 is endogenous screening RTN4BSR62 fragment protein electrophoresis figure.Wherein, 6 is RTN4BSR62, the negative contrast of NS.

Fig. 5 is the cell levels proof diagram of RTN4BSR62 to the consumption of RTN4B genetic expression.Wherein, the negative contrast of NS.

Fig. 6 for SMMC-7721 inoculation nude mice tumorigenesis after the Lentivims of injection RTN4BSR62.As seen RTN4BSR62 can obviously suppress tumor growth.

Embodiment

Embodiment 1RTN4B makes nude mice growth tumor experiment

1) the cell strain enlarged culturing of the cell strain SMMC-7721 of stable transfection RTN4B 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 of oxter, same nude mice left side injection stable transfection empty carrier, the cell strain of the right injection stable expression of exogenous RTN4B.

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 as Figure 1-3, obviously faster than the right in left side, this showed that expression RTN4B can promote tumor growth to the tumor growth on right side.

Embodiment 2 knurl body freezing microtome sections are analyzed

1) glucagonoma that grows of nude mice is cut into small pieces and is immersed in the embedding liquid, cuts into slices after quick freezing in the liquid nitrogen, and all slide glasss are handled through the anti-flake of poly-lysine

2) section is at (20 ℃) acetone internal fixing 20S of ice, then at 37 ℃ of baking sheets (but 12h).Slice, thin piece can be placed 1～2 year at-20 ℃

3) slice, thin piece is washed in PBS 2 times, 5min/time

4) sealing 15～30min (confining liquid: 5%BSA, 5%Goat serum)

5) PBS washes 3 times, 5min/time

6) 37 ℃ of 1.5h or 4 ℃ spend the night (background is few) among anti-(CD31) 0.5%BSA, volume 20～30 μ l

7) PBS washes 3 times, 5min/time

8) 37 ℃ of 30min of two anti-0.5%BSA (the available 1%BSA of the words that background is high, 1%Goat serum)

9) PBS washes 3 times, 5min/time

10) drip the 80% glycerine mounting that heat is crossed

11) 20 times of fluorescent microscopes detect down and take pictures

12) numeration blood vessel number, the blood vessel in the glucagonoma of the statistics left and right sides is counted difference

The result shows that in the section of the hepatoma that inoculation b6 (cell strain of stably express RTN4B) generates, obviously increase is many for the blood vessel number in the hepatoma section that blood vessel number ratio inoculation a6 (cell strain of stable transfection empty carrier) generates.

Embodiment 3 endogenous screening RTN4B inhibitor

According to the sequence of RTN4B, implementation sequence is that the nucleic acid molecule of 5 '-GGCACAGAUAGAUCAUUAUdTdT-3 ' is as the RTN4B candidate inhibitor.Be called RTN4BSR62.

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 RTN4BSR62 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 cell 5*10 ⁴As sample.Western detects, antibody Nogo (N-18) SC-11027 (santa cruz company) RTN4B intrinsic protein changes of expression level.

Conclusion: as shown in Figure 4, relatively, 72h is that visible RTN4B protein expression level reduces about 50% after the RTN4BSR62 transfection with the negative fragment NS of siRNA (5 '-UUCUCCGAACGUCACGUdTdT-3 ').

The cell levels screening experiment of the consumption of 4 pairs of RTN4B genetic expressions of embodiment

Experimental procedure:

1. plant the 293T cell in 96 orifice plates, plant plate density 0.8*10 ⁴/ hole

2.24h after will carry RTN4BSR62 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 RTN4B intrinsic protein changes of expression level of positive cell and negative cells.

Conclusion: as shown in Figure 5,

A.48h observe GFP fluorescence, positive rate is 50%, illustrates that virus transduction experimental technique is correct.

B. flow cytometry sorting transducer cell, the transduction rate of positive group: 94%.(cell that contains negative fragment NS) transduction rate of negative group: 97% (FCM results show data).

C.Western detects positive cell RTN4B protein expression level and reduces by 90% than negative cells.

Embodiment 5RTN4B inhibitor suppresses 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.

The SMMC-7721 cell is centrifugal after with trysinization 3., remove 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 as shown in Figure 6, compare with control group, the nude mice knurl bulk-growth of injecting virus (containing RTN4BSR62) obviously slows down.

Claims (8)

1. a nucleic acid molecule is characterized in that, its sequence comprises 5 '-ATAATGATCTATCTGTGCC-3 ' or 5 '-GGCACAGAUAGAUCAUUAU-3 '.

2. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence comprises 5 '-GGCACAGAUAGAUCAUUAUdTdT-3 '.

3. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence is 5 '-ATAATGATCTATCTGTGCC-3 ' or 5 '-GGCACAGAUAGAUCAUUAU-3 '.

4. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence is 5 '-GGCACAGAUAGAUCAUUAUdTdT-3 '.

5. the preparation method as any nucleic acid molecule among the claim 1-4 is characterized in that, by the sequence of any nucleic acid molecule among the claim 1-4, with each Yeast Nucleic Acid group dehydrating condensation successively.

6. as the application of any nucleic acid molecule among the claim 1-4 in the preparation antitumor drug.

7. application as claimed in claim 6 is characterized in that the pharmaceutical composition that described antitumor drug is made up of any nucleic acid molecule among the claim 1-4 that contains effective therapeutic dose and carrier pharmaceutically or vehicle.

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

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