JP2002142778A - Suppression of expression of midkine using antisense oligonucleotide sequence and suppression of proliferation of cancer cell associated with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress the biosynthesis of proliferation molecular factor midkine, expression of which is highly enhanced in a cancer cell, and further to suppress the proliferation of the cancer cell. SOLUTION: The biosynthesis of midkine is suppressed using an antisense sequence corresponding to the mRNA of midkine to suppress the proliferation of a cancer cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an antisense nucleotide sequence that suppresses midkine expression, and to the use of the oligonucleotide sequence.

[0002]

2. Description of the Related Art Midkine (MK)
Is a growth / differentiation factor discovered as a product of a gene that is transiently expressed during the induction of differentiation of embryonic tumor cells (EC) by retinoic acid. is there. (Kado
Matsu, K .; et al. : Biochem. Bi
ophys. Res. Commun. , 151: 131.
2-1318; Tomomara, M .; et al. :
J. Biol. Chem. , 265: 10765-10.
770, 1990). MKs have been found from humans to Xenopus laevis, but protein conservation between species is high, and human and mouse MKs have 87% homology in amino acid sequence. MK has various biological activities. In other words, it is considered that they promote neurite outgrowth and survival of nerve cells, and are involved in the construction of the nervous system. It is also found in tissues at the time of injury, in many human cancers, and in senile plaques of Alzheimer's disease, and is also noted for its relationship to tissue repair, fibrinolytic activation, and pathology. In particular, the expression of MK is increased in many human cancers (Aridome, K. et al .:
Jap. J. Cancer Res. , 86: 655-
661, 1995), and cells become cancerous by introduction and expression of MK cDNA (Kadomatsu, K., e).
t al. : Brit. J. Cancer, 75:35
4-359, 1997), which is presumed to be deeply involved in the development and progression of human cancer. Moreover, in knockout mice lacking the MK gene, neointimal formation is significantly reduced in a balloon injury model, and thus MK is considered to be involved in the occurrence of vascular lesions (Horiba, Me.e).
t al. : J. Clin. Invest. , 105:
489-495, 2000). Therefore, it is thought that suppressing or inhibiting the action of MK can contribute to treatment of some diseases, particularly cancer.

In order to suppress the activity of MK, an anti-MK antibody may be considered. In fact, the anti-MK antibody partially inhibited the growth of cultured Wilms tumor cells (Muramatsu, H .;
et al. : Dev. Biol, 159: 392-40.
2, 1993). However, because antibodies cannot contact all MK molecules, efficient MK suppression requires M
It is necessary to suppress the biosynthesis of K itself. to date,
This task has not been achieved.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a substance which specifically inhibits the biosynthesis of MK protein, and to further suppress the growth of cancer cells using this substance.

[0005]

In order to inhibit the biosynthesis of a specific protein, it is conceivable to target a messenger RNA (mRNA) which directs the synthesis of the protein. That is, an antisense oligonucleotide complementary to the sequence of the mRNA (Gene Handbook for Research and Development of Gene Therapy, Section 5, Genetic Information Knockout Strategy by Antisense Method, Kazuhisa Yokoyama, p.584-595,19)
99 NTT, Inc., antisense RNA containing antisense nucleotide sequence (Gene Therapy Research and Research Handbook Section 5 Genetic Information Knockout Strategy by Antisense Method) Kazuhisa Yokoyama p.584-59
5, 1999 NTT Corporation) Ribozyme containing antisense nucleotide sequence (Gene Therapy Development Handbook, Section 6, Ribozyme Technology, p. 596-60)
4, 1999 Kazuko Kuwahara, Shiori Koseki, Kazumasa Tahira (NTS Corporation) are candidates. In each case, determining the specific antisense sequence is the most important task. For this purpose, it is necessary to predict the secondary structure of MK mRNA, select some portions forming a relatively loose structure, and search for the effectiveness of each antisense sequence.

That is, the present invention comprises (1) determination of an antisense sequence that suppresses MK biosynthesis, and (2) control of cancer cell proliferation using the antisense sequence.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the accompanying drawings. FIG. 1 is a graph showing a Western blot for examining MK expression suppression, and FIG. 2 is an antisense DN.
A is a graph showing the inhibitory effect of A on the growth of cancer cells, and FIG. 3 is a graph showing the inhibition of colony formation of cancer cells by antisense DNA.

[0008] 1. The molecular design of the antisense oligonucleotide will be described. Mouse MK mRNA (To
momura, M .; et al. : J. Biol. Ch
em. 265: 10765-10770, 1990) or human MK mRNA (Tstsui, J. et.
al. : Biochem. Biophys. Res.
Commun. 176: 792-797, 1991).
Has already been reported. The secondary structure of RNA predicts the secondary structure having the minimum energy with a computer program. Based on the predicted secondary structure, a loop portion that does not form a base pair is selected as a candidate antisense oligonucleotide sequence. An antisense oligo DNA molecule is synthesized based on the antisense sequence.
In the present invention, a phosphorothioate-type skeleton (commonly known as S-oligo) in which one of the oxygens of the phosphodiester bond is substituted with sulfur was selected. The phosphorothioate-type skeleton has the property of being hardly decomposed by nucleases, and thus has the advantage that not only a higher dose can be expected with a small amount of administration, but also its synthesis is relatively easy. However, the scope of the present invention is not limited to this. For example, if an effective antisense oligonucleotide sequence is found, it can be converted into a hammerhead ribozyme (Gene therapy development handbook, Section 6, Ribozyme technology, p. 596-60).
4, 1999 Kazuko Kuwahara, Shiori Koseki, Kazumasa Tahira, NTT Co., Ltd.) and antisense RNA expression vector (Gene Handbook for Research and Development of Gene Therapy Section 5 Genetic Information Knockout Strategy by Antisense Method Kazuhisa Yokoyama p.584- 595, 1999 NTT Corporation
S) to cleave or inactivate MK mRNA.

[0009] 2. The determination of the effect of inhibiting the synthesis of MK by the antisense oligonucleotide sequence will be described. A substance having an antisense oligonucleotide sequence was
Is administered to cells secreting and secreting, and MK secreted into the culture solution is quantified immunobiochemically. As an immunobiochemical quantification method of MK, enzyme immunoassay (Muramatsu, H .;
et al. : J. Biochem. , 119: 117
1-1175, 1996) or Western blotting after SDS polyacrylamide gel electrophoresis (M
uramatsu, H .; et al. : Dev. Bio
l. , 159; 392-402, 1993) and quantification with a densitometer is conceivable.

[0010] 3. The determination of the anti-proliferative effect of the antisense oligonucleotide sequence on cancer cells is described. A substance having an antisense oligonucleotide sequence is administered to cultured cancer cells that secrete MK, and the effect on the growth ability of the cancer cells is determined by first quantifying the number of cells. Furthermore, the ability to form colonies in soft agar which is strongly associated with tumorigenicity in individuals (Chen, JY et al.
l. : EMBO J .: , 14: 1187-1197.
1995).

[0011]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Example 1) Antisense S oligo DNA
Was created. In order to determine the sequence of the antisense DNA, first, the target gene, that is, mRN of mouse MK
A (GenBank Accession No. M3
4327: Tomomura M. et al. : J. B
iol. Chem. , 265: 10765-1077.
0, 1990) by computer analysis. The program is GENETYX-MAC. Ve
r. 10 (Software Development Co., Ltd.) was used. The outline of the secondary structure prediction of the nucleic acid by this program is as follows. In other words, all possible stacking regions are extracted from the nucleic acid sequence data, and from among the combinations,
A program that searches for the secondary structure with the lowest energy and estimates the stable secondary structure of RNA. The following four types of parameter values required for the analysis, that is, the values defining the “stacking area”, “hairpin loop”, “bulge loop”, and “inner loop” were all default values built in the program. As a result, a predicted secondary structure of mouse MK mRNA was obtained. The target site of the antisense DNA is composed of a loop region that does not form a duplex and one of the base sequence that includes the first loop region as viewed from the initiation codon side.
The 5th to 32nd 18 base pair portions were selected. Designed antisense DNA (SEQ ID NO: 1), sense DNA (SEQ ID NO: 2) as control, reverse DNA
(SEQ ID NO: 3) is shown.

SEQ ID NO: 1 5′-AGGGCGAGAAGGAAGA
AG-3 'SEQ ID NO: 25'-CTTCTCTCCTTCTCGCC
CT-3 'SEQ ID NO: 3 5'-GAAGAAGGAAGAGCGG
GA-3 '

Several other antisense sequences were candidates, but these were not effective in the tests described below. DNA synthesizer, Expdite8900
Nucleic Acid Synthesis Sy
stem (Applied Biosystems)
Synthesizes antisense S oligo DNA by Wako
High performance liquid chromatography (H) on a pak Handy ODS column (Waters).
PLC) purification was performed and used for the study. As control sequences, sense and reverse DNAs corresponding to the antisense site were similarly synthesized and purified and used. Similarly, mRNA of human MK (GenBank Accession)
No. M69148: Tsutsui J.M. et a
l. Biochem. Biophys. Res. Com
mun. , 176: 792-797, 1991) by computer analysis. For humans, antisense DNA (SEQ ID NO: 4) corresponding to mouse, (based on nucleotides 15 to 32 of the human MK mRNA sequence) and antisense DNA corresponding to a sequence that does not form a loop near the 5 ′ end ( SEQ ID NO: 5) and (based on the nucleotide numbers 1 to 18 of the human MK mRNA sequence) were designed.

SEQ ID NO: 4 5′-AGGGTGAGGAGGAGGAGA
AG-3 ′ SEQ ID NO: 5 5′-GAAGCCTCGGTGCTGCC
AT-3 '

Example 2 MK production was suppressed by antisense oligo DNA. Mouse rectal cancer cells secreting MK (CMT-93, American Ty
peCulture Collection; ATCC
Number CCL-223) was accurately spread on a 35 mm dish (FALCON, 3001) at a cell density of 3 × 10 ⁵ cells / dish [unfetal serum (FBS) 10% / Dulbecco's modified minimum required medium (DMEM)],
Preculture was performed overnight at 37 ° C in the presence of 5% CO ₂ . The next day, 3
ITS-heparin / DMEM (insulin 10 μg / ml, transferrin 5.5 μg / m
l, 6.7 ng / ml of selenous acid and 20 μ of heparin
g / ml each at the final concentration), and 1 ml of the same medium was added, followed by culturing for 2 hours. 30 μl of antisense DNA or control DNA whose concentration has been adjusted to 1 mM in advance, and 630 μl of DMEM
And Lipofectamine-plus k
60 μl of it plus reagent was added and reacted at room temperature for 15 minutes. Furthermore, Lipofectamine reagent 24
μl, and allowed to react at room temperature for 15 minutes.
A. Preparation of the liposome treatment solution was completed. The above prepared amount is an amount that can be administered to five plates (for a quintuple assay) in a 35 mm dish. Subsequently, the CMT-93 cells were filled with 0.8 ml of fresh DMEM, 0.2 ml of the above-mentioned mouse MK antisense oligo DNA / liposome treatment solution was added, and transfection was performed at 37 ° C. for 3 hours. At this time, the administration concentration of the antisense oligo DNA is 5 μM. 1 ml of 10% FBS / DMEM was added, and the cells were cultured for 7 hours. The cells are washed with DMEM and
1 ml of S-heparin / DMEM was added, the culture was continued for further 16 hours, and the supernatant was collected. SDS gel electrophoresis (SDS-PAGE) (15%
Gel), Western blot using anti-MK antibody was performed according to the method of Muramatsu et al. (Muramatsu, H. et al:
Dev. Biol. 159: 392-402,199
3). The result is shown in FIG. 1 (FIG. 1A).
Only in the lane 1 antisense DNA administration, MK
Attenuation of specific bands was observed. Even if the control sequence sense and reverse DNA were administered, the MK
Production was not suppressed. This is antisense DNA
This shows that the administration reduced the amount of MK production / secretion of CMT-93 cells. In addition, the concentration of each band was measured with a densitometer, and it was found that the amount of MK production of the cells to which antisense DNA had been administered was reduced to 13% of that of the untreated cells (FIG. 1B).

Example 3 The growth of CMT-93 cells was inhibited by administration of antisense oligo DNA. According to Example 2, CMT-93 cells transfected with MK antisense oligo DNA by a liposome method were prepared. The cells were treated with 0.25% trypsin 0.0
The cells were detached from the dish using a 2% EDTA-2Na solution to prepare a cell suspension. Count the number of cells, 24
3 × 10 ⁴ cells / well (10% FBS / D
MEM), and the cells were washed with DMEM twice after culturing at 37 ° C. for 3 hours. DMEM in well
Fill 0.5 ml and use Cell counting k
After adding 50 μl of it (Dohjin Chemical) reagent and culturing at 37 ° C. for 2 hours, the absorbance (450 nm: subwavelength 630 nm) was measured. The same operation was repeated for 5 days thereafter, with n = 4 in each group, to obtain absorbance values for each culture day. Antisense D
An experimental system in which MK was exogenously added to the medium as a rescue molecule to NA-administered cells was also examined. That is, from the first day of culture, MK was added every day at a concentration of 5 ng / ml (final concentration). As a result, as shown in FIG. 2 (FIG. 2), when the day of antisense administration was set to day 0, proliferation began to be suppressed from day 2 in the antisense administration group, and the difference became significant after day 3. On the other hand, in the sense administration group, the growth of cancer cells was not suppressed. These phenomena can be considered to be the result of the remarkable decrease in the amount of MK produced by the cancer cells themselves due to the administration of antisense DNA, and the inhibition of their own growth. In order to actually replace the decreased MK in the cells to which antisense DNA had been administered, exogenously added MK restored the growth of the cells (FIG. 2).

Example 4 The colony formation of CMT-93 cells was inhibited by administration of antisense oligo DNA. CMT transfected with MK antisense oligo DNA by liposome method according to Example 2.
-93 cells were prepared. A cell suspension was prepared according to Example 3, and the number of cells was counted. First, a soft agar dish was prepared by partially modifying the conventional method (Kamata, Nobuyuki, New Cultured Cell Experiment Method for Molecular Biology Research, Yodosha, 2nd revised edition)
P. 256-258, 1999). That is, Nobl
e agar (Difco) 1g in 100ml of Milli-Q water
, And autoclaved to prepare a sterile 1% agar solution. This was diluted with a culture medium to prepare 10% FBS / DMEM containing 0.5% agar, and 1.5 ml was dispensed into a 35 mm dish to form a base layer (5% until solidified).
〜１０10 minutes). Solutions containing agar are always 42-45
The temperature was kept at 0 ° C. so as not to solidify during the operation. The previously prepared CMT-93 cells to which antisense oligo DNA had been administered were adjusted to 1.5 × 10 ⁴ cells / ml with 10% FBS.
/ DMEM was made 2 ml, 4 ml of 10% FBS / DMEM containing 0.5% agar was added thereto, mixed well, and 1 ml was gently laminated on the base layer. The number of swallowing cells per dish was 5 × 10 ³ . Once the agar has set, 10% FBS to prevent the upper part of the soft agar from drying out
/ DMEM 1 ml. After culturing at 37 ° C. in an incubator for 9 to 11 days, the total number of colonies per dish was counted under a microscope. N =
The average value of 5 dishes, ie, 5 dishes, was calculated. As a result, as shown in FIG. 3 (FIG. 3), the number of colonies formed was significantly suppressed in the cells to which the antisense oligo DNA had been administered. The number of colonies formed in the sense and reverse oligo DNA administration groups was not suppressed, and the specific effect of the antisense form was confirmed. When CMT-93 cells to which antisense oligo DNA had been administered were cultured in soft agar to which MK had been added in advance, the number of colonies formed recovered. The degree of recovery of the number of colonies formed was dependent on the concentration of MK added to the soft agar. These results strongly suggested that MK is deeply involved in anchorage-independent growth of cancer cells.

[0019]

As described above, the antisense nucleotide sequence of the present invention can suppress the expression of MK and suppress the growth of cancer cells.

[0020]

[Sequence List] <110> Takashi Muramatsu <120> Suppression of midkine expression using antisense oligonucleotide sequence and associated suppression of cancer cell proliferation <130> TM-001 <141> 2000-11-10 <160 > 5

<210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <400> 1 agggcgagaagagagaag

<210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <400> 2cttcttcccttctcgccct

<210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <400> 3 gaagaaggaagagggggga

<210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <400> 4 agggtgaggaggagag

<210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <400> 5gaagcctcggtgctgcat

[0026]

[Brief description of the drawings]

FIG. 1 shows the suppression of MK production in CMT-93 cells administered with antisense oligo DNA or the like according to one embodiment of the present invention. FIG. 1A is a Western blot, and FIG. 1B is specific to MK detected by A. 7 is a graph in which the density of a typical band is quantified by densitometer analysis.

FIG. 2 is a graph showing a growth curve of CMT-93 cells according to one example of the present invention.

FIG. 3 is a graph showing the number of colonies formed in soft agar of CMT-93 cells according to one example of the present invention.

Continued on front page F term (reference) 4B024 AA01 CA01 DA02 GA13 HA17 4B065 AA91X AA91Y AB01 BA05 CA44 4C084 AA13 NA14 ZB212 ZB261 ZC012 4C086 AA01 AA03 EA16 MA01 MA04 NA14 ZB21 ZB26 ZC01

Claims (2)

[Claims] 1. An antisense nucleotide sequence that suppresses midkine expression. 2. The antisense nucleotide sequence of claim 1, which is capable of suppressing the growth of cancer cells.