JP2005046064A - Arf6 siRNA INHIBITING MOVEMENT AND INVASION OF HUMAN CANCER CELL - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicine for preventing invasion and metastasis of a cancer. <P>SOLUTION: The siRNA of human Arf6 which comprises a sense chain composed of 21-23 nucleotides derived from the nucleotide sequence of the translation region of mRNA of human Arf6 and an antisense chain composed of nucleosides complementary to the nucleotides, in which both the chains are bonded to UN sequences at 3' ends, respectively and which inhibits movement and invasion of cancer cell is administered to a cancer patient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an Arf6 siRNA capable of preventing cancer metastasis by inhibiting cancer cell movement and invasion.

In order to diagnose and treat cancer more efficiently, it is essential to elucidate the molecular mechanisms of its onset and malignant acquisition. However, human knowledge of cancer and further understanding of the basic mechanism of normal cell growth control before canceration are still insufficient. In particular, the main cause of the great threat of malignant cancer is its invasion and metastasis. In the case of epithelial cancer, many metastases occur through infiltration of cancer cells into the tissue. The basic molecular mechanisms that lead to cell invasion and metastasis, as well as ways to inhibit cancer invasion through its application, were still under research and development at the world level (Hanahan, D. & Weinberg, RA (2000)). The hallmarks of cancer.
Cell 100: 57-70; Kong et al. (2003). A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3: 537-549).
Arf6 is a low molecular weight G protein belonging to the Ras superfamily. It is thought to be mainly involved in the regulation of endosomal recycling of cell plasma membrane components and recycling of cell membrane receptors (Moss, J. & Vaughan, M. (1998) Molecules in the ARF orbit. J. Biol. Chem. 273, 21431-21434; Roth, MG (2000) Arf. In “GTPases”, eds. Hall, A. (Oxford University Press), pp176-197). It is unknown whether Arf6 is involved in cancer cell motility.
Moss, J. & Vaughan, M. (1998) Molecules in the ARF orbit. J. Biol. Chem. 273, 21431-21434

  As described above, the present invention is to provide a means for suppressing the invasiveness of cancer treatment that is still incomplete. Accordingly, an object of the present invention is to provide an unprecedented new and more effective treatment for cancer.

The present inventor has conducted extensive research to solve the above problems. As a result, the present inventors have found that cancer cell movement and invasive activity can be prevented by treating cancer cells with siRNA (small interfering RNA) that specifically acts on Arf6 mRNA and significantly reducing the expression of Arf6. In addition, it has been found that the motility and invasive activity of cancer cells can also be inhibited by forcibly expressing an active mutant of Arf6, and the present invention has been completed based on these findings.
The present invention comprises a sense strand consisting of 21 to 23 nucleotides derived from the nucleotide sequence of mRNA of the translation region of human Arf6 shown in SEQ ID NO: 1, and an antisense strand consisting of nucleotides complementary thereto, both strands Relates to a human Arf6 siRNA that binds a UU sequence to each 3 ′ end and inhibits cancer cell movement and invasion.
The target cancer is cancer derived from epithelial tissues of all organs, but breast cancer is most preferable.

(1) Selection of siRNA A preferred siRNA of the present invention is exemplified.
1) siRNA having 22 nucleotides 162 to 183 of SEQ ID NO: 1, the antisense strand is composed of nucleotides complementary thereto, and both strands bind UU sequences to their 3 ′ ends;
2) An siRNA having 21 nucleotides of 223-243 of SEQ ID NO: 1, the antisense strand is composed of complementary nucleotides thereof, and both strands bind UU sequences to their 3 ′ ends;
3) siRNA having 21 nucleotides of 271 to 291 of SEQ ID NO: 1, the antisense strand is composed of nucleotides complementary thereto, and both strands bind UU sequences to their 3 ′ ends;
4) The sense strand is composed of 21 to 23 consecutive nucleotides among the 42 nucleotides of 309-350 of SEQ ID NO: 1, the antisense strand is composed of a complementary nucleotide thereto, and both strands are at their 3 ′ ends. SiRNA that binds the UU sequence, preferred among these is a siRNA whose sense strand has 21 nucleotides of 309-329 of SEQ ID NO: 1;
5) The sense strand is composed of 21 to 23 consecutive nucleotides among the 35 nucleotides of 392 to 426 of SEQ ID NO: 1, the antisense strand is composed of complementary nucleotides thereof, and both strands are at their 3 ′ ends. SiRNA that binds a UU sequence;
6) The sense strand is composed of 21 to 23 consecutive nucleotides out of 40 nucleotides 489 to 528 of SEQ ID NO: 1, the antisense strand is composed of complementary nucleotides, and both strands are at their 3 ′ ends. SiRNA that binds a UU sequence;
Can be mentioned. These siRNAs have low homology to other Arf isoforms and human genes, and are effective in suppressing Arf6 expression by the siRNA method.

(2) siRNA production method siRNA "double-stranded oligoribonucleotides. First, sense strand oligoribonucleotides and antisense strand oligoribonucleotides are respectively synthesized. Generally, four types of ribosomes with protecting groups are synthesized. Synthesized by organic synthesis using a synthesizer using nucleotides, RNA is a polymer molecule in which four nucleosides (adenosine, guanosine, cytidine, uridine) are linked by a phosphodiester bond, and the synthesis forms a backbone in the amidite A 2′-bis (acetoxyethoxy) -methyl ether (2′-ACE) protecting group is added to the 2 ′ end of the sugar to be phosphate-coupled one base at a time. 100 mM TEMED-Acetate (pH 3.8) buffer at 60 ° C. Deprotection is carried out by reacting for 30 minutes, and quality check is performed using HPLC.If high purity RNA is required, RNA can be purified from the gel after denaturing polyacrylamide gel electrophoresis, Purify.
Next, both strands are annealed. An aqueous solution of both strands having a concentration of about 50 μM (50 pmol / μL) was prepared. Heat at 1 ° C. for 1 minute, then hold at 37 ° C. for 60 minutes. This causes the sense and antisense oligonucleotides to anneal to each other into a duplex.

(3) Formulation of siRNA siRNA can be formulated using a lipofection method. Usually, liposomes composed of phosphatidylserine (PS) are used. Instead of this negatively charged phospholipid (PS), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-triethylammonium chloride (DOTMA) can be easily produced. It is preferable to use a cationic lipid (trade name: Transfectam, Lipofectamine). When a complex of these cationic lipids and negatively charged siRNA is formed, positively charged liposomes are adsorbed on the surface of negatively charged cells and fused with the cell membrane, so that siRNA is fused to cells. Can be introduced in.

Production of siRNA (i) Production of sense RNA and antisense RNA 23-mer sense RNA having the following sequences:
GCA CCG CAU UAU CAA UGA CCG UU (SEQ ID NO: 2)
And a 23-mer antisense RNA having the following sequence:
CGG UCA UUG AUA AUG CGG UGC UU (SEQ ID NO: 3)
Was manufactured by the following method.
Using an amidite with a 2′-ACE protecting group, organic synthesis was performed by an amidite method using an original synthesizer (UltraFast Parallel Synthesizer) manufactured by GENSET OLIGOS. After deprotection by reacting with 100 mM TEMED-Acetate (pH 3.8) buffer at 60 ° C. for 30 minutes, 23-mer sense RNA antisense RNA was purified by HPLC.
(Ii) Production of siRNA by annealing An aqueous solution was prepared so that each of the sense and antisense oligonucleotides produced in (i) had a concentration of 50 μM (50 pmol / μl). 30 μl of each aqueous solution was mixed with 15 μl of 5 × annealing buffer (500 mM potassium acetate, 150 mM HEPES-KOH pH 7.4, 10 mM magnesium acetate) (75 μl in total). The solution was heated at 90 ° C. for 1 minute and then incubated at 37 ° C. for 60 minutes. This causes the sense and antisense oligonucleotides to anneal to each other into a duplex. At that time, the concentration of the double-stranded oligonucleotide was 20 μM (20 pmol / μl).

Introduction of siRNA into cancer cells A complex of lipofectamine (Lipofectamine 2000 (GIBCO BRL)) and Arf6 siRNA was formed at room temperature. The amount of lipofectamine used and the method of complex formation were in accordance with the manufacturer's instructions. Specifically, 45 μl of Lipofectamine 2000 and 750 μl of Opti-MEM buffer (Gibco) were incubated at room temperature for 5 minutes and then mixed with 750 μl of Opti-MEM containing 200 pmol of double-stranded oligonucleotide. A complex of Lipofectamine (Lipofectamine 2000 (GIBCO BRL)) and Arf6 siRNA was formed at room temperature. This mixed solution (1.5 ml) was added to 1 × 10 ⁶ human breast cancer cells MDA-MB-231 cells cultured in 8.5 ml of growth medium, and siRNA was introduced into the cells.

Arf6 siRNA treatment by Arf6 siRNA treatment Arf6 siRNA was prepared by the method of Example 1 and introduced into human breast cancer cells MDA-MB-231 cells at a concentration of 20 nM by the method of Example 2. Thereafter, the cells were cultured as they were in a growth medium for 24 hours. After 24 hours, a cell extract was prepared, separated by electrophoresis as usual, and the expression level of Arf6 protein was measured by Western blotting using an anti-Arf6 antibody to examine the effect of Arf6 siRNA. siRNA-treated cells (+) and untreated cells (-), 20 μg of each extract were used. As a control, the results of immunoblotting using an anti-β-actin antibody are shown in the lower panel. Arf6 siRNA treatment suppressed the expression level of Arf6 to 1/10 or less (see FIG. 1).

Inhibition of invasive activity of MDA-MB-231 cells by treatment with Arf6 siRNA Arf6 siRNA was introduced into MDA-MB-231 cells in the same manner as in Example 2 to examine invasive activity. Invasion activity was evaluated by transferring cells cultured for 20 hours after introduction of siRNA onto gelatin labeled with a fluorescent dye (Alexa-594), and then measuring the degradation activity of gelatin in 15 hours according to the method described below. . For invasive activity, cells cultured for 20 hours after introduction of siRNA were transferred onto gelatin labeled with a fluorescent dye (Alexa-594), and the degradation activity of gelatin was measured after 15 hours. Specifically, a cell having a gelatin degrading activity is observed as a dark portion when gelatin under the cell is lost and the gelatin is observed with a fluorescence microscope. Cells in which dark areas were observed were evaluated as having gelatinolytic activity. mock is a control that was operated in the same manner without addition of siRNA. The invasive activity of MDA-MB-231 cells was suppressed to 1/10 or less by introducing Arf6 siRNA. The difference from the control was P <0.001 by statistical significance test (FIG. 2).

Inhibition of motor activity of MDA-MB-231 cells by treatment with Arf6 siRNA Arf6 siRNA was introduced into MDA-MB-231 cells in the same manner as in Example 2 to examine motor activity. The locomotor activity was evaluated by transferring cells cultured for 20 hours after introduction of siRNA to a Boyden Chamber with collagen applied on the lower surface, and measuring the movement to the lower surface after 3 hours as usual. mock is a control that was operated in the same manner without addition of siRNA. By introducing Arf6 siRNA, the motor activity of MDA-MB-231 cells was suppressed to about 1/4. The difference from the control was P <0.001 by statistical significance test (FIG. 3).

Inhibition of invasion activity of MDA-MB-231 cells by forced expression of Arf6 and expression of its mutant cDNA Introducing wild-type (WT) or mutant (Q67L, T27N) cDNA of Arf6 into MDA-MB-231 cells The invasive activity was examined. Lipofectamine 2000 was used for introduction. Invasion activity was evaluated by transferring cells cultured for 14 hours after gene transfer onto gelatin labeled with a fluorescent dye (Alexa-594), and then measuring the degradation activity of gelatin in 15 hours according to a conventional method. mock is a control that was operated in the same manner with no gene added. In each trial, significant inhibition of invasive activity was observed, and the difference from the control was P <0.001 by statistical significance test (FIG. 4).

Inhibition of motor activity of MDA-MB-231 cells by forced expression of Arf6 and expression of its mutant cDNA Arf6 wild-type (WT) or mutant (Q67L, T27N) cDNA was introduced and expressed in MDA-MB-231 cells. The motor activity was examined. Lipofectamine 2000 was used for introduction. The locomotor activity was evaluated by transferring cells cultured for 20 hours after gene transfer to a Boyden Chamber with collagen applied to the lower surface, and measuring the movement to the lower surface after 3 hours as usual. mock is a control that was operated in the same manner with no gene added. In each trial, significant inhibition of motor activity was observed, and the difference from the control was P <0.001 by statistical significance test (FIG. 5).

According to the present invention, a specific means by which invasiveness and motility can be effectively suppressed has been shown. In particular, siRNA is a low molecular weight compound and can actually be administered to a living body as a drug. This time, by showing siRNA sequences that can suppress invasion, it is possible to promote the development of more effective treatments for cancer, particularly breast cancer.

FIG. 6 is an electrophoretogram showing suppression of Arf6 protein expression by Arf6 siRNA treatment. It is a graph which shows the expression suppression of Arf6 protein by Arf6 siRNA process. It is a graph which shows inhibition of the invasion activity of MDA-MB-231 cell by Arf6 siRNA treatment. It is a graph which shows inhibition of the invasion activity of MDA-MB-231 cell by forced expression of Arf6 and its mutant cDNA expression. It is a graph which shows inhibition of the motor activity of MDA-MB-231 cell by forced expression of Arf6 and its mutant cDNA expression.

Claims (8)

It consists of a sense strand consisting of 21 to 23 nucleotides derived from the nucleotide sequence of the translation region of human Arf6 mRNA shown in SEQ ID NO: 1, and an antisense strand consisting of nucleotides complementary thereto. 'A human Arf6 siRNA that inhibits cancer cell motility and invasion, binding a UU sequence to the terminus. The siRNA of claim 1, wherein the sense strand has 22 nucleotides 162-183 of SEQ ID NO: 1. The siRNA of claim 1, wherein the sense strand has 21 nucleotides of 223-243 of SEQ ID NO: 1. The siRNA of claim 1, wherein the sense strand has 21 nucleotides of 271-291 of SEQ ID NO: 1. The siRNA according to claim 1, wherein the sense strand has 21 to 23 consecutive nucleotides among 42 nucleotides of 309 to 350 of SEQ ID NO: 1. 6. The siRNA of claim 5, wherein the sense strand has 21 nucleotides 309-329 of SEQ ID NO: 1. The siRNA according to claim 1, wherein the sense strand has 21 to 23 consecutive nucleotides among 35 nucleotides of 392 to 426 of SEQ ID NO: 1. The siRNA according to claim 1, wherein the sense strand has 21 to 23 consecutive nucleotides out of 40 nucleotides of 489-528 of SEQ ID NO: 1.

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