JP7224652B2 - Pancreatic cancer cell invasion and metastasis inhibitor - Google Patents

Description

The present invention encodes a pancreatic cancer cell invasion and metastasis inhibitor that can effectively suppress the invasion and metastasis of pancreatic cancer cells using RNA interference, and an RNA that is an active ingredient of the pancreatic cancer cell invasion and metastasis inhibitor. The present invention relates to DNA and vectors containing the DNA that can be used to suppress the invasion and metastasis of pancreatic cancer cells.

"Tumor" refers to abnormally proliferating cells, and refers to a state in which cell proliferation continues even after the cause of the abnormal proliferation has disappeared or been removed. Among tumors, benign tumors grow slowly and do not metastasize. Therefore, in general, there is no problem if it is excised, and even if it is left without excision, it can be said that there is no danger to life. On the other hand, malignant tumors, that is, cancers, unlike benign tumors, grow rapidly and metastasize to lymph nodes and other organs. Therefore, even if it is removed by a surgical operation, even a small amount of residual cancer cells or cancer cells that have already metastasized to lymph nodes or other organs may start to proliferate again. Therefore, cancer has a poor prognosis once treatment is completed, and the survival rate after 5 years has been investigated for each type of cancer. Cure is only considered if there is no recurrence by then.

Pancreatic cancer is said to have the worst prognosis among cancers. The reason for this is that early detection is difficult because the pancreas is a retroperitoneal organ, and pancreatic cancer cells have extremely high motility. , the gastrointestinal tract, nerves, etc., metastasis to nearby lymph nodes, and distant metastasis to the liver, etc.

As described above, the reason why cancer is a malignant tumor is that it invades or metastasizes to other tissues.

In recent years, a phenomenon called RNA interference has been discovered in which a double-stranded RNA having specificity for a target mRNA degrades the target mRNA, thereby inhibiting its translation. Therefore, there is a possibility that the invasion and metastasis of cancer cells can be suppressed by knocking down at the stage of mRNA by using RNA interference against a group of molecules related to the invasion and metastasis of cancer cells.

For example, in Patent Document 1, since invasion and metastasis of cancer cells such as breast cancer cells is enhanced by overexpression of angiopoietin-like factor 2 (ANGPTL2), invention that suppresses the expression of ANGPTL2 by siRNA or shRNA that causes RNA interference is disclosed.

Patent Document 2 describes an invention that inhibits invasion and metastasis of breast cancer cells and the like by cleaving, by RNA interference, mRNA related to a protein that specifically activates ADP-ribosylation factor.

Patent Document 3 discloses an invention in which the tissue invasion of leukemic cells is suppressed by inhibiting the expression of genes involved in the expression of the cell surface antigen CD43 and the selectin ligand sugar chain expressed on CD43 using RNA interference. It is

Patent Documents 4 and 5 disclose siRNAs that inhibit ARF6 and AMAP1/PAG2/ASAP1, which are proteins related to cancer cell motility and invasiveness, respectively.

By the way, insulin-like growth factor 2 mRNA-binding protein 3 (Insulin-like Growth Factor 2 mRNA-Binding Protein 3, hereinafter sometimes abbreviated as "IGF2BP3") is normally present in the nucleolus and acts as an insulin-like growth factor. It binds to the 5'UTR of the 5' untranslated region 3 of the RNA of insulin-like growth factor II and inhibits translation of insulin-like growth factor II. The gene encoding IGF2BP3 is cited as one of the genes whose overexpression is used for cancer detection in the invention described in Patent Document 6.

JP 2011-93896 A JP-A-2007-97421 Japanese Patent Application Laid-Open No. 2006-304716 JP 2005-46064 A JP 2005-224149 A Japanese Patent Publication No. 2011-516077

As described above, various techniques for suppressing invasion and metastasis of cancer cells have been investigated. However, the reason why pancreatic cancer cells are particularly prone to invasion and metastasis has not been shown, and it has not necessarily been effective in suppressing the invasion and metastasis of pancreatic cancer cells.

Therefore, the present invention provides an inhibitor of pancreatic cancer cell invasion and metastasis that can particularly effectively suppress the invasion and metastasis of pancreatic cancer cells, a DNA encoding an RNA that is an active ingredient of the pancreatic cancer cell invasion and metastasis inhibitor, and An object of the present invention is to provide a vector that contains the DNA and that can be used to suppress the invasion and metastasis of pancreatic cancer cells.

The inventor of the present invention has made intensive studies to solve the above problems. As a result, human insulin-like growth factor 2 mRNA-binding protein 3, which is normally found in the nucleolus, is localized in cell membrane protrusions, which are essential intracellular structures for invasion and metastasis in pancreatic cancer cells. We obtained experimental data to show that In addition, human insulin-like growth factor 2 mRNA-binding protein 3 in cell membrane protrusions regulates the local translation of specific mRNAs in cell membrane protrusions by binding to them. Therefore, the present invention was completed by experimentally demonstrating that invasive metastasis of pancreatic cancer cells can be suppressed by inhibiting the translation of the mRNA in cell membrane protrusions by RNA interference.

The present invention is shown below.

[1] A pancreatic cancer cell invasion and metastasis inhibitor comprising RNA (1) having one or more nucleotide sequences selected from SEQ ID NOs: 1 to 92.

[2] The pancreas according to [1] above, wherein the RNA (1) has at least one base sequence selected from SEQ ID NOs: 57 to 60 or one or more base sequences selected from SEQ ID NOs: 73 to 76 Cancer cell invasion and metastasis inhibitor.

[3] The inhibitor of pancreatic cancer cell invasion and metastasis of [1] or [2] above, further comprising RNA (2) having a sequence that hybridizes with RNA (1) under stringent conditions.

[4] The pancreatic cancer cell invasion and metastasis inhibitor according to [3] above, which contains a double-stranded RNA in which the above RNA (1) and the above RNA (2) are hybridized.

[5] the RNA (1), the RNA (2), and a linker RNA that binds the RNA (1) and the RNA (2), wherein the RNA (1) and the RNA (2) hybridize; The inhibitor of pancreatic cancer cell invasion and metastasis according to [3] above, which is soybean and contains a single-stranded RNA in which the linker RNA has a circular structure.

[6] A DNA characterized by having a base sequence encoding RNA (1) having one or more sequences selected from SEQ ID NOS: 1-92.

[7] A vector comprising the DNA of [6] above.

[8] Use of RNA (1) having one or more nucleotide sequences selected from SEQ ID NOs: 1 to 92 for inhibiting invasion and metastasis of pancreatic cancer cells.

[9] A method for inhibiting invasion and metastasis of pancreatic cancer cells, comprising the step of administering siRNA containing RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 92.

Human insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) according to the present invention is usually found in the nucleolus, whereas in pancreatic cancer cells it is present in cell membrane protrusions, and various mRNAs are present in this IGF2BP3. Bound and accumulated in cell membrane protrusions. After being translated, the mRNA in such cell membrane protrusions is involved in enhancing motility and invasion of pancreatic cancer cells through the formation of cell membrane protrusions, and as a result, it is thought to play a major role in invasion and metastasis. Therefore, if mRNA binding to IGF2BP3 in cell membrane protrusions of pancreatic cancer cells is inhibited by RNA interference, invasion and metastasis of pancreatic cancer cells can be effectively suppressed. On the other hand, the RNA interference according to the present invention inhibits the action of mRNA accumulated in cell membrane protrusions of pancreatic cancer cells, so it is considered harmless and safe for normal cells. Therefore, the pancreatic cancer cell invasion and metastasis inhibitor, DNA and vector according to the present invention are very useful as agents capable of safely suppressing the invasion and metastasis of pancreatic cancer cells.

FIG. 1 is a photograph of immunocytochemically fluorescently labeled human pancreatic ductal adenocarcinoma cells S2-013 and PANC-1, which are cultured on fibronectin to promote the formation of cell membrane protrusions. Green areas indicate areas labeled with anti-IGF2BP3 antibody and red areas indicate actin filaments labeled with phalloidin. Arrows indicate areas where IGF2BP3 is localized to cell membrane protrusions, bar length is 10 μm. FIG. 2 shows photographs of S2-013 cell lines cultured on fibronectin and stained with anti-IGF2BP3 antibody (green) or anti-G3BP antibody (red), actin filaments labeled with phalloidin (purple), and nuclei. are stained with DAPI and the results are integrated. Arrows point to IGF2BP3 localized in plasma membrane protrusions. FIG. 3 is a photograph showing the results of immunoprecipitation of IGF2BP3 or G3BP from extracts of S2-013 cells cultured on fibronectin and analysis by Western blotting using anti-IGF2BP3 antibody and anti-G3BP antibody. Rabbit or mouse anti-IgG isotype control antibodies were used as isotype controls. FIG. 4 shows IGF2BP3-RNAi S2-013 strain cells (siIGF-1 and siIGF-2) introduced with siRNA targeting IGF2BP3 mRNA and control RNAi S2-013 strain cells (Scr-1 and Scr-2). 1 is a photograph showing the results of Western blot analysis of proteins expressed in . FIG. 5 is a set of photographs showing the results of a motility assay of IGF2BP3-RNAi S2-013 cell line and control RNAi S2-013 cell line to the wound area. FIG. 6 is a graph showing the number of cells that migrated to the wound area in a motility assay to the wound area of IGF2BP3-RNAi S2-013 cell line and control RNAi S2-013 cell line. "*" indicates significant difference from control at p<0.001 in t-test. Fig. 7 shows that a wound area is formed by removing a portion of the S2-013 cell line that proliferates confluently in a monolayer with a pipette tip, and the advanced site of pancreatic cancer cells that begin to migrate toward the wound area is immunized. This is a stained photograph. Green areas indicate areas stained with anti-IGF2BP3 antibody. Arrows indicate IGF2BP3 present in cell membrane protrusions formed at advanced sites of S2-013 cell lines that have begun to migrate. The bar length is 10 μm. FIG. 8 is a graph showing the number of cells that migrated from the upper chamber to the lower chamber in the Matrigel invasion assay of the IGF2BP3-RNAi S2-013 cell line and the control RNAi S2-013 cell line. "*" indicates significant difference from control at p<0.001 in t-test. FIG. 9 shows photographs showing the results of Western blot analysis using an anti-IGF2BP3 antibody after the myc-tagged IGF2BP3 recovery construct or mock control vector was introduced into control RNAi cells or IGF2BP3-RNAi cells for 48 hours. Black arrows indicate endogenous IGF2BP3, white arrows indicate exogenous IGF2BP3. FIG. 10 is a graph showing the number of cells that migrated from the upper chamber to the lower chamber after introduction of myc-tagged IGF2BP3 restoration constructs or a mock control vector into control RNAi cells or IGF2BP3-RNAi cells followed by a dual-chamber invasion assay. be. "*" indicates that the number of cells transfected with IGF2BP3-pCMV6 is significantly higher than cells transfected with mock vector with p<0.005 in t-test. FIG. 11 is a hematoxylin-eosin stained photograph of tumors formed in the pancreas of nude mice transplanted with control RNAi S2-013 cell line or IGF2BP3-RNAi S2-013 cell line into the pancreas. "R" indicates cavity peritoneal tissue, "P" indicates muscle tissue, and "N" indicates normal tissue. FIG. 12 shows Z in confocal immunofluorescence microscopy studies of fibronectin-stimulated control RNAi S2-013 cell line, IGF2BP3-RNAi S2-013 cell line, and IGF2BP3-RNAi S2-013 cell line with restored IGF2BP3 expression. - stack photograph. Blue areas indicate DAPI-stained nuclei and red areas indicate phalloidin-labeled actin filaments. Arrows indicate actin filaments present in cell membrane protrusions. The lower and right panels show cross-sections of the yellow line, respectively. The bar length is 10 μm. FIG. 13 shows cell membrane protrusions versus total cell numbers for fibronectin-stimulated control RNAi S2-013 cell line, IGF2BP3-RNAi S2-013 cell line, and IGF2BP3-RNAi S2-013 cell line with restored IGF2BP3 expression. 1 is a graph showing percentages of cells; “*” indicates significant difference at p<0.001 from control RNAi S2-013 cell line or IGF2BP3-RNAi S2-013 cell line with restored IGF2BP3 expression in t-test. FIG. 14 is a photograph of the morphology of the control RNAi S2-013 cell line and the IGF2BP3-RNAi S2-013 cell line stimulated with fibronectin, taken using a phase-contrast microscope. FIG. 15 shows a scatter plot of the concentration of mRNA contained in IGF2BP3 or control IgG immunoprecipitated samples from extracts of S2-013 cell line cultured on fibronectin. Figure 16 shows the comparison between IGF2BP3-immunoprecipitated samples or control IgG-immunoprecipitated samples from extracts of S2-013 cells cultured on fibronectin, ribosomal RNA ((1), rRNA) and small nuclear RNA ( (2), snRNA) shows a linear regression plot of the number. In FIG. 16, the dotted line indicates the straight line of x=y. FIG. 17 shows that ARF6 and ARHGEF4 were selected from among the mRNAs bound to IGF2BP3 in cell membrane protrusions, and anti-IGF2BP3 antibody, isotype control IgG, or negative control CD63 was extracted from the S2-013 strain cell extract cultured on fibronectin. Fig. 10 is a photograph showing the expression levels of ARF6, ARHGEF4, and ubiquitin C mRNA, which is an internal quantification control, after purification of RNA from immunoprecipitation samples using antibodies. FIG. 18 is an immunofluorescence photograph of IGF2BP3 in cell membrane protrusions, ARF6 and ARHGEF4 among mRNAs bound to the IGF2BP3, and ubiquitin C mRNA as a control. The bar length is 10 μm. FIG. 19 shows photographs of fibronectin-stimulated control RNAi S2-013 cells and IGF2BP3-RNAi S2-013 cells stained with anti-ARF6 antibody (green) or anti-ARHGEF4 antibody (green), and actin filaments stained with phalloidin (red). ) and nuclei stained with DAPI (blue) and the results integrated. Arrows point to localization at membrane protrusions. The bar length is 10 μm. FIG. 20 shows photographs of staining with anti-myc antibody (green) or anti-ARF6 antibody (purple) after the myc-tagged IGF2BP3 restoration construct or mock control vector was introduced into IGF2BP3-RNAi cells for 48 hours, and actin filaments were stained with phalloidin (red). ) and nuclei stained with DAPI (blue) and the results integrated. Arrows point to localization at membrane protrusions. The bar length is 10 μm. FIG. 21 shows photographs of staining with an anti-myc antibody (green) or an anti-ARHGEF4 antibody (purple) after the myc-tagged IGF2BP3 restoration construct or mock control vector was introduced into IGF2BP3-RNAi cells for 48 hours. ) and nuclei stained with DAPI (blue) and the results integrated. Arrows point to localization at membrane protrusions. The bar length is 10 μm. FIG. 22 is a set of photographs showing the results of Western blot analysis of the amount of ARF6 or ARHGEF4 produced in S2-013 cell line transfected with a control vector or a vector producing ARF6-RNAi or ARHGEF4-RNAi. FIG. 23 is a graph showing the ratio of cells in which cell membrane protrusions were formed between cells in which ARF6-mRNA or ARHGEF4-mRNA was knocked down by RNA interference and control cells. FIG. 24 is confocal micrographs of cells in which ARF6-mRNA was knocked down by RNA interference and control cells. Green indicates areas labeled with anti-ARF6 antibody, red indicates phalloidin-stained actin filaments, and blue indicates DAPI-stained nuclei. The bar length is 10 μm. FIG. 25 is confocal micrographs of cells in which ARHGEF4-mRNA was knocked down by RNA interference and control cells. Green indicates areas labeled with anti-ARHGEF4 antibody, red indicates phalloidin-stained actin filaments, and blue indicates DAPI-stained nuclei. The bar length is 10 μm. Figure 26 shows ARF6-RNAi S2-013 cell line, ARF6-RNAi PANC-1 cell line, ARHGEF4-RNAi S2-013 line cell, ARHGEF4-RNAi PANC-1 cell line, control RNAi S2-013 line cell, and control Fig. 10 is a graph showing the number of cells that migrated to the wounded area in the motility assay of RNAi PANC-1 cell line to the wounded area. "*" indicates significant difference from control at p<0.001 in t-test. Figure 27 shows ARF6-RNAi S2-013 cell line, ARF6-RNAi PANC-1 cell line, ARHGEF4-RNAi S2-013 line cell, ARHGEF4-RNAi PANC-1 line cell, control RNAi S2-013 cell line, and control FIG. 4 is a graph showing the number of cells that migrated from the upper chamber to the lower chamber in the Matrigel invasion assay of RNAi PANC-1 cell line. "*" indicates significant difference from control at p<0.001 in t-test. FIG. 28 shows photographs of folic acid chitosan nanoparticles alone and folic acid chitosan nanoparticle-added control siRNA labeled with FITC fluorescence (green) and added to the culture medium of S2-013 cells, and cell nuclei stained with DAPI (blue). It is a photograph obtained by integrating these results taken using a confocal fluorescence microscope. It can be seen that the folic acid-chitosan nanoparticle-attached siRNA is taken up into cells in the same manner as the folic acid-chitosan nanoparticles alone. FIG. 29 shows the results of semi-quantitative RT-PCR analysis of mRNA expressed in S2-013 cells incorporating CCDC88A siRNA and WASF2 siRNA according to the present invention to which folic acid chitosan nanoparticles were added and control siRNA ( A) is a photograph showing the result of Western blot analysis of the lysate of the cells (B). Both methods confirmed the knockdown effect of CCDC88A siRNA and WASF2 siRNA of the present invention on mRNA. FIG. 30 shows the results of Matrigel invasion assay using CCDC88A siRNA and WASF2 siRNA according to the present invention to which folate-chitosan nanoparticles were added and control siRNA. FIG. 31 shows a human pancreatic cancer invasion/metastasis mouse model in which S2-013 cells were transplanted into the pancreas, and control siRNA, CCDC88A siRNA, or WASF2 siRNA added with folic acid chitosan nanoparticles were administered. It is a photograph showing siRNA accumulation in . FIG. 32 is a photograph of a primary S2-013 tumor removed from a mouse after photographing in FIG. 31 and stained with hematoxylin and eosin.

A pancreatic cancer cell invasion and metastasis inhibitor according to the present invention is characterized by comprising RNA (1) having one or more base sequences selected from SEQ ID NOS: 1-92. In addition, the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention contains siRNA or shRNA as an active ingredient, part of the siRNA or shRNA is RNA (1), and the base sequence of the RNA (1) is the sequence It is characterized by being one or more selected from numbers 1-92.

The nucleotide sequences of SEQ ID NOs: 1 to 92 are part of the nucleotide sequence of mRNA binding to human insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) localized in cell membrane protrusions of pancreatic cancer cells. , it becomes possible to degrade the above-mentioned mRNA and inhibit its translation by using RNA having these nucleotide sequences in RNA interference.

The present inventor found that in highly motile pancreatic cancer cells, IGF2BP3, which is normally present in the nucleolus, gathers in cell membrane protrusions, and that specific mRNA binds to IGF2BP3 in the cell membrane protrusions. It was experimentally demonstrated that the invasion and metastasis of pancreatic cancer cells can be very effectively suppressed by inhibiting the mRNA by RNA interference. Since the base sequences shown in SEQ ID NOs: 1 to 92 correspond to partial base sequences of such mRNA, RNA (1) can be used as a sense RNA for RNA interference.

Insulin-like growth factor (IGF) is a polypeptide whose amino acid sequence is highly similar to insulin, and in cell culture, it induces responses such as mitogenesis, similar to insulin. IGF-2 is considered to be the first growth factor required for early development, secreted by brain, kidney, pancreas and muscle in mammals, has more specific actions than IGF-1, and insulin in adults. is found at a concentration 600 times higher than While the actions of IGFs are regulated by a family of genes known as IGF-binding proteins, IGF gene expression is regulated by proteins that bind to their mRNAs.

IGF2BP3 is one of the regulatory factors of IGF gene expression, and normally exists in the nucleolus, whereas the present inventors found that in pancreatic cancer cells, the cell membrane is an essential site for its motility. We found that it is accumulated in the processes and is greatly involved in the invasion and metastasis of pancreatic cancer cells. For example, when the IGF2BP3 gene is knocked out in pancreatic cancer cells, they lose motility, significantly inhibit invasion, and prevent metastasis.

RNA interference uses siRNA or shRNA. These RNAs are safe because they are not incorporated into chromosomes and do not mutate when introduced into cells. In addition, siRNA is relatively easy to chemically synthesize and is stable in a double-stranded state.

siRNA is a double-stranded RNA in which a sense RNA that is homologous to a partial nucleotide sequence of the target mRNA and an antisense RNA that can hybridize with the sense RNA are hybridized. The 5'-end may be phosphorylated, and the 3'-end side may protrude by 1 or more bases and 4 or less bases. A specific protein binds to siRNA to form a complex called RISC (RNA-Induced-Silencing-Complex). RISC recognizes and binds to mRNA having a sequence homologous to the base sequence of sense RNA, and cleaves the mRNA with an RNase III-like enzymatic activity.

The shRNA consists of a single-stranded RNA, in which a sense RNA region homologous to a partial nucleotide sequence of the target mRNA and an antisense RNA region capable of hybridizing with the sense RNA are bound by a linker RNA exhibiting a circular structure, and the whole is a hairpin. It has a similar structure. The 3' end of the shRNA may protrude from 1 base to 4 bases, and the 3' protruding end may be composed of DNA. shRNAs are degraded intracellularly and, like siRNAs, cause RNA interference.

RNA (1) contained in the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention has one or more nucleotide sequences selected from SEQ ID NOS: 1-92. The nucleotide sequences of SEQ ID NOs: 1 to 92 are part of the nucleotide sequences of 19 types of mRNA contained in cell membrane protrusions of pancreatic cancer cells. A portion of siRNA or shRNA used as an active ingredient in the present invention corresponds to RNA (1). However, even an RNA having a base sequence containing deletion, substitution or addition of one base in the above sequence may exhibit RNA interference activity as well. Such equivalent inventions are intended to be included within the scope of the present invention.

In the present invention, "having (having a base sequence)" means that an RNA has only a predetermined base sequence, or may have a base sequence other than the predetermined base sequence. However, it is preferable that the nucleotide sequence of the RNA is substantially the same as the predetermined nucleotide sequence, except for the modifications at the 5' end and the overhanging RNA or DNA at the 3' end as described above.

As the base sequence of RNA (1), at least one base sequence selected from SEQ ID NOs: 57 to 60 or at least one base sequence selected from SEQ ID NOs: 73 to 76 is particularly preferred. The base sequences of SEQ ID NOS:57-60 are part of the base sequence of CCDC88A, and the base sequences of SEQ ID NOS:73-76 are part of the base sequence of WASF2. CCDC88A and WASF2 are mRNAs contained in cell membrane protrusions of pancreatic cancer cells, but their functions in pancreatic cancer cells were unknown. The present inventors have experimentally found that among mRNAs contained in cell membrane protrusions of pancreatic cancer cells, CCDC88A and WASF2 have the highest ability to promote invasion and metastasis of pancreatic cancer cells. Therefore, the preferred RNA (1) can suppress the actions of CCDC88A and WASF2 and most effectively inhibit the invasion and metastasis of pancreatic cancer cells.

The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention preferably contains, in addition to RNA (1), RNA (2) having a nucleotide sequence that hybridizes with RNA (1) under stringent conditions. RNA(2) can further stabilize RNA(1).

The base sequence of RNA (2) is preferably completely complementary to the base sequence of RNA (1), but may not be completely complementary as long as it hybridizes under stringent conditions. . In the present invention, the term “stringent conditions” means sodium chloride concentration of 150 mM or more and 900 mM or less, sodium citrate concentration of 15 mM or more and 90 mM or less, that is, 1 to 6×SSC, 0.1% by mass or more, 0.1% by mass or more, It refers to the condition of washing once or twice in an aqueous solution of SSD of 5% by mass or less at a temperature of 42° C. or more and 55° C. or less for 5 minutes or more and 15 minutes or less. Further, the homology of the nucleotide sequence of RNA(2) to the completely complementary sequence with the nucleotide sequence of RNA(1) is preferably 90% or higher, more preferably 92% or higher, and even more preferably 95% or higher. A person skilled in the art can easily determine nucleotide sequence homology using commercially available sequence analysis software.

In one aspect of the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention, RNA (1) and RNA (2) are contained independently, and these form a double-stranded RNA, that is, siRNA. .

In another aspect of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention, a single-stranded RNA in which RNA (1) and RNA (2) are bound via a linker RNA is included as an active ingredient, The single-stranded RNA, ie shRNA, has a hairpin structure, RNA(1) and RNA(2) are hybridized, and the linker RNA has a circular structure.

siRNA containing RNA(1) and RNA(2), and shRNA containing RNA(1), linker RNA and RNA(2) may be chemically synthesized or synthesized in vitro using T7 RNA polymerase. good.

The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention may contain only one type of RNA (1), or the above siRNA or the above shRNA as an active ingredient, or may contain two or more types. be good. When two or more types are included, the number is preferably 2 or more and 8 or less, and more preferably 3 or more and 5 or less.

The dosage form of the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention is not particularly limited as long as the active ingredient is delivered to the target site. . Although water is preferable as a solvent for these preparations, it is preferable to use physiological saline, PBS, serum albumin solution, or the like so that the preparations are finally an isotonic or approximately isotonic solution. Alternatively, the RNA of the present invention may be directly or indirectly bound to folic acid. Since folate receptors are highly expressed on the surface of pancreatic cancer cell membranes, such RNA-folate complexes may be selectively delivered and bound to pancreatic cancer cells. Also, the RNA according to the present invention may be bound to chitosan nanoparticles. Chitosan nanoparticles have the effect of suppressing enzymatic degradation of siRNA intravenously administered into the body. Furthermore, it is preferable to bind the RNA of the present invention to the folic acid-chitosan composite nanoparticles.

The target site of the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention may be not only the pancreas but also lymph nodes and other organs to which pancreatic cancer cells have metastasized. Injections are preferable as the dosage form in order to more reliably deliver the active ingredient to the target site. Also, known drug delivery technologies such as liposomes and polymeric micelles may be used.

The DNA according to the present invention is characterized by having a nucleotide sequence encoding RNA (1). Furthermore, RNA(2) may be encoded at a separate site, or RNA(1), linker RNA and RNA(2) may be encoded consecutively.

The DNA according to the present invention may further contain regulatory regions such as promoters, enhancers, silencers, splicing donors, acceptors and poly(A) so that the nucleotide sequence (gene) encoding the above RNA (1) is expressed. good. In particular, it is preferable that the coding region has a promoter at the 5' end and a terminator for terminating transcription at the 3' end.

As described above, the DNA of the present invention has a base sequence that encodes RNA (1) and the like. Therefore, when introduced into a cell, RNA (1) and the like are produced in the cell, and the target mRNA is produced by RNA interference. is decomposed. Therefore, a vector into which the DNA of the present invention is inserted can serve as an active ingredient of pancreatic cancer cell invasion and metastasis inhibitors.

The vectors used in the present invention may be appropriately selected from known vectors such as plasmid vectors, detoxified virus vectors, and liposome vectors.

The vector according to the present invention can be transferred to target cells by known methods such as lipid-mediated carrier transport methods such as the lipofectamine method, chemical substance-mediated methods such as calcium phosphate, microinjection, injection into genes, and electroporation. can be delivered inwards.

In pancreatic cancer cells with high motility, invasiveness, and metastasis, mRNA that binds to and accumulates with IGF2BP3 is present in cell membrane protrusions. It was experimentally confirmed that this mRNA promotes invasion and metastasis of pancreatic cancer cells by being locally translated in cell membrane protrusions. In the present invention, inhibition of translation of the mRNA in cell membrane protrusions by RNA interference makes it possible to suppress invasion and metastasis of pancreatic cancer cells.

The dose and administration frequency of DNA or vector such as RNA(1) according to the present invention may be appropriately adjusted according to each dosage form, severity of patient, age, sex, body weight and the like.

This application claims the benefit of priority based on Japanese Patent Application No. 2014-138217 filed on July 4, 2014. The entire contents of the specification of Japanese Patent Application No. 2014-138217 filed on July 4, 2014 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be modified appropriately within the scope that can conform to the gist of the above and later descriptions. It is of course possible to implement them, and all of them are included in the technical scope of the present invention.

Example 1: Localization of IGF2BP3 in Metastatic Pancreatic Cancer Cells Immunocytochemical techniques were used to localize IGF2BP in pancreatic ductal adenocarcinoma (PDAC) cells. Two types of PDAC cells were used: moderately differentiated PDAC cell line S2-013 and undifferentiated PDAC cell line PANC-1.

(1) Culture of pancreatic cancer cells S2-013 strain, which is a subline of human PDAC cell strain SUIT-2, was provided by Dr. Takeshi Iwamura of Miyazaki University. In addition, the PDAC cell strain PANC-1 was purchased from the American Type Culture Collection. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL) containing 10% heat-inactivated fetal calf serum (FCS) in a humid atmosphere containing 5% CO ₂ .

(2) Confocal immunofluorescence microscopy A glass coverslip was coated with 10 μg/mL fibronectin (Sigma-Aldrich) for 1 hour at room temperature. The S2-013 cell line or PANC-1 cell line was plated on the fibronectin-coated coverslip and incubated for 5 hours. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, coated with blocking solution (3% BSA/PBS), and incubated with anti-IGF2BP3 primary antibody, etc. for 1 hour. . Further, secondary antibodies conjugated to fluorescent dyes Alexa488-, Alexa546-, Alexa594- or Alexa647-conjugated (Cytoskeleton) were used in the presence or absence of rhodamine-conjugated phalloidin (Cytoskeleton). Several primary antibodies were conjugated with green or red fluorophores using commercially available antibody labeling technology (Life technologies "Zenon"). Visualization was performed with a microscope ("Zeiss LSM 510 META microscope" manufactured by Carl Zeiss).Photographs of the obtained microscopic images are shown in Figures 1 and 2. In Figures 1 and 2, IGF2BP3 is labeled with a green dye, and actin filaments are stained red with phalloidin, and arrows indicate IGF2BP3 present in cell membrane protrusions.

(3) Immunoprecipitation The above S2-013 strain cells were incubated on fibronectin for 5 hours and added with lysis buffer (50 mM Tris (pH 7.4), 150 mM sodium chloride, 1 mM magnesium chloride, 0.5% NP-40, and Protease inhibitor cocktail tablets (Roche)) were used to lyse, and the resulting lysates were immunoprecipitated with 2 μg anti-IGF2BP3 antibody or rabbit IgG isotype control antibody and Dynabeads protein G (Dynal). To test the interaction of endogenous IGF2BP3 and G3BP, immune complexes were analyzed by Western blot. The results are shown in FIG.

(4) Results and Discussion Pancreatic ductal adenocarcinoma cells S2-013 strain or PANC-1 strain were cultured on fibronectin to promote the formation of cell membrane protrusions. It was accumulated in cell membrane protrusions where filaments were present. In addition, as shown in FIG. 2, IGF2BP3 was found to co-localize with G3BP, which is a marker protein for stress granules, in granules accumulated in cell membrane protrusions. Furthermore, as shown in Figure 3, extracts of S2-013 cell lines cultured on fibronectin were subjected to immunoprecipitation experiments with anti-IGF2BP3 or anti-G3BP antibodies, which showed that IGF2BP3 co-precipitated with G3BP. .

Stress granules contain 40S subunit ribosomal proteins and various translation initiation factors, and are involved in regulation of translation from mRNA to protein. Stress granules also contain several RNA-binding proteins, which are involved in stabilizing the encapsulated mRNA. Therefore, IGF2BP3, as an RNA-binding protein localized in stress granules, is thought to regulate translation of specific mRNAs in cell membrane protrusions.

Example 2: Knockdown test of IGF2BP3 in metastatic pancreatic cancer cells (1) IGF2BP3-RNA interference In order to clarify the effect of IGF2BP3 on invasion and metastasis of pancreatic cancer cells, siRNA specific to IGF2BP3 is expressed. Using the vector, clonal cells were established in which the production of IGF2BP3 in S2-013 cells was constitutively suppressed.

Specifically, scrambled negative control (OriGene Technologies "TR30013") or siRNA targeting IGF2BP3 mRNA (OriGene Technologies "TG312221") is incorporated into rapidly proliferating GP2-293 packaging cells (Clontech). They were then transiently infected with the pGFP-V-RS vector (OriGene Technologies), which generates replication-deficient lentiviruses. Along with transient infection of the vector into packaging cells, replication-defective virus was obtained and used to infect S2-013 cell lines. Infected S2-013 strain cells were transferred to flasks 48 hours after infection and then cultured in DMEM medium containing 0.5 μg/mL puromycin to stably express siRNA targeting IGF2BP3 mRNA. A clonal cell derived from the S2-013 cell line was established. These cells were cultured for an additional 10 days after reaching confluency. During culture, the medium was replaced with a new one every two days. Only cells in which IGF2BP3 suppression was observed by Western blot analysis were used. IGF2BP3-RNAi clones (siIGF-1 and siIGF-2) of S2-013 cells infected with siRNA targeting IGF2BP3 and control RNAi clones (Scr-1 and Scr-2) were treated with an anti-IGF2BP3 antibody. FIG. 4 shows the results of analysis by Western blotting. Knockdown of IGF2BP3 was confirmed by immunoblotting, as shown in FIG.

(2) Wound Area Motility Assay For each wound healing assay, a cross-shaped incision was made as a wound area in a confluent monolayer of cells using a plastic pipette tip. Photographs were taken using a phase-contrast microscope after several markings were made on the wound area to be observed. Cells migrating into the marked wound area were observed and photographed continuously for 1 to 8 hours. A photograph of the cells is shown in FIG. The degree of occlusion of the wound area by migrated cells was also quantified by counting migrated cells. Quantitative values are shown in FIG. In FIG. 6, "*" indicates a significant difference with p<0.001 in the t-test from Scr-1 or Scr-2.

As the results shown in FIG. 5, suppression of IGF2BP3 gene expression inhibited cell migration to the wound area of the confluent cultured strain S2-013. Also, as shown in FIG. 6, the number of cells migrating to the wound area was significantly reduced by inhibiting the expression of the IGF2BP3 gene by RNA interference.

(3) Immunocyte Staining of Leading Regions of Cell Motility Using a plastic pipette tip, a confluent S2-013 cell line was cross-shaped incised, and then migration of the cells to the wound area was promoted. After 4 hours, cells were immunostained with primary antibody and incubated with fluorophore-conjugated secondary antibody as in Example 1 above. The leading edge of the movement of cells that started to migrate into the wound area was observed with a confocal laser scanning fluorescence correlation spectroscopy microscope (Carl Zeiss "Zeiss LSM 510 META microscope"). The results are shown in FIG. As shown in FIG. 7, IGF2BP3 was accumulated in the cell membrane protrusions formed at the leading edge of the cells of the S2-013 strain that started to migrate.

(4) Matrigel Invasion Assay 4.0×10 ⁴ cells suspended in serum-free medium were seeded in the upper chamber of Matrigel Invasion Chamber (24-well plate, pore size: 8 μm, Becton Dickinson). A solvent containing 5% chemoattractant was added to the lower chamber. Cells were incubated in the upper chamber for 20 hours. Three independent areas were then observed microscopically and cells that invaded the lower chamber were counted. The same experiment was repeated three times, and S2-013 control clones (Scr-1 and Scr-2) that constantly expressed scrambled negative control siRNA and IGF2BP3-specific siRNA were constantly expressed to suppress IGF2BP3 expression. A comparison was made between the generated S2-013 clones (siIGF-1 and siIGF-2). The results are shown in FIG. In FIG. 8, "*" indicates a significant difference with p<0.001 in t-test from Scr-1 or Scr-2.

As shown in FIG. 8, as a result of the Matrigel invasion assay using Matrigel Invasion Chamber, the invasion of the S2-013 strain (siIGF-1 and siIGF-2), in which the expression of IGF2BP3 was inhibited by RNA interference, was higher than that of the control (Scr -1 and Scr-2).

In addition, in order to amplify the cDNA encoding all amino acids of IGF2BP3, RT-PCR was performed using RNA of S2-013 cell line as a template. The resulting PCR product was inserted into the pCMV6-Entry vector (Origene) which generates a myc-DDK tag at the C-terminus. The resulting vector is hereinafter referred to as "IGF2BP3-pCMV6". In order to restore IGF2BP expression in siIGF-1 and siIGF-2 clone cells, IGF2BP3-pCMV6 was transfected into siIGF-1 and siIGF-2 clone cells using X-tremeGENE HP DNA Transfection Reagent (manufactured by Roche). rice field. Western blot analysis using an anti-IGF2BP3 antibody was performed 48 hours later. The results are shown in FIG. As shown in FIG. 9, the forced expression of exogenous IGF2BP was confirmed.

A mock control vector or IGF2BP3-pCMV6 was transiently introduced into control RNAi cells or IGF2BP3-RNAi cells, and after 48 hours, Matrigel invasion assay was performed in the same manner as above. The results of Western blot analysis using anti-IGF2BP3 antibody are shown in FIG. In FIG. 9, black arrows indicate endogenous IGF2BP3 and white arrows indicate exogenous IGF2BP3. "GAPDH" is glyceraldehyde-3-phosphate dehydrogenase as an internal control to indicate that equal amounts of each sample are used in Western blot analysis. Figure 10 shows the number of cells that invaded the lower chamber. “*” in FIG. 10 indicates that the number of cells transfected with IGF2BP3-pCMV6 is significantly higher than that of cells transfected with the mock vector, with a significance level of p<0.005 in the t-test. show.

As the results shown in FIGS. 9 and 10 show, transfection of IGF2BP3-pCMV6 into IGF2BP3-RNAi S2-013 cells abrogates the suppression of cell invasion caused by IGF2BP3-RNAi by restoring IGF2BP3 expression. It was found that

(5) Orthotopic Implantation of Mice and Tumor Cells 6-week-old sterile female athymic nude mice (BALB/cSlc-nu/nu) were purchased from Japan SLC Co., Ltd. and followed the animal care and use guidelines within Kochi University research institutions. handled. S2-013 control clones (Scr-1 and Scr-2) that constantly express scrambled negative control siRNA and S2-013 clones that constantly express IGF2BP3-specific siRNA and suppress IGF2BP3 expression (siIGF -1 and siIGF-2) were surgically and orthotopically implanted into the pancreas of each mouse at 8.0×10 ⁵ each to form tumors in the mouse pancreas. Forty-two days after transplantation, the mice were sacrificed and hematoxylin-eosin staining was performed to examine the presence or absence of invasion of the tumor formed in the mouse pancreas into the retroperitoneum and the presence or absence of metastasis to the lung and liver. Tumors formed in the mouse pancreas were also weighed. Table 1 shows the weighing results and the presence or absence of retroperitoneal infiltration and distant metastasis, and staining photographs are shown in FIG. In FIG. 11, "R" indicates cavity peritoneal tissue, "P" indicates muscle tissue, and "N" indicates normal tissue.

As the results shown in Table 1, the invasion of the tumor formed in the mouse pancreas into the retroperitoneum was observed in clone cells with suppressed IGF2BP3 expression (siIGF- 1 and siIGF-2) were significantly lower. In addition, pancreatic cancer tissue derived from control clone cells metastasized to the liver and lung, whereas pancreatic cancer tissue derived from clone cells in which IGF2BP3 expression was suppressed did not metastasize.

Moreover, as shown in FIG. 11, the control clone cell-derived pancreatic cancer tissue infiltrated the entire mouse pancreatic tissue. Notably, the boundary between tumor and normal tissue was not clear in control samples. In contrast, most of the pancreatic cancer tissue derived from clone cells in which IGF2BP3 expression was suppressed was encased in mouse stromal cells and clearly separated from normal mouse pancreatic tissue. In mice transplanted with control clone cells, the peritoneal surface was covered with a relatively thick layer of cancer cells disseminated from control clone cell-derived pancreatic cancer tissue, and the cancer cells invaded into the muscle layer. was On the other hand, no cancer cells were observed in most of the peritoneum of mice transplanted with clonal cells in which IGF2BP3 expression was suppressed. Furthermore, mice transplanted with control clone cells showed metastatic lesions in the lung and liver, whereas mice transplanted with clone cells in which IGF2BP3 expression was suppressed showed no metastatic lesions in the lung and liver. rice field. From the above results, it can be said that suppressing the expression of IGF2BP3 in pancreatic cancer cells can completely prevent metastasis to the lung and liver, and that IGF2BP3 can be a novel therapeutic target molecule for pancreatic cancer. .

(6) Discussion The above results show that IGF2BP3 particularly promotes the invasion and metastasis of pancreatic cancer cells, and that a reduction in the expression level of IGF2BP3 is associated with 1) an increase in tumors in the pancreas in pancreatic tumorigenesis in vivo. 2) invasion into adjacent tissues of the pancreas, and 3) metastasis to other tissues.

Example 3: Role of IGF2BP3 in formation of cell membrane protrusions in pancreatic cancer cells Using a confocal microscope, the polymerization of actin filaments and the three-dimensional structure of cell membrane protrusions in fibronectin-stimulated S2-013 cells were investigated. The detailed conditions of the confocal microscope were the same as in Example 1 above. The results are shown in Figures 12-14.

FIG. 12 is a photograph of actin filaments formed directly under the cell membrane. As shown in FIG. 12, S2-013 in which IGF2BP3 expression was suppressed by constantly expressing IGF2BP3-specific siRNA compared to the S2-013 control clone (Scr-1) that constantly expressed the scrambled negative control siRNA. The amount of polymerized actin filaments in the clone (siIGF-1) is low. These results indicate that transfection of IGF2BP3-pCMV6 into IGF2BP3-RNAi S2-013 cell line restores IGF2BP3 expression and restores actin filament polymerization just below the plasma membrane.

FIG. 13 shows the percentage of cells with cell membrane protrusions in all cells. As shown in FIG. 13, the proportion of cells with cell membrane protrusions in the clone (siIGF-1) in which IGF2BP3 expression was suppressed was significantly lower than in the control clone (Scr-1). Furthermore, the introduction of IGF2BP3-pCMV6 into the IGF2BP3-RNAi S2-013 cell line restored the expression of IGF2BP3 and restored the actin filament just below the cell membrane.

FIG. 14 shows photographs of control clone cells (Scr-1) and clone cells in which IGF2BP3 expression was suppressed (siIGF-1) using a phase-contrast microscope. As shown in FIG. 14, the control clone cells were spindle-shaped and exhibited a fibroblast-like morphology, while the clone cells in which IGF2BP3 expression was suppressed were rounded and exhibited an epithelial cell-like morphology. Since the fibroblast-like morphology often has a stronger tendency to invasion and metastasis than the epithelial cell-like morphology, such results suggest that clone cells with suppressed IGF2BP3 expression show higher invasion than control clone cells without suppressed IGF2BP3 expression. Indicates low metastatic potential.

Example 4: Identification of Transcripts Bound to IGF2BP3 (1) RNA Immunoprecipitation, Sequencing and Bioinformatics Analysis S2-013 line cells were plated on fibronectin and incubated for 5 hours. Cells were washed twice with PBS and then lysed with NP2 buffer containing 50 mM Tris (pH 7.4), 150 mM sodium chloride, 1 mM magnesium chloride, 0.5% NP-40, and RNase inhibitor (Roche). Extracts were immunoprecipitated by treatment with Dynabeads Protein G (Dynal) and anti-IGF2BP3 antibody or rabbit IgG isotype control antibody for 2 hours at 4°C. Beads were pelleted on a magnetic rack (Dynal). The precipitated complex was treated with proteinase K, extracted with a phenol-chloroform mixed solvent, and then the nucleic acid was precipitated with ethanol. The precipitated nucleic acids were then treated with DNase I (Promega) and RNA was purified using the RNeasy kit (Qiagen). The identification of the RNA samples obtained by the next-generation sequencer was entrusted to Hokkaido System Science, Inc., and the bioinformatics analysis of the obtained data was entrusted to World Fusion. A scatter plot of the concentration of mRNA contained in the IGF2BP3 immunoprecipitated sample or the control IgG immunoprecipitated sample is shown in FIG. Also, linear regression plots of the numbers of ribosomal RNA (rRNA) and small nuclear RNA (snRNA) between the IGF2BP3 immunoprecipitated sample and the control IgG immunoprecipitated sample are shown in FIGS. 16(1) and 16(2), respectively. ). In FIG. 16, the dotted line indicates the straight line of x=y.

As shown in FIGS. 15 and 16, the validity of sequencer analysis using RNA samples from which IGF2BP3 immunoprecipitation samples and control IgG immunoprecipitation samples were purified was objectively demonstrated.

In the above experiment, the expression ratio was calculated by performing unique processing after surrogate processing of RPKM values. Specifically, RPKM values were converted to a log ₂ [(RPKM value of IGF2BP3 sample)/(RPKM value of isotype control sample)] ratio and RNA >1.0 (>2-fold as expression ratio) was converted to IGF2BP3 were considered to be transcripts bound to As a result, we identified 2,826 RNAs with significantly higher concentrations in anti-IGF2BP3 immunoprecipitates compared to rabbit IgG isotype control immunoprecipitates.

(2) RT-PCR
Among the IGF2BP3-binding mRNAs identified in (1) above, two mRNAs, ADP-ribosylation factor 6 (ARF6) and Rhoguanine nucleotide exchange factor 4 (ARHGEF4), were confirmed to bind to IGF2BP3. Specifically, using StrataScript reverse transcriptase (Agilent) and oligo d(T) _12-18 primer, anti-IGF2BP3 antibody, rabbit IgG isotype control antibody or negative control from S2-013 cell lysate inducing cell membrane protrusion formation RNA immunoprecipitated with anti-CD63 antibody was purified and RT-PCR was performed. Ubiquitin C mRNA was used as an internal quantitative control. The results are shown in FIG. As shown in Figure 17, the two transcripts formed immunoprecipitation complexes with anti-IGF2BP3 antibody, but not with isotype control and anti-CD63 antibodies.

(3) Immunofluorescence QuantiGene View RNA plate-based for in situ hybridization of each sequence-specific fluorescent probe to the endogenous ARF6 mRNA and ARHGEF4 mRNA present in the S2-013 cells that induced the formation of cell membrane protrusions. An assay kit (manufactured by Panomics) was used. Fibronectin-stimulated S2-013 cells were fixed with 8% formaldehyde, dehydrated with 50%, 70% and 100% ethanol, and left at 4° C. overnight. Cells were then dehydrated again, permeabilized and hybridized with fluorescent probes. Target mRNAs were ARF6 and ARHGEF4, and control RNA was ubiquitin C mRNA. After in situ hybridization, sections were washed with PBS, blocked with blocking buffer, 4% goat serum in PBS for 1 hour, and incubated with anti-IGF2BP3 antibody in blocking buffer for 3 hours. Secondary antibodies were allowed to act in blocking buffer at room temperature for 30 minutes, and nuclei were stained with DAPI for 3 minutes and embedded in Aqua Polymount (manufactured by Polysciences). Confocal fluorescence images were obtained using a confocal laser scanning fluorescence correlation spectroscopy microscope ("Zeiss LSM 510 META microscope" manufactured by Carl Zeiss). The results are shown in FIG.

As shown in FIG. 18, ARF6 mRNA and ARHGEF4 mRNA co-localized with IGF2BP3 in granules present in the cytoplasm, which assembled in cytoplasmic processes. In contrast, control ubiquitin C mRNA did not co-localize with IGF2BP3. IGF2BP3 granules were also concentrated around the nucleus. These granules are considered to have been transported from the perinucleus to the cell membrane projections together with ARF6 mRNA and ARHGEF4 mRNA.

(4) Conclusion The above experimental results indicate that stress granules containing IGF2BP3 and IGF2BP3-binding mRNA are accumulated in cell membrane protrusions.

Example 5: Confirmation of the relationship between local translation of mRNA in cell membrane protrusions and motility invasion of pancreatic cancer cells (1)
Control RNAi cells (Scr-1) and IGF2BP3-RNAi cells (siIGF-1) of strain S2-013 were incubated on fibronectin and stained with anti-ARF6 antibody (green) or anti-ARHGEF4 antibody (green). Actin filaments were labeled in red using phalloidin. Nuclei were also stained blue with DAPI. The results are shown in FIG.

As shown in FIG. 19, in the IGF2BP3-RNAi cells, the production of ARF6 and ARHGEF4 decreased only in cell membrane protrusions as the expression of IGF2BP3 was suppressed.

(2)
The myc-tagged IGF2BP3 expression restoration construct vector was introduced into IGF2BP3-RNAi cells (siIGF-1). After 48 hours, cells were incubated on fibronectin. Cells were stained with anti-myc antibody (green) or anti-ARF6 antibody (purple). Actin filaments were labeled in red using phalloidin. Nuclei were also stained blue with DAPI. The results are shown in FIG.

As shown in FIG. 20, restoring IGF2BP3 gene expression in IGF2BP3-RNAi cells (siIGF-1) regenerated ARF6 protein in cell membrane protrusions.

(3)
The experiment was performed in the same manner as in (2) above, except that the anti-ARHGEF4 antibody (purple) was used instead of the anti-ARF6 antibody. The results are shown in FIG.

As shown in FIG. 21, similar to the production of ARF6 protein, restoration of IGF2BP3 gene expression in IGF2BP3-RNAi cells (siIGF-1) regenerated ARHGEF4 protein in cell membrane protrusions.

(4)
Functional analysis of ARF6 protein and ARHGEF4 protein in pancreatic cancer cells was performed. Double-stranded RNA consisting of RNA having the base sequences of SEQ ID NO: 1 and SEQ ID NO: 5 corresponding to the partial base sequences of ARF6 RNA and ARHGEF4 RNA, respectively, and its complementary RNA (hereinafter referred to as "siARF6" and "siARHGEF4", respectively) ), or control siRNA oligos were introduced into S2-013 cell lines. These cells were cultured on fibronectin and analyzed for peripheral actin filaments in cell membrane protrusions. 48 hours after vector introduction, the production of ARF6 and ARHGEF4 proteins was analyzed by Western blotting. The results are shown in FIG. In FIG. 22, "Scr", "siARF6" and "siARHGEF4" indicate the results of cells transfected with the control siRNA oligo, the siRNA having the nucleotide sequence of SEQ ID NO:1, and the siRNA having the nucleotide sequence of SEQ ID NO:5, respectively.

As shown in FIG. 22, the production of ARF6 protein or ARHGEF4 protein was clearly suppressed in ARF6-RNAi cells or ARHGEF4-RNAi cells compared to control cells.

In addition, the ratio of cells in which cell membrane protrusions were formed between cells (siARF6 or siARHGEF4) in which the translation of ARF6-mRNA or ARHGEF4-mRNA was inhibited by RNA interference and control cells (Scr) was calculated. The results are shown in FIG. In FIG. 23, "*" indicates a significant difference at p<0.001 in the t-test.

As shown in FIG. 23, inhibition of translation of ARF6-mRNA and ARHGEF4-mRNA significantly inhibited the formation of fibronectin-mediated cell membrane protrusions.

Further, confocal micrographs of cells in which translation of ARF6-mRNA or ARHGEF4-mRNA was inhibited by RNA interference (siARF6 or siARHGEF4) and control cells (Scr) are shown in FIGS. 24 and 25, respectively.

As shown in FIGS. 24 and 25, in pancreatic cancer cells in which the translation of ARF6-mRNA and ARHGEF4-mRNA was inhibited, the formation of peripheral actin filaments indicating the formation of cell membrane protrusions was clearly suppressed.

(5)
In the same manner as in (4) above, by transiently introducing siRNA oligos specific to the respective mRNAs of ARF6 and ARHGEF4 and control siRNA oligos into S2-013 cell lines and PANC-1 cell lines, control RNAi cells, ARF6-RNAi cells and ARHGEF4-RNAi cells were obtained.

Using each cell, a wound area motility assay was performed under the same conditions as in Example 2(2) above, and the number of cells that migrated to the wound area was counted. The results are shown in FIG. In addition, FIG. 26 shows the average values of 4 examples, and "*" indicates a significant difference at p<0.001 in the t-test.

As the results shown in FIG. 26 , it was revealed that the motility of pancreatic cancer cells was significantly suppressed when the translation of ARF6 mRNA and ARHGEF4 mRNA was inhibited.

(6)
Using the cells of Example 5(5) above, Matrigel invasion assay was performed in the same manner as in Example 2(4) above, and the cells that invaded from the upper chamber to the lower chamber were counted. A similar experiment was repeated four times, and the average value was calculated. The results are shown in FIG. In FIG. 27, "*" indicates a significant difference with p<0.001 in the t-test.

As shown in FIG. 27, when the translation of ARF6 mRNA and ARHGEF4 mRNA was inhibited, invasiveness was significantly suppressed compared to the control (Scr).

(7) Conclusion Based on the above experimental results, mRNA bound to IGF2BP3 is selectively translated locally at cell membrane protrusions to produce proteins. It is thought to be involved in the motility invasion of cancer cells.

Example 6: In vitro experiment using folic acid chitosan nanoparticle-added siRNA for confirming the effects of the present invention (1) Experiment to confirm cellular uptake of siRNA It was examined whether or not chitosan nanoparticle-attached siRNA was taken up into cells. Chitosan nanoparticles suppress enzymatic degradation of siRNA intravenously administered into the body, and folic acid enables siRNA to bind to folate receptors highly expressed on the surface of pancreatic cancer cell membranes.

As a control that is not mRNA in pancreatic cancer cell membrane protrusions, scrambled siRNA was added with folic acid chitosan nanoparticles and fluorescently labeled with FITC. The control siRNA was added to the culture medium of the pancreatic cancer cell line S2-013. After 48 hours, uptake into S2-013 cells was observed with a confocal microscope. The results are shown in FIG.

As shown in FIG. 28, it was confirmed that the control siRNA to which folic acid-chitosan nanoparticles were added was taken up by the S2-013 cells and localized in granular form in the cytoplasm.

(2) Confirmation experiment of knockdown effect by folic acid-chitosan nanoparticle-added siRNA according to the present invention Next, the knockdown effect of siRNA against CCDC88A and siRNA against WASF2 was examined. The present inventor recently revealed that proteins encoded by CCDC88A and WASF2 are involved in cell morphological changes, motility, or invasion by forming complexes with different actin-binding proteins. CCDC88A siRNA contains the base sequence of SEQ ID NO:57. WASF2 siRNA comprises the base sequence of SEQ ID NO:73.

Control siRNA, CCDC88A siRNA, and WASF2 siRNA to which folic acid chitosan nanoparticles were added were each added to the culture medium of strain S2-013, and the cells were collected after 48 hours. A semi-quantitative RT-PCR method was performed using the harvested cellular RNA. Western blotting was also performed using cell lysates. The semi-quantitative RT-PCR results are shown in FIG. 29A, and the Western blot results are shown in FIG. 29B. As the results shown in FIG. 29, the knockdown effect of CCDC88A siRNA and WASF2 siRNA on each mRNA could be confirmed by both semi-quantitative RT-PCR and Western blotting.

(3) Confirmation experiment of cell motility inhibitory effect by folic acid-chitosan nanoparticle-added siRNA according to the present invention Control siRNA, CCDC88A siRNA, and WASF2 siRNA to which folic acid-chitosan nanoparticles were added were each added to the culture medium of S2-013. , Matrigel invasion assay was performed 48 hours later. A graph showing the number of cells that migrated from the upper chamber to the lower chamber in the Matrigel invasion assay is shown in FIG. In FIG. 30, "*" indicates a significant difference from control at p=0.00073 in t-test, and "**" indicates a significant difference at p=0.00852. .

As shown in FIG. 30, cell invasion was significantly suppressed in S2-013 cells into which CCDC88A siRNA or WASF2 siRNA was transfected compared to S2-013 cells into which control siRNA was transfected.

From the above results, the folic acid chitosan nanoparticle-added siRNA according to the present invention added to the medium of cultured cells inhibits the expression of mRNA that is taken up into S2-013 cells and involved in cell motility. could be confirmed.

Example 7: In vivo experiment using folic acid chitosan nanoparticle-added siRNA to confirm the effect of the present invention Pancreatic cancer invasion and metastasis model in which human pancreatic cancer cell line S2-013 was transplanted into the pancreas of 6-week-old nude mice was used. Specifically, on the first day, 12 nude mice were anesthetized by inhalation anesthesia using isoflurane, and the abdomen was opened to expose the pancreas. One million human pancreatic cancer cells S2-013 were suspended in 0.1 mL of PBS and implanted into the pancreas of each mouse. As in the in vitro experiment of Example 6 above, siRNAs against CCDC88A and WASF2 and scrambled siRNA as a control were used. Each synthesized siRNA was attached to folic acid chitosan nanoparticles. Each group of 4 mice implanted with human pancreatic cancer cells was intravenously administered with each folic acid-chitosan nanoparticle-added siRNA into the tail vein once a week. Systemically administered siRNA to mice is passively delivered to human pancreatic cancer tissue via chitosan nanoparticles, and folic acid is expected to promote efficient endocytosis via folate receptors on pancreatic cancer cells. can. A total of five siRNA injections were administered, during which the S2-013 cells implanted into the mouse pancreas on the first day formed tumors and metastasized distantly to the liver and lungs. Tumors exposed from the pancreas also cause peritoneal dissemination and retroperitoneal invasion.

In order to confirm whether the folic acid chitosan nanoparticle-added siRNA that was intravenously administered to the S2-013 tumor in the pancreas of the mouse is accumulated, one week after a total of five siRNA intravenous injections, one animal in each group folate-chitosan nanoparticle-loaded siRNA labeled with Alexa546 was administered intravenously. 3 hours or 24 hours after administration of the labeled siRNA, accumulation in the tumor was observed using an in vivo imaging system (“IVIS spectrum imaging system” manufactured by Caliper Life Science) under inhalation anesthesia. The results of siRNA accumulation in mice are shown in FIG. 31A, and the results of siRNA accumulation in excised tumors are shown in FIG. 31B.

As shown in FIG. 31, it was confirmed that all of the control siRNA, CCDC88A siRNA and WASF2 siRNA accumulated in the S2-013 cell tumor formed in the mouse pancreas. Importantly, the accumulation of control siRNA in S2-013 tumors was markedly increased 24 hours after administration of labeled folate-chitosan nanoparticles-loaded control siRNA compared to 3 hours. Each siRNA did not accumulate in the excised mouse heart, but specifically accumulated in the S2-013 tumor. Mice to which control siRNA was administered tended to have multiple peritoneal disseminations, and specific accumulation of control siRNA in the peritoneally disseminated tissues was observed. In addition, fluorescence development of primary pancreatic cancer lesions in mice administered with CCDC88A siRNA or WASF2 siRNA was weaker than in mice administered with control siRNA. The reason for this will be discussed with reference also to FIG. 32 below.

Mice were dissected after imaging with an in vivo imaging system. The presence or absence of peritoneal dissemination in the mouse peritoneal cavity was confirmed, and the primary tumor, liver and lung were formalin-fixed. Each sample was stained with hematoxylin/eosin, and the presence or absence of retroperitoneal infiltration from the primary pancreatic cancer focus and metastasis to the lung/liver was compared with a control siRNA-administered mouse group. A magnified photograph of the primary tumor tissue is shown in FIG. 32, and Table 2 shows the results of confirming the tumor in each sample.

As the results shown in Table 2, retroperitoneal infiltration was significantly suppressed in the mouse group to which CCDC88A siRNA and WASF2 siRNA according to the present invention were administered. Peritoneal dissemination also tended to be less in the group of mice administered with CCDC88A siRNA and WASF2 siRNA. Of particular note, no liver and lung metastases were observed in the group of mice that received CCDC88A siRNA. In the WASF2 siRNA-administered mouse group, there was no difference in lung metastasis from that in the control, but no liver metastasis was observed. In addition, as shown in the results shown in FIG. 32, the tumors in the mouse group administered with the control siRNA had few necrotic areas and were solid tumors, whereas the tumors in the mouse group administered with CCDC88A siRNA and WASF2 siRNA showed S2 There were many necrotic parts in the -013 tumor. Moreover, in FIG. 31, the fluorescence development of primary pancreatic cancer lesions in mice administered with CCDC88A siRNA or WASF2 siRNA was weaker than in mice administered with control siRNA. The reason for this is thought to be that there were many necrotic areas in the S2-013 tumors of mice administered with CCDC88A siRNA or WASF2 siRNA. This is considered to be the effect of folic acid-chitosan nanoparticle-added siRNA.

Claims (4)

A pancreatic cancer cell invasion and metastasis inhibitor comprising RNA (1) having one or more nucleotide sequences selected from SEQ ID NOs: 29-32 and 77-80. The inhibitor of pancreatic cancer cell invasion and metastasis according to claim 1, further comprising RNA (2) having a sequence that hybridizes with said RNA (1) under stringent conditions. The inhibitor of pancreatic cancer cell invasion and metastasis according to claim 2, wherein said RNA (1) and said RNA (2) comprise a hybridized double-stranded RNA. The RNA (1), the RNA (2), and a linker RNA that binds the RNA (1) and the RNA (2), wherein the RNA (1) and the RNA (2) hybridize 3. The inhibitor of pancreatic cancer cell invasion and metastasis according to claim 2, wherein the linker RNA contains a single-stranded RNA having a circular structure.

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