WO2016002844A1 - Agent for inhibiting invasion and metastasis of pancreatic cancer cells - Google Patents

Abstract

　The purpose of the present invention is to provide: an agent for inhibiting the invasion and metastasis of pancreatic cancer cells, the agent being capable of particularly effectively suppressing the invasion and metastasis of pancreatic cancer cells; DNA encoding RNA that is the active ingredient of this agent for inhibiting the invasion and metastasis of pancreatic cancer cells; and a vector including this DNA that can be used to suppress the invasion and metastasis of pancreatic cancer cells. The agent for inhibiting the invasion and metastasis of pancreatic cancer cells according to the present invention is characterized by including RNA (1) having one or more base sequences selected from SEQ ID NOS: 1-92.

Description

Pancreatic cancer cell invasion metastasis inhibitor

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

“Tumor” refers to a cell that proliferates abnormally, and refers to a state in which cell proliferation continues even if the cause of the abnormal growth disappears or is removed. Among tumors, benign tumors grow slowly and do not metastasize. Therefore, in general, there is no problem if it is excised, and it can be said that there is no difference in life even if it is left without being excised. On the other hand, unlike benign tumors, malignant tumors, that is, cancers, proliferate rapidly and metastasize to lymph nodes and other organs. Therefore, for example, even if removed by surgical operation, cancer cells that remain even slightly, or cancer cells that have already metastasized to lymph nodes or other organs may start to proliferate again. Therefore, cancer has a poor prognosis after treatment is completed, and the survival rate of each cancer has been investigated after 5 years. Generally, after 5 years since the cancer disappeared by treatment. If there is no recurrence before, it is finally considered a cure.

Pancreatic cancer is said to have the worst prognosis among cancers. This is because the pancreas is a retroperitoneal organ and is difficult to detect at an early stage, and the pancreatic cancer cells have extremely high motility. Invasion into the digestive tract, nerves, etc., metastasis to nearby lymph nodes, and distant metastasis to the liver.

As mentioned above, the reason why cancer is a malignant tumor is that it infiltrates and metastasizes to other tissues in particular, and it is thought that the prognosis will be good if there is no invasion and metastasis.

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

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

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

Patent Document 3 discloses an invention for suppressing tissue infiltration of leukemia cells by inhibiting the expression of CD43, which is a cell surface antigen, and a gene involved in the expression of a selectin ligand sugar chain expressed on CD43 by RNA interference. Has been.

Patent Documents 4 and 5 disclose siRNAs that inhibit ARF6 and AMAP1 / PAG2 / ASAP1, which are proteins related to motility and invasiveness of cancer cells, 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 usually present in nucleolus and is an insulin-like growth factor. It binds to the 5 ′ UTR of the 5 ′ untranslated region 3 of the RNA of II and inhibits the translation of insulin-like growth factor II. In the invention described in Patent Document 6, a gene encoding IGF2BP3 is listed as one of genes whose overexpression is used for detection of cancer.

JP 2011-93896 A JP 2007-97421 A JP 2006-304716 A JP 2005-46064 A JP 2005-224149 A Special table 2011-516077 gazette

As described above, various techniques for suppressing cancer cell infiltration and metastasis have been studied. However, there is no particular reason why pancreatic cancer cells tend to infiltrate and metastasize, and it has not always been effective in suppressing pancreatic cancer cell invasion and metastasis.

Therefore, the present invention provides a pancreatic cancer cell invasion and metastasis inhibitor capable of particularly effectively suppressing invasion and metastasis of pancreatic cancer cells, a DNA encoding RNA that is an active ingredient of the pancreatic cancer cell invasion and metastasis inhibitor, and It aims at providing the vector which contains the said DNA and can be used for suppression of invasion and metastasis of pancreatic cancer cells.

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

Hereinafter, the present invention will be described.

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

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

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

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

[5] The RNA (1), the RNA (2), and a linker RNA that binds the RNA (1) and the RNA (2). The RNA (1) and the RNA (2) are hybridized. The pancreatic cancer cell invasion and metastasis inhibitor according to [3] above, wherein the linker RNA contains a single-stranded RNA having a circular structure.

[6] DNA comprising a base sequence encoding RNA (1) having one or more sequences selected from SEQ ID NOs: 1 to 92.

[7] A vector comprising the DNA according to [6] above.

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

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

The 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 exists in cell membrane processes, and various mRNAs are present in this IGF2BP3. It binds and accumulates in cell membrane processes. It is considered that mRNA in such cell membrane processes is translated and then involved in the enhancement of motor invasion of pancreatic cancer cells through the formation of cell membrane processes, and as a result, plays an important role in invasive metastasis. Therefore, if the mRNA binding to IGF2BP3 in the plasma membrane process of pancreatic cancer cells is inhibited by RNA interference, invasion and metastasis of pancreatic cancer cells can be effectively suppressed. On the other hand, RNA interference according to the present invention inhibits the action of mRNA accumulated in cell membrane protrusions of pancreatic cancer cells, and thus is considered to be 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 those capable of safely suppressing invasion and metastasis of pancreatic cancer cells.

FIG. 1 is an immunocytochemical fluorescence-labeled photograph of human pancreatic ductal adenocarcinoma cells S2-013 and PANC-1 which have been promoted to form cell membrane protrusions by culturing on fibronectin. The green part shows the part labeled with the anti-IGF2BP3 antibody, and the red part shows the actin filaments labeled with phalloidin. The arrow indicates the part where IGF2BP3 is localized in the cell membrane projection, and the length of the bar is 10 μm. FIG. 2 shows a photograph of S2-013 cell line cultured on fibronectin and stained with anti-IGF2BP3 antibody (green) or anti-G3BP antibody (red), actin filaments labeled with phalloidin (purple), and nucleus. Is a photograph in which these results are integrated by staining with DAPI. The arrow points to IGF2BP3 localized in the cell membrane process. FIG. 3 is a photograph showing the results of immunoprecipitation of IGF2BP3 or G3BP from the extract of S2-013 strain cells cultured on fibronectin and analysis by Western blot using anti-IGF2BP3 antibody and anti-G3BP antibody. As an isotype control, a rabbit or mouse anti-IgG isotype control antibody was used. FIG. 4 shows IGF2BP3-RNAi S2-013 cell line (siIGF-1 and siIGF-2) into which siRNA targeting IGF2BP3 mRNA was introduced, and control RNAi S2-013 cell line (Scr-1 and Scr-2). It is a photograph which shows the result of having analyzed the protein expressed in <3> by Western blot. FIG. 5 is a photograph showing the results of a motility assay of the IGF2BP3-RNAi S2-013 cell line and the control RNAi S2-013 cell line into the wound area. FIG. 6 is a graph showing the number of cells migrated to the wound area in the motility assay of the IGF2BP3-RNAi S2-013 cell line and the control RNAi S2-013 cell line to the wound area. “*” Indicates that there is a significant difference at p <0.001 with respect to the control in the t-test. FIG. 7 shows that a wound region is formed by removing a part of the S2-013 cell line grown confluently in a monolayer with a pipette tip, and an advanced site of pancreatic cancer cells that begin to migrate toward the wound region is immunized. It is a dyed photograph. The green part shows the part dye | stained with the anti- IGF2BP3 antibody. The arrow indicates IGF2BP3 present in the cell membrane protrusion formed at the advanced site of the S2-013 cell line that has begun to migrate. The length of the bar is 10 μm. FIG. 8 is a graph showing the number of cells migrated from the upper chamber to the lower chamber in the Matrigel invasion assay of IGF2BP3-RNAi S2-013 cell line and control RNAi S2-013 cell line. “*” Indicates that there is a significant difference at p <0.001 with respect to the control in the t-test. FIG. 9 is a photograph showing the results of Western blot analysis using anti-IGF2BP3 antibody after introducing myc-tagged IGF2BP3 recovery construct or mock control vector into control RNAi cells or IGF2BP3-RNAi cells for 48 hours. The black arrow indicates endogenous IGF2BP3 and the white arrow indicates exogenous IGF2BP3. FIG. 10 is a graph showing the number of cells that have migrated from the upper chamber to the lower chamber after introducing a myc-tagged IGF2BP3 recovery construct or mock control vector into control RNAi cells or IGF2BP3-RNAi cells and then performing a bilayer chamber invasion assay. is there. “*” Indicates that the number of cells transfected with IGF2BP3-pCMV6 is significantly greater at p <0.005 in the t-test versus cells transfected with the mock vector. FIG. 11 is a photograph of a tumor formed in the pancreas stained with hematoxylin-eosin in a nude mouse transplanted with control RNAi S2-013 cell line or IGF2BP3-RNAi S2-013 cell line into the pancreas. “R” indicates luminal peritoneal tissue, “P” indicates muscle tissue, and “N” indicates normal tissue. FIG. 12 shows Z in a confocal immunofluorescence microscope test of fibronectin-stimulated control RNAi S2-013 cell line, IGF2BP3-RNAi S2-013 cell line, and IGF2BP3-RNAi S2-013 cell line restored to IGF2BP3 expression. -Stack (multi-layer capture) photo. The blue part shows nuclei stained with DAPI, and the red part shows actin filaments labeled with phalloidin. Arrows indicate actin filaments present in cell membrane processes. The lower and right panels each show a cross section of the yellow line. The length of the bar is 10 μm. FIG. 13 shows cell membrane protrusions relative to the total number of cells of control RNAi S2-013 cells stimulated with fibronectin, IGF2BP3-RNAi S2-013 cells, and IGF2BP3-RNAi S2-013 cells restored to IGF2BP3 expression. It is a graph which shows the ratio of a cell. “*” Indicates that there is a significant difference at p <0.001 with respect to the control RNAi S2-013 cell line or the IGF2BP3-RNAi S2-013 cell cell in which IGF2BP3 expression was restored in the t-test. FIG. 14 is a photograph of the morphology of control RNAi S2-013 cells and IGF2BP3-RNAi S2-013 cells stimulated with fibronectin using a phase contrast microscope. FIG. 15 shows a scatter plot of the concentration of mRNA contained in an IGF2BP3 immunoprecipitation sample or control IgG immunoprecipitation sample derived from an extract of S2-013 strain cells cultured on fibronectin. FIG. 16 shows ribosomal RNA ((1), rRNA) and small nuclear RNA ((1), rRNA) and an IGF2BP3 immunoprecipitation sample or control IgG immunoprecipitation sample derived from the extract of S2-013 cell line cultured on fibronectin ( (2) shows a linear regression diagram of the number of snRNA). In FIG. 16, a dotted line shows a straight line of x = y. FIG. 17 shows that ARF6 and ARGGEF4 are selected from mRNAs bound to IGF2BP3 in cell membrane processes, and an anti-IGF2BP3 antibody, isotype control IgG, or negative control CD63 is extracted from an extract of cell line S2-013 cultured on fibronectin. It is the photograph which refine | purified RNA from the immunoprecipitation sample using an antibody, and investigated the expression level of ubiquitin C mRNA which is ARF6, ARGGEF4, and internal quantitative control. FIG. 18 is an immunofluorescence photograph of IGF2BP3 in cell membrane processes, ARF6 and ARGGEF4 among mRNAs bound to the IGF2BP3, and ubiquitin C mRNA as a control. The length of the bar is 10 μm. FIG. 19 shows photographs of control RNAi S2-013 and IGF2BP3-RNAi S2-013 cells stimulated with fibronectin stained with anti-ARF6 antibody (green) or anti-ARHGEF4 antibody (green), and actin filaments as phalloidin (red). ) And a photograph in which nuclei are stained with DAPI (blue) and the results are integrated. Arrows indicate localization at cell membrane processes. The length of the bar is 10 μm. FIG. 20 shows a photograph of a myc-tagged IGF2BP3 recovery construct or mock control vector introduced into IGF2BP3-RNAi cells for 48 hours and then stained with anti-myc antibody (green) or anti-ARF6 antibody (purple), and actin filaments as phalloidin (red). ) And a photograph in which nuclei are stained with DAPI (blue) and the results are integrated. Arrows indicate localization at cell membrane processes. The length of the bar is 10 μm. FIG. 21 shows a photograph in which a myc-tagged IGF2BP3 recovery construct or mock control vector was introduced into IGF2BP3-RNAi cells for 48 hours and then stained with anti-myc antibody (green) or anti-ARHGEF4 antibody (purple), and actin filaments were treated with phalloidin (red). ) And a photograph in which nuclei are stained with DAPI (blue) and the results are integrated. Arrows indicate localization at cell membrane processes. The length of the bar is 10 μm. FIG. 22 is a photograph showing a Western blot result obtained by analyzing the amount of ARF6 or ARGGEF4 produced in the S2-013 cell line into which a control vector, or a vector that produces ARF6-RNAi or ARHGEF4-RNAi was introduced. FIG. 23 is a graph showing the ratio of cells in which cell membrane projections were formed in cells in which ARF6-mRNA or ARHGEF4-mRNA was knocked down by RNA interference and in control cells. FIG. 24 is a confocal micrograph of cells in which ARF6-mRNA was knocked down by RNA interference and control cells. Green indicates a labeled portion with anti-ARF6 antibody, red indicates an actin filament stained with phalloidin, and blue indicates a nucleus stained with DAPI. The length of the bar is 10 μm. FIG. 25 is a confocal photomicrograph of cells in which ARGGEF4-mRNA was knocked down by RNA interference and control cells. Green indicates a labeled portion with an anti-ARHGEF4 antibody, red indicates an actin filament stained with phalloidin, and blue indicates a nucleus stained with DAPI. The length of the bar is 10 μm. FIG. 26 shows ARF6-RNAi S2-013 cell, ARF6-RNAi PANC-1 cell, ARHGEF4-RNAi S2-013 cell, ARGGEF4-RNAi PANC-1 cell, control RNAi S2-013 cell, and control It is a graph which shows the cell number which moved to the wound area | region in the motility assay to the wound area | region of RNAi PANC-1 strain cell. “*” Indicates that there is a significant difference at p <0.001 with respect to the control in the t-test. FIG. 27 shows ARF6-RNAi S2-013 cell line, ARF6-RNAi PANC-1 cell, ARHGEF4-RNAi S2-013 cell, ARGGEF4-RNAi PANC-1 cell, control RNAi S2-013 cell, and control 2 is a graph showing the number of cells migrated from the upper chamber to the lower chamber in the matrigel invasion assay of RNAi PANC-1 strain cells. “*” Indicates that there is a significant difference at p <0.001 with respect to the control in the t-test. FIG. 28 shows a photograph of a folic acid chitosan nanoparticle alone and a folate chitosan nanoparticle-added control siRNA labeled with FITC fluorescence (green), added to the culture medium of S2-013 cells, and the cell nucleus stained with DAPI (blue). Then, the photographs are taken using a confocal fluorescence microscope, and these results are integrated. It can be seen that the folate chitosan nanoparticle-added siRNA is taken into the cells in the same manner as the folate chitosan nanoparticle alone. FIG. 29 shows the result of analysis of mRNA expressed in S2-013 cells incorporating CCDC88A siRNA and WASF2 siRNA according to the present invention to which folate chitosan nanoparticles were added and control siRNA by semi-quantitative RT-PCR ( A) A photograph showing the results (B) of analyzing the lysate of the cells by Western blotting. In any method, the knockdown effect on the mRNA of CCDC88A siRNA and WASF2 siRNA according to the present invention could be confirmed. FIG. 30 shows the results of a Matrigel invasion assay using CCDC88A siRNA and WASF2 siRNA according to the present invention to which folate chitosan nanoparticles are added, and a control siRNA. FIG. 31 shows that when a control siRNA, CCDC88A siRNA or WASF2 siRNA added with folate chitosan nanoparticles was administered to a human pancreatic cancer infiltrating / metastasized mouse model in which S2-013 cells were transplanted into the pancreas, the S2-013 tumor It is a photograph which shows siRNA accumulation | storage in. FIG. 32 is a photograph of the primary S2-013 tumor that was removed from the mouse after the photography of FIG. 31 and stained with hematoxylin and eosin.

The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention comprises RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 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 a sequence. It is one or more selected from the numbers 1 to 92.

The base sequences of SEQ ID NOs: 1 to 92 are part of the base sequence of mRNA bound to human insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) that is localized in the plasma membrane process of pancreatic cancer cells. If RNAs having these base sequences are used for RNA interference, it is possible to degrade the mRNA and inhibit its translation.

The present inventor has found that in pancreatic cancer cells with high motility, IGF2BP3, which is usually present in the nucleolus, gathers in the cell membrane process, and specific mRNA is bound to IGF2BP3 in the cell membrane process, It was experimentally proved that if the mRNA is inhibited by RNA interference, invasive metastasis of pancreatic cancer cells can be suppressed very effectively. 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 that of insulin, and causes a mitogenic reaction in the cell culture similar to insulin. IGF-2 is believed to be the first growth factor required for early development, secreted by the brain, kidneys, pancreas and muscle in mammals and acts more specifically than IGF-1 and in adults by insulin It can be seen at a concentration 600 times that of. The function of IGF is regulated by a family of genes known as IGF binding proteins, but IGF gene expression is regulated by proteins that bind to its mRNA.

IGF2BP3 is one of the regulators of IGF gene expression and is normally present in the nucleolus, whereas the present inventor has found that the plasma membrane is a site essential for motility in pancreatic cancer cells. It has been 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, motility is lost, invasion is significantly suppressed, and metastasis does not occur.

In RNA interference, siRNA or shRNA is used. Even if these RNAs are introduced into cells, they are safe because they are not integrated into chromosomes and do not cause mutation. Moreover, siRNA is relatively easy to synthesize chemically 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 base sequence of a target mRNA and an antisense RNA that can hybridize with the sense RNA are hybridized. Usually, the 3 ′ end remains OH. The 5 ′ end may be phosphorylated, and the 3 ′ end side may protrude from 1 base to 4 bases. A specific protein binds to the 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 the sense RNA, and cleaves the mRNA by RNaseIII-like enzyme activity.

shRNA is composed of single-stranded RNA, and a sense RNA region that is homologous to the partial base sequence of the target mRNA and an antisense RNA region that can hybridize with the sense RNA are linked by a linker RNA that exhibits a circular structure, and is a hairpin as a whole. The structure has a shape. The shRNA may have a 3 ′ end protruding from 1 to 4 bases, and the 3 ′ protruding end may be composed of DNA. shRNA is degraded in the cell and causes RNA interference in the same way as siRNA.

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

In the present invention, “having (with base sequence)” means that RNA has only a predetermined base sequence, or may have other base sequences in addition to the predetermined base sequence. However, it is preferable that the base sequence of the RNA is substantially the same as the predetermined base sequence except for the modification of the 5 ′ end as described above and the protruding RNA or protruding DNA at the 3 ′ end.

The nucleotide sequence of RNA (1) is particularly preferably at least one selected from SEQ ID NOs: 57 to 60 or at least one selected from SEQ ID NOs: 73 to 76. The base sequences of SEQ ID NOs: 57 to 60 are a part of the base sequence of CCDC88A, and the base sequences of SEQ ID NOs: 73 to 76 are a 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 inventor has experimentally found that, among mRNAs contained in cell membrane protrusions of pancreatic cancer cells, CCDC88A and WASF2 have the highest ability to enhance invasion and metastasis of pancreatic cancer cells. Thus, the preferred RNA (1) suppresses the functions of CCDC88A and WASF2 and can most effectively inhibit invasion and metastasis of pancreatic cancer cells.

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

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, “stringent conditions” means a sodium chloride concentration of 150 mM or more and 900 mM or less, a sodium citrate concentration of 15 mM or more and 90 mM or less, that is, 1 to 6 × SSC, 0.1 mass% or more, 0. This refers to the condition of washing once or twice in an aqueous solution of 5% by mass or less of SSD at a temperature of 42 ° C. or more and 55 ° C. or less for 5 minutes or more and 15 minutes or less. In addition, the homology of the base sequence of RNA (2) to the completely complementary sequence with the base sequence of RNA (1) is preferably 90% or more, more preferably 92% or more, and further preferably 95% or more. In addition, the homology of a base sequence can be easily determined by those skilled in the art using commercially available sequence analysis software.

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

Moreover, in another aspect of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention, single-stranded RNA in which RNA (1) and RNA (2) are bound via a linker RNA is contained as an active ingredient, The single-stranded RNA, ie, shRNA has a hairpin structure, RNA (1) and RNA (2) are hybridized, and 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 kind of RNA (1), the above siRNA or the above shRNA as an active ingredient, or may contain two or more kinds. Be good. The number of the two or more types is preferably 2 or more and 8 or less, more preferably 3 or more and 5 or less.

The dosage form of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention is not particularly limited as long as the active ingredient is delivered to the target site, and can be, for example, an injection, a liquid, or a sustained release agent. . As a solvent for these preparations, water is preferable, but it is preferable to use physiological saline, PBS, serum albumin solution or the like so that the preparation finally becomes an isotonic solution or a substantially isotonic solution. In addition, the RNA according to the present invention may be bound directly or indirectly to folic acid. Since the folate receptor is highly expressed on the surface of the pancreatic cancer cell membrane, such an RNA-folate complex may be selectively delivered and bound to pancreatic cancer cells. Further, the RNA according to the present invention may be bound to chitosan nanoparticles. Chitosan nanoparticles have an action of suppressing enzymatic degradation of siRNA intravenously administered into the body. Furthermore, it is preferable to bind the RNA according to the present invention to folic acid-chitosan composite nanoparticles.

The target site of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention may be not only the pancreas but also a lymph node or other organ to which pancreatic cancer cells have metastasized. Moreover, in order to deliver an active ingredient more reliably to a target site, an injection is preferable as a dosage form. Moreover, you may use well-known drug delivery techniques, such as a liposome and a polymer micelle.

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

The DNA according to the present invention may further contain regulatory regions such as a promoter, an enhancer, a silencer, a splicing donor, an acceptor, and poly A so that the base sequence (gene) encoding the RNA (1) is expressed. Good. In particular, it is preferable that a promoter is present on the 5 'end side of the coding region and a terminator for terminating transcription is linked on the 3' end side.

Since the DNA according to the present invention has a base sequence encoding RNA (1) as described above, RNA (1) and the like are produced in the cell by being introduced into the cell, and the target mRNA is caused by RNA interference. Is disassembled. Therefore, the vector in which the DNA according to the present invention is inserted can be an active ingredient of an inhibitor of pancreatic cancer cell invasion and metastasis.

The vector 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 obtained by a known method such as a carrier transport method using a lipid medium such as the lipofectamine method, a method of mediating a chemical substance such as calcium phosphate, a microinjection method, a gene implantation method, or an electroporation method. Can be delivered into.

In pancreatic cancer cells with high motility, invasiveness, and metastasis, mRNA accumulated in combination with IGF2BP3 is present in cell membrane processes. It was experimentally confirmed that this mRNA enhances invasion and metastasis of pancreatic cancer cells by being translated locally in cell membrane processes. In the present invention, it is possible to suppress the invasion and metastasis of pancreatic cancer cells by inhibiting the translation of the mRNA in the cell membrane process by RNA interference.

The dose and administration frequency of DNA or vector, such as RNA (1) according to the present invention, may be appropriately adjusted depending on the dosage form, patient severity, age, sex, 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 content of the specification of Japanese Patent Application No. 2014-138217 filed on July 4, 2014 is incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1: Confirmation of location of IGF2BP3 in metastatic pancreatic cancer cells The location of IGF2BP in pancreatic ductal adenocarcinoma (PDAC) cells was confirmed using an immunocytochemical technique. Two types of PDAC cells were used: S2-013 strain cells, which are moderately differentiated PDAC cells, and PANC-1 strain cells, which are undifferentiated PDAC cells.

(1) Culture of pancreatic cancer cells The S2-013 strain, a sub-line of the human PDAC cell SUIT-2 strain, was obtained from Professor Takeshi Iwamura of Miyazaki University. The PDAC cell PANC-1 strain was purchased from the American Type Culture Collection. These cells were cultured in a Dulbecco's modified Eagle medium (DMEM, manufactured by Gibco-BRL) containing 10% of fetal calf serum (FCS) inactivated by heating in a humid atmosphere containing 5% CO ₂ .

(2) Confocal Immunofluorescence Microscopy Glass coverslips were coated with 10 μg / mL fibronectin (Sigma-Aldrich) for 1 hour at room temperature. The S2-013 cell line or the PANC-1 cell line was seeded on the fibronectin-coated coverslip and incubated for 5 hours. Next, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, coated with a blocking solution (3% BSA / PBS), and then incubated with an anti-IGF2BP3 primary antibody or the like for 1 hour. . Furthermore, fluorescent dyes Alexa488-, Alexa546-, Alexa594- or Alexa647-conjugated secondary antibody (Cytoskeleton) were used in the presence or absence of rhodamine-conjugated phalloidin (Cytoskeleton). Some primary antibodies were coupled with green or red fluorophores using commercially available antibody labeling technology ("Zenon (R)" from Life technologies). It was visualized with a microscope ("Zeiss LSM 510 META microscope" manufactured by Carl Zeiss), and photographs of the obtained microscopic images are shown in Fig. 1 and 2. In Fig. 1 and Fig. 2, IGF2BP3 is labeled with a green dye, and is an actin filament. Is stained in red with phalloidin, and the arrow indicates IGF2BP3 present in the cell membrane process.

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

(4) Results and Discussion When S2-013 strain or PANC-1 strain, which is a pancreatic ductal adenocarcinoma cell, was cultured on fibronectin to promote the formation of cell membrane protrusions, as shown in FIG. 1, IGF2BP3 contained actin in it. Filaments were accumulated in the existing cell membrane processes. Further, as shown in FIG. 2, it was found that IGF2BP3 co-localizes with G3BP, which is a marker protein of stress granules, in the granules accumulated in cell membrane processes. Furthermore, as shown in FIG. 3, when the extract of cell line S2-013 cultured on fibronectin was subjected to an immunoprecipitation experiment with anti-IGF2BP3 antibody or anti-G3BP antibody, it was shown that IGF2BP3 co-precipitated with G3BP. .

The stress granule contains 40S subunit ribosomal protein and various translation initiation factors, and is involved in regulation of translation from mRNA to protein. The stress granule also contains several RNA-binding proteins, which are involved in stabilizing the contained mRNA. Therefore, it is considered that IGF2BP3 regulates the translation of specific mRNA in cell membrane processes as an RNA binding protein localized in stress granules.

Example 2: IGF2BP3 knockdown test in metastatic pancreatic cancer cells (1) IGF2BP3-RNA interference To elucidate the effect of IGF2BP3 on invasion and metastasis of pancreatic cancer cells, an siRNA specific for IGF2BP3 is expressed Using the vector, a clonal cell in which the production of IGF2BP3 in the S2-013 cell line was constantly suppressed was established.

Specifically, the rapidly growing GP2-293 packaging cells (Clontech) incorporate scrambled negative controls (OriGene Technologies "TR30013") or IGF2BP3 mRNA targeting siRNAs (OriGeneTechnG3Technology3) A pGFP-V-RS vector (produced by OriGene Technologies) that generates replication-defective lentivirus was temporarily infected. Along with the transient infection of the packaging cells with the vector, a replication defective virus was obtained and infected with the S2-013 cell line. The infected S2-013 cell line is transferred to a flask 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 further cultured for 10 days after becoming confluent. During the culture, the medium was replaced with a new one every two days. Only cells in which suppression of IGF2BP3 was recognized by Western blot analysis were used. IGF2BP3-RNAi clones (siIGF-1 and siIGF-2) and control RNAi clones (Scr-1 and Scr-2) of S2-013 cell line infected with siRNA targeting IGF2BP3 were used for anti-IGF2BP3 antibody The results of analysis by Western blotting are shown in FIG. As shown in FIG. 4, knockdown of IGF2BP3 was confirmed by immunoblotting.

(2) Wound area motility assay In each wound healing assay, a plastic pipette tip was used to make a cruciform cut as a wound area in a confluent monolayer cell. After marking some wound areas to be observed, photographs were taken using a phase contrast microscope. Cells that migrated to the marked wound area were observed for 1 to 8 hours and photographed continuously. A photograph of the cells is shown in FIG. Also, the degree of blockage of the wound area by the migrated cells was quantified by counting the migrated cells. The quantitative values are shown in FIG. In FIG. 6, “*” indicates a case where there is a significant difference at a risk rate of p <0.001 in the t-test with respect to Scr-1 or Scr-2.

As shown in the results shown in FIG. 5, by suppressing the expression of the IGF2BP3 gene, the cell migration to the wound region of the confluently cultured S2-013 strain was inhibited. Moreover, as shown in FIG. 6, the number of cells that migrated to the wound area was significantly reduced by inhibiting the expression of the IGF2BP3 gene by RNA interference.

(3) Immune Cell Staining at Advanced Cell Movements Using a plastic pipette tip, a cross-shaped cut was made in confluent S2-013 cell line, and then cell migration to the wound area was promoted. After 4 hours, the cells were immunostained with a primary antibody and incubated with a phosphor-conjugated secondary antibody as in Example 1 above. The cell movement advanced part that began to migrate to the wound region was observed with 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. 7, IGF2BP3 was accumulated in cell membrane processes formed in the cell advanced part of the S2-013 cell line that started migration.

(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, manufactured by Becton Dickinson). A solvent containing 5% chemoattractant was added to the lower chamber. Cells were incubated for 20 hours in the upper chamber. Three independent areas were then observed with a microscope and cells invading into the lower chamber were counted. The same experiment was repeated three times, and the S2-013 control clone (Scr-1 and Scr-2) constitutively expressing scrambled negative control siRNA and IGF2BP3-specific siRNA constitutively expressed to suppress IGF2BP3 expression. Comparison was made between the S2-013 clones (siIGF-1 and siIGF-2). The results are shown in FIG. In FIG. 8, “*” indicates a case where there is a significant difference at a risk rate of p <0.001 in the t-test with respect to Scr-1 or Scr-2.

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

Also, in order to amplify cDNA encoding all amino acids of IGF2BP3, RT-PCR was performed using RNA of S2-013 cell line as a template. The obtained PCR product was inserted into a pCMV6-Entry vector (produced by Origen) that generates a myc-DDK tag at the C-terminus. Hereinafter, the obtained vector is referred to as “IGF2BP3-pCMV6”. In order to restore IGF2BP expression in siIGF-1 and siIGF-2 clone cells, IGF2BP3-pCMV6 was introduced into siIGF-1 and siIGF-2 clone cells using X-tremeGEN HP DNA Transfection Reagent (Roche). It was. 48 hours later, Western blot analysis using anti-IGF2BP3 antibody was performed. The results are shown in FIG. As shown in FIG. 9, the expression of exogenous IGF2BP that was forcibly expressed was confirmed.

Mock control vector or IGF2BP3-pCMV6 was temporarily introduced into control RNAi cells or IGF2BP3-RNAi cells, and 48 hours later, Matrigel invasion assay was performed as described above. The results of Western blot analysis using anti-IGF2BP3 antibody are shown in FIG. In FIG. 9, the black arrow indicates endogenous IGF2BP3, and the white arrow indicates exogenous IGF2BP3. “GAPDH” is glyceraldehyde-3-phosphate dehydrogenase as an internal control indicating that an equal amount of each sample is used in Western blot analysis. Further, FIG. 10 shows the number of cells infiltrating the lower chamber. “*” In FIG. 10 indicates that the number of cells transfected with IGF2BP3-pCMV6 is significantly higher at the risk of p <0.005 in the t-test compared to the cells transfected with the mock vector. Show.

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

(5) Orthotopic transplantation of mice and tumor cells Six-week-old sterile female athymic nude mice (BALB / cSlc-nu / nu) were purchased from Japan SLC Co., Ltd., according to the Kochi University Research Institute Animal Care and Use Guidelines Handled. S2-013 control clones (Scr-1 and Scr-2) constitutively expressing scrambled negative control siRNA and S2-013 clones (siIGF) in which IGF2BP3-specific siRNA was constitutively expressed and IGF2BP3 expression was suppressed −1 and siIGF-2) were transplanted surgically and orthotopically into each mouse pancreas, 8.0 × 10 ⁵ , respectively, to form tumors in the mouse pancreas. At 42 days after transplantation, the mice were sacrificed, stained with hematoxylin-eosin, and examined for the presence of invasion of the retroperitoneum from the tumor formed in the pancreas of the mice and the presence of metastasis to the lung and liver. In addition, tumors formed in the mouse pancreas were weighed. Table 1 shows the measurement results and the presence or absence of infiltration into the retroperitoneum and distant metastasis, and FIG. 11 shows a stained photograph. In FIG. 11, “R” indicates a cavity peritoneum tissue, “P” indicates a muscle tissue, and “N” indicates a normal tissue.

As shown in the results shown in Table 1, invasion from the tumor formed in the mouse pancreas to the retroperitoneum was caused by clonal cells (siIGF−) in which IGF2BP3 expression was suppressed compared to control clonal cells (Scr-1 and Scr-2). 1 and siIGF-2) were significantly lower. In addition, pancreatic cancer tissue derived from control clonal cells metastasized to the liver and lung, whereas clonal cell derived pancreatic cancer tissue in which IGF2BP3 expression was suppressed did not metastasize.

In addition, as shown in the results shown in FIG. 11, the pancreatic cancer tissue derived from the control clone cells invaded the entire pancreatic tissue of the mouse. In particular, the boundary between tumor tissue and normal tissue was not clear in the control sample. On the other hand, most of the clonal cell-derived pancreatic cancer tissue in which IGF2BP3 expression was suppressed was encased in mouse stromal cells and clearly separated from mouse normal pancreatic tissue. In mice transplanted with control clone cells, the surface of the peritoneum is covered with a relatively thick cancer cell layer seeded from pancreatic cancer tissue derived from control clone cells, and the cancer cells infiltrate to the muscle layer. It was. On the other hand, in the mice transplanted with clonal cells in which IGF2BP3 expression was suppressed, cancer cells were not observed in most of the peritoneum. In addition, mice transplanted with control clone cells showed metastases in the lung and liver, but mice transplanted with clone cells with suppressed IGF2BP3 expression had no metastases to the lungs and liver. It was. From the above results, it can be said that IGF2BP3 can be a novel therapeutic target molecule for pancreatic cancer by inhibiting the expression of IGF2BP3 in pancreatic cancer cells and completely preventing metastasis to the lung, liver and the like. .

(6) Discussion From the above results, IGF2BP3 particularly promotes invasion and metastasis of pancreatic cancer cells, and in the formation of pancreatic tumors in vivo, the decrease in the expression level of IGF2BP3 is 1) the increase of tumors in the pancreas And 2) it affects the invasion of adjacent tissues of the pancreas and 3) affects the metastasis to other tissues.

Example 3 Role of IGF2BP3 in Formation of Plasma Membrane Process of Pancreatic Cancer Cell Using a confocal microscope, the polymerization of actin filaments and the three-dimensional structure of the plasma membrane process in S2-013 cell line stimulated with fibronectin were examined. The detailed conditions of the confocal microscope were the same as in Example 1 above. The results are shown in FIGS.

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

FIG. 13 shows the ratio of cells having cell membrane protrusions in all cells. As shown in FIG. 13, the proportion of cells having cell membrane protrusions in the clone in which IGF2BP3 expression was suppressed (siIGF-1) was significantly smaller than that in the control clone (Scr-1). Furthermore, by introducing IGF2BP3-pCMV6 into IGF2BP3-RNAi S2-013 cell line, the expression of IGF2BP3 was restored, and actin filaments directly under the cell membrane were restored.

FIG. 14 is a photograph using a phase contrast microscope of a control clonal cell (Scr-1) and a clonal cell (siIGF-1) in which IGF2BP3 expression is suppressed. As shown in FIG. 14, the control clonal cells were spindle-shaped and exhibited a fibroblast-like morphology, while the clonal cells in which IGF2BP3 expression was suppressed were round and exhibited an epithelial cell-like morphology. Since fibroblast-like morphology is often more prone to invasion and metastasis than epithelial cell-like morphology, this result indicates that clonal cells with suppressed IGF2BP3 expression infiltrated more than control clonal cells with suppressed IGF2BP3 expression. Shows low metastatic potential.

Example 4: Identification of transcripts bound to IGF2BP3 (1) RNA immunoprecipitation, sequencing and bioinformatics analysis S2-013 strain cells were seeded on fibronectin and incubated for 5 hours. The 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). The extract was immunoprecipitated by treatment with Dynabeads Protein G (manufactured by Dynal) and anti-IGF2BP3 antibody or rabbit IgG isotype control antibody at 4 ° C. for 2 hours. The 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 nucleic acid was precipitated with ethanol. Next, the precipitated nucleic acid was treated with DNase I (manufactured by Promega), and RNA was purified using RNeasy kit (manufactured by Qiagen). Identification of the RNA sample obtained by the next-generation sequencer was entrusted to Hokkaido System Science, and further bioinformatics analysis of the obtained data was entrusted to World Fusion. A scatter plot of the concentration of mRNA contained in the IGF2BP3 immunoprecipitation sample or the control IgG immunoprecipitation sample is shown in FIG. In addition, linear regression diagrams of the numbers of ribosomal RNA (rRNA) and small nuclear RNA (snRNA) between the IGF2BP3 immunoprecipitation sample and the control IgG immunoprecipitation sample are shown in FIGS. 16 (1) and 16 (2), respectively. ). In FIG. 16, a dotted line shows a straight line of x = y.

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

In the above experiment, the expression ratio was calculated by performing the unique processing after surrogate processing of the RPKM value. Specifically, the RPKM value is converted to a log ₂ [(RPKM value of IGF2BP3 sample) / (RPKM value of isotype control sample)] ratio, and RNA of more than 1.0 (over 2 times as the expression ratio) is converted into IGF2BP3. Was considered to be a transcript bound to. As a result, 2,826 types of RNA having a significantly higher concentration in the anti-IGF2BP3 immunoprecipitate than the rabbit IgG isotype control immunoprecipitate were identified.

(2) RT-PCR
Among the IGF2BP3-binding mRNAs identified in (1) above, ADP-ribosylation factor 6 (ARF6) and Rho guanine nucleotide exchange factor 4 (ARHGEF4) were confirmed to bind to IGF2BP3. Specifically, anti-IGF2BP3 antibody, rabbit IgG isotype control antibody or negative control from S2-013 cell lysate that induced the formation of cell membrane protrusion using StrataScript reverse transcriptase (manufactured by Agilent) and oligo d (T) _12-18 primer The 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 FIG. 17, the two transcripts formed an immunoprecipitation complex with the anti-IGF2BP3 antibody, but did not form an isotype control antibody and an anti-CD63 antibody.

(3) Immunofluorescence In order to make each sequence-specific fluorescent probe in situ hybridize with endogenous ARF6 mRNA and ARHGEF4 mRNA present in S2-013 cells that have induced the formation of cell membrane protrusions, Quanti Gene ViewRNA plate-based Assay kit (manufactured by Panomics) was used. S2-013 cells stimulated with fibronectin were fixed with 8% formaldehyde, dehydrated with 50%, 70% and 100% ethanol, and allowed to stand at 4 ° C. overnight. Cells were then dehydrated again, permeabilized, and hybridized with fluorescent probes. The target mRNAs were ARF6 and ARGGEF4, and the control RNA was ubiquitin C mRNA. After in situ hybridization, the cross section was washed with PBS, blocked for 1 hour with a blocking buffer that was a 4% solution of goat serum in PBS, and incubated with anti-IGF2BP3 antibody in blocking buffer for 3 hours. The secondary antibody was allowed to act at room temperature for 30 minutes in blocking buffer, and the 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” from Carl Zeiss). The results are shown in FIG.

As shown in FIG. 18, ARF6 mRNA and ARHGEF4 mRNA co-localized with IGF2BP3 in granules existing in the cytoplasm, and these were assembled in cytoplasmic processes. On the other hand, the control ubiquitin C mRNA was not colocalized with IGF2BP3. IGF2BP3 granules also accumulated around the nucleus. These granules are probably transported from the periphery of the nucleus to the cell membrane process along 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 processes.

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

As shown in FIG. 19, in IGF2BP3-RNAi cells, the production of ARF6 and ARGGEF4 decreased only in the cell membrane process with the suppression of IGF2BP3 expression.

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

As shown in FIG. 20, by restoring the expression of the IGF2BP3 gene in IGF2BP3-RNAi cells (siIGF-1), ARF6 protein was again generated in the cell membrane process.

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

As shown in FIG. 21, similarly to the production of ARF6 protein, ARGGEF4 protein was produced again in the cell membrane process by restoring the expression of IGF2BP3 gene in IGF2BP3-RNAi cells (siIGF-1).

(4)
Functional analysis of ARF6 protein and ARGGEF4 protein in pancreatic cancer cells was performed. Double-stranded RNAs (hereinafter referred to as “siARF6” and “siARHGEF4”, respectively) consisting of RNA having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5 corresponding to the partial nucleotide sequences of ARF6 RNA and ARHGEF4 RNA, respectively ) Or a control siRNA oligo was introduced into the S2-013 cell line. These cells were cultured on fibronectin, and peripheral actin filaments in cell membrane processes were analyzed. Forty-eight hours after the introduction of the vector, the production of ARF6 protein and ARHGEF4 protein was analyzed by Western blot. The results are shown in FIG. In FIG. 22, “Scr”, “siARF6”, and “siARHGEF4” show the results of cells into which a control siRNA oligo, siRNA having the base sequence of SEQ ID NO: 1, and siRNA having the base sequence of SEQ ID NO: 5 were introduced, respectively.

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

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

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

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

As shown in FIG. 24 and FIG. 25, in the 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)
As in the above (4), control RNAi cells by transiently introducing siRNA oligos and control siRNA oligos specific to the respective mRNAs of ARF6 and ARHGEF4 into the S2-013 cell line and the PANC-1 cell line, ARF6-RNAi cells and ARHGEF4-RNAi cells were obtained.

Using each cell, a wound region motility assay was performed under the same conditions as in Example 2 (2) above, and the number of cells that migrated to the wound region was counted. The results are shown in FIG. FIG. 26 shows the average value of four cases, and “*” indicates that there is a significant difference at p <0.001 in the t-test.

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

(6)
Using the cells of Example 5 (5), Matrigel invasion assay was performed in the same manner as in Example 2 (4) above, and cells infiltrating from the upper chamber to the lower chamber were counted. The same experiment was repeated 4 times, and the average value was calculated. The results are shown in FIG. In FIG. 27, “*” indicates a case where there is a significant difference in the risk rate of 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 relative to the control (Scr).

(7) Conclusion According to the above experimental results, mRNA binding to IGF2BP3 is selectively translated locally at the cell membrane projections to produce proteins, and these proteins promote the formation of cell membrane projections, and the pancreas It may be involved in the motor infiltration of cancer cells.

Example 6: In vitro experiment using siRNA with folate chitosan nanoparticles added to confirm the effect of the present invention (1) Confirmation of uptake of siRNA into cells Using pancreatic cancer cell line S2-013, folic acid was used. It was examined whether or not chitosan nanoparticle-added siRNA was taken up into cells. Chitosan nanoparticles suppress enzymatic degradation of siRNA administered intravenously into the body, and folic acid enables siRNA to bind to a folate receptor that is highly expressed on the surface of pancreatic cancer cell membranes.

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

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

(2) Confirmation experiment of knockdown effect by siRNA with folate chitosan nanoparticle added 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 the proteins encoded by CCDC88A and WASF2 are involved in cell morphological change, movement or invasion by forming complexes with different actin-binding proteins. CCDC88A siRNA comprises the nucleotide sequence of SEQ ID NO: 57. WASF2 siRNA comprises the base sequence of SEQ ID NO: 73.

Control siRNA, CCDC88A siRNA, and WASF2 siRNA added with folate chitosan nanoparticles were added to the culture medium of S2-013 strain, and the cells were collected 48 hours later. Semi-quantitative RT-PCR was performed using the recovered RNA of the cells. In addition, Western blotting was performed using cell lysate. The result of the semi-quantitative RT-PCR method is shown in FIG. 29A, and the result of the Western blot is shown in FIG. 29B. As shown in FIG. 29, the knockdown effect on each mRNA of CCDC88A siRNA and WASF2 siRNA could be confirmed in both the semi-quantitative RT-PCR method and the Western blot.

(3) Confirmation Experiment of Cell Motility Inhibitory Effect by Addition of Fotochitosan Nanoparticles Added siRNA According to the Present Invention Each of control siRNA, CCDC88A siRNA and WASF2 siRNA added with folate chitosan nanoparticles was added to the culture medium of S2-013 48 hours later, a Matrigel invasion assay was performed. 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 that there was a significant difference at p = 0.00073 in the t-test, and “**” indicates that there was a significant difference at p = 0.00852. .

As shown in FIG. 30, S2-013 cells incorporated with CCDC88A siRNA or WASF2 siRNA significantly suppressed cell infiltration compared to S2-013 cells incorporated with control siRNA.

From the above results, the siRNA with folate chitosan nanoparticles added according to the present invention added to the culture medium of cultured cells is incorporated into S2-013 cells and inhibits the expression of mRNA involved in cell motility. Was 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 a 6-week-old nude mouse Was used. Specifically, 12 nude mice were anesthetized by inhalation anesthesia using isoflurane on the first day, 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 transplanted into the pancreas of each mouse. As in the in vitro experiment of Example 6 above, siRNA was used for CCDC88A and WASF2, and scrambled siRNA was used as a control. Each synthesized siRNA was added to folate chitosan nanoparticles. Each group of 4 mice transplanted with human pancreatic cancer cells were intravenously administered with the chitosan nanoparticle-added siRNA added intravenously into the tail vein once / week. SiRNA administered systemically to mice is passively delivered to human pancreatic cancer tissue by chitosan nanoparticles, and folic acid is expected to promote efficient endocytosis via folate receptors on pancreatic cancer cells it can. A total of 5 siRNA intravenous injections were performed. During this period, S2-013 cells transplanted into the mouse pancreas on day 1 formed tumors and distantly metastasized to the liver and lungs. Tumors exposed from the pancreas also cause peritoneal dissemination and retroperitoneal infiltration.

One week after each administration of 5 total siRNA intravenous injections, one group was administered to each mouse to confirm whether siRNA with folate chitosan nanoparticles added intravenously to S2-013 tumor in mouse pancreas was accumulated. The folate chitosan nanoparticle-added 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 accumulation result of siRNA in the mouse is shown in FIG. 31A, and the accumulation result of siRNA in the excised tumor is shown in FIG. 31B.

As shown in the results shown in FIG. 31, it was confirmed that all of control siRNA, CCDC88A siRNA and WASF2 siRNA were accumulated in the S2-013 cell tumor formed in the mouse pancreas. Importantly, the accumulation of the control siRNA in the S2-013 tumor was significantly increased 24 hours later, compared to 3 hours after administration of the labeled folate chitosan nanoparticle-added control siRNA. Each siRNA did not accumulate in the isolated mouse heart and accumulated specifically in the S2-013 tumor. Mice administered with control siRNA tended to have frequent peritoneal seeding, and specific accumulation of control siRNA in peritoneal seeding tissue was observed. In addition, the fluorescent color development of the primary pancreatic cancer in mice administered with CCDC88A siRNA or WASF2 siRNA was weaker than that in mice administered with control siRNA. The reason will be discussed with reference to FIG.

The mouse was dissected after photographing with the in vivo imaging system. The presence or absence of peritoneal dissemination in the mouse abdominal cavity was confirmed, and the primary tumor, liver and lung were fixed in formalin. Each sample was stained with hematoxylin and eosin, and the presence or absence of retroperitoneal invasion from the pancreatic cancer primary lesion and metastasis to the lung / liver was compared with a group of mice administered with control siRNA. An enlarged photograph of the primary tumor tissue is shown in FIG. 32, and the results of confirming the tumor in each sample are shown in Table 2.

As shown in Table 2, retroperitoneal infiltration was significantly suppressed in the group of mice administered with the CCDC88A siRNA and WASF2 siRNA according to the present invention. Peritoneal dissemination also tended to be less common in the group of mice administered with CCDC88A siRNA and WASF2 siRNA. It should be noted that no metastasis to the liver and lung was observed in the group of mice administered with CCDC88A siRNA. In the group of mice administered with WASF2 siRNA, lung metastasis was not different from control, but no liver metastasis was observed. Moreover, as shown in the results shown in FIG. 32, the mice in the group of mice administered with the control siRNA had a solid tumor with few necrotic parts, whereas S2 of the group of mice administered with the CCDC88A siRNA and WASF2 siRNA. -013 Many tumors were necrotic. Further, in FIG. 31, the fluorescence development of the primary pancreatic cancer lesion in the mice administered with CCDC88A siRNA or WASF2 siRNA was weaker than that in the mice administered with control siRNA. The reason for this is considered to be that there were many necrotic portions in the S2-013 tumor of mice administered with CCDC88A siRNA or WASF2 siRNA, and the present invention was administered once / week. This is considered to be an effect of siRNA added with folate chitosan nanoparticles.

Claims (9)

A pancreatic cancer cell invasion metastasis inhibitor comprising RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 92. The pancreatic cancer cell invasion according to claim 1, wherein the RNA (1) has at least one base sequence selected from SEQ ID NOs: 57 to 60 or one or more selected from SEQ ID NOs: 73 to 76. Metastasis inhibitor. Furthermore, the pancreatic cancer cell invasion metastasis inhibitor of Claim 1 or 2 containing RNA (2) which has a sequence | arrangement which hybridizes with said RNA (1) on stringent conditions. The pancreatic cancer cell invasion and metastasis inhibitor according to claim 3, comprising a double-stranded RNA in which the RNA (1) and the RNA (2) are hybridized. The RNA (1), the RNA (2), and a linker RNA that binds the RNA (1) and the RNA (2), and the RNA (1) and the RNA (2) are hybridized. The pancreatic cancer cell invasion and metastasis inhibitor according to claim 3, wherein the linker RNA comprises a single-stranded RNA having a circular structure. DNA having a base sequence encoding RNA (1) having one or more sequences selected from SEQ ID NOs: 1 to 92 A vector comprising the DNA according to claim 6. Use of RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 92 for inhibiting invasive metastasis of pancreatic cancer cells. A method for inhibiting invasion and metastasis of pancreatic cancer cells, comprising a step of administering RNA comprising RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 92.

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