WO2016002844A1 - Inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas - Google Patents

Inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas Download PDF

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WO2016002844A1
WO2016002844A1 PCT/JP2015/069013 JP2015069013W WO2016002844A1 WO 2016002844 A1 WO2016002844 A1 WO 2016002844A1 JP 2015069013 W JP2015069013 W JP 2015069013W WO 2016002844 A1 WO2016002844 A1 WO 2016002844A1
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rna
cells
igf2bp3
pancreatic cancer
sirna
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恵介 谷内
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国立大学法人高知大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • 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.
  • 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.
  • cancer is a malignant tumor
  • 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.
  • 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.
  • 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.
  • IGF2BP3 insulin-like growth factor 2 mRNA binding protein 3
  • a gene encoding IGF2BP3 is listed as one of genes whose overexpression is used for detection of cancer.
  • 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.
  • a pancreatic cancer cell invasion metastasis inhibitor comprising RNA (1) having one or more base sequences selected from SEQ ID NOs: 1 to 92.
  • 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.
  • DNA comprising a base sequence encoding RNA (1) having one or more sequences selected from SEQ ID NOs: 1 to 92.
  • 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 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.
  • 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 ⁇ m.
  • 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
  • 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. 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.
  • 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.
  • 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 ⁇ m.
  • 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).
  • 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 ⁇ m.
  • 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 ⁇ m.
  • 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 ⁇ m.
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • IGF2BP3 insulin-like growth factor 2 mRNA binding protein 3
  • RNA (1) can be used as a sense RNA for RNA interference.
  • IGF Insulin-like growth factor
  • 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.
  • 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.
  • 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 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).
  • 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.
  • RNA has only a predetermined base sequence, or may have other base sequences in addition to the predetermined base sequence.
  • 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.
  • 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).
  • 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.
  • 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.
  • RNA (1) and RNA (2) are independently contained, and these form double-stranded RNA, ie, siRNA. .
  • 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
  • linker RNA has a circular structure
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • DMEM Dulbecco's modified Eagle medium
  • FCS fetal calf serum
  • fluorescent dyes Alexa488-, Alexa546-, Alexa594- or Alexa647-conjugated secondary antibody 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.
  • 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.
  • 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.
  • the rapidly growing GP2-293 packaging cells incorporate scrambled negative controls (OriGene Technologies "TR30013") or IGF2BP3 mRNA targeting siRNAs (OriGeneTechnG3Technology3)
  • a pGFP-V-RS vector produced by OriGene Technologies
  • 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 ⁇ g / mL puromycin to stably express siRNA targeting IGF2BP3 mRNA.
  • DMEM medium containing 0.5 ⁇ g / 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.
  • 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 ⁇ m, 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.
  • IGF2BP3-pCMV6 pCMV6-Entry vector (produced by Origen) that generates a myc-DDK tag at the C-terminus.
  • IGF2BP3-pCMV6 the obtained vector is referred to as “IGF2BP3-pCMV6”.
  • 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.
  • FIG. 9 shows the results of Western blot analysis using anti-IGF2BP3 antibody.
  • the black arrow indicates endogenous IGF2BP3, and the white arrow indicates exogenous IGF2BP3.
  • GPDH is glyceraldehyde-3-phosphate dehydrogenase as an internal control indicating that an equal amount of each sample is used in Western blot analysis.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • FIG. 11 shows a stained photograph.
  • “R” indicates a cavity peritoneum tissue
  • P indicates a muscle tissue
  • N indicates a normal tissue.
  • pancreatic cancer tissue derived from the control clone cells invaded the entire pancreatic tissue of the mouse.
  • the boundary between tumor tissue and normal tissue was not clear in the control sample.
  • 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.
  • 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.
  • Example 5 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.
  • 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.
  • CCDC88A siRNA comprises the nucleotide sequence of SEQ ID NO: 57.
  • WASF2 siRNA comprises the base sequence of SEQ ID NO: 73.
  • S2-013 cells incorporated with CCDC88A siRNA or WASF2 siRNA significantly suppressed cell infiltration compared to S2-013 cells incorporated with control siRNA.
  • 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.
  • control siRNA 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.
  • 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.
  • 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.

Abstract

L'invention a pour objet de fournir un inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas qui permet d'empêcher efficacement et spécifiquement une infiltration cellulaire et une métastase du cancer du pancréas, un ADN qui code un ARN constituant le principe actif de cet inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas, et un vecteur qui contient cet ADN, et qui permet d'être mis en œuvre pour empêcher une infiltration cellulaire et une métastase du cancer du pancréas. L'inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas de l'invention est caractéristique en ce qu'il contient l'ARN (1) possédant une séquence de bases ou plus choisie parmi les SEQ ID NO : 1 à 92.
PCT/JP2015/069013 2014-07-04 2015-07-01 Inhibiteur d'infiltration cellulaire et de métastase du cancer du pancréas WO2016002844A1 (fr)

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