WO2014097875A1 - Development of pluripotent stem cell employing novel dedifferentiation induction method - Google Patents

Abstract

The purpose of the present invention is to obtain: a novel pluripotent stem cell production method; or a therapeutic agent for malignant tumors. A method for inducing the dedifferentiation of a cell, which involves a step of inhibiting the expression or function of a miR-520d-5p-controlled gene, is employed. Or, a therapeutic agent for malignant tumors, which comprises an inhibitor of the expression or function of the miR-520d-5p-controlled gene, is used.

Description

Pluripotent stem cells using a novel dedifferentiation induction method

The present invention relates to a method for inducing cell dedifferentiation, a method for producing pluripotent stem cells, a therapeutic agent for malignant tumors, and the like.

Pluripotent stem cell production technology is a field that has received particular attention in the medical industry in recent years. As a representative technique for producing pluripotent stem cells, the method described in Patent Document 1 can be mentioned. This document describes that pluripotent stem cells were prepared by introducing four genes (Oct3 / 4, Klf4, Sox2, c-Myc) into cells. These cells are called iPS cells, and since the development of this technology, the number of reports on research results related to pluripotent stem cells has been increasing rapidly. For example, Patent Document 2 describes that pluripotent stem cells were prepared by introducing three genes (Oct3 / 4, Klf4, Sox2) and one miRNA (hsa-miR-372 etc.) into the cells. Has been. Non-Patent Document 1 describes that the efficiency of producing pluripotent stem cells increased when the p53 gene of cells to be pluripotent stem cells was deleted when the above four or three genes were introduced. Has been. Patent Documents 3 and 4 describe that pluripotent stem cells were prepared by introducing specific RNA strands (such as miR-520d-5p) into cells.

On the other hand, the field in which pharmaceutical companies have invested in recent years is the malignant tumor field. Malignant tumors have many complicated and unclear points, and there are few effective therapeutic agents compared to other diseases. Under such circumstances, the present inventor has reported in Non-Patent Document 2 that hTERTTERmRNA can be applied as a biomarker of cancer. Patent Documents 3 and 4 report that specific RNA strands can be used for the treatment of malignant tumors.

International Publication No. 2007/069666 International Publication No. 2009/075119 International Publication No. 2012/008301 International Publication No. 2012/008302

'Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.' Hong et al., Nature. 2009 Aug 27; 460 (7259): 1132-5. Epub 2009 Aug 9. 'A novel biomarker TERT mRNA is applicable for early detection of hepatoma.' Miura et al., BMC Gastroenterol. 2010 May 18; 10: 46.

However, as described above, there are examples of successful production of pluripotent stem cells, but there are no products that have been approved by the authorities for therapeutic use. This is due to the fact that research does not progress easily due to limited methods for producing pluripotent stem cells. Except for a few of the few methods that have confirmed obvious side effects (for example, canceration in the early stage of culture by c-Myc), the effective methods are further limited.

In addition, although the mechanism of malignant tumors has gradually been clarified in the above-mentioned literature, it is not sufficient as it is, and malignant tumors are considered to be the top cause of human death in the future. Therefore, it is necessary to identify new anti-malignant tumor substances and accumulate information on malignant tumors in order to develop new drugs or treatment strategies for malignant tumors.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel dedifferentiation induction method, a pluripotent stem cell production method, a malignant tumor therapeutic agent, and the like.

As described in Examples described later, the present inventor has found that inhibition of ELAVL2 expression in normal cells or malignant tumor cells causes the cells to dedifferentiate and become pluripotent stem cells. Furthermore, when the obtained pluripotent stem cells were transplanted subcutaneously into mice, no malignant tumor formation was observed over a long period of 4 months or longer. From this, it became clear that the technique found by the present inventor is very excellent in safety.

In addition, the present inventors also tried to inhibit the expression of TEAD1 and GATAD2B in normal cells or malignant tumor cells. As a result, in this case as well, the cells were dedifferentiated to become pluripotent stem cells. All of these genes were genes whose expression could be controlled by miR-520d-5p. Similar to these genes, other genes whose expression can be controlled by miR-520d-5p include SBF2.

That is, according to one aspect of the present invention, there is provided a method for inducing cell dedifferentiation, which comprises the step of inhibiting the expression or function of a miR-520d-5p regulated gene.

Also, according to one aspect of the present invention, there is provided a method for producing pluripotent stem cells, comprising a step of inducing cell dedifferentiation by the above method. Moreover, according to this invention, the pluripotent stem cell or population of a pluripotent stem cell obtained through said production method is provided. Moreover, according to this invention, the material for regenerative medicine obtained through the process of culture | cultivating the said pluripotent stem cell is provided.

Also, according to one embodiment of the present invention, there is provided a therapeutic agent for malignant tumor, comprising an expression or function inhibitor for miR-520d-5p controlled gene.

Moreover, according to one aspect of the present invention, there is provided a dedifferentiation inducing agent comprising an expression or function inhibitor for miR-520d-5p controlled gene.

Moreover, according to one aspect of the present invention, there is provided a pluripotent stem cell inducer comprising an expression or function inhibitor for miR-520d-5p regulated gene.

Further, according to one embodiment of the present invention, the miR-520d-5p regulated gene is ELAVL2, TEAD1, GATAD2B, SBF2, PUM2, NBEA, UBE2E3, CPEB3, GNAO1, WNK1, MEF2A, SEH1L, HOOK3, KLHL23, PAPOLG Or one or more genes selected from the group consisting of JAZF1, JAG1, CPEB2, ZCRB1, ZFC3H1, ZEB2, EPHA7, UBN2, PELI2, PRKD3, and FCHO2. Moreover, according to one aspect of the present invention, the inhibition may be inhibition by an RNAi molecule against mRNA of a miR-520d-5p controlled gene. Moreover, according to one aspect of the present invention, the inhibitor may be an RNAi molecule against mRNA of a miR-520d-5p-controlled gene, or a polynucleotide encoding the RNAi molecule.

According to the present invention, it is possible to induce dedifferentiation, produce pluripotent stem cells, or treat malignant tumors by a novel method.

FIG. 1 shows the result of observing the pluripotent stem cells according to the example with a microscope. FIG. 2 shows the results of evaluating the gene expression profile of pluripotent stem cells according to the Example at the transcription level. FIG. 3 shows the results of examining the ELAVL2 expression suppression rate for the ELAVL2 siRNA-introduced HLF cells according to the example. FIG. 4 shows the results of evaluating the gene expression profile of pluripotent stem cells according to the example at the translation level. FIG. 5 shows the results of observing the protein level of pluripotent stem cells according to Example by cell immunostaining. FIG. 6 shows the results of observing the proliferation of pluripotent stem cells according to the example. FIG. 7 shows the results of AP staining of pluripotent stem cells according to the example. FIG. 8 shows the results of transplanting pluripotent stem cells according to the Example into mice. FIG. 9 shows the results of reporter assay performed on ELAVL2 according to the example. FIG. 10 shows the results of examining the ELAVL2 expression suppression rate for the HLF cells into which the ELAVL2 siRNA according to the example was introduced. FIG. 11 shows the results of examining the suppression rate of TEAD1 expression in HLF cells into which TEAD1 siRNA according to the example was introduced. FIG. 12 shows the results of examining the GATAD2B expression suppression rate for HLF cells into which the GATAD2B siRNA according to the example was introduced. FIG. 13 shows the results of examining the DNA methylation level of HLF cells into which ELAVL2 siRNA, TEAD1 siRNA, or GATAD2B siRNA according to the example was introduced. FIG. 14 shows the results of examining the DNA methylation level of Huh7 cells into which ELAVL2 siRNA, TEAD1 siRNA, or GATAD2B siRNA according to the example was introduced.

Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.

One embodiment of the present invention is a novel dedifferentiation induction method. This method is, for example, a method for inducing cell dedifferentiation, which includes a step of inhibiting the expression or function of a miR-520d-5p-controlled gene. According to this method, pluripotent stem cells can be produced by inducing cell dedifferentiation, as demonstrated in the examples described later. The inhibition can be performed by, for example, RNAi against mRNA of miR-520d-5p controlled gene.

In one embodiment of the present invention, the “miR-520d-5p regulated gene” is a gene whose expression is controlled by miR-520d-5p. The miR-520d-5p controlled gene may have, for example, a miR-520d-5p binding site on the 3 ′ UTR. The miR-520d-5p regulated gene is ELAVL2, TEAD1, GATAD2B, SBF2, PUM2, NBEA, UBE2E3, CPEB3, GNAO1, WNK1, MEF2A, SEH1L, HOOK3 from the viewpoint of more stably causing cell dedifferentiation. , KLHL23, PAPOLG, JAZF1, JAG1, CPEB2, ZCRB1, ZFC3H1, ZEB2, EPHA7, UBN2, PELI2, PRKD3, and FCHO2 (hereinafter also referred to as "one or more of ELAVL2") Preferably). Further, the miR-520d-5p controlled gene is preferably ELAVL2, TEAD1, or GATAD2B from the viewpoint of safety to living bodies. The control includes suppression or inhibition.

In addition, the details of the nucleotide sequence etc. regarding ELAVL2 etc. can be seen from the website of HGNC (HUGO Gene Nomenclature Committee) or NCBI (National Center for Biotechnology Information). HGNC IDs listed on HGNC are HGNC: 3313 for ELAVL2, HGNC: 11714 for TEAD1, HGNC: 30778 for GATAD2B, HGNC: 2135 for PBF2, HGNC: 14958 for PUM2, HGNC: 7648 for UBE2E3, HGNC for UBE2E3: 12479, CPEB3 is HGNC: 21746, GNAO1 is HGNC: 4389, WNK1 is HGNC: 14540, MEF2A is HGNC: 6993, SEH1L is HGNC: 30379, HOOK3 is HGNC: 23576, KLHL23 is HGNC: 27506, PAPOLG is HGNC: 14982, JAZF1 is HGNC: 28917, JAG1 is HGNC: 6188, CPEB2 is HGNC: 21745, ZCRB1 is HGNC: 29620, ZFC3H1 is HGNC: 28328, ZEB2 is HGNC: 14881, EPHA7 is HGNC: 3390, UBN2 is HGNC: 21931, PELI2 is HGNC: 8828, PRKD3 is HGNC: 9408, and FCHO2 is HGNC: 25180. ELAVL2 mRNA may contain the base sequence of SEQ ID NO: 29. TEAD1 mRNA may contain the nucleotide sequence of SEQ ID NO: 30. GATAD2B mRNA may contain the base sequence of SEQ ID NO: 31. Each gene may be called differently, and other names are listed on the HGNC website. Therefore, each gene may be called by another name. For example, ELAVL2 contains a gene called HEL-N1 or HuB. Further, miR-520d-5p includes, for example, hsa-miR-520d-5p whose miRBase accession number is MI0003164.

One embodiment of the present invention is a method for producing pluripotent stem cells, which comprises a step of inhibiting the expression or function of a miR-520d-5p regulated gene. By using this production method, a pluripotent stem cell or a pluripotent stem cell population can be obtained. In addition, a regenerative medical material (for example, a regenerative medical organ) can be obtained through the step of culturing the pluripotent stem cells. The pluripotent stem cells obtained by using this production method demonstrate that endogenous p53 is highly expressed in Examples described later. Since p53 is a gene classified as a malignant tumor suppressor gene, it can be said that the pluripotent stem cells that highly express this p53 have a low risk of malignant tumor formation.

One embodiment of the present invention is a dedifferentiation inducing agent comprising an expression or function inhibitor for miR-520d-5p regulated genes. If this dedifferentiation inducer is used, cell dedifferentiation can be induced. Moreover, as a result of dedifferentiating the cells, for example, pluripotent stem cells can be obtained. Or if this dedifferentiation inducer is applied to a malignant tumor cell, a malignant tumor can be treated. It can also be used as an additive for assisting animal growth in livestock through effects such as suppression of malignant tumors.

One embodiment of the present invention is a method for treating a malignant tumor, comprising a step of inhibiting the expression or function of a miR-520d-5p controlled gene. Yet another embodiment is a therapeutic agent for a malignant tumor comprising an expression or function inhibitor for a miR-520d-5p regulated gene. Another embodiment is the use of an expression or function inhibitor against the miR-520d-5p regulated gene to produce a therapeutic agent for malignancy. A conventional therapeutic agent comprising a low molecular weight compound is one that induces apoptosis to treat a malignant tumor, but the therapeutic method, therapeutic agent, or inducer of this embodiment induces a malignant tumor in the direction of dedifferentiation. By doing so, a therapeutic effect can be exhibited.

One embodiment of the present invention is a method for transforming a cell into a pluripotent stem cell, comprising a step of inhibiting the expression or function of a miR-520d-5p regulated gene. Yet another embodiment is a pluripotent stem cell inducer comprising an expression or function inhibitor for a miR-520d-5p regulated gene. If this method or a pluripotent stem cell inducer is used, pluripotent stem cells can be prepared. Or if this method or a pluripotent stem cell inducer is applied to a malignant tumor cell, a malignant tumor can be treated. It can also be used as an additive for assisting animal growth in livestock through effects such as suppression of malignant tumors.

One embodiment of the present invention is a method of increasing the proportion of pluripotent stem cells in a cell population, comprising a step of contacting an expression or function inhibitor for a miR-520d-5p regulated gene with the cell population. Yet another embodiment is a method of producing a cell population with an increased proportion of pluripotent stem cells, comprising the step of contacting an expression or function inhibitor for a miR-520d-5p regulated gene with the cell population.

One embodiment of the present invention is a research or medical kit containing the inhibitor described above. Examples of this kit include dedifferentiation induction, pluripotent stem cell preparation, artificial organ preparation, malignant tumor treatment, undifferentiation marker expression regulation, or p53-expressing cell preparation. The kit may further include, for example, a buffer solution, a package insert describing information on active ingredients, a container for containing the active ingredients, or a package.

One embodiment of the present invention comprises a step of selecting a test substance that decreases the expression or functional amount of a miR-520d-5p regulated gene, a dedifferentiation inducer, a pluripotent stem cell inducer, or a malignant tumor This is a screening method for therapeutic drugs. According to this method, a dedifferentiation inducer, a pluripotent stem cell inducer, or a malignant tumor therapeutic agent can be obtained. This method may include a step of introducing a test substance into a cell and a step of measuring the expression or functional amount of a miR-520d-5p controlled gene.

Each method according to the above embodiment further includes (a) a step of introducing an expression or function inhibitor for the miR-520d-5p controlled gene into the cell, or (b) a cell into which the expression or function inhibitor has been introduced. A step of culturing or proliferating may be included.

In one embodiment of the present invention, “dedifferentiation” includes changing a cell into a cell with a lower differentiation state. For example, if a cell that has undergone differentiation has changed to a pluripotent stem cell, it can be said that it has been dedifferentiated. In one embodiment of the present invention, the “dedifferentiation inducer” is a substance having an action of bringing a cell closer to a cell having a lower differentiation state. In one embodiment of the present invention, the “pluripotent stem cell inducer” is a substance having an action of bringing a cell closer to a pluripotent cell such as a pluripotent stem cell. In one embodiment of the present invention, “reprogramming” refers to the action of bringing a cell closer to a pluripotent cell such as a pluripotent stem cell.

In one embodiment of the present invention, a “pluripotent stem cell” is a pluripotent stem cell. The pluripotent stem cell includes, for example, a cell expressing any undifferentiated marker at the same level or higher than hiPSC (HPS0002S253G1) which is a human induced pluripotent stem cell. Artificially produced pluripotent stem cells are sometimes referred to as iPS cells. Moreover, the pluripotent stem cell obtained by the production method of the above-mentioned embodiment may highly express p53. This p53 expression level is significantly higher than the p53 expression level of, for example, control samples (eg, hiPSC (HPS0002 253G1), p53 knockout cells, normal cells, or samples derived therefrom). It is preferable. In addition, the expression level of p53 is, for example, 1.05, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 3.0, 4.0, 5.0, 10, 100, or 1000 times or more that of the control sample. May be. The method for measuring the expression level of p53 is preferably real-time PCR in terms of measurement accuracy and simplicity. In the pluripotent stem cell obtained by the production method of the above-described embodiment, a protruding structure may be generated around the cell.

In the embodiment of the present invention, the “cell” used for dedifferentiation, pluripotent stem cell formation and the like may be a somatic cell. These somatic cells are other cells excluding germ cells, and include skin-derived cells or fibroblasts. Somatic cells usually have limited or disappeared pluripotency. Alternatively, the cell may be a malignant tumor cell. Malignant tumors include, for example, tumors that arise when normal cells are mutated. Malignant tumors can arise from all organs and tissues throughout the body, and when malignant tumor cells proliferate, they are known to clump together and invade and destroy surrounding normal tissues. This malignant tumor is, for example, carcinoma, sarcoma, hematological malignancy, lung cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, cervical cancer, uterus Body cancer, ovarian cancer, bladder cancer, prostate cancer, ureteral cancer, renal pelvic cancer, ureteral cancer, penile cancer, testicular cancer, brain tumor, cancer of central nervous system, cancer of peripheral nervous system, head and neck cancer (oral cancer, Pharyngeal cancer, laryngeal cancer, nasal cavity / sinus cancer, salivary gland cancer, thyroid cancer, etc.), glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, or malignant lymphoma.

In one embodiment of the present invention, the “undifferentiation marker” is a general term for compounds such as DNA strands, RNA strands or proteins that are specifically expressed in undifferentiated cells. Examples include Klf4, c-Myc, Oct4, Sox2, PROM1, Nanog, SSEA-1, ALP, eRas, Esg1, Ecat1, Fgf4, Gdf3, or REX-1. Sometimes referred to as a pluripotent stem cell marker.

In the embodiment of the present invention, “endogenous” means that the test substance is derived from the internal mechanism of the cell. For example, a protein that is constitutively expressed in a cell is endogenously contained only in the expressed protein.

In one embodiment of the present invention, “inhibiting gene expression” includes, for example, inhibiting a transcription mechanism from a gene to mRNA, or inhibiting a translation mechanism from mRNA to protein or polypeptide. In addition, for example, it includes inhibiting by inducing the degradation of a gene, mRNA, or protein. In the biochemical field, the role of a gene includes, for example, producing mRNA derived from the gene, producing a protein derived from the gene, and causing the protein derived from the gene to exhibit activity. Therefore, in one embodiment of the present invention, “inhibiting gene function” includes a decrease in the amount of mRNA or protein produced as a result of inhibiting gene expression. Alternatively, “inhibiting the function of a gene” includes reducing the activity of mRNA derived from the gene or protein derived from the gene.

In one embodiment of the present invention, the “state in which expression is inhibited” includes a state in which the expression level is significantly reduced as compared with the normal state. The above “significantly reduced” may be, for example, a state where the expression level is reduced to 0.35, 0.3, 0.2, 0.1, 0.05, 0.01, or 0 times or less, and the range of any two values thereof It may be in a state of decreasing to the inside. From the viewpoint of causing cell dedifferentiation more strongly or stably, it is preferably 0.2 times or less, more preferably 0.1 times or less. The expression level may be determined using the mRNA level or protein level as an index. Further, in one embodiment of the present invention, “significantly” may include, for example, a case where statistical significance is evaluated using Student's t test (one-sided or two-sided) and p <0.05. Or the state in which the difference has arisen substantially is included. In addition, the inhibitory strength in the “state in which the function is inhibited” is the same as in the embodiment of the inhibitory strength of expression inhibition.

In one embodiment of the present invention, the “form of the inhibitor” is not particularly limited, and may be, for example, an RNA chain, a DNA chain, a polynucleotide, a low molecular organic compound, an antibody, or a polypeptide. As the RNA strand, for example, an RNAi molecule against mRNA of a miR-520d-5p controlled gene can be used. As the DNA strand or polynucleotide, a DNA strand or polynucleotide encoding the RNA strand can be used. The form of this DNA strand or polynucleotide may be, for example, a vector. The low molecular weight organic compound may be obtained by utilizing combinatorial chemistry or HTS (High Throughput Screening). For the combinatorial chemistry, for example, an automatic synthesizer L-COS series (Shoko Scientific Co., Ltd.) may be used. For example, an Octet system (ForteBio) may be used for HTS. The antibody is, for example, an antibody against a miR-520d-5p controlled protein. The above antibody may be produced by a known antibody production method (for example, the method described in Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628.), Or a contract company (for example, EVEC, Inc.). At this time, it is preferable to prepare an antibody library and select one having a high inhibitory activity. The polypeptide can be purchased from a trust company (for example, Wako Pure Chemical Industries, Ltd.). The inhibitor includes a substance that inhibits the expression of the subject or a substance that inhibits the function. The inhibitor is preferably a substance having low cytotoxicity or substantially no cytotoxicity. In this case, side effects can be suppressed when the inhibitor is administered in vivo. From the viewpoint of suppressing cytotoxicity or side effects and stably causing dedifferentiation, the inhibitor is preferably an RNAi molecule or a polynucleotide encoding the RNAi molecule. From the viewpoint of stably causing dedifferentiation, RNAi molecules against ELAVL2651mRNA correspond to positions 631 to 651, 721 to 741, 1158 to 1178, or 1281 to 1299 of SEQ ID NO: 29. RNAi molecules that target RNA sequences are preferred. From the viewpoint of stably causing dedifferentiation, RNAi molecules against TEAD1699 mRNA correspond to positions 681 to 699, 1021 to 1038, 1349 to 1367, or 1429 to 1447 of SEQ ID NO: 30. RNAi molecules that target RNA sequences are preferred. From the viewpoint of stably causing dedifferentiation, the RNAi molecule for GATAD2B mRNA has an RNA sequence corresponding to positions 368 to 286, 854 to 872, 1009 to 1027, or 1166 to 1184 of SEQ ID NO: 31. The target RNAi molecule is preferred. Here, the target includes being capable of being combined.

In one embodiment of the present invention, the “RNAi molecule” is an RNA strand having an RNAi action, and examples thereof include siRNA, shRNA, miRNA, and small RNA having an RNAi action.

In one embodiment of the present invention, “RNAi” is a function of a target gene, mRNA, or the like by one or more of siRNA, shRNA, miRNA, short or long one or multiple strand RNA, or a modification thereof. Including the phenomenon that is suppressed or silenced. In general, the suppression mechanism by RNAi is sequence-specific and exists in various biological species. The mechanism of RNAi in a typical mammal when siRNA or shRNA is used is as follows. First, a vector capable of expressing siRNA or shRNA is introduced into cells. Then, after siRNA or shRNA is expressed in the cell, siRNA or shRNA becomes single-stranded, and then RISC (RNA-induced Silencing Complex) is formed. RISC uses the incorporated single-stranded RNA as a guide molecule and recognizes a target RNA strand having a sequence highly complementary to this single-stranded RNA. The target RNA strand is cleaved by an enzyme such as AGO2 in RISC. Thereafter, the cleaved target RNA strand is degraded. The above is an example of the mechanism. A plurality of RNAi molecules may be introduced into a cell at the same time, for example, 1, 2, 3, 4, 5, 6, or 10 may be introduced, and any one of these two values is included. Numbers may be introduced. The RNAi molecule is preferably single-stranded or double-stranded from the viewpoint of more stably suppressing the function of the target gene or mRNA.

For the design of RNAi molecules, Stealth RNAi designer (Invitrogen), siDirect 2.0 (Naito et al., BMC Bioinformatics. 2009 2009 Nov 30; 10: 392.), Etc. can be used. Further, it may be entrusted to a trust company (for example, Thermo Scientific). The RNAi action can be confirmed by quantifying the expression level of the RNA strand by real-time RT-PCR. Alternatively, it can also be performed by methods such as analysis of RNA strand expression level by Northern blot, analysis of protein amount by Western blot, and observation of phenotype. In particular, the method using real-time RT-PCR is efficient.

In one embodiment of the present invention, “siRNA” includes an RNA strand capable of inducing RNAi. In general, the duplex of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC. The guide strand incorporated into RISC is used to recognize the target RNA. Artificially produced RNAi research is mainly used in RNAi research, but some that exist endogenously in the living body are also known. The guide strand may be composed of RNA having 15 or more bases. If it is 15 bases or more, the possibility of binding to the target polynucleotide with high accuracy increases. The guide strand may be composed of RNA having 40 bases or less. If it is 40 bases or less, the risk that disadvantageous phenomena such as interferon response occur will be lower.

In one embodiment of the present invention, “shRNA” includes a single-stranded RNA strand that can induce RNAi and can form a hairpin-like structure (hairpin-like structure). Typically, shRNA is cleaved by Dicer in the cell, and siRNA is excised. It is known that target RNA is cleaved by this siRNA. The shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility that a hairpin-like structure peculiar to shRNA can be formed with high accuracy increases. The shRNA may be composed of RNA of 100 bases or less. If it is 100 bases or less, the risk that disadvantageous phenomena such as interferon response occur will be reduced. However, since many pre-miRNAs that are generally similar in structure and function to shRNA have a length of about 100 nucleotides or more, the length of shRNA is not necessarily 100 bases or less. However, it is thought that it can function as shRNA.

In one embodiment of the present invention, “miRNA” includes an RNA strand having a function similar to that of siRNA, and is known to suppress or degrade the translation of a target RNA strand. The difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.

In one embodiment of the present invention, “small RNA” refers to a relatively small RNA strand, and examples thereof include siRNA, shRNA, miRNA, antisense RNA, and single- or multiple-stranded small RNA. , But not limited to them. When small RNA is used, disadvantageous phenomena such as interferon response can be suppressed.

The RNA strand may contain an overhang consisting of 1 to 5 bases at the 5 ′ end or 3 ′ end. In this case, it is considered that the efficiency of RNAi increases. This number may be, for example, 5, 4, 3, 2, or 1 base, and may be within the range of any two of them. The overhang can be, for example, ac, c, uc, ag, aa, or uu. From the viewpoint of stably exhibiting RNAi action, the overhang is preferably 3'-terminal ac, c, or uc. When the RNA strand is a double strand, mismatch RNA may exist between the RNA strands. The number may be, for example, 1, 2, 3, 4, 5, or 10 or less, and may be in the range of any two of them. The RNA strand may include a hairpin loop, and the number of bases of the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, and any two values thereof. It may be within the range. The base sequence of the hairpin loop may be, for example, gugcuc or cucuuga. As long as this base sequence has a desired effect, one or a plurality of base sequences may be deleted, substituted, inserted, or added. In addition, the notation of each base sequence is the 5 ′ end on the left side and the 3 ′ end on the right side.

The length of the RNA strand is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. , 40, 50, 60, 80, 100, 200, or 500 bases, or any of these two values. If this number is 15 or more, the possibility of being able to bind to the target polynucleotide with high accuracy increases. Further, if this number is 100 or less, the risk of adverse phenomena such as interferon response occurring when RNA strands are administered in vivo is reduced. Interferon response is generally known as a phenomenon in which cells enter an antiviral state by sensing dsRNA.

In one embodiment of the present invention, the “polynucleotide” includes those in which a plurality of nucleotides, nucleobases, or their equivalents are combined. The polynucleotide includes a DNA strand or an RNA strand. In one embodiment of the present invention, the “RNA strand” includes those in which a plurality of RNAs or equivalents thereof are combined. Further, in one embodiment of the present invention, the “DNA strand” includes those in which a plurality of DNAs or their equivalents are combined. This RNA strand or DNA strand includes an RNA strand or a DNA strand in the form of a single strand or a plurality of strands (for example, a double strand). The RNA strand or DNA strand may be a cell uptake promoting substance (eg, PEG or a derivative thereof), a label tag (eg, a fluorescent label tag), a linker (eg, a nucleotide linker), or a chemotherapeutic agent (eg, an anti-malignant). A tumor substance or the like). The RNA strand or DNA strand can be synthesized using a nucleic acid synthesizer. In addition, it can also be purchased from a trust company (for example, Invitrogen). In vivo RNA strands or DNA strands may form salts or solvates. In addition, RNA strands or DNA strands in vivo may be subjected to chemical modification. The term RNA strand or DNA strand includes, for example, an RNA strand or DNA strand that forms a salt or solvate, or an RNA strand or DNA strand that has undergone chemical modification. The RNA strand or DNA strand may be an RNA strand analog or a DNA strand analog. The “salt” is not particularly limited, but includes, for example, an anion salt formed with any acidic (eg, carboxyl) group, or a cation salt formed with any basic (eg, amino) group. The salts include inorganic salts or organic salts, for example, salts described in “Berge” et al., “J. Pharm. Sci.,” 1977, 66, 1-19. Examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and the like. The “solvate” is a compound formed by a solute and a solvent. As for the solvate, for example, J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953) can be referred to. If the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers. Examples of the “chemical modification” include modification with PEG or a derivative thereof, fluorescein modification, or biotin modification.

In addition, from the viewpoint of stably exhibiting an RNAi action, the RNA strand preferably includes a base sequence complementary to a part of the base sequence of mRNA derived from the target gene. The “part” is, for example, 5, 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 50 bases or more. It may be within the range of any two of these values.

A plasmid that generates siRNA or shRNA for a specific gene can be purchased from, for example, a trust company (for example, Thermo Scientific, GeneCopoeia, etc.). The nucleotide sequences of the four plasmids capable of generating ELAVL2 siRNA used in Examples described later are SEQ ID NO: 25, 26, 27, or 28. The eight plasmids capable of generating TEAD1 siRNA or GATAD2B siRNA used in Examples described later are those in which DNA sequences encoding TEAD1 shRNA or GATAD2B shRNA are mounted on the psiLv-U6 ^TM vector.

In addition, shRNAs containing the nucleotide sequences of SEQ ID NOs: 5, 6, 7, or 8 are respectively generated from the four plasmids that can generate ELAVL2 siRNA used in Examples described later. These shRNAs are thought to be cleaved by enzymes in cells to generate siRNA. These siRNAs contain the nucleotide sequences of SEQ ID NO: 1 (uuauugguguuaaagucacgg), 2 (aauacgagaaguaauaaugcg), 3 (uuuguuugucuuaaaggag), or 4 (auuugcaucucugauagaagc), respectively. This base sequence of SEQ ID NO: 1, 2, 3, or 4 is a base sequence that is complementary to a part of ELAVL2 mRNA, and is considered to be a part that functions as a guide strand.

In addition, shRNAs containing the nucleotide sequence of SEQ ID NO: 13, 14, 15, or 16 are generated from the four plasmids that can generate TEAD1 siRNA used in the examples described later. These shRNAs are thought to be cleaved by enzymes in cells to generate siRNA. These siRNAs each contain the nucleotide sequence of SEQ ID NO: 9 (uuggcuuaucugcagaguc), 10 (gcuuguuaugaauggcag), 11 (guaagaaugguuggcaugc), or 12 (aguuccuuuaagccaccuu). The base sequence of SEQ ID NO: 9, 10, 11, or 12 is a base sequence complementary to a part of TEAD1EAD mRNA, and is considered to be a part that functions as a guide strand.

In addition, shRNAs containing the nucleotide sequence of SEQ ID NO: 21, 22, 23, or 24 are generated from the four plasmids that can generate GATAD2B siRNA used in the examples described later. These shRNAs are thought to be cleaved by enzymes in cells to generate siRNA. These siRNAs each contain the nucleotide sequence of SEQ ID NO: 17 (caacagauucaagcgaaga), 18 (caauagaugcugcauucug), 19 (caucaacauguguggaagg), or 20 (aggauguuguacgcugaca). The base sequence of SEQ ID NO: 17, 18, 19, or 20 is a base sequence that is complementary to a part of GATAD2B mRNA, and is considered to be a part that functions as a guide strand.

In one embodiment of the present invention, ELAVL2 siRNA is a base sequence complementary to the base sequence of SEQ ID NO: 1, 2, 3, or 4 (for example, the base sequence at positions 1 to 21 of the base sequence of SEQ ID NO: 5, the sequence The base sequence at positions 1-21 of the base sequence of No. 6, the base sequence at positions 1-19 of the base sequence of SEQ ID NO: 7, or the base sequence of positions 1-21 of the base sequence of SEQ ID NO: 8) Good. In one embodiment of the present invention, TEAD1 siRNA is a base sequence complementary to the base sequence of SEQ ID NO: 9, 10, 11, or 12 (for example, the base sequence at positions 1-19 of the base sequence of SEQ ID NO: 13, sequence The base sequence at positions 1-18 of the base sequence of number 14, the base sequence at positions 1-19 of the base sequence of sequence number 15, or the base sequence at positions 1-19 of the base sequence of sequence number 16) Good. In one embodiment of the present invention, GATAD2B siRNA is a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17, 18, 19, or 20 (for example, 1- 1 of the nucleotide sequence of SEQ ID NO: 21, 22, 23, or 24). 19-base sequence).

In one embodiment of the present invention, as long as the base sequences represented by SEQ ID NOs: 1 to 31 have a desired effect, (c) one or a plurality of base sequences is deleted or substituted in any base sequence, Inserted or added base sequence, (d) a base sequence having 90% or more homology with any base sequence, (e) a polymorphic base sequence complementary to any base sequence It may be one or more base sequences selected from the group consisting of a base sequence encoded by a polynucleotide that specifically hybridizes to a nucleotide under stringent conditions. In one embodiment of the present invention, the “complementary base sequence” is a base sequence possessed by another highly complementary polynucleotide capable of hybridizing to one polynucleotide. Hybridization refers to the property that a base pair can be formed by hydrogen bonding between bases between a plurality of polynucleotides. Base pairs can occur in Watson-Crick base pairs, Hoogsteen base pairs, or any other sequence specific form. A state in which two single strands are hybridized is called a double strand.

The “plurality” may be, for example, 10, 8, 6, 4, or 2, or may be less than any of these values. From the viewpoint of causing cell dedifferentiation more strongly or stably, the smaller the number, the better. It is known to those skilled in the art that an RNA strand that has undergone deletion, addition, insertion, or substitution of one or more bases maintains its biological activity.

The above “90% or more” may be, for example, 90, 95, 97, 98, 99, or 100% or more, and may be in the range of any two of them. From the viewpoint of causing cell dedifferentiation more powerfully or stably, a larger number is preferable. The above-mentioned “homology” may be calculated according to a method known in the art in the ratio of bases that are homologous in two or more base sequences. Before calculating the ratio, the bases of the base sequence groups to be compared are aligned, and a gap is introduced into a part of the base sequence if necessary to maximize the ratio of the same bases. Methods for alignment, percentage calculation, comparison methods, and related computer programs are well known in the art (eg, BLAST, GENETYX, etc.). In the present specification, “homology” can be expressed by a value measured by NCBI BLAST unless otherwise specified. Blastn can be used as the default algorithm for comparing sequences with BLAST.

For example, the following conditions can be adopted as the “stringent conditions”. (1) Use low ionic strength and high temperature for washing (e.g., 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C), (2) during hybridization Use a denaturing agent such as formamide (for example, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, And 750 mM sodium chloride, 75 mM sodium citrate), or (3) 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denaturation Incubate overnight at 37 ° C. in a solution containing sheared salmon sperm DNA, then wash the filter with 1 × SSC at approximately 37-50 ° C. The formamide concentration may be 50% or higher. The wash time may be 5, 15, 30, 60, or 120 minutes, or longer. Multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization reaction. For details, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995) .

The introduction of the inhibitor into cells and the method for culturing the cells can be performed according to methods known in the art. For introduction into cells, for example, infection introduction using a viral vector, calcium phosphate method, lipofection method, electroporation method, or microinjection can be used. Also, only introduced cells can be selected using drug resistance, cell sorter or the like. As the medium, for example, ReproStem, a medium for primate ES cells (Cosmo Bio), a medium for maintaining undifferentiation, a medium for normal human cells (for example, a medium based on DMEM or RPMI), and the like can be used. For example, the cells may be cultured in a ReproStem under conditions of 37 ° C., 5% CO ₂ and 10% FBS. This numerical value may be moved back and forth within a range of, for example, plus or minus 10, 20, or 30%. It may be established or cultured in the absence of feeder cells. The number of days of culture when establishing pluripotent stem cells is not particularly limited, and may be, for example, 1, 2, 3, 4, 5, 6, 8, 10, 15, 30, or 40 days or more, It may be within the range of any two of them. From the viewpoint of more stably establishing pluripotent stem cells, the cancer cells to be dedifferentiated are preferably undifferentiated cancer cells. In addition, when the RNA chain is introduced into a cell, a plurality of RNA chains may be introduced into the cell. In addition, when the DNA strand is introduced into a cell, a plurality of DNA strands may be introduced into the cell. Here, the number of “plural RNA strands” or “plurality of DNA strands” may be, for example, 2, 3, 4, 5, 6, 10, 12, 16, or 20 or more, It may be within the range of any two values. In one embodiment of the present invention, the “cell population” may be a substantially uniform cell population.

In one embodiment of the present invention, the “inhibitor” may be an RNA strand that binds to one or more 3′UTRs such as ELAVL2 and inhibits the transcription mechanism, but excludes miR-520d-5p. In addition, in the case of inhibiting the expression or function of one or more of ELAVL2, etc., the act of inhibiting using miR-520d-5p is excluded.

In one embodiment of the present invention, the “vector” refers to a viral (eg, lentivirus, adenovirus, retrovirus, or HIV) vector, a plasmid derived from E. coli (eg, pBR322), a plasmid derived from Bacillus subtilis (eg, pUB110). ), Yeast-derived plasmids (eg, pSH19), bacteriophages such as lambda phage, psiCHECK-2, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, pSUPER (OligoEngine), BLOCK-it Inducible H1 RNAi Entry Vector (Invitrogen), pRNATin-H1.4 / Lenti (GenScript, corp., NJ, USA) and the like can be used. The vector may contain components necessary for the expression of a DNA strand, such as a promoter, a replication origin, or an antibiotic resistance gene. The vector may be a so-called expression vector.

In one embodiment of the present invention, the “cell population” is a population including a plurality of cells. This cell population may be, for example, a population containing substantially uniform cells. The cell population may also be a cell preparation. A cell preparation may contain, for example, cells and buffer or media components. The pluripotent stem cell population may contain pluripotent stem cells, for example, 80, 90, 95, 96, 97, 98, 99, or 100% or more, and any one of these two values. May be.

In one embodiment of the present invention, the “therapeutic agent for malignant tumor” may further include DDS (Drug-Delivery System). In this case, the polynucleotide can be more efficiently introduced into the cell. Examples of DDS include gelatin hydrogel and atelocollagen.

In one embodiment of the present invention, “treatment” refers to the ability to exert a symptom improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. In one embodiment of the present invention, a “therapeutic agent” may be a pharmaceutical composition comprising one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing an active ingredient (for example, an inhibitor against one or more of ELAVL2 and the like) and the carrier. In addition, the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution). In addition, the therapeutic agent of a malignant tumor contains the drug (preventive agent) used for the prevention of a malignant tumor, the benign improvement inducer of normal malignant tumor, or a normal stem cell induction agent.

The administration route of the therapeutic agent is preferably one that is effective in the treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, granule or the like. When administering a polynucleotide, it is effective to use it as an injection. An aqueous solution for injection may be stored in, for example, a vial or a stainless steel container. The aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.

The dose is not particularly limited, but may be, for example, 0.0001 mg to 1000 mg / kg body weight per dose. The dosing interval is not particularly limited, but may be administered once every 1 to 10 days, for example. The dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. It may also be administered in combination with other appropriate chemotherapeutic drugs. The therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker decreases after administration, it may be determined that there was a therapeutic effect.

This therapeutic agent may be reprogrammed in vivo and further differentiated in vivo. In addition, the characteristics of malignant tumor cells may be changed in vivo and assimilated into surrounding tissues. In addition, malignant tumor cells may be changed to non-malignant tumor cells in vivo.

In one embodiment of the present invention, a “patient” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset. , Monkey or chimpanzee).

The various embodiments when the expression or function of the miR-520d-5p regulated gene is inhibited are also applied to the case where the activity of the RNA chain or protein derived from the miR-520d-5p regulated gene is inhibited. Yes, except for embodiments specific to gene function. Such an embodiment is also included in one embodiment of the present invention. For example, an embodiment of the present invention includes a method for inducing dedifferentiation, a method for inducing pluripotent stem cells, or a treatment for malignant tumor, which comprises a step of inhibiting the activity of an RNA chain or protein derived from a miR-520d-5p-controlled gene. Including methods. One embodiment of the present invention also includes a method for producing pluripotent stem cells, which comprises the step of inhibiting the activity of an RNA chain or protein derived from a miR-520d-5p-controlled gene. In addition, one embodiment of the present invention provides a dedifferentiation inducer, a pluripotent stem cell inducer, a malignant tumor therapeutic agent, or the like containing an activity inhibitor of an RNA chain or protein derived from a miR-520d-5p-controlled gene. Including.

In this specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to “or”. In this specification, when “in the range of two values” is specified, the range includes the two values themselves.

As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above can also be employ | adopted. Moreover, it is also possible to adopt a combination of the configurations described in the above embodiments.

Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

<Example 1> Production and evaluation of pluripotent stem cells by inhibition of ELAVL2 ELAVL2 is a gene encoding an RNA binding protein according to a summary published by NCBI. The present inventor discovered that when the expression of this gene was inhibited, the cells became pluripotent stem cells. Specific experimental procedures and results are described below.

(1) Introduction of ELAVL2 siRNA into HLF cells Four plasmids (lentiviral expression vectors) expressing siRNA against ELAVL2 mRNA were purchased from Thermo Scientific (B-9 (SEQ ID NO: 25), B-10 (sequence) No. 26), B-11 (SEQ ID NO: 27), B-12 (SEQ ID NO: 28)). These plasmids are DNAs encoding ELAVL2 siRNA incorporated into the pLKO.1 vector. Each of these plasmids was introduced into 293FT cells (Clontech, human embryonic kidney cell line). Thereafter, virus particles released to the culture supernatant were purified by ultracentrifugation (27000 rpm) and stored at -80 degrees. Thereafter, the stock was thawed. After measuring the virus titer of the four types of solutions obtained, the HLF cells (human hepatoma-derived cells) were simultaneously sprinkled at a concentration such that one copy of virus was contained in one cell, and the infection was introduced. Through the above procedure, four types of ELAVL2 siRNA (hereinafter also referred to as “ELAVL2 siRNAs”) were introduced into HLF cells by co-introduction. This ELAVL2 siRNA s has a base sequence complementary to ELAVL2 mRNA (SEQ ID NOs: 1, 2, 3, and 4 respectively).

As a result, an average of 92% suppression of ELAVL2 expression in HLF cells was confirmed. Furthermore, iPS cell-like globular cell populations could be induced 5 days after the introduction of ELAVL2 siRNAs (FIG. 1, upper left × 40, upper right × 100). FIG. 1 shows the result of observation with an inverted microscope manufactured by Olympus. The ELAVL2 siRNAs-introduced HLF cells obtained by the above procedure are hereinafter referred to as siELAVL2-HLF. Five days later, siELAVL2-HLF grew and proliferated little by little in places where it depended on the scaffold, unlike cancer cells. In addition, radially-like protrusion-like structures were observed from the spherical cell population over the entire circumference (FIG. 1, bottom x40).

In addition to the above, the present inventor conducted ELAVL2 siRNAs introduction into HLF cells and cultured for 1 month once a week for a total of 4 times. As a result, 4/4 = 100% of HLF cells were stemmed in an undifferentiated maintenance environment. The result of having become sex cell is obtained. In addition, when ELAVL2 siRNAs were introduced into well-differentiated cancer cells, after introduction of ELAVL2 siRNAs, normal undifferentiated cells were induced in vivo via moderately differentiated cancer, poorly differentiated cancer, and undifferentiated cancer. Yes. In addition, even when ELAVL2 siRNAs were introduced into 293FT cells instead of the above HLF cells, iPS cell-like spherical cells were observed.

(2) Evaluation of transcription level After obtaining about 1 × 10 ⁶ siELAVL2-HLF, the gene profile was evaluated at the transcription level. This evaluation was performed by RT-PCR. At this time, OneStep RT-PCR kit manufactured by QIAGEN was used. As a result, suppression of ELAVL2 and high expression of Oct4 and Nanog as undifferentiated markers and p53 as a tumor suppressor gene were confirmed (FIG. 2). In the figure, β-actin is a control, and * is P0.05.

In addition, HLF into which the plasmids B-9, B-10, B-11, and B-12 were respectively introduced was prepared, and the expression suppression rate of ELAVL2 was examined by RT-PCR. The results are as shown in FIG. 3, and in all cases, the expression of ELAVL2 was suppressed.

(3) Evaluation of translation level After obtaining about 1 × 10 ⁶ siELAVL2-HLF, the gene profile was evaluated at the translation level. This evaluation was performed by Western blot. At this time, iBlot from Invitrogen and an antibody from Abnova were used. As a result, it was reconfirmed that Oct4, Nanog and P53 were highly expressed (FIG. 4). In addition, ELAVL2 expression was down. That is, the expression pattern was maintained at the transcription level and the translation level.

(4) Cellular immunostaining Cell immunostaining was performed on siELAVL2-HLF by 3D Matrigel culture (Clontech). At this time, Human Embryonic Stem Cell Marker Antibody Panel from R & D Systems, Inc. was used. As a result, both Oct4 and Nanog were stained red with rhodamine, reconfirming the same level of expression at the protein level (FIG. 5). In the figure, bar represents 200 μm.

(5) Cell cycle analysis Cell cycle analysis was performed on HLF and siELAVL2-HLF introduced HLF, mock (purchased from pLKO.1: Addgene) (contract analysis to the Tottori University Biofunction Search Center). At this time, siELAVL2-HLF was cultured separately when RPMI 1640 (WAKO) was used as the culture medium and when undifferentiated maintenance culture medium ReproStem (ReproCELL) was used. As a result of DNA content comparison, siELAVL2-HLF was not a heterogeneous cell population peculiar to cancer cells, but had a special growth direction similar to normal cells toward a homogeneous growth direction. Specifically, in siELAVL2-HLF, the S period increased and the GoG1 period decreased. Since siELAVL2-HLF has few apoptotic cells unlike cancer cells, it can be considered that ELAVL2 siRNA is different from conventional anticancer components.

(6) Growth form siELAVL2-HLF and mock-introduced HLF were cultured in ReproStem for 14 days and then observed with a microscope. The results are shown in FIG. siELAVL2-HLF proliferated while spreading so as to be connected to the protrusions radiating from the spherical cells. In the figure, the upper row shows the growth form of mock-HLF and the lower row shows the growth form of siELAVL2-HLF. In addition, siELAVL2-HLF was confirmed to be stained by AP staining (alkaline phosphatase staining) (FIG. 7). AP is an undifferentiated cell marker. Therefore, it was suggested that the cell population in FIG. 6 is an undifferentiated cell population.

(7) Transplantation siELAVL2-HLF was cultured in ReproStem for 1 week and then implanted subcutaneously in immunodeficient mice (KSN / Slc, Charles River). The transplanted tissue was observed 2 months after transplantation (FIG. 8). As a result, all the cases were non-tumorous. In the figure, the upper photograph is an appearance of a mouse, the middle photograph is a photograph when the liver tissue is observed after incision of the abdomen, and the lower photograph is an enlarged photograph of the liver tissue. Scars were seen in the transplanted tissue. This becomes a trace in the transplanted living body after transplantation.

<Example 2> ELAVL2 reporter assay The present inventor has confirmed that miR-520d-5p binds to the 3 'UTR (SEQ ID NO: 10) of ELAVL2 mRNA (SEQ ID NO: 9), and the expression of ELAVL2 can be inhibited. , Found in the luciferase reporter assay. Specific experimental procedures and results are described below.

Synthetic miR-520d-5p (consigned to IDT) and pMIR-520d-5p expression lentivirus were co-introduced into HLF cells, and assayed with the Promega psiCHECK2 reporter vector. For evaluation, the expression level (RLU) of luciferin was measured with a microplate reader (TECAN), and the value corrected by dividing lenilla (RLU) with firefly (RLU) was used as a control (synthetic miR-520d-3p (IDT) ) Or pLKO.1 lentiviral vector (Addgene)). The results are shown in FIG. In the figure, * is P0.05 and ** is P0.01. In addition, “+” in miR-520d-5p or pMIR-520d-5p means that those genes were expressed. “-” Means not expressed. In addition, “+” in binding site 1 or 2 means that ELAVL2 3′UTR is a base sequence having no mismatch. “-” Means that ELAVL2 3′UTR is a base sequence having a mismatch. This result shows that miR-520d-5p specifically binds to ELAVL2 3 'UTR region 1 (3'UTR: 508-525) and 2 (3'UTR: 880-894) and suppresses ELAVL2 expression. It means that it was done. This indicates that ELAVL2 3 ′ UTR is the target of miR-520d-5p.

<Example 3> Production of pluripotent stem cells by inhibition of TEAD1 The present inventor used the database of DIANA-MICROT, miRDB, MicroRNA.org, and TargetScan-VERT, and the 3 ′ UTR of TEAD1 was miR-520d- We identified it as a target of 5p. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of TEAD1 will be described. First, a plasmid expressing siRNA against TEAD1 mRNA is purchased from GeneCopoeia. This plasmid is introduced into malignant tumor cells. Thereafter, pluripotent stem cells can be obtained by culturing for 5 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ. In addition, at least 8000 or more of candidate gene candidates for miR-520d-5p are considered, including those that are not actually targeted. In Example 3-9, target genes such as TEAD1 were identified using DIANA-MICROT, miRDB, MicroRNA.org, or TargetScan-VERT. Specifically, in each database, the miR- Genes were identified on the basis of the fact that there are many bases that match 520d-5p and excellent overall complementarity.

Example 4 Production of Pluripotent Stem Cells by Inhibition of GATAD2B The present inventors have identified that 3 ′ UTR of GATAD2B can be a target of miR-520d-5p using miRDB and TargetScan-VERT. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of GATAD2B will be described. First, a plasmid expressing siRNA or shRNA against GATAD2B mRNA is purchased from GeneCopoeia. This plasmid is introduced into malignant tumor cells. Subsequently, pluripotent stem cells can be obtained by culturing for 7 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 5> Production of pluripotent stem cells by inhibition of SBF2, PUM2, or NBEA The present inventor used MicroRNA.org and TargetScan-VERT, and SBF2, PUM2, and NBEA 3'UTR was miR- It was identified that it could be a target of 520d-5p. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of SBF2, PUM2, or NBEA will be described. First, a plasmid expressing siRNA or shRNA against SBF2 mRNA, PUM2 mRNA, or NBEA mRNA is purchased from Thermo Scientific. This plasmid is introduced into malignant tumor cells. Subsequently, pluripotent stem cells can be obtained by culturing for 7 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 6> Production of pluripotent stem cells by inhibition of each gene shown in Table 1 The present inventor used DIANA-MICROT to target that the 3 'UTR of each gene shown in Table 1 is miR-520d-5p Identified that could be. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of each gene shown in Table 1 will be described. First, a plasmid expressing siRNA or shRNA against mRNA derived from each gene shown in Table 1 is purchased from Thermo Scientific. This plasmid is introduced into malignant tumor cells. Subsequently, pluripotent stem cells can be obtained by culturing for 7 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 7> Production of pluripotent stem cells by inhibition of each gene shown in Table 2 The present inventor can use miRDB to target the 3R UTR of each gene shown in Table 2 as miR-520d-5p It was identified. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of each gene shown in Table 2 will be described. First, a plasmid expressing siRNA or shRNA against mRNA derived from each gene shown in Table 2 is purchased from Thermo Scientific. This plasmid is introduced into malignant tumor cells. Subsequently, pluripotent stem cells can be obtained by culturing for 7 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 8> Production of pluripotent stem cells by inhibition of each gene shown in Table 3 The present inventor used MicroRNA.org to target that the 3 'UTR of each gene shown in Table 3 is miR-520d-5p Identified that could be. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of each gene shown in Table 3 will be described. First, a plasmid expressing siRNA or shRNA against mRNA derived from each gene shown in Table 3 is purchased from Thermo Scientific. This plasmid is introduced into malignant tumor cells. Subsequently, pluripotent stem cells can be obtained by culturing for 7 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 9> Production of pluripotent stem cells by inhibition of each gene shown in Table 4 The present inventor used TargetScan-VERT to target the 3 'UTR of each gene shown in Table 4 as miR-520d-5p Identified that could be. Hereinafter, a method for producing pluripotent stem cells using the inhibitory effect of each gene shown in Table 4 will be described. First, plasmids that express siRNA or shRNA against mRNA derived from each gene shown in Table 4 are purchased from Thermo Scientific. This plasmid is introduced into malignant tumor cells. Thereafter, pluripotent stem cells can be obtained by culturing for 5 days. When the cultured cells are observed with a microscope, a spherical cell population can be observed. When this cell population is transplanted into a mouse, tumor non-formation or differentiation can occur in the transplanted organ.

<Example 10> Evaluation of inhibitory effect of ELAVL2, TEAD1, and GATAD2B Four types of plasmids expressing siRNA against TEAD1 mRNA and four types of plasmids expressing siRNA against GATAD2B mRNA were purchased from GeneCopoeia. These plasmids are obtained by incorporating DNA strands encoding TEAD1 siRNA or GATAD2B siRNA into a psiLv-U6 ^™ vector. Then, in the same procedure as in Example 1, HLF cells co-introduced with 4 types of ELAVL2 siRNA, HLF cells co-introduced with 4 types of TEAD1 siRNA, and HLF cells co-introduced with 4 types of GATAD2B siRNA Produced. The TEAD1 siRNA has a base sequence complementary to TEAD1 mRNA (SEQ ID NOs: 9, 10, 11, 12 respectively). The GATAD2B siRNA has a base sequence complementary to GATAD2B mRNA (SEQ ID NOs: 17, 18, 19, and 20 respectively).

Next, for each HLF cell, the expression suppression rate of ELAVL2, TEAD1, and GATAD2B was examined by RT-PCR. The results are as shown in FIGS. 10 to 12, and the expression of any gene was suppressed. In the figure, D is the number of culture days, R1 is a group of cells that have formed liver tissue or teratoma in about 1 month after in vivo, and R2 is a group of cells that has not shown tumor formation after about 1 month in in vivo. I mean.

<Example 11> Evaluation of DNA methylation level The DNA methylation level as an index of pluripotent stem cell formation was examined by the following procedure. In the same procedure as in Example 10, 4 types of ELAVL2 siRNA co-introduced HLF cells (siE), 4 types of TEAD1 siRNA co-introduced HLF cells (siT), and 4 types of GATAD2B siRNA were co-introduced. HLF cells (siG) were prepared. Also, 4 types of ELAVL2 siRNA and 4 types of TEAD1 siRNA co-introduced HLF cells (siET), 4 types of ELAVL2 siRNA and 4 types of GATAD2B siRNA co-introduced HLF cells (siEG), 4 types of TEAD1 HLF cells (siTG) into which siRNA and 4 types of GATAD2B siRNA were co-introduced were prepared. In addition, HLF cells (siETG) into which four types of ELAVL2 siRNA, four types of TEAD1 siRNA, and four types of GATAD2B siRNA were co-introduced were prepared.

For each HLF cell 7 days after co-introduction, the DNA methylation level was measured with MethylFlash-Methylated-DNA-Quantification-Kit (EPIGENTEK). The results are shown in FIG. The scramble in the figure means an miRNA-sized RNA base sequence that is assumed not to affect any gene. The data were compared after measuring the absolute quantitative value and setting the methylation level in HLF cells to 1.

Furthermore, HLF cells were changed to Huh7 cells (well-differentiated liver cancer cells), and the DNA methylation level was examined by the same procedure. The results are shown in FIG.

In HLF cells, a reduction in methylation level was observed with the introduction of each siRNA. In particular, siETG was able to induce a demethylation level equivalent to hiPSC. In Huh7 cells, each siRNA was introduced to induce a demethylation level equivalent to hiPSC. In addition, when HLF cells into which siETG had been introduced were transplanted subcutaneously or intraperitoneally into immunodeficient mice (KSN / Slc), no tumor was formed.

<Example 12> Evaluation of tumorigenicity In the same procedure as in Example 11, HLF cells into which four types of ELAVL2 siRNA, four types of TEAD1 siRNA, and four types of GATAD2B siRNA were co-introduced were prepared. Two days after co-introduction, HLF cells (1 × 10 ⁷ cells or more) were transplanted subcutaneously (12 mice) and intraperitoneally (12 mice) into immunodeficient mice. As a result, tumor formation ability was not observed in all cases in observations over 4 months.

In the same procedure as in Example 11, 4 types of ELAVL2 siRNA and 4 types of TEAD1 siRNA co-introduced HLF cells, 4 types of ELAVL2 siRNA and 4 types of GATAD2B siRNA co-introduced HLF cells, 4 types of HLF cells into which TEAD1 siRNA and 4 types of GATAD2B siRNA were co-introduced were prepared. Two days after co-introduction, HLF cells (1 × 10 ⁷ cells or more) were transplanted subcutaneously (8 mice) and intraperitoneally (8 mice) into immunodeficient mice. Again, no tumor-forming ability was observed in all cases over 4 months of observation.

HLF cells into which four types of ELAVL2 siRNAs were co-introduced were prepared by the same procedure as in Example 11. Two days after co-introduction, HLF cells (1x10 ⁷ cells or more) were transplanted subcutaneously into immunodeficient mice, but only 2 of 12 cases formed subcutaneous tumors, but not malignant tumors, such as "adipocytes and collagen fibers. It was a diagnosis of “non-undifferentiated tumor tissue having a differentiating tendency including normal cells”, and it was an unprecedented benign tumor difficult to make a clear diagnosis on the pathological tissue. In the other 10 cases, no tumor formation was observed after 4 months of observation.

The results of Examples 1 to 12 above show that cells are reprogrammed into pluripotent stem cells by inhibiting the expression of one or more of the genes listed above. In particular, reprogramming of malignant tumor cells is an area where little research results have been made worldwide, and it has new possibilities for the treatment of malignant tumors in the future. In addition, the technique described in this example can be used for the production of pluripotent stem cells for research and regenerative medicine. Furthermore, the pluripotent stem cells obtained in this example did not become malignant even after a long period of 4 months or longer after in vivo administration. Therefore, it can be said that the pluripotent stem cell obtained in the present Example is very excellent in safety.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (14)

A method of inducing cell dedifferentiation, which comprises a step of inhibiting the expression or function of a miR-520d-5p controlled gene. The miR-520d-5p controlled genes are ELAVL2, TEAD1, GATAD2B, SBF2, PUM2, NBEA, UBE2E3, CPEB3, GNAO1, WNK1, MEF2A, SEH1L, HOOK3, KLHL23, PAPOLG, JAZF1, JAG1, CPEB2, ZCRB1 2. The method according to claim 1, wherein the gene is one or more genes selected from the group consisting of ZEB2, EPHA7, UBN2, PELI2, PRKD3, and FCHO2. The method according to claim 1 or 2, wherein the inhibition is inhibition by an RNAi molecule against mRNA of a miR-520d-5p controlled gene. The inhibition includes ELAVL2 mRNA, TEAD1 mRNA, GATAD2B mRNA, SBF2 mRNA, PUM2 mRNA, NBEA mRNA, UBE2E3 mRNA, CPEB3 mRNA, GNAO1 mRNA, WNK1 mRNA, MEF2A mRNA, SEH1L mRNA, FOH3 Inhibition by RNAi molecules against one or more mRNAs selected from the group consisting of mRNA, JAG1 mRNA, CPEB2 mRNA, ZCRB1 mRNA, ZFC3H1 mRNA, ZEB2 mRNA, EPHA7 mRNA, UBN2 mRNA, PELI2 mRNA, PRKD3 mRNA, and FCHO2 mRNA The method according to any one of claims 1 to 3. A method for producing pluripotent stem cells, comprising a step of inducing cell dedifferentiation by the method according to any one of claims 1 to 4. A pluripotent stem cell or a pluripotent stem cell population obtained through the production method according to claim 5. A regenerative medical material obtained through the step of culturing the pluripotent stem cells according to claim 6. Remedies for malignant tumors, including inhibitors of expression or function of miR-520d-5p regulated genes. The miR-520d-5p controlled genes are ELAVL2, TEAD1, GATAD2B, SBF2, PUM2, NBEA, UBE2E3, CPEB3, GNAO1, WNK1, MEF2A, SEH1L, HOOK3, KLHL23, PAPOLG, JAZF1, JAG1, CPEB2, ZCRB1 9. The therapeutic agent according to claim 8, which is one or more genes selected from the group consisting of, ZEB2, EPHA7, UBN2, PELI2, PRKD3, and FCHO2. The therapeutic agent according to claim 8 or 9, wherein the inhibitor is an RNAi molecule against mRNA of a miR-520d-5p controlled gene, or a polynucleotide encoding the RNAi molecule. The inhibitor is ELAVL2 mRNA, TEAD1 mRNA, GATAD2B mRNA, SBF2 mRNA, PUM2 mRNA, NBEA mRNA, UBE2E3 mRNA, CPEB3 mRNA, GNAO1 mRNA, WNK1 mRNA, RNA HL23mRNA, SEH1L mRNA One or more RNAi molecules selected from the group consisting of JAZF1 mRNA, JAG1 mRNA, CPEB2 mRNA, ZCRB1 mRNA, ZFC3H1 mRNA, ZEB2 mRNA, EPHA7 mRNA, UBN2 mRNA, PELI2 mRNA, PRKD3 mRNA, and FCHO2 mRNA, or RNAi thereof The therapeutic agent according to any one of claims 8 to 10, which is a polynucleotide encoding an RNAi molecule. The therapeutic agent according to any one of claims 8 to 11, which induces malignant tumor cells in a dedifferentiation direction in vivo. De-differentiation inducer containing an expression or function inhibitor for miR-520d-5p controlled gene. A pluripotent stem cell inducer containing an expression or function inhibitor for miR-520d-5p regulated gene.

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