WO2021157689A1 - Control agent for initial stage of g1 phase of cell cycle - Google Patents

Abstract

The present invention addresses the problem of providing a cell cycle control agent based on a novel mechanism, by identifying a novel gene involved in a control mechanism of a cell cycle for canceration or differentiation of cells and using the gene and a translation product thereof.　The present invention provides a control agent for the initial stage of the G1 phase of the cell cycle, said control agent containing: C7orf26 gene having the activity of arresting the cell cycle at the initial stage of the G1 phase and causing a transition from the G1 phase to the G0 phase; and a translation product thereof.

Description

G1 phase initial regulator of cell cycle

The present invention relates to the use of a new factor having an activity of arresting the cell cycle in the early G1 phase and shifting from the early G1 phase to the G0 phase.

The cell cycle refers to the cell division cycle in eukaryotes, which is the G1 phase, which is the preparatory phase for DNA replication, the S phase, which is the preparatory phase for DNA replication, the G2 phase, which is the preparatory phase for mitosis, and mitosis. It forms one cell cycle in four phases, the M phase, which is the mitotic phase. Cells undergo self-renewal by accurately repeating G1-S-G2-M-G1. In addition, differentiated cells such as nerve cells and septal muscle cells and many somatic cells lose their ability to divide and go to the quiescent phase (G0 phase). In the G0 phase, general cells become unique morphology and perform their original activities (stem cell differentiation, endocrine cell hormone production, nerve fiber signal transduction, muscle contraction, etc.). Cancer cells are abnormal cells that do not transition to G0 phase and continue to proliferate indefinitely, and it is thought that the difference between cancer cells and normal cells lies in their ability to transition to G0 phase.

In the cell cycle, there is a cell cycle checkpoint that monitors whether cells are proceeding correctly in order to achieve orderly proliferation and differentiation of cells and accurate replication of genetic information possessed by cells. Cell cycle checkpoints include G1 / S checkpoints, S phase checkpoints, G2 / M checkpoints, and M phase checkpoints. At the S-phase checkpoint, normal replication is performed, at the G2 / M checkpoint, DNA replication is completed without problems, and at the M-phase checkpoint, whether the chromosome is bound to the mitotic spindle, etc. are checked. .. Of these, DNA damage is checked in G1, S phase, S phase, and M phase invasion. If the DNA is damaged or replicates poorly, the cell cycle is stopped or slowed down at checkpoints and adjusted so that the next step does not begin before it is ready. It is the complex of cyclins and cyclin-dependent kinases (CDKs) that are activated by the binding of cyclins that advance the cell cycle. The transition from G1 to S phase is triggered by the cyclin D / CDK4 (or CDK6) complex, which activates the cyclin E / CDK2 complex and the cells transition to S phase. After that, activation of cyclin A / CDK2 causes cells to move from S phase to G2 phase, and activation of cyclin B / CDK1 causes cells to move to M phase. Several proteins are known that block the action of the cyclin-CDK complex and stop the cell cycle when DNA damage is found, and p53 and its downstream p21 are typical examples. At the four checkpoints, monitor whether there is any problem in advancing the cell cycle, and if any abnormality is found, immediately stop the cell cycle. For example, at the G1 / S checkpoint, when DNA damage is confirmed, p53 is expressed to express downstream p21, which binds to the cyclin-CDK complex and causes the loss of phosphorylation of CDK. As a result, the cell cycle is arrested in the G1 phase. In the biological reaction of the checkpoint of the cell cycle, genes are transcribed and translated to produce new proteins only in p53 and p21, and the production of p53 and p21 is a very short reaction called "immediately early gene expression". It is done in. The generated p21 binds to the cyclin-CDK complex and inhibits the phosphorylation and dephosphorylation function of CDK, and as a result, the cell cycle is arrested (Non-Patent Document 1). When abnormalities occur in these factors related to P53, p21, and cyclin-CDK complex, checkpoints cannot be controlled, and even if DNA abnormalities occur, the cell cycle does not stop, damaged DNA is replicated as it is, and cells It is thought that disordered growth occurs and becomes cancerous. Therefore, a typical disease caused by abnormal cell cycle is cancer. On the other hand, the molecular mechanism of the transition from the G1 phase to the G0 phase has not been fully elucidated so far, but it promotes the differentiation of stem cells and progenitor cells, the growth and maintenance of organs and individuals, and the role that cells originally have. Is important.

The present invention identifies a new gene involved in the control mechanism of the cell cycle for canceration and differentiation of cells and its pathway, and utilizes the gene, its translation product, and a conjugate substance to develop a novel mechanism. To provide a cell cycle regulator based on.

The present inventors have discovered that a missense mutation (p.A425V) due to a single nucleotide substitution of the C7orf26 gene exists in a large family of congenital abnormalities of the eye, and the function of the C7orf26 gene, which is the causative gene of the congenital abnormality of the eye. Upon analysis, the C7orf26 gene was a novel member of the integrator complex and was involved in mRNA maturation. Surprisingly, as a new function, it is highly expressed in the M to early G1 phase of the cell cycle, has the activity of arresting the cell cycle in the early G1 phase, and the cell cycle arrest is a tumor suppressor gene. Achieved through certain p53 upregulation, the C7orf26 gene acts as a transcriptional cofactor for the transcription factor E2F family members (E2F4, E2F1, etc.) in the promoter region of p53, increasing p53 expression, and in that case, the integrator. We found that the complex subunit (INTS) also acts as a transcriptional cofactor in collaboration with C7orf26. It was also shown that each member of INTS also has the above-mentioned cell cycle arresting action of C7orf26 alone. In addition, it was confirmed that cell death was induced by long-term expression or high expression of p53 in cells in which C7orf26 gene was continuously or highly expressed at a physiological level for a long period of time. Furthermore, when iPS cells into which the C7orf26 gene was introduced were induced to differentiate into ectoderm, mesodermal, and endoderm cells, they were transformed from normal iPS cells into ectodermal, mesodermal, and endoderm cells in the early stages of development. In the differentiation, or in the later differentiation into the brain and retina, the number of differentiated cells increased and the specific gene was highly expressed. Therefore, the C7orf26 gene stopped the cell cycle in the early G1 phase and then G0. It was also clarified that it has the activity of shifting to the phase and can promote the differentiation of cells. The present invention has been completed based on such findings.

That is, the present invention includes the following.
[1] A G1 phase initial regulator of the cell cycle, which comprises any of the following proteins (A) to (C).
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(B) The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted, and the cell cycle is stopped in the early G1 phase, and from the early G1 phase to the G0 phase. Protein with activity to transfer;
(C) An activity consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Protein to have.
[2] A G1 phase early regulator of the cell cycle containing any of the following genes (D) to (F).
(D) A gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(E) An activity consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Genes containing DNA encoding the proteins they have;
(F) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, arrests the cell cycle in the early G1 phase, and shifts from the early G1 phase to the G0 phase. A gene that contains DNA that encodes a protein that has the activity to cause it.
[3] The G1 phase early regulator of the cell cycle according to [2], wherein the gene is inserted into a vector.
[4] A drug for treating cancer, which comprises the G1 phase initial regulator of the cell cycle according to any one of [1] to [3].
[5] A differentiation-promoting agent for undifferentiated cells, which comprises the G1 phase initial regulator of the cell cycle according to any one of [1] to [3].
[6] The agent for promoting differentiation of undifferentiated cells according to [5], wherein the undifferentiated cells are stem cells or progenitor cells.
[7] A method for promoting differentiation of undifferentiated cells, which comprises the step of expressing any of the following genes (D) to (F) in undifferentiated cells.
(D) A gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(E) An activity consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Genes containing DNA encoding the proteins they have;
(F) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, arrests the cell cycle in the early G1 phase, and shifts from the early G1 phase to the G0 phase. A gene that contains DNA that encodes a protein that has the activity to cause it.
This application claims the priority of Japanese Patent Application No. 2020-020019 filed on February 7, 2020, and includes the contents described in the specification of the patent application.

In the present invention, the C7orf26 gene, which has been newly identified to have an activity of arresting the cell cycle in the early G1 phase and shifting from the early G1 phase to the G0 phase, is used for randomly proliferating cells such as cancer cells. The cell cycle can be stopped in the early G1 phase to induce cell death. Therefore, the C7orf26 gene and its translation products are effective as pharmaceuticals for the prevention and / or treatment of diseases caused by or related to abnormal cell cycle control such as cancer. In addition, when the C7orf26 gene is expressed in stem cells and progenitor cells of each cell such as muscle, nerve, and endocrine cells, it stops cell division and proliferation in the state of stem cells and progenitor cells, and shifts to the G0 phase for differentiation. Can be promoted. Furthermore, stem cells and progenitor cells that do not transition to the GO phase are thinned out by cell death, and the expression of the C7orf26 gene allows cells that can preserve or differentiate such thinned stem cells and progenitor cells in a quiescent state. It is expected to be directed to functional cells. Therefore, according to the present invention, the efficient use of stem cells as raw materials for reproductive medical materials, the increase in yield of regenerative medicine / cell medicine materials, the increase in yield of agricultural products / livestock products / marine products, and the efficiency of differentiation of produced cells are used to improve immunological preparations and hormones. It is effective for increasing the yield of preparations, treating fertility by promoting the differentiation of germ cells, promoting the reproduction and reproduction of beneficial organisms, and suppressing the reproduction and / or reproduction of harmful organisms (including harmful insects, beasts, and harmful plants). be.

FIG. 1A shows the results of flow cytometry analysis of HeLa cells whose cell cycle was synchronized by administration of hydroxyurea (HU) for 24 hours after 0 hours, 16 hours, and 20 hours. FIG. 1B shows the relative expression levels of C7orf26 mRNA and Ki67 mRNA in HeLa cells at each stage of the cell cycle (error bars ± SD, n = 3). FIG. 1C shows immunostaining of C7orf26 in HeLa cells at each phase of the cell cycle (scale bar: 5 μm). Figure 2 shows HeLa / pPB-CuO-C7orf26 cells (HeLa cells in which the C7orf26 gene was transposoned into the genome using the PiggyBac Cumate Switch Inducible Vector) cultured in a medium supplemented with a cumate whose concentration was gradually increased. , C7orf26 is expressed depending on the concentration of cumate added) shows the changes in the relative expression levels of C7orf26, p53, p21, and Ki67 mRNA. FIG. 3A shows the C7orf26 gene transposoned into the genome using HeLa / pPB-CuO-C7orf26 cells (PiggyBac Cumate Switch Inducible Vector) cultured in a medium supplemented with cumate continuously (5th, 6th, 8th). The flow cytometric analysis results of (C7orf26 is expressed according to the concentration of cumate added) in the integrated HeLa cells are shown. FIG. 3B shows the results of flow cytometric analysis of HeLa / pPB-CuO-C7orf26 cells tuned with hydroxyurea (HU) or Ro3306. FIG. 3C shows HeLa / pPB-CuO-C7orf26 cells tuned with hydroxyurea (HU), Ro3306 treatment, or serum-free medium (serum (-)), and serum-recovered medium after 24-hour serum starvation (serum (-)). The relative expression levels of C7orf26, p53, p21, and Ki67 mRNA at each stage of the cell cycle in HeLa / pPB-CuO-C7orf26 cells cultured in serum recovery) are shown. It is a schematic diagram showing the scheme of C7orf26 expression in the cell cycle and the site where transcriptional regulation by p53 is possible. A and B in FIG. 5-1 are transcriptions of p53 by C7orf26 and integrator complex subunits (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14). The results of a reporter assay to elucidate the control mechanism are shown. On the contrary, FIG. 5C shows the results of a reporter assay for elucidating that p53 has a transcriptional regulation mechanism of C7orf26 (*: p <0.05, Mann-Whitney U-test, error bar: ± SD, in each figure). n = 3) Figure 5-2 shows C7orf26 and INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14 during the cell cycle in HeLa cells tuned with hydroxyurea (HU). The relative expression level of is shown. A in FIG. 6-1 shows the relative expression levels of E2F1, E2F4, and E2F6 during the cell cycle in HeLa cells tuned with hydroxyurea (HU). B in FIG. 6-1 shows the results of a reporter assay for p53 expression in HeLa cells in which E2F was knocked down using siRNA (*: p <0.05, Mann-Whitney U-test, error bars: ± SD, n). = 3). C in FIG. 6-1 shows the results of Western blot analysis using an anti-E2F4 antibody (a-E2F4) of a HeLa cell extract transfected with pCMV-GFP or pCMV-C7orf26-Flag. A in Figure 6-2 shows the results of a reporter assay for C7orf26 expression in HeLa cells transfected with pCMV-E2F4 or pCMV-E2F6 (*: p <0.05, Mann-Whitney U-test, error bars: ± SD, n = 3). B in FIG. 6-2 shows the relative expression level of C7orf26 during the cell cycle in HeLa cells synchronized with hydroxyurea (HU) and acted on E2F4 and E2F6 siRNA (*: p <0.05, Mann-Whitney U-). test, error bar: ± SD, n = 3). C in FIG. 6-2 shows the change in the expression level of C7orf26 during the cell cycle in HeLa cells by administration of a CDK1 inhibitor (CDK1 inhibitor). FIG. 7A shows the C7orf26 gene transposoned into the genome using HeLa / pPB-CuO-C7orf26 cells (PiggyBac Cumate Switch Inducible Vector) cultured in a medium supplemented with cumate continuously (5th, 6th, 8th). The flow cytometric analysis results (decrease in live cells and increase in dead cells) of C7orf26 expressed in the integrated HeLa cells depending on the concentration of cumate added are shown. FIG. 7B shows the results of flow cytometry analysis (decrease in live cells and increase in dead cells) of HeLa / pPB-CuO-C7orf26 cells cultured in a medium supplemented with cumate continuously (5 days, 8 days). .. FIG. 8 shows the cell death induction test results of HeLa cells (uterine cancer cell line) and glioma cells U-251MG in which C7orf26 was forcibly expressed [ad: HeLa cells, eg: glioma cells U-251MG, h: C7orf26 non- Mitochondrial staining of introduced HeLa cells (control)]. FIG. 9A shows human iPS cells HiPSC / pPB-CuO-C7orf26 cells (human iPS cells in which C7orf26 is incorporated into the genome by PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed according to the concentration of cumate added). Relative expression levels of C7orf26, p53, p21, Ki67 mRNA in cells (Cum +) induced to differentiate into ectoderm with (cumate) -added medium and cells (Cum-) induced to differentiate into ectoderm with cumate-free medium (The expression level in Cum- is 1). FIG. 9B shows a group of cells (Cum +) in which HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into ectoderm in a medium supplemented with cumate, and a group of cells in which differentiation was induced into ectoderm in a medium not supplemented with cumate (cumate). The flow cytometry analysis result of Cum-) is shown. FIG. 9C shows cells in which HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into ectoderm with a medium supplemented with cumate (Cum +) and cells induced to differentiate into ectoderm with a medium without cumate (Cum-). ) Shows the relative expression levels of C7orf26, Pax6, Pax3, and Sox1 mRNA (the expression level in Cum- is 1). FIG. 10A shows human iPS cells HiPSC / pPB-CuO-C7orf26 cells (human iPS cells in which C7orf26 is incorporated into the genome by PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed according to the concentration of cumate added). C7orf26, p53, p21, Ki67 mRNA in cells (Cum +) induced to differentiate into retinal ganglion cells in (cumate) -added medium and cells (Cum-) induced to differentiate into retinal ganglion cells in a medium without cumate (cumate) Indicates the relative expression level (the expression level in Cum- is 1). In FIG. 10B, HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into retinal ganglion cells (Cum +) in a medium supplemented with cumate, and into retinal ganglion cells in a medium not supplemented with cumate (cumate). The relative expression levels of Brn3B, Math5, and C7orf26 mRNA in cells (Cum-) are shown (the expression level in Cum- is 1). FIG. 10C shows cells in which HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into the brain with a medium supplemented with cumate (Cum +) and cells induced to differentiate into the brain with a medium not supplemented with cumate (Cum-). The relative expression levels of Otx2, En1 and C7orf26 mRNA are shown (the expression level in Cum- is 1). FIG. 11A shows human iPS cells HiPSC / pPB-CuO-C7orf26 cells (human iPS cells in which C7orf26 is incorporated into the genome by PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed according to the concentration of cumate added). The flow cytometry analysis results of the cell group (Cum +) induced to differentiate into the mesoderm with the (cumate) -added medium and the cell group (Cum-) induced to differentiate into the mesoderm with the cumate-free medium are shown. FIG. 11B shows cells in which HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into mesoderm in a medium supplemented with cumate (Cum +) and cells induced to differentiate into mesoderm in a medium not supplemented with cumate (Cum-). ) Shows the relative expression levels of C7orf26 and TBXT mRNA (the expression level in Cum- is 1). FIG. 12A shows human iPS cells HiPSC / pPB-CuO-C7orf26 cells (human iPS cells in which C7orf26 is incorporated into the genome by PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed according to the concentration of cumate added). The flow cytometry analysis results of the cell group (Cum +) induced to differentiate into endometrial follicle with (cumate) -added medium and the cell group (Cum-) induced to differentiate into endometrial follicle with medium without cumate (cumate) are shown. FIG. 12B shows cells in which HiPSC / pPB-CuO-C7orf26 cells were induced to differentiate into endoderm in a medium supplemented with cumate (Cum +) and cells induced to differentiate into endoderm in a medium without cumate (Cum-). ) Shows the relative expression levels of C7orf26 and Sox17 mRNA (the expression level in Cum- is 1).

Hereinafter, the present invention will be described in detail.
1. 1. G1 phase early regulator of the cell cycle In the present invention, the human C7orf26 gene, which was newly identified to have the activity of arresting the cell cycle in the early G1 phase and shifting from the early G1 phase to the G0 phase, is derived from 449 amino acids. Encodes the protein. The nucleotide sequence of human C7orf26 cDNA (SEQ ID NO: 1) and the amino acid sequence of the translation product (SEQ ID NO: 2) are registered in the National Center of Biotechnology Information (NCBI) database as NCBI accession numbers NM_024067 and NP_076972, respectively. Further, the C7orf26 gene used in the present invention may be a C7orf26 gene derived from an animal species other than human, and the C7orf26 gene derived from another animal or a translation product thereof is the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 By using the amino acid sequence shown in (1) as a query and searching the genome and / or cDNA database of non-human mammals or the protein database using BLAST or FASTA, etc., it is derived from the desired animal species. The nucleotide sequence and amino acid sequence of C7orf26 can be obtained.

Therefore, the G1 phase initial regulator of the cell cycle of the present invention includes any of the following proteins (sometimes referred to as "C7orf26 protein" in the present specification).
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(B) The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted, and the cell cycle is stopped in the early G1 phase, and from the early G1 phase to the G0 phase. Protein with activity to transfer;
(C) An activity consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Protein to have.

Regarding (B) above, the range of "1 to several" in the "amino acid sequence in which one or several amino acids are added, deleted, or substituted in the amino acid sequence" includes the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The number is not particularly limited as long as it retains the activity of stopping the cell cycle in the early G1 phase and shifting from the early G1 phase to the G0 phase, but for example, 1 to 10, preferably 1 to 7, and more. It preferably means 1 to 5, and more preferably 1 to 3. Amino acid deletion means that an arbitrary amino acid is selected and deleted from the amino acid sequence shown in SEQ ID NO: 2. Further, the addition of an amino acid means adding one to several amino acids to the N-terminal or C-terminal side of the amino acid sequence shown in SEQ ID NO: 2. Examples of amino acid substitutions include conservative amino acid substitutions.

The above-mentioned conservative amino acid substitutions include polar, electrical, and structural properties such as hydrophobic amino acids, polar amino acids, acidic amino acids, basic amino acids, amino acids having branched side chains, and aromatic amino acids. Refers to the substitution of amino acids with similar properties. Examples of hydrophobic (non-polar) amino acids are glycine, alanine, valine, leucine, isoleucine, proline, etc. Examples of polar amino acids are serine, threonine, cysteine, methionine, aspartic acid, glutamine, etc. Examples of acidic amino acids are aspartic acid. Examples of acids and glutamic acids, basic amino acids are lysine, arginine and histidine, examples of amino acids with branched side chains are valine, isoleucine and leucine, examples of aromatic amino acids are phenylalanine, tyrosine, tryptophan and histidine. be. Preferred conservative amino acid substitutions include substitutions between amino acids selected from valine and leucine and isoleucine, phenylalanine and tyrosine, lysine and arginine, alanine and valine, and asparagine and glutamine.

Amino acid addition, deletion, or substitution can be performed by modifying the gene encoding the above protein by a method known in the art. Introducing a mutation into a gene can be carried out by a known method such as the Kunkel method or the Gapped duplex method, or a method similar thereto.

Regarding (C) above, "amino acid sequence having 90% or more sequence identity" means that the sequence identity of the amino acid sequence is at least 90% or more, preferably 95% or more, more preferably 97%. As mentioned above, it is most preferably 98% or more. The sequence identity of amino acids can be determined by using a method well known to those skilled in the art, sequence analysis software, or the like. For example, the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm can be mentioned. Here, the sequence identity of the amino acid sequence is a value obtained by comparing the amino acid sequence to be evaluated with respect to the amino acid sequence shown in SEQ ID NO: 2 and expressing the frequency of appearance of the same amino acid at the same site in%.

The "cell" in the present invention includes all cells of animals including humans, for example, germ cells such as sperm and egg, somatic cells constituting the living body, stem cells, precursor cells, cancer cells, and immortalizing ability separated from the living body. Includes cells (cell lines) that are acquired and stably maintained in vitro, cells that have been isolated from the living body and artificially modified. The "body cells that make up the living body" are, for example, skin, muscle, bone, cartilage, connective tissue, blood vessels, blood (including umbilical cord blood), bone marrow, heart, eye, brain, nerve, thoracic gland, spleen, adrenal gland, lung. , Pancreas, liver, stomach, small intestine, large intestine, liver, bladder, prostate, testis, ovary, uterus, etc. The cells of the present invention also include cells other than animals, such as plant cells and microbial cells.

Examples of somatic cells constituting the living body include fibroblasts, bone marrow cells, erythrocytes, granulocytes (neutrophils, eosinophils, basophils), monospheres, and lymphocytes (T cells, B cells, NK cells). ), Thrombosis, macrophages, dendritic cells, bone cells, osteoblasts, cartilage cells, epidermal cells, keratinocytes (keratinocytes), fat cells, mesenchymal cells, epithelial cells, endothelial cells, hepatic parenchymal cells, oval cells, Examples thereof include dendritic cells, glial cells, neurons, oligodendrocytes, microglia, stellate glial cells (astrosites), heart cells, muscle cells, pancreatic beta cells, and the like. Stem cells include pluripotent stem cells and somatic stem cells. "Pluripotent stem cells" are stem cells derived from ectoderm, mesoderm, and endoderm that have the ability to differentiate into all cells existing in the living body (pluripotency) and also have proliferative ability. say. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells (ntES cells) derived from cloned embryos obtained by transplantation, and pluripotent germ stem cells (mGS). Cells), sperm stem cells (GS cells), adult pluripotent stem cells (MAPC cells), muse cells (MUSE cells) and the like, but iPS cells and ES cells are preferable. "Somatic stem cells" are somatic stem cells present in bone marrow, blood, skin (epidermal, dermal, subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidneys, muscles and other tissues. For example, hematopoietic stem cells, vascular endothelial stem cells, mesenchymal stem cells, hepatic stem cells, nerve stem cells, pancreatic stem cells, intestinal stem cells, reproductive stem cells and the like can be mentioned. "Cancer cell" refers to a cell derived from the somatic cell and acquired infinite proliferative capacity. "Progenitor cells" refer to cells in the process of differentiating from the stem cells into specific somatic cells or germ cells, and are hematopoietic progenitor cells and myeloid progenitor cells (erythroblasts, myeloid blasts, progenitor cells). , Giant nuclei, etc.), lymphocytic progenitor cells (monoblasts, lymphoblasts, etc.) and the like. "Cell line" refers to a cell that has acquired infinite proliferative capacity by artificial manipulation in vitro.

The "G1 phase early control" in the present invention means that the cell cycle is stopped in the early G1 phase through the upregulation of p53, and the transition from the early G1 phase to the G0 phase is defined as "early G1 phase". , Means the period until cycloin D-cyclin-dependent kinase (CDK) 4/6, which acts as an engine to move from the M phase of mitosis to the S phase of the next cell cycle, begins to work. The action of Cyclin D-CDK4 / 6 can be suppressed by p53 and p21, and the cell cycle is stopped at this time, which enables the transition to the G0 phase. "Activity to arrest the cell cycle in the early G1 phase and shift from the early G1 phase to the G0 phase" is caused by introducing the C7orf26 gene into cells, overexpressing it, and enhancing the expression of p53 and p21. , PCR and FACS analysis. Further, the above-mentioned stop includes not only a complete stop but also a deceleration. Further, "having an activity of stopping the cell cycle in the early G1 phase and shifting from the early G1 phase to the G0 phase" is substantially the same as the activity retained by the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. It means that they are equivalent.

The above C7orf26 protein can be produced by a genetic engineering method using a polynucleotide containing a gene encoding it. For example, a method of preparing RNA by in vitro transcription from a recombinant vector having a polynucleotide containing a gene encoding the C7orf26 protein and performing in vitro translation using this as a template, or a polynucleotide containing a gene encoding the C7orf26 protein is suitable. It can be produced by operably linking to a vector, incorporating it, introducing it into a host cell to prepare a transformed cell, and expressing the target C7orf26 protein from the transformed cell. As the above vector, a vector suitable for the host cell into which the vector is introduced can be appropriately selected and used. In addition, the vector contains a polynucleotide containing a gene encoding a C7orf26 protein operably linked to a suitable promoter, and preferably contains a transcription termination signal, that is, a terminator region, downstream of the polynucleotide. Furthermore, a selectable marker gene for selecting transformed cells (drug resistance gene, gene complementing auxotrophic mutation, etc.) can also be included. In addition, a sequence encoding a tag sequence useful for separating and purifying the expressed protein may be included. The vector may also be integrated into the genome of the host cell. The vector can be introduced into a host cell by a transformation method known per se, such as a competent cell method, a protoplast method, or a calcium phosphate coprecipitation method.

The host cell into which the vector is introduced and used for the expression of the recombinant protein may be any cell as long as the vector can be expressed, and commonly used known microorganisms such as bacteria, yeast, fungi, and mammalian cells can be mentioned. Be done. Examples of bacteria include Gram-negative bacteria such as Escherichia coli and Gram-positive bacteria such as Bacillus or Streptomyces. Recombinant cells can be cultured by a method known per se that is suitable for the host cell.

For the expressed protein, the cells collected by centrifugation or the like are ground from the culture supernatant of the host cell by ultrasound or glass beads, and then solid substances such as cell fragments are removed by centrifugation or the like. After preparing the crude enzyme solution, known methods used for protein and peptide purification, such as ammonium sulfate, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric point chromatography, Gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrate or antibody, reversed-phase column chromatography, chromatography such as HPLC, precision filtration, ultrafiltration, back-penetration filtration, etc. It is possible to purify by using one or more combinations such as filtration treatment.

The C7orf26 protein can also be produced by a method of chemically synthesizing based on its amino acid sequence. The C7orf26 protein can be chemically synthesized by a known chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method).

Further, according to another aspect of the present invention, the cell cycle G1 phase early regulator of the present invention comprises a gene encoding a C7orf26 protein. Specific examples of the gene include any of the following genes (D) to (F) (may be referred to as "C7orf26 gene" in the present specification).
(D) A gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(E) It consists of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, and has an activity of arresting the cell cycle in the early G1 phase and shifting from the G1 phase to the G0 phase. Genes containing DNA encoding proteins;
(F) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, arrests the cell cycle in the early G1 phase, and shifts from the G1 phase to the G0 phase. A gene containing DNA that encodes an active protein.

Regarding (E) above, "a base sequence having 90% or more sequence identity" means that the sequence identity of the base sequence is at least 90% or more, preferably 95% or more, more preferably 97%. As mentioned above, it is most preferably 98% or more. The sequence identity of the base sequence can be determined by using a method well known to those skilled in the art, sequence analysis software, or the like. For example, the blastn program of the BLAST algorithm and the fasta program of the FASTA algorithm can be mentioned. Here, the sequence identity of the base sequence is a value obtained by comparing the base sequence to be evaluated with respect to the base sequence shown in SEQ ID NO: 1 and expressing the frequency of appearance of the same base at the same site in%.

Regarding (F) above, the "stringent condition" means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, Green and Sambrook, Molecular Cloning, 4th Ed (2012) , Cold Spring Harbor Laboratory Press can be referred to and determined as appropriate. Specifically, stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, for example, the condition that the reaction temperature of hybridization is in the range of 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 68 ° C., and / or the formamide concentration in the hybridization solution. Is in the range of 0 to 50%, preferably 20 to 50%, and more preferably 35 to 45%. Further, the sodium salt concentration in washing the filter after hybridization is 15 to 750 mM, preferably 15 to 500 mM, more preferably 15 to 300 mM, 15 to 200 mM or 15 to 100 mM.

The method for obtaining the C7orf26 gene is not particularly limited. For example, a human cDNA library (prepared according to a conventional method from an appropriate cell expressing the gene of the present invention) is synthesized using a known method, and a probe DNA prepared based on the nucleotide sequence of SEQ ID NO: 1 is obtained. It can be used to isolate the cDNA of interest. The obtained cDNA can be amplified by a conventional gene amplification method such as a PCR method, a NASBN method, or a TMA method. In addition, cDNA can also be obtained by the RT-PCR method using mRNA isolated from human cells as a template using a primer set prepared based on the nucleotide sequence of SEQ ID NO: 1. Other animals also have homologous genes, which can be used.

In order to make the above C7orf26 protein or C7orf26 gene into a form that can be introduced into cells, for example, there are the following means. Here, the cells can be either in vitro cells (cultured cells) or in vivo cells (cells in vivo).

The C7orf26 protein can be made into a form that can be introduced into cells without changing its structure or function, for example, by mixing a protein molecule with a pharmacologically acceptable carrier solution and formulating it. .. Such a drug can be introduced into cells, for example, into in vitro cells by a microinjection method. Alternatively, an intracellular introduction method using lipids can be adopted.

As another aspect, the protein can be introduced into the cell by forming a fusion protein in which a cell membrane-passing peptide is linked to the N-terminal side of the C7orf26 protein. By including this transmembrane peptide, the C7orf26 protein passes through the cell membrane and is taken up into the cell. As the cell membrane-transparent peptide, PTD (protein transmission domain) of HIV-1 / TAT or PTD of Homeobox protein antennapedia of Drosophila can be used, and a DNA fragment encoding a region corresponding to PTD is referred to as the above-mentioned cDNA. By linking to prepare a fused DNA fragment and expressing this fused DNA fragment in a host cell such as Escherichia coli, a fused protein in which a PTD peptide is linked to the N-terminal side can be prepared. Alternatively, a fusion protein in which the transmembrane peptide is linked can be prepared by a method of binding the C7orf26 protein and the PTD peptide via a divalent cross-linking agent (for example, EDC, β-alanine, etc.).

On the other hand, the C7orf26 gene or C7orf26 mRNA can be made into a form that can be introduced into cells by, for example, incorporating it into an expression vector. As the expression vector, a known expression vector for eukaryotic cells having a promoter, splicing region, poly (A) addition site, etc. can be used, and the C7orf26 gene or C7orf26 mRNA should be inserted into the cloning site of this expression vector. Can construct a C7orf26 gene expression vector.

This expression vector can be introduced into in vitro cells (cultured cells) by a known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method.

In addition, for in vivo cells (cells in vivo), for the purpose of promoting uptake into cells and enhancing the directivity toward target cells, for example, means such as a viral or non-viral gene transfer vector. Can be introduced into cells. The drug in such a form can be introduced into a living body for gene therapy. Here, examples of the viral vector include an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, a lentivirus vector, a vaccinia virus vector, a baculovirus vector, and the like. It is particularly desirable to use a retrovirus vector because the viral genome is integrated into the host chromosome after the cells are infected and the foreign gene incorporated into the vector can be expressed stably and for a long period of time. Examples of the non-viral vector include liposomes, artificial lipid vesicles, hollow nanoparticles, polymer compounds such as dendrimers, and the like.

2. Drugs for Cancer Treatment The G1 phase initial regulator of the cell cycle of the present invention arrests the cell cycle in the early G1 phase of cancer cells and increases the expression of p53, as will be specifically shown in Examples described later. Since it induces cell death, it can be used as a drug for treating and / or preventing a disease caused by or related to cell cycle dysregulation, particularly as a drug for treating cancer. A typical disease caused by dysregulation of the cell cycle is a malignant tumor (cancer), the type of which is not particularly limited, and is gastric cancer, breast cancer, lung cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer. , Kidney cancer, bladder cancer, skin cancer, uterine cancer, brain tumor, osteosarcoma, bone marrow tumor, leukemia, malignant lymphoma, etc. In particular, intractable cancers such as glioma and undifferentiated cancers are effective in combination with the well-differentiated stage G0 phase. The presence or absence of cell death induction and its degree can be detected by methods such as TUNEL method, DNA ladder detection method, quantification of DNA fragmentation rate, and measurement of cell size distribution. In addition, cell proliferation of diseases in which cells overproliferate can be suppressed. The disease is not particularly limited and includes adenomas such as ptosis, hyperthyroidism, pheochromocytoma, polycystic ovary syndrome and the like.

When the G1 phase initial regulator of the cell cycle of the present invention is used for the treatment of cancer or cell hyperproliferative disease, it is administered in a therapeutically effective amount to a mammal having cancer or cell hyperproliferative disease. .. Here, examples of mammals include humans, dogs, cats, sheep, goats, cows, horses, pigs and the like. "Therapeutically effective amount for cancer and hyperproliferative disease" means that administration of this drug to proliferating cancer and hyperproliferative cells stops the growth of cancer and hyperproliferative cells, and reduces or eliminates the size of the tumor. The amount to bring. The specific dose should be appropriately increased or decreased depending on the route of administration, the age and weight of the patient, the type and malignancy of the cancer, the presence or absence of metastasis or recurrence, the type and growth rate of hyperproliferative cells, the size of the tumor, and the like.

Examples of the administration form include intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, local, intramass, oral, transdermal, rectal, intravaginal, intranasal, and sublingual administration. Specifically, for example, solid tumors in various organs that can be easily accessed by surgery may be administered by local injection into or near the tumor using a stereotaxic needle or the like, and leukemia. Non-solid tumors such as brain tumors, cancers in sites that are difficult to access by surgical operations such as brain tumors, and metastatic cancers can be administered by intravenous injection. In addition, the above-mentioned administration method can be appropriately selected and used depending on the type and site of cancer.

When the G1 stage initial control agent of the cell cycle of the present invention is formulated as a pharmaceutical for treating cancer, the C7orf26 protein, the C7orf26 gene, and the C7orf26 mRNA are mixed with a pharmacologically and pharmaceutically acceptable additive. Tablets, powders, granules, fine granules, capsules, internal liquids (suspendories, syrups, emulsions, etc.), external liquids (injections, sprays / aerosols, inhalants, etc.) by methods known in the field. , Coating agent, etc.), injection, drip, suppository, etc. can be formulated into various formulations. Examples of pharmacologically and pharmacologically acceptable additives include pharmaceutical substrates and carriers, excipients, diluents, binders, preservatives, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegrants, stabilizers, preservatives, preservatives, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, colorants Etc. may be appropriately added to prepare various pharmaceutical forms that can be orally or parenterally administered systemically or topically by various known methods. The drug for treating cancer of the present invention prepared in various pharmaceutical forms can be orally or parenterally administered systemically or topically. When the drug for treating cancer of the present invention is orally administered, it is formulated or used in tablets, capsules, granules, powders, pills, liquid solutions for internal use, suspensions, emulsions, syrups, etc. It may be a dry product to be redissolved. When the pharmaceutical agent for treating cancer of the present invention is administered parenterally, intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, suppository, etc. In the case of an injectable formulation, it is provided in the form of a unit-dose ampoule or a multi-dose container.

As a form of gene therapy, there are an in vitro method (ex vivo method) in which a target cell is taken out of the body and a gene is introduced, and an in vivo method (in vivo method) in which a gene is introduced into the body. Early stage control agents are applied to any form of treatment. In the in vitro method, cells derived from the patient may be cultured once in vitro, the above gene transfer process may be performed, and then administered to the patient. In the in vitro method, the above gene transfer vector may be directly administered into the patient body (organ tissue, skin, muscle, etc.). ) May be administered. In addition, such cancer and hyperproliferative disease treatments may be combined with well-known cancer and hyperproliferative disease treatment means, including surgery, chemotherapy, and radiation therapy.

3. 3. Differentiation promoter for undifferentiated cells and method for promoting differentiation of undifferentiated cells The G1 phase initial regulator of the cell cycle of the present invention sets the cell cycle to G1 phase with respect to stem cells, as specifically shown in Examples described later. It can be used as a differentiation-promoting agent for undifferentiated cells because it can be stopped in the early stage and then transferred to the G0 phase to induce differentiation into target cells.

In the present invention, the "undifferentiated cell" refers to an undifferentiated cell capable of differentiating into ectodermal cells, mesenchymal cells, endometrial cells or promoting their repair, and refers to stem cells, precursor cells, and the like. Includes dedifferentiated (juvenile) cells of mature cells. The stem cells are not particularly limited as long as they meet the object of the present invention, and examples thereof include the above-mentioned somatic stem cells and pluripotent stem cells (ES cells, iPS cells, etc.). The stem cells may be either primary cultured cells, subcultured cells or frozen cells. Here, the ectodermal cells include sensory organ cells (cells of the retina and inner ear), neural cells (nerve cells (for example, forearn nerve cells, middle brain nerve cells, cerebral nerve cells, posterior brain nerve cells, spinal cord nerve cells). Etc.), neural tube cells, neural ridge cells), epidermal cells (epidermal cells, crystalline epithelial cells), and mesenchymal cells include muscular cells (myosblasts, muscle satellite cells, etc.), skeletal system Cells (osteoblasts, bone cells, cartilage cells, etc.), fat cells, dermal cells, cardiovascular cells (myocardial cells, hematopoietic stem cells, erythrocytes, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymph Spheres, etc.), urogenital cells (tubular cells, mesangial cells, parafilamental cells, testis, ovaries, etc.), connective tissues, etc. Examples of endometrial cells include digestive cells (hepatocellular, bile duct, etc.) Examples include cells of tissues such as cells, pancreatic endocrine cells, adenocarcinoma cells, pancreatic duct cells, absorptive cells, cup cells, panate cells, intestinal endocrine cells, etc.), lungs, and thyroid gland.

The differentiation-promoting agent for undifferentiated cells of the present invention can be applied to all undifferentiated cells as long as it has the same characteristics regarding the direction of differentiation and the process of differentiation. For example, the differentiation-promoting agent for undifferentiated cells according to the present invention includes mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, birds, reptiles, and both sexes. It can exert its effect on undifferentiated cells of species, fish, reptiles, crustaceans, and soft animals.

The application of the differentiation-promoting agent for undifferentiated cells according to the present invention to the cells may be in vitro or in vivo, and in either case, the action can be exerted. For example, when the differentiation-promoting agent for undifferentiated cells of the present invention is applied in vitro, an expression vector having the target C7orf26 gene downstream of the expression promoter is typically introduced into the undifferentiated cells of interest. It is carried out by the method of culturing.

Examples of the expression promoter used here include CMV promoter, SV40 promoter and the like. Further, as the expression vector, for example, a plasmid vector or a liposome can be used as the non-viral vector, and an adenoviral vector, a retroviral vector or the like can be used as the viral vector. Commercially available products from each manufacturer can also be used as these expression vectors. The expression vector may be integrated into the genome of the host cell. Further, as a method for introducing the expression vector into cells, for example, a lipofection method, an electroporation method, a method of incorporating a gene into a viral vector and infecting the virus vector can be used.

The medium and additives used in culturing undifferentiated cells into which the above gene has been introduced are not particularly limited, and for example, media and additives generally used for culturing stem cells may be used. Specifically, basic media containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glassgow Minimum Essential Medium (Glasgow MEM), Hanks Liquid salt solution) etc. In addition, the medium contains cell growth factors (for example, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epithelial growth factor (EGF), hepatocellular growth factor (eg,) depending on the target cell for inducing differentiation. HGF), vascular endothelial growth factor (VEGF), insulin, etc.), cytokines (eg, interleukins, chemokines, interferons, colony stimulators, tumor necrosis factors, etc.), neurotrophic factors (nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), etc.) and other physiologically active substances, such as amino acids (eg, glycine, phenylalanine, lysine, aspartic acid, glutamate, etc.), vitamins, etc. (For example, biotin, pantothenic acid, vitamin D, etc.), serum albumin, antibiotics, etc. may be contained.

The incubator used for culturing undifferentiated cells is not particularly limited, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles.

The incubator may be cell non-adhesive or adhesive, and is appropriately selected according to the purpose. As the cell adhesion incubator, one treated with a cell support substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells. Examples of the cell-supporting substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

The culture conditions for undifferentiated cells may follow the usual conditions used for culturing stem cells and the like, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples do not limit the present invention.
The materials and test methods used in each example are shown.

(vector)
Human C7orf26 (NM_024067) ORF clones (Origene #: RC219786; pCMV C7orf26-Flag) and negative control GFP-expressing clones (TaKaRa # Z2470N; pAcGFP1-C1) with a C-terminal Myc-DKK tag were purchased. These expression vectors were prepared using the EndoFree plasmid Maxi Kit (QIAGEN #: 12362).

(Primer)
The following is a list of primers used for gene expression analysis by quantitative PCR (real-time RT-PCR).
C7orf26_F: 5'-CCGAGATTCTGCTGCCAGTTC-3' (SEQ ID NO: 3)
C7orf26_R: 5'-GTGACAATCATTTCAAGCAAGTCCA-3' (SEQ ID NO: 4)
p53_F: 5'-CAGCACATGACGGAGGTTGT-3' (SEQ ID NO: 5)
p53_R: 5'-TCATCCAAATACTCCACACGC-3' (SEQ ID NO: 6)
P21_F: 5'-TGTCCGTCAGAACCCATGC-3' (SEQ ID NO: 7)
P21_R: 5'-AAAGTCGAAGTTCCATCGCTC-3' (SEQ ID NO: 8)
Ki67_F: 5'-GCCTGCTCGACCCTACAGA-3' (SEQ ID NO: 9)
Ki67_R: 5'-GCTTGTCAACTGCGGTTGC-3' (SEQ ID NO: 10)
Pax6_F: 5'-TTTAAAGATCCTGGAGGTGGACATA-3' (SEQ ID NO: 11)
Pax6_R: 5'-GCTCAGGTGCTCGGGTTCTA-3' (SEQ ID NO: 12)
Brn3b_F: 5'-TGACACATGAGCGCTCTCACTTAC-3' (SEQ ID NO: 13)
Brn3b_R: 5'-ACCAAGTGGCAAATGCACCTA-3' (SEQ ID NO: 14)
Math5_F: 5'-CCCTAAATTTGGGCAAGTGAAGA-3' (SEQ ID NO: 15)
Math5_R: 5'-CAAAGCAACTCACGTGCAATC-3' (SEQ ID NO: 16)
Pax3_F: 5'-CCATACGTCCTGGTGCCATC-3' (SEQ ID NO: 17)
Pax3_R: 5'-TGAACATGCCCGGGTTCTC-3' (SEQ ID NO: 18)
Sox1_F: 5'-ATGCACAACTCGGAGATCA-3' (SEQ ID NO: 19)
Sox1_R: 5'-GCGAGTACTTGTCCTTCTTG-3' (SEQ ID NO: 20)
En1 F: 5'-GAGCGCAGGGCACCAAATA-3' (SEQ ID NO: 21)
En1_R: 5'-CGAGTCAGTTTTGACCACGG-3' (SEQ ID NO: 22)
Otx2_F: 5'-CAAAGTGAGACCTGCCAAAAAGA-3' (SEQ ID NO: 23)
Otx2_R: 5'-TGGACAAGGGATCTGACAGTG-3' (SEQ ID NO: 24)
TBXT_F: 5'-TATGAGCCTCGAATCCACATAGT-3' (SEQ ID NO: 25)
TBXT_R: 5'-CCTCGTTCTGATAAGCAGTCAC-3' (SEQ ID NO: 26)
Sox17_F: 5'-AGCAGAATCCAGACCTGCACAA-3' (SEQ ID NO: 27)
Sox17_R: 5'-TCAGCGCCTTCCACGACTT-3' (SEQ ID NO: 28)

(Cell culture)
HeLa cells were purchased from ATCC, 293T cells from Takara Bio, glioma cells U-251MG from DS Biofferma Medical, and human iPS 409B02 cells from RIKEN BRC. HeLa cells were grown in DMEM medium (Thermo Fisher Scientific CAT #: 11965092) supplemented with 10% FBS and 1% Pen Strep. 293T cells were grown in DMEM medium (Thermo Fisher Scientific CAT #: 35050061) supplemented with 10% FBS and 1 × GlutaMAX ^{TM Supplement.} Human iPS cells were grown on a feeder layer (inactivated MEFs) in a medium supplemented with Prime ES medium (ReproCELL) at 10 ng / mL and bFGF (Invitrogen).

(Cell cycle analysis)
Cell cycle analysis was performed in combination with FxCycle PI / RNase staining solution (Thermo Fisher Scientific, # F10797) and according to the recommended staining protocol using the Click-iT Plus EdU flow cytometry assay kit (Thermo Fisher Scientific, # C10632). went. HeLa cells were incubated with 10 μMEdU for 1.5 hours at 37 ° C and harvested. Cells were fixed with Click-iT fixative for 15 minutes, followed by permeabilization with Click-iT saponin-based permeabilization and detergent for 15 minutes in the dark. A click-IT reaction with Alexa Fluor 488 pycolyl azaide was performed in a reaction volume of 0.6 mL for 30 minutes. After washing, the cell pellet was suspended in 0.2 mL of FxCycle PI / RNase staining solution and incubated in the dark at room temperature for 30 minutes. The cells were then analyzed with an Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific). EdU was detected using Alexa Fluor 488 pycolyl azaide using 488 nm excitation using a green emission filter (530 / 30 nm), and PI was detected using 488 nm excitation using an orange emission filter (574/26 nm). used. Compensation analysis was performed using unstained HeLa cells and monostained cells with either EdU or PI. The cell cycle population ratio was analyzed using Attune Cytometric Software v2.1.

(Synchronization of cell cycle)
For HeLa cells, the cell cycle was synchronized by culturing in a medium containing 10 mM hydroxyurea (Sigma # H8627) or 10 μM Ro3306 (Abcam # ab141491) or in a serum-free medium for 24 hours. Then, the HeLa cells tuned with hydroxyurea were continuously cultured in a normal medium, and collected after a lapse of a predetermined time for expression analysis and cell cycle analysis.

(Luciferase Reporter Assay)
The luciferase reporter assay was performed using the LightSwitch Luciferase Assay Kit (Active Motif # 32031) according to the manufacturer's protocol. As a reporter vector, LightSwitch promoter reporter GoClone (Active Motif # S717666, # S721662) containing the c7orf26 and p53 promoter regions and the 3'-RenSP luciferase region downstream was used. The reporter vector was co-transfected into HeLa cells with FuGENE HD transfection reagent (Promega # E2311) together with c7orf26, p53, and the cDNA clone encoding INTS, and 48 hours after transfection, the cells in the 96-well plate were cotransfected. Frozen. After thawing, LightSwitch Luciferase Assay Reagent was added to the wells, and after 30 minutes of reaction, it was transferred to a 1/2 Area Plate-96 plate (PerkinElmer # 6002350). Chemiluminescent signals were read using a 2030 ARVO X multi-label counter device (PerkinElmer). The signal ratio for each promoter activity was calculated in comparison with GFP-transfected cells.

(Cell death assay)
HeLa cells were transfected with the pU6-shC7orf26-GFP vector, transfected, cultured in 96-well plates or 8-well glass chambers, and immobilized 36 hours later. Immunostaining with M30 CytoDEATH antibody (Roche # 12140322001) was performed on fixed cells to detect cell death. After washing twice with PBS containing 0.1% Tween 20, cells were incubated with M30 CytoDEATH antibody solution (1:50) for 60 minutes at room temperature. Cells were washed twice and incubated with Goat anti-Mouse IgG (H + L) secondary antibody and Alexa Fluor Plus 555 solution (1: 500) (Thermo Fisher Scientific # A32727) for 30 minutes at room temperature. ProLong with DAPI After mounting with Gold Antifade reagent (Thermo Fisher Scientific # A32727), M30 CytoDEATH-positive cells were observed with DeltaVision ELITE (CORNES Technologies).

(Immunofluorescence)
Specimens were fixed in 4% paraformaldehyde (pH 7.0) at room temperature for 20 minutes. After rinsing twice with PBS, the specimen was incubated with 0.1% Triton X-100 at room temperature for 15 minutes and rinsed twice with PBS. The sample was then incubated with 3% BSA for 60 minutes at room temperature, followed by a primary antibody reaction at 4 ° C. for 16 hours. After washing 3 times with PBS for 5 minutes, a secondary antibody reaction was carried out by incubating with the corresponding species-species-specific Alexa Fluor-conjugated antibody at room temperature for 1 hour in the dark. After washing 4 times with PBS for 5 minutes, the sample was mounted with ProLong Gold Antifade Reagent (Thermo Fisher Scientific # A32727) containing DAPI and observed with DeltaVision ELITE (CORNES Technologies).

(E2F knockdown by siRNA)
E2F4 specific siRNA (Human E2F4 (1874) siRNA-SMARTpool L-003262-00-0005), E2F6 specific siRNA (Human E2F6 (1876) siRNA-SMARTpool L-003264-00-0005), E2F1 Control siRNAs containing specific siRNA (E2F1 (1869) siRNA-SMARTpool, 5 nmol L-003259-00-0005) and scrambled sequences (ON-TARGETplus Non-targeting Pool, # D-001810-10; NC siRNA) Purchased from Dharmacon. HeLa cells were seeded in 24-well plates 2 days prior to transfection. HeLa cells with a cell density of 50-70% were transfected with 50 μL Opti-MEMI medium containing 5 pmol of siRNA using Lipofectamine ^TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific # 13778150) according to the manufacturer's instructions. For analysis, cells were harvested and fixed at different time points after transfection.

(Immunoprecipitation)
293 T cells with 70-80% confluency in a 10 cm dish were transfected with 1.5 mL Opti-MEM I medium containing 15 μg pC7orf26-myc-Flag using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific # L3000001). .. After 72 hours of culturing, proteins were extracted from cells using CelLytic M Cell Lysis Reagent (Sigma #: C2978) containing a protease inhibitor cocktail (Roche #: 04693116001) according to the manufacturer's instructions.

Immunoprecipitation was performed using EZview ^TM Red ANTI-FLAG M2 Affinity Gel (Sigma #: F2426) and FLAG Immunoprecipitation Kit (Sigma #: FLAGIPT1). The affinity gel was suspended in 1% BSA wash buffer and allowed to rotate at room temperature for 30 minutes. After washing 4 times with wash buffer, the affinity gel was suspended in cell lysates and rotated at 4 ° C. for 16 hours. The C7orf26 Flag-fusion protein was isolated from the affinity gel by washing 4 times with a wash buffer and then eluting with a 3xFlag peptide solution. After concentrating the volume using Amicon Ultra-0.5 (Millpore #: UFC5003), the amount of ^{protein was measured using Qubit TM} R Protein Assay (Thermo Fisher Scientific #: Q33211).

(Immune blot analysis)
Approximately 5 μg of the extract and 1 μg of the clarified eluate were boiled at 95 ° C. for 5 minutes, separated by 7.5% acrylamide SDS-PAGE gel, and transferred to a PVDF membrane (Bio-Rad # 1620174) by wet transfer. Membranes were blocked with Blocking One (NAKARAI # 03953), incubated for 20 minutes at room temperature, and washed with TBST. Membranes were incubated overnight with a primary antibody (anti-E2F4 antibody) at 4 ° C., washed with TBST, incubated with HRP-conjugated secondary antibody for 1 hour at room temperature, and eluted with Blocking One. Immune response bands were visualized with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientifi # 34095).

(Real-time RT-PCR)
Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). The expression level of mRNA in each RNA sample was measured using the Step ONE Sequence Detection System (Applied Biosystems). Reverse transcription polymerase chain reaction (RT-PCR) was ^{performed using the One Step SYBR TM} PrimeScript ^TM PLUS RT-PCR Kit (Takara Bio). Real-time PCR was performed using the primers to measure the expression level of each gene. The PCR conditions were as follows: after holding at 42 ° C. for 5 minutes, 95 ° C. for 10 seconds, followed by 95 ° C. for 5 seconds and 60 ° C. for 31 seconds for 40 cycles. The expression level of mRNA was evaluated by the Ct (Threshold cycle) value.

(Example 1) Cell cycle expression fluctuation of C7orf26 In order to analyze the expression fluctuation of C7orf26 during the cell cycle, the cell cycle of HeLa cells was synchronized by adding hydroxyurea (HU) to the medium for 24 hours. Later, the cell cycle was restarted by removing HU from the medium. HeLa cells were analyzed by flow cytometry after a certain period of time after the cell cycle was restarted. At the time of cell cycle resumption (0 hours), it was confirmed that most HeLa cells were in the early S phase, progressed to the G2 / M phase in 16 hours, and the number of cells in the early G1 phase increased in 20 hours (Fig.). 1A). The expression of C7orf26 mRNA gradually increased with the progress of the cell cycle, reached a peak in the M phase 16 hours after HU removal, and continued to be highly expressed in the early G1 phase 20 hours after HU removal. On the other hand, the expression of the cell proliferation marker Ki67 mRNA peaked in the G2 phase 12 hours after HU removal and gradually decreased in the M phase (Fig. 1B). Immunostaining of HeLa cells at each phase of the cell cycle revealed that C7orf26 was present in the nucleus as expected (Fig. 1C; upper left panel, white dot) and was prominently expressed in the cytoplasm during metaphase M. (Fig. 1C; upper right panel, white part). High expression of C7orf26 continued until the end of M phase (Fig. 1C; lower left panel, white part), and staining was still confirmed until G1 stage (Fig. 1C; lower right panel, white part). These results indicate that the housekeeping gene is stably expressed during the cell cycle, whereas the expression of the C7orf26 gene fluctuates dramatically. C7orf26 expression was expected to be high from G2 to M phase because various alternative transcriptional variants and their maturation are required for dynamic cell behavior in mitosis, but cell division has already ended and cell division has already ended. The role of C7orf26 in the cell cycle should be noted in that high expression continues until early G1 phase, when many transcriptional variants are no longer needed.

(Example 2) Cell cycle control by C7orf26 In order to investigate the role of C7orf26 in the cell cycle, which is characterized by high expression even in the G1 phase, it functions using the Cumate-Switch System in which a physiological level of C7orf26 increase is achieved. An acquisition experiment was conducted. The starting agent cumate used was SBI (System Biosciences, LLC) Water-soluble cumate solution (300 mg / mL, 10,000x), product number QM150A-1.

HeLa cells were transfected with a pPB-Cuo-C7orf26 vector containing the C7orf26 coding region using a PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC # PBQM812A-1) to obtain a stable cell line, HeLa / pPB-Cuo-C7orf26. Cloning (the cell line expresses C7orf26 depending on the concentration of cumate added). The cell line is seeded in a medium, and on the second day of seeding, 1/1000 amount (final concentration 0.3 mg / ml) of the above-mentioned cumate is added to 10x and cultured to express C7orf26 mRNA. rice field. As a result, as shown in FIG. 2, C7orf26 was expressed in the cells in a concentration-dependent manner of the cumate added to the culture medium. In addition, the expression level of p53 increased in response to C7orf26, and the expression level of p21 downstream of p53 also increased in response to the expression of p53. On the other hand, in contrast to the expression of p53 and p21 up-regulated by C7orf26, the expression level of Ki67 mRNA, which is a cell proliferation marker, was significantly reduced. Thus, it was confirmed that C7orf26 increases the expression of p53, which is a transcription factor that suppresses the cell cycle, and p21, which is downstream of it, and arrests the cell cycle seen in the decrease in Ki67.

Cells in which C7orf26 was continuously expressed in HeLa / pPB-Cuo-C7orf26 by continuous supplementation with cumate were analyzed by flow cytometry. As shown in FIG. 3A, continuous expression of C7orf26 by continuous supplementation of cumate for 5 days increased the relative amount of cells in the M phase with respect to the control without supplementation with cumate. After 6 days of continuous expression, the number of cells in G1 phase increased and the relative amount of cells in S phase and G2 / M phase decreased. After 8 days of continuous expression, the number of cells decreased in all stages of G1, S, and G2 / M, but most of them were only cells in G1 and cells in G1 without cell cycle progression. The cycle has stopped.

The expression profiles of C7orf26, p53, p21, and Ki67 at each stage of the cell cycle were analyzed. In order to arrest the cell cycle at each phase, HeLa cells were cultured in a medium supplemented with a cell cycle arresting agent or a serum-free medium, synchronized, and then washed to restart the cell cycle. As the cell cycle terminator, hydroxyurea (HU), which arrests the cell cycle in the S phase, and Ro3306, which arrests the cell cycle in the M phase, were used. When HeLa cells were analyzed by flow cytometry, the cell cycle of HeLa cells was stopped by HU in the early S phase (Fig. 3B, central panel) compared to the control (Fig. 3B, upper panel). ), Ro3306 stopped in phase M (Fig. 3B, lower panel).

As shown in FIG. 3C, expression of C7orf26 was minimal in early S phase in cells that resumed cell cycle after arresting in S phase with HU, compared to control, at p53 and p21. It was accompanied by a decrease in expression. In cells in which the cell cycle was stopped in the M phase by Ro3306 and then resumed, the expression of C7orf26 was the highest in the M phase, and p53 and p21 were up-regulated as C7orf26 increased. In addition, 24-hour serum starvation (corresponding to cell cycle arrest in early G1 phase), which is a condition for inducing forced arrest in G0 phase in human fibroblasts, also induces C7orf26 expression and C7orf26. Corresponding to the increased expression of p53, the expression of p53 and p21 also increased. In the late G1 phase, 4 hours after resuming the cell cycle from serum starvation, C7orf26 expression decreased, and p53 and p21 expression also decreased accordingly. On the other hand, the expression of Ki67 also decreased in the cell cycle arrested cells by HU in the early S phase, and in the cell cycle arrested cells by Ro3306, it increased only slightly during the cell division in the M phase, and the cells due to serum starvation. In cell-cycle arrested cells, it decreased in the early G1 phase, and in the late G1 phase due to serum recovery, it was almost the same as that of the control.

(Example 3) Induction mechanism of cell arrest in G1 phase via the p53 pathway of C7orf26 There are four cell cycle checkpoints in the cell cycle, and p53 controls the progression of the cell cycle at each checkpoint and the p53 pathway. Is thought to stop the cell cycle. Since C7orf26 was highly expressed from the M phase to the early G1 phase, Ki67 decreased, and the G1 phase was stopped by C7orf26, the main points at which C7orf26 acts on p53 are the start of cyclinD-CDK4 / 6 and cyclinE-CDK2. It was considered (restriction point). Of the two points, initiation of cyclinD-CDK4 / 6 showed significant expression of the C7orf26 protein in early G1 phase in immunohistochemistry, suggesting that C7orf26 may have a strong effect on p53 specifically in early G1 phase. The highest (Fig. 4).

Since the dose-dependent upregulation of p53 by C7orf26 confirmed in Example 2 was considered to be due to direct transcriptional regulation, the reporter vector containing the promoter region of p53 and pCMV-C7orf26-Flag were co-transfected. A reporter assay was performed on the HeLa cells. Compared with transfection of the control vector pCMV-GFP, the p53-Luc reporter signal was significantly increased by the expression of C7orf26 (Fig. 5-1 and A).

In addition, HeLa cells were co-transfected with a reporter vector containing the promoter region of p53 and pCMV-INTSX-FLAG (X = 1-14). Compared to transfection of the control vector pCMV-GFP, the p53-Luc reporter signal is an integrator complex subunit (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13. , INTS14) was also significantly increased (Fig. 5-1 and B).

These results indicate that C7orf26 upregulates p53 in collaboration with the integrator complex subunit. Neither the integrator complex subunit nor C7orf26 contained a known promoter-binding domain, suggesting that they may be transcription coactivators of unknown transcription factors that regulate the transcription of p53.

Surprisingly, the C7orf26-Luc reporter signal was also significantly increased by transfection with pCMV-p53-HA, indicating the presence of positive feedback between C7orf26 and p53 (Fig. 5). -1, C).

In addition, the expression variation analysis of the integrator complex subunits (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14) during the cell cycle is described in Example 1. I went in the same way. Similar to C7orf26, the expression of mRNA in each member of the integrator complex subunit gradually increased with the progression of the cell cycle, most peaked in the M phase 16 hours after HU removal, and 20 hours after HU removal. High expression continued even in the early G1 phase (Fig. 5-2).

Next, when the Web database and JASPAR were searched using the promoter region of p53, which is the target of C7orf26, members of the E2F family (E2F1, E2F4, E2F6) were found among the candidate transcription factors. Therefore, we analyzed the expression profile of the transcription factor E2F family at each stage of the cell cycle. In order to arrest the cell cycle at each phase, HeLa cells were cultured in a medium supplemented with a cell cycle arresting agent or a serum-free medium, synchronized, and then washed to restart the cell cycle. Hydroxyurea (HU), which arrests the cell cycle in the S phase, was used as the cell cycle terminator. After resumption of the cell cycle by removal of HU, E2F1 expression gradually decreased from S phase to G2 phase (0 to 8 hours), but E2F4 expression gradually increased in G2 / M phase. This suggests that increased E2F4 may act on the upregulation of C7orf26 during the G2 / M phase. The expression of E2F6 was relatively stable (Fig. 6-1 and A).

The change in p53 reporter activity by C7orf26 was investigated by knockdown of E2F (E2F1, E2F4, E2F6) using siRNA. Upregulation of p53 by C7orf26 was significantly reduced by siRNAs of transcription factors E2F1 and E2F4. Therefore, E2F1 and E2F4 were considered to be candidate transcription factors that work in collaboration with C7orf26 and INTS to activate the transcription of p53 (Fig. 6-1 and B). The Flag affinity eluate from HeLa cells transfected with pCMV-GFP or pCMV-C7orf26-Flag was subjected to Western blot analysis using an antibody against human E2F4. As a result, it was confirmed that C7orf26 and E2F4 bind to each other, and it was supported that C7orf26 in FIGS. 6-1 and B is a transcriptional cofactor for E2F4 (Fig. 6-1 and C).

Regarding E2F expressing the C7orf26 gene, E2F4 significantly increased the reporter activity of C7orf26-Luc in HeLa cells overexpressing E2F4 and E2F6 (Fig. 6-2, A). In addition, when E2F4 and E2F6 siRNAs were allowed to act on HeLa cells whose cell cycle was synchronized, the expression of C7orf26 was suppressed in the G2 and M phases (Fig. 6-2, B). Therefore, the upstream gene that expresses the C7orf26 gene is presumed to be E2F4 / 6.

In HeLa cells whose cell cycle was synchronized, C7orf26 mRNA showed a cell cycle-dependent expression pattern, and C7orf26 mRNA and protein expression peaked in the M phase (FIGS. 1B and 1C), so CDK1 was C7orf26. It was expected to be a transcriptional repressor upstream of. Therefore, HeLa cells were cultured in a medium containing or without a CDK1 inhibitor, synchronized with the S phase by HU, and after 24 hours, the HU was removed from the medium and the cell cycle was restarted, and the cells were cultured in a medium containing a CDK1 inhibitor. In cells, C7orf26 mRNA levels were clearly increased 12 and 16 hours after HU removal (Fig. 6-2, C). From this result, it was shown that CDK1 is an upstream transcriptional repressor of C7orf26. As a result, at the checkpoint in the early stage of M, C7orf26 is suppressed and the expression of p53 does not increase, the cells escape cell cycle arrest, and after entering the G1 phase in which CDK1 is degraded after cell division, C7orf26 It is presumed that the G1 phase early cell cycle arrest function is exerted.

Based on the above series of experiments, the C7orf26 protein is a transcription coactivator of p53 in the M to G1 phase that works together with INTS and E2F1 / E2F4, and rapidly upregulates p53 in the G1 phase with a rapid cell cycle. It was revealed that it would cause an outage and introduction into the G0 phase. The function of C7orf26 is suppressed by CDK1 until the M phase, and the expression increase is released in the G1 phase, and the expression is rapidly increased by the positive feedback of p53. In addition, the integrator complex subunit protein, which co-operates with the transcription factor E2F4 / 1, is rapidly formed during the M phase (about 30 minutes) and works with C7orf26 in the early G1 phase to arrest the cell cycle in the early G1 phase. .. These are thought to be the mechanism by which C7orf26 specifically arrests the cell cycle at early G1 phase checkpoints.

(Example 4) Confirmation of cell death induction (1) Cell death induction by continuous expression of C7orf26 HeLa / pPB-Cuo- in which C7orf26 was continuously expressed by cumate supplementation according to the same method as in Example 2 above. The flow cytometry analysis result of C7orf26 is shown in FIG. Positive cells (488 nm) were analyzed after staining for 30 minutes using ^{LIVE / DEAD TM} Fixable Red Dead Cell Stain Kit Part No. L34971 manufactured by Thermo Fisher Scientific as a staining reagent. Weak fluorescence (low numbers) indicates cells or cell debris containing dead cells. After 8 consecutive days of continuous expression of C7orf26, cells in G1 phase decreased, accompanied by an increase in dead cells and cell debris (FIG. 7A, arrowhead).

In HeLa / pPB-Cuo-C7orf26, in which C7orf26 was continuously expressed for 5 days by cumate supplementation, the cells were almost alive except for a few dead cells, and the control (without cumate supplementation) It was the same as HeLa / pPB-CuO-C7orf26 cells (Fig. 7B, upper panel). HeLa / pPB-Cuo-C7orf26, in which C7orf26 was continuously expressed for 8 days by cumate supplementation, was partially alive compared to control (cumate-free) HeLa / pPB-CuO-C7orf26 cells. The cells were contained, whereas most were dead (Fig. 7B, lower panel).

From these results, it was shown that C7orf26 induces G1 arrest in HeLa / pPB-CuO-C7orf26 cells, which ultimately induces cell death.

(2) Induction of cell death of cancer cells by forced expression of C7orf26 The C7orf26 gene was introduced into HeLa cells (uterine cancer cell line) and glioma cell line 251 (malignant tumor line of the brain) and forcedly expressed. When C7orf26 is forcibly expressed in HeLa cells, C7orf26 (white part in the figure) is collected in the nucleus 24 hours later (Fig. 8a, b; a is C7orf26 single staining, b is merged with nuclear staining by DAPI). After 36 hours (Fig. 8c, d), it was confirmed that the nucleus was atrophied by autophagy leading to cell death. Similarly, in the glioma cell line 251 after 24 hours (Fig. 8e, f; e is C7orf26 single staining, f is merged with nuclear staining by DAPI), C7orf26 (white part in the figure) is highly concentrated in the nucleus. At 36 hours (Fig. 8g), the nuclei were thawed by autophagy. At the same time, it was confirmed that organelles such as mitochondria in the cytoplasm were also thawed by autophagy, leading to cell death (the part surrounded by the broken line in the figure). On the other hand, according to the mitochondrial staining of HeLa cells (control) by forcibly expressing the GFP gene instead of the C7orf26 gene, it was observed that the mitochondria were arranged as beautiful granules in the living cells (Fig. 8h). , White point).

(Example 5) Confirmation of promotion of stem cell differentiation (1) Induction of differentiation from human iPS cells to ectoderm A pPB-Cuo-C7orf26 vector containing a C7orf26 coding region was used as a PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC # PBQM812A-). It was introduced into a human iPS 409B02 cell line using 1), and a cumate-induced c7orf26-expressing iPS cell line was obtained by buromycin drug selection. 6 cm adhesive culture in which a cumate-induced c7orf26-expressing iPS cell line grown by feeder-free culture using Stem Fit medium (Ajinomoto # AK02N) was coated with iMatrix-511 solution (0.5 mg / mL PBS Nippi # 892012). 40,000 seeds were sown in a BD Falcon, and after culturing for 24 hours, 10,000x Cumate Solution (System Biosciences, LLC # PBQM100A-1) was administered to a final concentration of 2x. The next day, the cells were exchanged with the ectoderm induction medium of the STEM diff Trilineage differentiation kit (STEMCELL Technologies # ST-05230), and induction was started (Day 0). Then, the medium was changed every day, and the cells were collected and fixed on the 7th day (Day 7).

On the 5th day after administration of cumate (4th day from the start of differentiation induction), cultured cells were collected and the expression levels of C7orf26, p53, and p21 genes were analyzed by quantitative PCR. As a result, the expression level of each gene in the cells (Cum-) induced to differentiate in the cumate-free medium was set to 1, and the relative expression level of each gene in the cells (Cum +) induced to differentiate in the medium added with cumate was set to 1. Indicated. The expression levels of p53 and p21 increased in response to the increase in the expression level of C7orf26 by administration of cumate, but the expression level of Ki67 decreased (Fig. 9A). This result was the same as the gene expression profile in Hela cells described above.

The state of differentiation was evaluated by flow cytometry. Sample preparation was performed using the Human Pluripotent Stem Cell Transcription Factor Analysis Kit (BD # 560589) according to the attached protocol. Separately, Alexa Fluor (registered trademark) 647 Mouse Anti-Human Pax6 (BD Biosciences # 562249) was purchased for ectoderm marker analysis and used for staining. Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific) was used as the measuring device, and the attached analysis software was used for data analysis.

Compared to the cell group (Cum-) that was induced to differentiate in the medium without cumate, the cell group (Cum +) that was induced to differentiate in the medium without cumate showed a peak in the number of cells at high fluorescence intensity, and the ectoderm appeared. The number of differentiated cells increased. From this result, it was shown that iPS cells whose cell cycle was arrested in the G0 phase due to increased expression of C7orf26 transitioned to the G1 phase and differentiated into ectoderm. In addition, on the 7th day from the start of differentiation induction, cultured cells were collected and the expression levels of C7orf26 and Pax6, Pax3, and Sox1 genes were analyzed by quantitative PCR. As a result, the expression levels of Pax6, Pax3, and Sox1 also increased (Fig. 9C).

(2) Induction of differentiation of human iPS cells into retinal ganglion cells and brain A pPB-Cuo-C7orf26 vector containing the C7orf26 coding region was used in human iPS using the PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC # PBQM812A-1). The iPS cell line was introduced into a 409B02 cell line, and a cumate-induced C7orf26-expressing iPS cell line was obtained by buromycin drug selection. Using iPS cell lines expressing cumate-induced C7orf26 grown by feeder-free culture in Stem Fit medium (Ajinomoto # AK02N), literature (Tanaka T et al., Sci Rep. 2015 Feb 10; 5: According to the method of 834410.103), induction of retinal ganglion differentiation was carried out as follows. Commercially available serum-free medium containing 20% KSR (G-MEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid additive, 1 mM sodium pyruvate, 20 μM Y-27636,) on a non-adhesive 96-well plate. The above iPS cells suspended in 3 μM IWR-1e) were seeded at 9,000 per well. On the 2nd day after sowing, Matrigel was added to a final concentration of 0.5%, and on the 12th day, IWR-1e was removed from the previous medium composition and replaced with a medium to which FBS (final concentration 1%) was newly added. did. On the 15th day, CHIR99021 (final concentration 3 μM) and SAG (final concentration 100 nM) were added and cultured for 3 days. Replaced with serum-free retinal culture medium (composition: D-MEM / F12 with GlutaMAX, N2 supplement) on the 18th day, and after culturing for 6 days, serum retinal maturation medium (composition: D-MEM / F12 with GlutaMAX) on the 24th day. , N2 supplement, 1% FBS, 0.5 μM retinoic acid), and then the culture was continued for 3 days. On day 27, cells were transferred to a plate coated with poly-D-lysine and laminin to initiate differentiation into retinal ganglion cells, and at the same time, a final concentration of 10,000x Cumate Solution (System Biosciences, LLC # PBQM100A-1) was applied. It was administered to 2x and cultured in the presence of cumate. On the 7th day from the start of differentiation induction, colonies were collected and RNA was extracted.

Using this RNA as a template, the expression level of retinal ganglion markers (Brn3b, Math5) was evaluated by quantitative PCR. As a result, the expression level of each gene in the cells (Cum-) induced to differentiate in the cumate-free medium was set to 1, and the relative expression level of each gene in the cells (Cum +) induced to differentiate in the medium added with cumate was set to 1. Indicated. The expression levels of p53 and p21 increased in response to the increase in the expression level of C7orf26 by administration of cumate, but the expression level of Ki67 decreased (Fig. 10A). In addition, compared to cells (Cum-) that were induced to differentiate in a medium without cumate, cells (Cum +) that were induced to differentiate in a medium without cumate increased C7orf26, resulting in differentiation of retinal ganglion cells. It was confirmed that the expression levels of specific Brn3b and Math5, especially Math5, were increased, and the differentiation into retinal ganglion cells was enhanced (Fig. 10B).

The center of the above differentiated colonies was recovered and RNA was recovered in the same manner, and the expression level of brain markers (Otx2, En1) was evaluated by a quantitative PCR method using this RNA as a template. Compared to cells (Cum-) that were induced to differentiate in a medium without cumate, cells (Cum +) that were induced to differentiate in a medium without cumate increased C7orf26, and Otx2, which is specific to brain differentiation, It was confirmed that the expression level of En1 was increased and the differentiation into the brain was enhanced (Fig. 10C).

(3) Induction of differentiation of human iPS cells into mesoderm Cumate-induced C7orf26-expressing iPS cell lines grown by feeder-free culture using Stem Fit medium (Ajinomoto # AK02N) in iMatrix-511 solution (0.5 mg / mL PBS nippi) 15,000 cells were seeded on a 24-well multiplate (BD Falcon) coated with # 892012), cultured for 3 days, and then replaced with a mesoderm-inducing medium of STEMdiff Trilineage differentiation kit (STEMCELL Technologies # ST-05230). Induction was started (Day 0). At the same time, 10,000 x Cumate Solution (System Biosciences, LLC # PBQM100A-1) was administered to a final concentration of 5 x. After that, the medium was changed every day, and on the 5th day (Day 5), the cells were collected and RNA was extracted. The expression of the mesoderm marker TBXT was analyzed by quantitative PCR, and the progress of differentiation was confirmed. In addition, FACS was performed in the same manner, and the number of cells differentiated into mesoderm was measured by the mesoderm marker TBXT. Compared to the cell group (Cum-) that was induced to differentiate in the medium without cumate, the cell group (Cum +) that was induced to differentiate in the medium without cumate showed a peak in the number of cells at high fluorescence intensity, and the mesoderm. The number of cells differentiated into mesoderm increased (Fig. 11A). In addition, compared to cells (Cum-) that were induced to differentiate in a medium without cumate, cells (Cum +) that were induced to differentiate in a medium without cumate were specific to differentiation into mesoderm as C7orf26 increased. It was confirmed that the expression level of TBXT was increased and the differentiation into mesoderm was enhanced (Fig. 11B).

(4) Induction of differentiation of human iPS cells into endoderm A Cumate-induced C7orf26-expressing iPS cell line grown by feeder-free culture using Stem Fit medium (Ajinomoto # AK02N) was used in iMatrix-511 solution (0.5 mg / mL PBS nippi). 15,000 cells were seeded on a 24-well multiplate (BD Falcon) coated with # 892012), cultured for 3 days, and then replaced with endoderm induction medium of STEMdiff Trilineage differentiation kit (STEMCELL Technologies # ST-05230). Induction was started (Day 0). At the same time, 10,000 x Cumate Solution (System Biosciences, LLC # PBQM100A-1) was administered to a final concentration of 5 x. After that, the medium was changed every day, and on the 5th day (Day 5), the cells were collected and RNA was extracted. The expression of the endoblast marker Sox17 was analyzed by quantitative PCR, and the progress of differentiation was confirmed. FACS was also performed in the same manner, and the number of cells differentiated into endoderm was measured by the endoderm marker Sox17. Compared to the cell group (Cum-) that was induced to differentiate in the medium without added cumate, the cell group (Cum +) that was induced to differentiate in the medium added with cumate showed a peak in the number of cells at a higher fluorescence intensity and endoderm. The number of cells differentiated into the medium increased (Fig. 12A). In addition, compared to cells (Cum-) that were induced to differentiate in a medium without cumate, cells (Cum +) that were induced to differentiate in a medium without cumate were specific to differentiation into endoderm as C7orf26 increased. It was confirmed that the expression level of Sox17 was increased and the differentiation into endoderm was enhanced (Fig. 12B).

INDUSTRIAL APPLICABILITY The present invention can be used in the fields of manufacturing pharmaceuticals for cancer treatment by controlling the cell cycle and materials for regenerative medicine and cell medicine.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (7)

1. A G1 phase initial regulator of the cell cycle containing any of the following proteins (A) to (C).
   (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
   (B) The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are added, deleted, or substituted, and the cell cycle is stopped in the early G1 phase, and from the early G1 phase to the G0 phase. Protein with activity to transfer;
   (C) An activity consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Protein to have.
2. A G1 phase initial regulator of the cell cycle containing any of the following genes (D) to (F).
   (D) A gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1;
   (E) An activity consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Genes containing DNA encoding the proteins they have;
   (F) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, arrests the cell cycle in the early G1 phase, and shifts from the early G1 phase to the G0 phase. A gene that contains DNA that encodes a protein that has the activity to cause it.
3. The G1 phase initial regulator of the cell cycle according to claim 2, wherein the gene is inserted into a vector.
4. A drug for treating cancer, which comprises the G1 phase initial control agent for the cell cycle according to any one of claims 1 to 3.
5. An agent for promoting differentiation of undifferentiated cells, which comprises the G1 phase initial regulator of the cell cycle according to any one of claims 1 to 3.
6. The differentiation-promoting agent for undifferentiated cells according to claim 5, wherein the undifferentiated cells are stem cells or progenitor cells.
7. A method for promoting differentiation of undifferentiated cells, which comprises the step of expressing any of the following genes (D) to (F) in undifferentiated cells.
   (D) A gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1;
   (E) An activity consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, arresting the cell cycle in the early G1 phase, and shifting from the early G1 phase to the G0 phase. Genes containing DNA encoding the proteins they have;
   (F) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, arrests the cell cycle in the early G1 phase, and shifts from the early G1 phase to the G0 phase. A gene that contains DNA that encodes a protein that has the activity to cause it.

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