Protein encoded by the upstream open reading frame of the Lin28B gene and its applications

Abstract

This invention relates to the field of biomedical technology, and proposes a protein encoded by the upstream open reading frame of the Lin28B gene and its applications. The polypeptide has the amino acid sequence shown in SEQ ID NO:2 or 4, or an amino acid sequence modified in its conserved form. According to embodiments of the present invention, the polypeptide is obtained by expression of the upstream open reading frame of the Lin28B gene. This polypeptide can effectively regulate the interconversion between the primordial and primitive states of pluripotent stem cells and has important significance in the treatment of diseases related to embryo implantation defects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the protein encoded by the upstream open reading frame of the Lin28B gene and its applications. Background Technology

[0002] Currently, existing functional reports on non-canonical open reading frames (ORFs) mainly focus on ORFs of long non-coding RNAs (lncRNAs). The upstream open reading frames (uORFs) of mRNAs mainly function as cis-regulatory elements to inhibit the translation of downstream main open reading frames (mORFs), while there are fewer functional reports on microproteins encoded by upstream open reading frames (uORFs) of mRNAs.

[0003] Pluripotent stem cells primarily exist in two pluripotency states: naive and primed, corresponding to different stages of embryonic development. Compared to primed cells, naive cells possess embryonic chimerism capability, making them an important source of seed cells for future xenogeneic organ regeneration. Regulating the transition between these two pluripotency states has significant research value for basic research and regenerative medicine. Existing effective factors promoting the transition between different pluripotency states are mostly transcription factors, and their efficiency is relatively low. Research on the regulation of the transition between the primed and naive states by microproteins encoded by uORF has not yet been reported. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems in the related art.

[0005] The upstream open reading frame (uORF) of mRNA primarily functions as a cis-regulatory element, inhibiting the translation of the downstream main open reading frame (mORF). There are few reports on the functions of microproteins encoded by the upstream open reading frame (uORF) of mRNA. However, in this application, the inventors, through extensive experimental research, discovered that Lin28B uORF can encode a novel microprotein (named 85aa / 87aa-uORF, where 87aa is found in humans and 85aa in mice) that regulates pluripotent stem cell fate. This protein can regulate the interconversion of primordial pluripotent stem cells to primitive pluripotent stem cells, with a regulatory efficiency approaching 100%, resulting in homogenized primitive or primordial pluripotent stem cells.

[0006] Therefore, in a first aspect, the present invention provides a polypeptide. According to an embodiment of the invention, the polypeptide has the amino acid sequence shown in SEQ ID NO:2 or 4, or an amino acid sequence modified in its conserved form. The polypeptide according to an embodiment of the invention is encoded by Lin28BuORF, and the polypeptide can effectively regulate the primitive and primordial states of pluripotent stem cells, playing an important role in embryo implantation and effectively treating diseases related to embryo implantation defects.

[0007] In a second aspect, the present invention provides a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes the polypeptide described in the first aspect. The polypeptide encoded by the nucleic acid according to an embodiment of the invention can effectively regulate the primitive and primordial states of pluripotent stem cells, and can effectively treat diseases related to embryo implantation defects.

[0008] According to embodiments of the present invention, the above-mentioned nucleic acid may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the nucleic acid has the nucleotide sequence shown in SEQ ID NO:1 or 3.

[0010] In a third aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector comprises the nucleic acid described in the second aspect, or expresses the polypeptide described in the first aspect. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the expression of the aforementioned polypeptide is effectively achieved, thereby enabling the large-scale in vitro production of the aforementioned polypeptide.

[0011] In a fourth aspect, the present invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the aforementioned nucleic acid or expression vector, or expresses the aforementioned polypeptide. Using this recombinant cell, under suitable conditions, the aforementioned polypeptide can be efficiently expressed intracellularly.

[0012] According to embodiments of the present invention, the recombinant cells may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the cell comprises mammalian pluripotent stem cells.

[0014] According to an embodiment of the present invention, the cells comprise mammalian embryonic stem cells or induced pluripotent stem cells.

[0015] In a fifth aspect, the present invention provides a biological model. According to an embodiment of the invention, the biological model does not express or expresses at low levels the polypeptide described in the first aspect. As previously mentioned, the polypeptide according to an embodiment of the invention is encoded by Lin28BuORF, and the polypeptide can effectively regulate the primitive and primordial states of pluripotent stem cells, playing an important role in embryo implantation. When the biological model does not express or expresses at low levels the polypeptide, pluripotent stem cells will transform into primitive pluripotent stem cells, easily leading to embryo implantation defects. In this case, the biological model can be a biological model related to embryo implantation defects, which can be used for subsequent disease treatment research or other scientific research.

[0016] According to an embodiment of the present invention, the biological model is a cell model, an organoid model, or an animal model. Those skilled in the art can choose to prepare a cell model, organoid model, or animal model according to their needs.

[0017] Cell models refer to the stylized structure and shape of cells, designed to facilitate the understanding of cell-cell interactions and the relationships between function and structure / morphology. They can also include artificial particles such as ribosomes that possess some cellular functions, or cells that retain the structures (contraction structures) involved in cell movement and function while removing membrane structures and soluble portions of the cytoplasm. Organoid models are miniature three-dimensional structures constructed using cell engineering techniques that mimic the key functions and microenvironments of animal or human organs. These miniature organs can be cultured from patient cells and can replicate the physiological and pathological characteristics of organs, providing researchers with new tools for studying complex disease mechanisms, drug screening, and treatment development. Animal models refer to animals established for various scientific research purposes that exhibit simulated human diseases.

[0018] In a sixth aspect, the present invention proposes the use of a reagent that modifies the polypeptide described in the first aspect of pluripotent stem cells in the preparation of high-purity nascent-state pluripotent stem cells or primitive-state pluripotent stem cells. As previously described, the polypeptide can effectively regulate the primitive and nascent-state states of pluripotent stem cells. When the expression of the polypeptide is downregulated or inhibited, it can effectively promote the transformation of pluripotent stem cells into primitive-state pluripotent stem cells, especially nascent-state pluripotent stem cells. Overexpression of the polypeptide can effectively transform pluripotent stem cells into nascent-state pluripotent stem cells, with a transformation efficiency of up to 100%. Thus, high-purity, uniformly conditioned primitive-state pluripotent stem cells or nascent-state pluripotent stem cells can be obtained.

[0019] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0020] According to an embodiment of the present invention, the modification includes overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells.

[0021] According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0022] According to an embodiment of the present invention, the reagent further includes an overexpression vector or agonist of the polypeptide.

[0023] According to an embodiment of the present invention, the agonist includes at least one of a small molecule, a drug, an enzyme, or a hormone.

[0024] According to an embodiment of the present invention, the nascent pluripotent stem cells are prepared by overexpressing the polypeptide described in the first aspect in the pluripotent stem cells. This yields high-purity nascent pluripotent stem cells.

[0025] According to an embodiment of the present invention, the primitive pluripotent stem cells are prepared by downregulating or inhibiting the expression of the polypeptide described in the first aspect in the pluripotent stem cells. Thus, high-purity primitive pluripotent stem cells can be obtained.

[0026] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0027] In a seventh aspect of the invention, the present invention discloses the use of a reagent for overexpressing the polypeptide described in the first aspect of pluripotent stem cells in the preparation of high-purity nascent pluripotent stem cells. According to embodiments of the present invention, the polypeptide can effectively transform pluripotent stem cells into nascent pluripotent stem cells, and, according to embodiments of the present application, the transformation efficiency can reach 100%. Therefore, overexpression of the polypeptide described in the first aspect can effectively prepare high-purity nascent pluripotent stem cells.

[0028] According to embodiments of the present invention, the reagent comprises an overexpression vector or agonist of the polypeptide. Those skilled in the art will understand that any reagent capable of overexpressing the polypeptide without affecting the survival of the pluripotent stem cells may be used.

[0029] According to an embodiment of the present invention, the agonist includes at least one of a small molecule, a drug, an enzyme, or a hormone.

[0030] According to embodiments of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells. Those skilled in the art will understand that any pluripotent cell can be used herein.

[0031] In an eighth aspect of the invention, the present invention discloses the use of a reagent for downregulating or inhibiting the expression of the polypeptide described in the first aspect in pluripotent stem cells in the preparation of naïve pluripotent stem cells. As previously described, the polypeptide can effectively regulate the naïve and primordial states of pluripotent stem cells. When the expression of the polypeptide is downregulated or inhibited, it can effectively promote the transformation of pluripotent stem cells into naïve pluripotent stem cells, especially primordial pluripotent stem cells into naïve pluripotent stem cells, with a transformation efficiency of up to 100%. Thus, highly pure, uniformly sized naïve pluripotent stem cells can be obtained.

[0032] According to an embodiment of the present invention, the reagent is based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0033] According to embodiments of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells. Those skilled in the art will understand that any mammalian cell with pluripotency can be used herein.

[0034] In a ninth aspect of the invention, a method for preparing high-purity nascent-state pluripotent stem cells or primitive-state pluripotent stem cells is provided. According to an embodiment of the invention, the method includes contacting pluripotent stem cells with a reagent that alters the polypeptide described in the first aspect. As previously mentioned, the polypeptide can effectively regulate the primitive and nascent states of pluripotent stem cells. When the expression of the polypeptide is downregulated or inhibited, it can effectively promote the transformation of pluripotent stem cells into primitive-state pluripotent stem cells, particularly converting nascent-state pluripotent stem cells into primitive-state pluripotent stem cells. Overexpression of the polypeptide can effectively transform pluripotent stem cells into nascent-state pluripotent stem cells, with a transformation efficiency reaching 100%. Thus, high-purity, uniformly sized primitive-state pluripotent stem cells or nascent-state pluripotent stem cells can be obtained.

[0035] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0036] According to an embodiment of the present invention, the modification includes overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells.

[0037] According to an embodiment of the present invention, the reagent is based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0038] According to embodiments of the present invention, the reagent further includes an overexpression vector or agonist of the polypeptide. Those skilled in the art will understand that any reagent capable of overexpressing the polypeptide without affecting the survival of the pluripotent stem cells may be used.

[0039] According to an embodiment of the present invention, the agonist includes at least one of a small molecule, a drug, an enzyme, or a hormone.

[0040] According to an embodiment of the present invention, the method includes: obtaining the nascent pluripotent stem cells by overexpressing the polypeptide described in the first aspect in the pluripotent stem cells or by contacting the pluripotent stem cells with the polypeptide described in the first aspect. Thus, high-purity nascent pluripotent stem cells can be obtained.

[0041] According to an embodiment of the present invention, the method includes obtaining the primitive pluripotent stem cells by downregulating or inhibiting the expression of the polypeptide described in the first aspect in the pluripotent stem cells. Thus, high-purity primitive pluripotent stem cells can be obtained.

[0042] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0043] In a tenth aspect of the invention, a method for preparing high-purity nascent pluripotent stem cells is provided. According to an embodiment of the invention, the method includes: contacting the pluripotent stem cells with the polypeptide described in the first aspect, or contacting the pluripotent stem cells with a reagent that overexpresses the polypeptide described in the first aspect. As previously mentioned, the polypeptide can effectively regulate the primitive and nascent states of pluripotent stem cells. Upregulation of the polypeptide expression, or contact between the primitive pluripotent stem cells and the polypeptide, can effectively promote the transformation of the primitive pluripotent stem cells into nascent pluripotent stem cells, thereby obtaining high-purity, uniformly conditioned primitive pluripotent stem cells.

[0044] According to embodiments of the present invention, the reagent comprises an overexpression vector or agonist of the polypeptide. Those skilled in the art will understand that any reagent capable of overexpressing the polypeptide without affecting the survival of the pluripotent stem cells may be used.

[0045] According to an embodiment of the present invention, the agonist includes at least one of a small molecule, a drug, an enzyme, or a hormone.

[0046] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0047] In an eleventh aspect, the present invention provides a method for preparing high-purity primordial pluripotent stem cells. According to an embodiment of the present invention, the method includes: contacting the pluripotent stem cells with a reagent that downregulates or inhibits the expression of the polypeptide described in the first aspect. As previously mentioned, the polypeptide can effectively regulate the primordial and nascent states of pluripotent stem cells. When the expression of the polypeptide is downregulated or completely inhibited, it can effectively promote the transformation of nascent pluripotent stem cells into primordial pluripotent stem cells, thereby obtaining high-purity, uniformly conditioned primordial pluripotent stem cells.

[0048] According to an embodiment of the present invention, the reagent is based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0049] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0050] In a twelfth aspect of the invention, the invention proposes the use of the biological model described in the fifth aspect in screening drugs for treating diseases related to implantation defects. According to embodiments of the invention, the diseases include at least one of threatened abortion, recurrent miscarriage, infertility, endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency. As previously stated, the peptide according to embodiments of the invention can effectively regulate the primitive and primordial states of pluripotent stem cells, playing an important role in embryo implantation. When the biological model does not express or expresses the peptide at low levels, the pluripotent stem cells will transform into primitive pluripotent stem cells, easily leading to implantation defects. In this case, the biological model is an implantation defect-related biological model, which can be used for subsequent disease treatment research or other scientific research, such as drug screening.

[0051] In a thirteenth aspect of the invention, the use of the polypeptide described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a medicament is provided. According to an embodiment of the invention, the medicament is used to prevent, alleviate, and / or treat diseases related to implantation defects. As previously stated, the polypeptide according to an embodiment of the invention can effectively regulate the primitive and primordial states of pluripotent stem cells, and when the polypeptide contacts the primitive-state pluripotent stem cells, it can effectively promote the transformation of primitive-state pluripotent stem cells into primordial-state pluripotent stem cells.

[0052] Primordial pluripotent stem cells (PSCs) and nascent pluripotent stem cells (NPSCs) are two distinct stem cell states, each corresponding to a different stage of embryonic development. Primordial PSCs exhibit greater plasticity and can mimic the pre-implantation embryonic development stage, which corresponds to day 3.5–4.5 after mouse zygote formation. Nascent PSCs, on the other hand, correspond to the post-implantation embryonic development stage, which corresponds to day 5.5–7.5 after mouse zygote formation. Primordial PSCs correspond to day 6–8 after human zygote formation, while nascent PSCs correspond to day 11–12 after human zygote formation. Therefore, drugs containing the aforementioned peptides, nucleic acids, expression vectors, or recombinant cells play a crucial role in embryo implantation.

[0053] According to an embodiment of the present invention, the disease includes at least one of threatened abortion, recurrent miscarriage, infertility, endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency.

[0054] In a fourteenth aspect, the present invention provides a medicament for the prevention, mitigation, and / or treatment of diseases related to implantation defects. According to embodiments of the invention, the medicament comprises the polypeptide described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect. As previously stated, the polypeptide can effectively regulate the transition between the primordial and primitive states of pluripotent stem cells, thereby facilitating embryo implantation. Therefore, products related to the polypeptide, such as the nucleic acid, expression vector, and recombinant cells, all have the same therapeutic effect as the polypeptide. Furthermore, a medicament comprising the above-mentioned products can also be used for diseases related to implantation defects.

[0055] According to embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, the drug comprises pharmaceutically acceptable excipients.

[0057] According to an embodiment of the present invention, the disease includes at least one of threatened abortion, recurrent miscarriage, infertility, endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency.

[0058] In a fifteenth aspect of the invention, the invention proposes the use of a formulation in the preparation of a medicament for the prevention, mitigation, and / or treatment of diseases related to implantation defects. According to an embodiment of the invention, the formulation causes the polypeptide described in the first aspect to be overexpressed in cells. As previously mentioned, the polypeptide can effectively regulate the transition from the primordial to the primitive state of pluripotent stem cells, thereby promoting embryo implantation. Therefore, a formulation that enables the overexpression of the polypeptide can promote the transformation of primitive pluripotent stem cells to the primordial state, thus promoting embryo implantation. Therefore, the formulation can be used for diseases related to implantation defects.

[0059] According to an embodiment of the present invention, the cell comprises mammalian pluripotent stem cells.

[0060] According to an embodiment of the present invention, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells.

[0061] According to an embodiment of the present invention, the formulation comprises an overexpression vector or agonist of the polypeptide;

[0062] According to an embodiment of the present invention, the agonist includes at least one of a small molecule, a drug, an enzyme, or a hormone.

[0063] In a sixteenth aspect of the invention, the invention provides the use of reagents for detecting the polypeptide described in the first aspect or nucleic acids encoding the polypeptide described in the first aspect in the preparation of a kit. According to an embodiment of the invention, the kit is used to detect embryo implantation defect-related diseases. As previously stated, the polypeptide can effectively regulate the transition between the primordial and primitive states of pluripotent stem cells. When the polypeptide is not expressed or is expressed at low levels in the pluripotent stem cells, the pluripotent stem cells will transform towards the primitive state, for example, from primordial to primitive. When the polypeptide is overexpressed in the pluripotent stem cells, the pluripotent stem cells will transform towards the primordial state, for example, from primitive to primordial. Therefore, reagents for detecting the polypeptide described in the first aspect or related products, such as nucleic acids, can effectively determine the state of pluripotent stem cells. This kit can be used for scientific research, such as studying changes in the expression levels of the polypeptide or nucleic acid in cells or tissues, or for qualitative or quantitative detection.

[0064] According to an embodiment of the present invention, the embryo implantation defect-related diseases include at least one of threatened miscarriage, recurrent miscarriage, infertility, endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency.

[0065] According to an embodiment of the present invention, the reagent includes antibodies, probes, primers, and mass spectrometry detection reagents targeting the polypeptide or the nucleic acid encoding the polypeptide.

[0066] In a seventeenth aspect, the present invention provides a kit, characterized in that the kit comprises reagents for detecting the polypeptide described in the first aspect, or the nucleic acid encoding the polypeptide described in the first aspect. The kit of the present invention is capable of specifically binding to the polypeptide, effectively detecting the polypeptide, or detecting the nucleic acid encoding the polypeptide, thereby determining the state or disease status of cells in the sample to be tested.

[0067] According to an embodiment of the present invention, the kit comprises an antibody for detecting the polypeptide described in the first aspect.

[0068] According to an embodiment of the present invention, the kit further includes probes, primers, and / or mass spectrometry detection reagents for amplifying nucleic acids encoding the polypeptide described in the first aspect. The kit can detect nucleic acids encoding the polypeptide. As those skilled in the art will know, when detecting nucleic acid sequences, primers corresponding to the nucleic acid are required to amplify the nucleic acid sequence.

[0069] The beneficial effects of this application are at least as follows:

[0070] (1) The polypeptide described in this application (the microprotein 85aa / 87aa-uORF encoded by Lin28B uORF, which is 87aa in humans and 85aa in mice) can regulate the primitive and primordial states of pluripotent stem cells with nearly 100% efficiency. When the polypeptide is not expressed or is expressed at low levels in the pluripotent stem cells, the pluripotent stem cells will transform into primitive pluripotent stem cells, for example, from primordial pluripotent stem cells to primitive pluripotent stem cells. When the polypeptide is overexpressed in the pluripotent stem cells, the pluripotent stem cells will transform into the primordial state, for example, from primitive pluripotent stem cells to primordial pluripotent stem cells, thereby obtaining homogenized primitive or primordial pluripotent stem cells.

[0071] (2) Primordial pluripotent stem cells and nascent pluripotent stem cells are two different stem cell states, corresponding to different stages of embryonic development. Primordial pluripotent stem cells have greater plasticity and can mimic the pre-implantation embryonic development stage, while nascent pluripotent stem cells correspond to the post-implantation embryonic development stage. Since the polypeptide can regulate the primordial and nascent states of pluripotent stem cells, it plays an important role in embryo implantation and can be used for scientific research, treatment, and diagnostic applications in related fields.

[0072] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0073] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0074] Figure 1 The endogenous expression and identification of the 85aa-uORF-encoded microprotein of this invention are shown in the embodiments of the present invention, wherein:

[0075] Figure A: A schematic diagram showing the location of 85aa-uORF on Lin28B mRNA.

[0076] Figure B: The amino acid sequence of 85aa-uORF is highly conserved in various mammals, including humans and mice.

[0077] Figure C: 85aa-uORF begins translation with ATG.

[0078] Figure D: 85aa-uORF is highly expressed in nascent pluripotent stem cells;

[0079] Figure 2 This invention demonstrates the establishment of an 85aa-uORF knockout cell line (KO) using the CRISPR-Cas9 system in an embodiment of the present invention, wherein:

[0080] Figure A: Schematic diagram of the location of sgRNA targeting the 85aa-uORF sequence.

[0081] Figure B: Sequencing results show that the 85aa-uORF knockout cell line (KO) has either an added base or a deleted 4 bases, resulting in a frameshift mutation.

[0082] Figure C: Western blot shows that 85aa-uORF was successfully knocked out in OG2-EpiSCs.

[0083] Figure D: Knockout of 85aa-uORF does not affect the translational expression of Lin28B;

[0084] Figure 3 The invention demonstrates that 85aa-uORF knockout in embodiments significantly promotes Primed-to- The transformation (PNT) has an efficiency close to 100%.

[0085] Figure 4 This invention demonstrates the effect of 87aa-uORF knockout promoting human pluripotent stem cell production in a Primed-to- The transformation demonstrates the consistency of Lin28B uORF function in humans and mice. The three AP-stained images on the left show the transformation from the control group (sgCTRL) and 87aa-uORF knockout (sguORF1 / 2) to... The clone diagram shows the state of the clones, and the statistical chart on the right shows the number of clones in the control group and the experimental group.

[0086] Figure 5 This invention demonstrates how overexpression of 85aa-uORF promotes... -to-Primed transition(NPT), where:

[0087] Figure A: Overexpression of 85aa-uORF promotes... A schematic diagram illustrating the transformation of ESC cells into Primed EpiSC cells.

[0088] Figure B: ESC and ESC overexpressing 85aa-uORF at day 5 transition The expression status of primed pluripotency marker genes;

[0089] Figure 6 The construction of the 85aa-uORF gene knockout (MP85- / -) model mouse in Example 7 of this invention is shown, wherein:

[0090] Figure A: Schematic diagram of sgRNA targeting sequence design.

[0091] Figure B: MP85- / - mouse genome sequencing shows a 4-base deletion, resulting in a frameshift mutation.

[0092] Figure C: Western blot shows that MP85- / - genotype mice were successfully constructed;

[0093] Figure 7 The embodiments of the present invention demonstrate that 85aa-uORF promotes normal development of mouse embryos, promotes blastocyst implantation, and improves offspring fertility. Detailed Implementation

[0094] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0095] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0096] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0097] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0098] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0099] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0100] It should be noted that the "nucleic acid" described in this application can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and its length is not particularly limited. For constructs used to build recombinant cells, the nucleic acid is preferably DNA because DNA is more stable and easier to manipulate than RNA. The "nucleic acid" described in this application actually includes any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed. For example, references to SEQ ID NO:1 and 3 actually include their complementary sequences. Those skilled in the art will also understand that one strand can be used to detect the other strand, and vice versa.

[0101] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0102] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0103] Single-base gene editing refers to gene editing technology that can cause a single base change in the genome. The basic principle is to fuse cytosine deaminase (APOBEC) or adenosine deaminase with an existing Cas9n (D10A) to form a gene editing technology that relies on the CRISPR principle to modify a single base at position 4 to 7 away from the PAM end.

[0104] Zinc finger nucleases (ZFNs) consist of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-his2 zinc finger proteins tandemly. Each zinc finger protein recognizes and binds to a specific triplet base. For example, the most classic zinc finger nuclease is the fusion of a non-specific endonuclease FokI with the zinc finger-containing domain, the purpose of which is naturally to cut a specific sequence. The cut DNA can be repaired by the excision repair mechanism, which deletes the single-stranded portion at the cut site and then rejoins it. This method can be used to delete specific segments on chromosomes, thereby creating mutants or performing therapeutic procedures.

[0105] Transcription activator-like (TAL) effector nucleases (TALENs) are enzymes that can target and modify specific DNA sequences. They use TAL effectors—natural proteins secreted by plant bacteria—to recognize specific DNA base pairs. TAL effectors can be designed to recognize and bind to all target DNA sequences. Adding a nuclease to a TAL effector generates TALENs. TAL effector nucleases can bind to DNA and cut the DNA strand at specific sites, thereby introducing new genetic material.

[0106] The CRISPR / Cas9 system is widely present in prokaryotic genes and is an acquired immune defense mechanism evolved by bacteria and archaea to cope with constant attacks from viruses and plasmids. In these organisms, exogenous genetic material from bacteriophages is acquired and integrated into CRISPR sites; these sequence-specific fragments are transcribed into short CRISPR RNA (CRISPR-derived RNA). The crRNA binds to tracrRNA (trans-activating RNA) through base pairing to form double-stranded RNA. Then, the tracrRNA / crRNA complex directs the Cas9 protein to cleave the double-stranded DNA, thus performing gene editing.

[0107] Human induced pluripotent stem cells (iPSCs) can be induced into almost any type of cell. Combining iPSCs with CRISPR / Cas9 gene editing technology can both repair disease-causing sites in patient-derived iPSCs and introduce disease-causing mutations into healthy WT iPSCs.

[0108] Adeno-associated virus (AAV) has low immunogenicity. Due to its high safety and stable long-term expression, it is regarded as the most promising gene therapy vector. Using AAV to deliver CRISPR / Cas9 system can achieve highly efficient gene editing.

[0109] In this document, the term "drug" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely pulverized solid carrier, or both.

[0110] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0111] For ease of understanding, the content of this application is summarized below:

[0112] An upstream open reading frame (uORF) is a sequence located in the 5' untranslated region (5'UTR) of mRNA, containing a start codon (usually AUG) and a stop codon, which can be recognized by ribosomes and initiate the translation process. In this application, the inventors discovered an upstream open reading frame (uORF) in the 5'UTR region of the Lin28B gene that encodes the microprotein 85aa / 87aa-uORF, which is 87aa in humans and 85aa in mice. The amino acid sequence of this microprotein is highly conserved in multiple mammals, including humans and mice, and is highly expressed in primordial Primed pluripotent stem cells. The Lin28B gene uORF can not only function as a cis-regulatory element but also encode novel proteins that can play a role.

[0113] Then, the inventors used CRISPR / Cas9 to introduce a frameshift mutation in a region where the uORF does not overlap with the main open reading frame (mORF), establishing a mouse EpiSC cell line with 85aa-uORF knockout in its primordial state. Western blot analysis demonstrated that 85aa-uORF knockout does not affect the translation of its Lin28B mORF. The inventors found that 85aa-uORF knockout promotes the primed-to-prime transition. This invention significantly improves PNT efficiency to nearly 100%. Furthermore, this invention establishes a Lin28B-uORF (87aa-uORF) knockout human primed pluripotent stem cell line (sguORF1 / 2), and finds that sguORF1 / 2 significantly promotes PNT, demonstrating that the function of Lin28B_uORF is highly conserved in humans and mice. This invention pioneers a new method for promoting the transition of pluripotent stem cells from their primed state to their primitive state through microproteins encoded by uORF, greatly improving efficiency to nearly 100%, and can efficiently obtain uniform and high-purity primitive pluripotent stem cells, which has important application value in the field of regenerative medicine.

[0114] This invention also established a naive cell line overexpressing 85aa-uORF, and found that overexpression of 85aa-uORF significantly promoted the naive-to-primed transition (NPT). This invention reveals that 85aa-uORF plays an important role in the pluripotency naive / primed transition. This invention explores a novel method for promoting the naive-to-primed transition using uORF-encoded microproteins.

[0115] Furthermore, the inventors constructed genetically engineered mice with 85aa-uORF knockout and found that the absence of 85aa-uORF led to embryo implantation defects in the mice and a reduction in the number of offspring. This invention discovered an important new protein, 85aa-uORF, which is related to embryo implantation. It not only provides a new research model for embryo implantation defects, but also has application value in the prevention, mitigation, or treatment of diseases related to embryo implantation defects.

[0116] Therefore, in some specific embodiments, the present invention provides the use of the aforementioned polypeptide in the preparation of high-purity naïve pluripotent stem cells and primordial pluripotent stem cells. As previously stated, the polypeptide can regulate the naïve and primordial states of pluripotent stem cells, with a transformation efficiency of 100%. Overexpression of the polypeptide in pluripotent stem cells yields the primordial pluripotent stem cells, while downregulation or inhibition of the polypeptide expression in pluripotent stem cells yields the naïve pluripotent stem cells, with a bidirectional transformation efficiency of 100%.

[0117] In some specific embodiments, the present invention also provides a biological model that highly expresses the aforementioned polypeptide. The biological model can be a cell model, organoid model, or animal model. As mentioned earlier, the polypeptide can effectively regulate the primordial and primitive states of pluripotent stem cells. According to embodiments of the present invention, the biological model can be a well-developed pluripotent stem cell or its tissue, or a fertilized egg with a development time of less than 14 days. This biological model can be used for scientific research in areas such as embryonic development, for example, to study the mechanisms of embryonic development or to screen for drugs that promote or inhibit further embryonic development.

[0118] The biological model described above may also include at least one of the following additional technical features:

[0119] According to a specific embodiment of the present invention, the cells comprise mammalian pluripotent stem cells.

[0120] According to a specific embodiment of the present invention, the cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0121] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0122] Example 185aa-uORF-encoded microprotein endogenous expression and identification

[0123] In this embodiment, an open reading frame (ORF) encoding an 85-amino acid microprotein was discovered in the 5'UTR region of the mouse Lin28B gene (NM_001031772.2) using the ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ). This ORF is named (Mus-)85aa-uORF, with the nucleotide sequence of 85aa-uORF as shown in SEQ ID NO:1 and the amino acid sequence of the microprotein encoded by 85aa-uORF as shown in SEQ ID NO:2. Similarly, an open reading frame encoding an 87aa microprotein also exists in the 5'UTR region of the human Lin28B gene. This ORF is named (Homo-)87aa-uORF, with the nucleotide sequence of 87aa-uORF as shown in SEQ ID NO:3 and the amino acid sequence of the microprotein encoded by 87aa-uORF as shown in SEQ ID NO:4. In this embodiment, an open reading frame upstream of mouse Lin28B (SEQ ID NO: 1) was constructed, and its start codon ATG was mutated to TAA. A flag-tagged mutant was added, and Western blot analysis was used to demonstrate that 85aa-uORF translation begins with ATG, while the mutated TAA cannot translate 85aa-uORF. Furthermore, a polyclonal antibody specifically recognizing this microprotein and detectable by Western blot was designed based on the amino acid sequence of 85aa-uORF. The expression of 85aa-uORF was detected in naïve and nascent pluripotent stem cells and somatic cells, confirming high expression of 85aa-uORF in naïve pluripotent stem cells. Specific experimental procedures are as follows:

[0124] A peptide segment ESFHLWSLK (SEQ ID NO:5) or the full amino acid sequence of 85aa-uORF was synthesized using a chemically synthesized peptide method and used as an immunogen. The immunogen was injected subcutaneously into New Zealand rabbits. Four weeks after the initial immunization, booster immunizations were administered every week for a total of four immunizations. When the antibody titer reached the expected level, blood was excised to prepare antiserum. Specific polyclonal antibodies were isolated from the antiserum using antigen-specific affinity purification. The immunization and polyclonal antibody isolation procedures were performed using standard experimental techniques in the field. The purified antibodies were analyzed by Western blotting to detect the endogenous expression and changes of the microprotein encoded by 85aa-uORF in different cells. Western blotting was performed using standard techniques in the field.

[0125] The nucleotide sequence of Mus-85aa-uORF (258nt) is as follows:

[0126] Atgcgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtccaaagggaaagttttcatctctgg agtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagccagaaaaactgcccgggctggca gaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggattcatctccatga (SEQ ID NO: 1).

[0127] The amino acid sequence (Lin28B-85aa) of the Mus-85aa-uORF microprotein is as follows:

[0128] MRTKFVIAQNQDVRLMLKITPVQRESFHLWSLKLRAGGATWPKAGQAKVKSQKNCPGWQRTNPRFCMALATVNGSTCAWDSDSSP (SEQ ID NO: 2).

[0129] The nucleotide sequence of Homo-87aa-uORF (264nt) is as follows:

[0130] ATGCGAGCTAAATTTGTGATCGCACAAAAATCAAGATGTTAGATTGATGCAGAAGATCACTCC

[0131] GTTCCAAAGGGAAAGTTTTCATCTCACGAGTTTGGAGCTGAGGGCCCGTGGGGCAACATGGCC

[0132] GAAGGCGGGGCTAGCAAAGGTGGTGGAGAAGAGCCCGGGAAGCTGCCGGAGCCGGCAGAGG

[0133] AGGAATCCCAGGTTTTGCGCGGAACTGGCCACTGTAAGTGGTTCAATGTGCGCATGGGATTTG

[0134] GATTCATCTCCATGA (SEQ ID NO:3).

[0135] The amino acid sequence (Lin28B-87aa) of the Homo-87aa-uORF microprotein is as follows:

[0136] MRAKFVIAQNQDVRLMQKITPFQRESFHLTSLELRARGATWPKAGLAKVVEKSPGSCRSRQRRNPRFCAELATVSGSMCAWDLDSSP (SEQ ID NO: 4).

[0137] 1) Construction of the start codon ATG mutant

[0138] The nucleotide sequence of the 5'UTR of Lin28B was analyzed using gene software, and 85aa-uORF and two mutant plasmids were constructed: a) the start codon ATG of 85aa-uORF was mutated to TAA (85aa-uORF-mut1); b) the UTR region before ATG of 85aa-uORF was retained (85aa-uORF-mut2); then, a 3Flag tag protein sequence was added to the C-terminus of the above mutants, and they were cloned into the pLVX-EF1α-IRES-Puro vector (purchased from Takara, 631988) using ECORI and BamHI restriction sites. The constructed plasmids pLVX-TAA-85aa-uORF-3Flag (85aa-uORF-mut1), UTR-85aa-uORF-3Flag (85aa-uORF-mut2), and 85aa-uORF-3Flag (wild-type) were transfected into HEK293T cells, respectively. The specific transfection steps were as follows: 1 mL of serum-free diluent Opti-MEM (purchased from Gibco), 8 μg of plasmid DNA, and 64 μL of polyethyleneimine (PEI) transfection reagent were mixed thoroughly to prepare a nucleic acid-PEI complex, which was then incubated at room temperature for 15 minutes. The prepared nucleic acid-PEI complex was then added to HEK293T cells that had been seeded in 10 cm cell culture dishes and grown to a density of 70%-80% one day prior. After culturing for 8-10 hours, the medium was replaced with fresh medium, and the cells were cultured for another 36 hours. Western blot was then used to detect the protein expression of Lin28B microprotein.

[0139] The nucleotide sequence of 85aa-uORF-mut1 is as follows:

[0140] Aagaaggaaagcacattagacctaacgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtc

[0141] caaagggaaagttttcatctctggagtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagcca

[0142] gaaaaactgcccgggctggcagaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggat

[0143] tcatctccatga(SEQ ID NO:9).

[0144] The nucleotide sequence of 85aa-uORF-mut2 is as follows:

[0145] aagaaggaaagcacattagaccatgcgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtc

[0146] caaagggaaagttttcatctctggagtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagcca

[0147] gaaaaactgcccgggctggcagaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggat

[0148] tcatctccatga (SEQ ID NO:10).

[0149] 2) Establishment of stable cell lines overexpressing 85aa-uORF

[0150] The 85aa-uORF-3Flag plasmid constructed as described above, along with the lentiviral packaging plasmids psPAX2 (purchased from Addgene, 12260) and pMD2g (purchased from Addgene, 12259), were transfected into HEK293T cells at a ratio of 4:3:1 as described above. Forty-eight hours after transfection, the lentiviral supernatant was collected, centrifuged, concentrated, and diluted with EpiSC medium before infecting EpiSC cells. Cells were screened using puromycin (approximately 3 days later) and then expanded normally.

[0151] 3) Cell culture

[0152] The transfected HEK-293T cells obtained in Part 2) were cultured in DMEM (purchased from Gibco) medium supplemented with 10% fetal bovine serum (FBS, NTC), GlutaMax (1x, Gibco), and NEAA (1x, Gibco). The cells were then incubated in a humidified cell culture incubator at 37°C with 5% CO2.

[0153] The Primed cells were derived from mouse embryos at 4.5 and 6.5 days of development, respectively. Cells were cultured in DMEM (Hyclone) + 15% fetal bovine serum (Gibco) + L-GlutaMax (1x, Gibco) + NEAA (1x, Gibco) + Sodium pyruvate (1x, Gibco) + Penicillin-Streptomycin antibiotic (1×, Hyclone) + 0.1mM β-mercaptoethanol (Gibco) + Lif (self-made) + 3μM CHIR99021 (Selleck) + 1μM PD0325901 (Selleck). Primed cells were cultured in 50% Neurobasal (Gibco) + 50% F12 (Gibco) + N2 (0.5×, Gibco) + B27 (0.5×, Gibco) + L-Glutamax glutamine (1×, Gibco) + non-essential amino acids NEAA (1×, Gibco) + 0.1mM β-mercaptoethanol (Gibco) + 5% BSA (Sigma) + 20ng / mL bFGF (PeproTech) + 20ng / mL activin A (PeproTech), with the addition of 1μM XAV939 (XAV; Selleck) or 0.5μM WR-endo (Selleck) to prevent cell differentiation.

[0154] 4) Western blot

[0155] Total cellular protein was extracted using the RIPA method and quantified using the BCA protein quantification method. A 15% lower gel and a 5% upper gel were prepared using the Yaxin one-step rapid gel preparation kit. Proteins were loaded after 30 minutes. Electrophoresis was performed at 80V for 30 minutes and 130V for 1 hour; transfer was performed at 250mA for 50 minutes; blocking was done with 5% skim milk for 1.5 hours; incubation was then performed overnight at 4°C with 85aa-uORF rabbit anti-antibody (human / mouse compatible) (dilution 1:500), Flag antibody (dilution 1:1000, purchased from Abcam), and β-actin antibody (dilution 1:3000, purchased from Abcam). The next day, incubation was performed at room temperature for 1 hour with rabbit secondary antibody (dilution 1:5000), followed by TBST washing for 5 minutes each time, for a total of 5 washes, and then chemiluminescence imaging.

[0156] Figure 1 A shows the location of the upstream open reading frame 85aa-uORF of Lin28B on mouse Lin28B mRNA. Figure 1B shows that the amino acid sequence of 85aa-uORF is highly conserved in various mammals, including humans and mice. Figure 1 C shows that the start codon for 85aa-uORF is ATG, and if it mutates to TAA, it cannot be translated. Figure 1 D showed that 85aa-uORF microprotein was highly expressed in mouse Primed pluripotent stem cells, while... It is almost not expressed in pluripotent stem cells and embryonic fibroblasts (MEF) cells, suggesting that this microprotein may play a role in maintaining the pluripotency of Primed cells and influencing cell fate transitions.

[0157] Example 285aa-uORF microprotein regulates the transition between the primordial and primitive states of pluripotent stem cells.

[0158] 1) Establishment of CRISPR / Cas9 knockout cell lines

[0159] Based on the 85aa-uORF sequence, several gRNAs targeting different regions of 85aa-uORF were designed. Using CRISPR / Cas9 technology, a frameshift mutation was introduced into a non-overlapping segment of Lin28B uORF and mORF, without affecting the translational expression of Lin28B mORF, thus obtaining an 85aa-uORF knockout EpiSC cell line. Similarly, based on the 87aa-uORF sequence, several gRNAs were designed, and lentiviral vector plasmids were constructed to infect human pluripotent stem cells, thereby constructing an 87aa-uORF knockout EpiSC cell line.

[0160] The specific gRNA sequence information is as follows:

[0161] 85aa-sgRNA1: 5'-TGAAGATCACTCCGGTCCAA-3' (SEQ ID NO: 5);

[0162] 85aa-sgRNA2: 5'-GAAGATCACTCCGGTCCAAA-3' (SEQ ID NO: 6);

[0163] 87aa-sgRNA1: 5'-AGAAGATCACTCCGTTCCAA-3' (SEQ ID NO: 7);

[0164] 87aa-sgRNA2: 5'-GAAGATCACTCCGTTCCAAA-3' (SEQ ID NO: 8);

[0165] After synthesizing the above sgRNAs (SEQ ID NO:5 and SEQ ID NO:6), they were cloned into the pSpCas9(BB)-2A-Puro(PX459)V2.0 vector (purchased from Addgene, 62988) via the BBSI restriction site. The vectors were then transfected into OG2-EpiSC cells (OG2: Oct4-ΔPE-GFP as the transfection site) using the transfection method described in Example 1. Pluripotent reporter lines were used. EpiSC cells transfected with 85aa-sgRNA1 and 2 were screened with puromycin for three days. Positive clones were then selected, and their genomes were extracted and sequenced to obtain two EpiSC cell lines with 85aa-uORF knockout. KO1 was a frameshift mutation with the insertion of one G base, and KO2 was a frameshift mutation with the absence of four GGTC bases. Similarly, the above-mentioned sgRNAs (SEQ ID NO:7 and SEQ ID NO:8) were synthesized and cloned into lentiCRISPR v2 (purchased from Addgene, 52961) via the BbsI restriction site. Lentiviral packaging was used to infect human pluripotent stem cells H9. After three days of puromycin screening of infected H9 cells, 87aa-uORF knockout cell lines were obtained.

[0166] 2) Flow cytometry detection of the transition from the primate to the primitive state of 85aa-uORF knockout mouse pluripotent stem cells (Primed-to- Transition efficiency (PNT)

[0167] OG2-mEpiSC (wild-type, WT) and 85aa-uORF knockout OG2-mEpiSC (KO) were seeded in 12-well plates, 30,000 cells per well, with 3 replicates per cell type, and the experiment was performed three times. WT and KO cells were induced to tropism using PNT medium containing BMP4+iCD1. A change of direction, a successful change The cells emitted green fluorescence, which could be detected by fluorescence microscopy and flow cytometry. WT and KO cells cultured for days 6 and 10 were analyzed and their efficiencies were statistically determined using a C6 flow cytometer (BD Biosciences).

[0168] 3) AP staining and qPCR detection of PNT efficiency in 87aa-uORF(sguORF1 / 2) knockout human pluripotent stem cells

[0169] Wild-type and 87aa-uORF knockout human pluripotent stem cell lines (sguORF1 and sguORF2) were seeded in 12-well plates, 30,000 cells per well, with three replicates per line, for three experiments. After culturing the culture system (PMID:29669738) for 9 days, the cells were stained using alkaline phosphatase (AP) detection reagent, and the number of clones of the three cell types was counted.

[0170] 4) Overexpression of 85aa-uORF promotes the transformation of mouse embryonic stem cells from the primitive state to the primordial state. -primedtransition, NPT)

[0171] Following the method described above, the constructed pLVX-85aa-uORF-3Flag and lentiviral packaging plasmid were transfected into HEK293T cells to package lentivirus. The lentiviral supernatant was collected, centrifuged, concentrated, diluted with culture medium, and used to infect mouse embryonic stem cells (mESCs). Cells were screened using puromycin (approximately 3 days later) and then expanded normally to obtain cells overexpressing 85aa-uORF. Cell lines, compared with controls Cells were seeded in 6-well plates, 50,000 cells per well, with 3 replicates per well. Primed cells were induced to transition to the Primed state using Primed cell culture medium. After 5 days, samples were photographed under an optical microscope, and RNA was extracted for analysis.

[0172] Sequencing and Western blot results showed that the 85aa-uORF knockout OG2-mEpiSC(KO) cell line was successfully constructed. Figure 2 (A-2C), and does not affect the translation and expression of the main open reading frame of Lin28B ( Figure 2 D). Flow cytometry experiments demonstrated that the PNT efficiency of 85aa-uORF KO was significantly higher than that of WT on day 6, and that the PNT-promoting efficiency of KO approached 100% on day 10. Figure 3 Knocking out 87aa-uORF in human pluripotent stem cells also significantly promoted PNT efficiency, demonstrating the high conservation of the amino acid sequence and function of Lin28B uORF in different species such as humans and mice. Figure 4 Furthermore, overexpression of 85aa-uORF significantly promoted... Differentiation of cells from the Primed state to the Primed state reduces Nanog, Klf4, Esrrb, Tfcp2l1, Dpp5a, Sox2, etc. Increase the expression of pluripotent marker genes, such as Fgf5, Otx2, and Dnmt3b, by enhancing the expression of Primed pluripotent marker genes. Figure 5 ).

[0173] Example 385aa-uORF microprotein regulates embryo implantation

[0174] 1) Western blot detection of 85aa-uORF microprotein expression in different organs of mice

[0175] Adult mice were euthanized by cervical dislocation. Tissues from the brain, heart, liver, lung, kidney, intestine, spleen, testis, and ovary were collected, ground, and sonicated. After centrifugation, the supernatant was used to prepare protein samples. Electrophoresis, membrane transfer, blocking, incubation with primary antibody (rabbit anti-85aa-uORF, 1:500) at 4°C overnight, washing with TBST, incubation with secondary antibody (mouse anti-1:5000) for 1 hour, washing, development, and photographic analysis were performed.

[0176] Experimental results are as follows Figure 6 As shown, the microprotein encoded by 85aa-uORF is normally expressed in the brain, heart, liver, kidney, intestine and testis of wild-type (MP85+ / +) mice, and a knockout model mouse of 85aa-uORF (MP85- / -) was successfully constructed.

[0177] 2) Construct 85aa-uORF knockout mice (MP85- / -)

[0178] sgRNAs were designed targeting the non-overlapping regions of Lin28B uORF and mORF. After verifying the cleavage efficiency in vitro, the most efficient sgRNA (ACTGATGCTGAAGATCACTCCGG (SEQ ID NO:11)) and Cas9 were transcribed in vitro. The mixture of Cas9 and sgRNA was then microinjected into mouse zygotes. Frameshift mutations with base deletions or insertions were introduced through non-homologous end joining (NHEJ) repair after double-strand breaks. F0 generation mice with frameshift mutations were obtained after genotyping, and F1 generation mice were further bred and used in experiments after genotyping.

[0179] 3) Chicago Blue 6B assay for the number of implantation sites in 85aa-uORF knockout mouse embryos.

[0180] Superovulation experiments were conducted on wild-type (MP85+ / +) female mice and 85aa-uORF knockout female mice (MP85- / -) aged 7-8 weeks. 7.5 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally, followed by 7.5 IU of human chorionic gonadotropin (hCG) 48 hours later. Each mouse was mated with an adult male mouse of the same genotype. The female mice with vaginal plugs were recorded as 0.5 dpc (0.5 days post-mating). 5.5 dpc mice were injected intravenously with 1% Chicago Blue 6B. Five minutes later, the mice were sacrificed, photographed, and the blastocyst implantation site was recorded.

[0181] Experimental results are as follows Figure 7As shown, using Chicago Blue 6B to stain mouse blastocyst implantation sites blue, and after photographing and statistical analysis, it was found that MP85- / - genotype mice had significantly fewer implantation sites (5.5 dpc) than MP85+ / + genotype mice, indicating that 85aa-uORF knockout affects the number of mouse blastocyst implantations. To eliminate the influence of MP85- / - female mice themselves, mating MP85- / - genotype female mice with wild-type MP85+ / + male mice did not significantly affect the number of female blastocyst implantation sites, indicating that 85aa-uORF deficiency leads to embryonic developmental defects and affects blastocyst implantation.

[0182] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0183] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:2 or 4.

2. An expression carrier, characterized in that, The expression vector expresses the polypeptide of claim 1.

3. A recombinant cell, characterized in that, It carries the expression vector of claim 2, or expresses the polypeptide of claim 1.

4. The recombinant cell according to claim 3, characterized in that, The cells include mammalian pluripotent stem cells.

5. The recombinant cell according to claim 3, characterized in that, The cells include mammalian embryonic stem cells or induced pluripotent stem cells.

6. Use of a reagent for overexpressing the polypeptide of claim 1 in pluripotent stem cells in the preparation of high-purity nascent state pluripotent stem cells, wherein the reagent is an overexpression vector for the polypeptide.

7. The use according to claim 6, characterized in that, The pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

8. Use of an agent that downregulates or inhibits the expression of the polypeptide of claim 1 in pluripotent stem cells in the preparation of high-purity primitive pluripotent stem cells, said agent being based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

9. The use according to claim 8, characterized in that, The pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

10. A method for preparing high-purity nascent pluripotent stem cells, characterized in that, include: The reagent for overexpressing the polypeptide of claim 1 is contacted with pluripotent stem cells, or the pluripotent stem cells are contacted with the polypeptide of claim 1 to obtain the nascent pluripotent stem cells, wherein the reagent is an overexpression vector of the polypeptide.

11. The method according to claim 10, characterized in that, The pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

12. A method for preparing high-purity, primitive pluripotent stem cells, characterized in that, include: The reagent that downregulates or inhibits the polypeptide of claim 1 is contacted with pluripotent stem cells, said reagent being at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

13. The method according to claim 12, characterized in that, The pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

14. The use of the polypeptide of claim 1, the nucleic acid encoding the polypeptide of claim 1, the expression vector of claim 2, or the recombinant cell of any one of claims 3 to 5 in the preparation of a drug, characterized in that, The drug is used to prevent, alleviate and / or treat diseases related to implantation defects.

15. The use according to claim 14, characterized in that, The disease includes at least one of threatened miscarriage, recurrent miscarriage, and infertility.

16. The use according to claim 14, characterized in that, The diseases mentioned include at least one of endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency.

17. A medicament for preventing, alleviating, and / or treating diseases related to implantation defects, characterized in that, The drug comprises the polypeptide of claim 1, the nucleic acid encoding the polypeptide of claim 1, the expression vector of claim 2, or the recombinant cell of any one of claims 3 to 5.

18. The medicament according to claim 17, characterized in that, The disease includes at least one of threatened miscarriage, recurrent miscarriage, and infertility.

19. The medicament according to claim 17, characterized in that, The diseases mentioned include at least one of endometriosis, thin endometrium, endometritis, uterine malformation, polycystic ovary syndrome, and luteal insufficiency.

20. The use of the formulation in the preparation of a medicament for the prevention, alleviation, and / or treatment of diseases related to implantation defects, characterized in that, The formulation enables the overexpression of the polypeptide of claim 1 in cells, and the formulation is an overexpression vector for the polypeptide.

21. The application according to claim 20, characterized in that, The cells include mammalian pluripotent stem cells.

22. The application according to claim 21, characterized in that, The pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells.

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