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WO2016119113A1 - Procédé de régulation, par un miarn, du niveau de modification de m6a et ses applications - Google Patents

Procédé de régulation, par un miarn, du niveau de modification de m6a et ses applications Download PDF

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WO2016119113A1
WO2016119113A1 PCT/CN2015/071582 CN2015071582W WO2016119113A1 WO 2016119113 A1 WO2016119113 A1 WO 2016119113A1 CN 2015071582 W CN2015071582 W CN 2015071582W WO 2016119113 A1 WO2016119113 A1 WO 2016119113A1
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mirna
modification
mir
rna
level
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Chinese (zh)
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周琪
杨运桂
王秀杰
陈同
张映
郝亚娟
李苗苗
王猛
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Institute of Genetics and Developmental Biology of CAS
Institute of Zoology of CAS
Beijing Institute of Genomics of CAS
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Institute of Genetics and Developmental Biology of CAS
Institute of Zoology of CAS
Beijing Institute of Genomics of CAS
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Priority to CN201580074289.4A priority Critical patent/CN107207557B/zh
Priority to PCT/CN2015/071582 priority patent/WO2016119113A1/fr
Publication of WO2016119113A1 publication Critical patent/WO2016119113A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs

Definitions

  • the invention belongs to the field of cell biotechnology, and in particular relates to a method for regulating the level of modification of 6 -methyladenosine (m 6 A) on an RNA molecule by miRNA and related further application.
  • m 6 A 6 -methyladenosine
  • RNA modification database updated in 2011, RNAMDB, contains a total of 109 RNA modifications, of which methylation modification accounts for 80%.
  • 6-methyladenosine (m 6 A) which is methylated at the sixth N atom of base A, is the most common post-transcriptional modification of RNA in eukaryotes because of its It is rich in content and highly conserved, and has received extensive attention and research in recent years.
  • m 6 A is produced by a multi-component methyltransferase complex comprising at least three core proteins, METTL3, METTL14 and WTAP.
  • RNA demethylase The two demethylases that have been identified are FTO and ALKBH5, respectively.
  • YTH domain protein YTHDF1-3
  • YTHDF2 Human YTHDF2 "reader" protein identified by m 6 A selective regulation of mRNA degradation.
  • m 6 A modification does not affect the ability of the modified adenine to encode and complement thymine or uracil, it affects non-classical adenine: guanine (A:G) pairing and may affect RNA secondary structure.
  • guanine (A:G) guanine pairing
  • RNA secondary structure In the m 6 A methylase and demethylase deficient cells, the expression level, translation efficiency, nuclear retention time and stability of messenger RNA are greatly affected, so the m 6 A modification is considered to mainly affect the messenger. The metabolism of ribonucleic acid.
  • RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
  • m 6 A-Seq sequencing technology With the birth of the m 6 A-Seq sequencing technology, the basic features of m 6 A modification in tissues or cells of human, mouse and yeast have been identified. More importantly, the researchers found that m 6 A is present in a large number of mRNAs encoded by genes associated with human diseases, including cancer and several brain diseases such as autism, Alzheimer's and schizophrenia, indicating This modification can be used as a target for the treatment of diseases (Fu, Y., et al.; 2014. Gene expression regulation mediated through reversible m 6 A RNA methylation. Nat Rev Genet 15, 293-306.). Subsequent studies have shown that this RNA modification has many important functions.
  • RNA modification For example, in January last year, researchers found that one of the main functions of this modification is to control the life and degradation of RNA, which is extremely important for healthy cell development. Prolonged RNA life will result in the production of more protein. If this demethylation mechanism is flawed, it is possible to greatly affect cellular protein levels. Some of these proteins are likely to be critical to the energy regulation of the human body and affect obesity. In order to accomplish the task of accurately silencing various mRNAs, the organism needs to ensure its stability and effectiveness through various fine-tuning mechanisms such as processing and post-mature processing. Understanding the process of RNA modification will help scientists analyze this mechanism of action and how to make up for it after a problem with this mechanism.
  • miRNAs are non-coding microRNAs of approximately 21 to 25 bases (nt) in length that are widely found in the genome of eukaryotes. It is generally transcribed from the miRNA gene located in the intergenic region and intron to form the original miRNA (pri-miRNA), which is processed into a 70 nt miRNA precursor (pre-miRNA) in the nucleus of the animal, and then transported to the cytoplasm. Processed into mature miRNAs. The mature miRNA enters the miRNA-induced silencing complex (miRISC) and is paired with the target mRNA to negatively regulate gene expression by degrading the target mRNA or hindering protein translation.
  • miRISC miRNA-induced silencing complex
  • m 6 A modification may affect miRNA binding to mRNA target regions (Wang, Y., et al.; 2014b. N(6)-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol 16,191- 198.), but whether miRNA directly regulates m 6 A has not been proven.
  • Object of the present invention is by analyzing the m 6 A modification in the distribution pattern of the plurality of pluripotent stem cells and differentiated cells, a cell type-specific identification of previously unreported and m 6 A modified features.
  • the m 6 A modification site was found to be a potential miRNA target region. Further studies showed that miRNA is involved in the regulation of abundance of m 6 A and the modification of target region-specific modification abundance in both mouse and human cells, changing miRNA The sequence can also generate new m 6 A modifications. Also confirmed m 6 A modified cell change affects reprogramming efficiency.
  • the present invention for the first time, links miRNA and m 6 A modifications together, confirming that regulation of cell state can be involved by altering m 6 A modification, and will provide new ideas for studying the regulatory mechanisms and functions of m 6 A and miRNA through miRNAs. Modulation of RNA modification may be a new regulatory aspect of cell fate regulation and disease treatment.
  • m 6 A enrichment sequencing (m 6 A-Seq)
  • the known functions of m 6 A modified genes stably expressed in 4 cell lines involve many important biological processes including transcriptional regulation, cell cycle regulation, ribonucleic acid (RNA) processing, chromosome modification, programmed death and cells. Internal signal path.
  • small interfering RNA (siRNA) knockdown or plasmid overexpression can be used to increase the expression of the miRNA-producing enzyme Dicer, which is responsible for the cleavage of the miRNA precursor. Decreasing or increasing m 6 A abundance, while also indicating that Dicer regulates the overall level of m 6 A is conservative in humans and rodents.
  • Dicer small interfering RNA
  • expression of an overexpressed miRNA or a knockdown miRNA can correspondingly increase or decrease the m 6 A abundance of the corresponding target region, indicating that the miRNA regulates the modification of a specific site of m 6 A.
  • the mutated RNA can be small molecules with mutations in the new small RNA complementary to mRNA
  • the present invention provides a m 6 A methylase inhibitor and a knockdown methylation transferase method for reducing iPS efficiency, indicating that the level of modification of m 6 A is associated with cell fate turnover.
  • the present invention provides a method of modulating the level of m 6 A modified reagent, said reagent comprising a miRNA, miRNA modulator or exogenously introduced miRNA similar small molecule RNA.
  • the modifying agent is capable of increasing or decreasing the level of m 6 A.
  • the small RNA is a small RNA molecule having m 6 A pair exogenous motif design. More preferably, the small molecule RNA is a small molecule RNA that is structurally mutated relative to an endogenous miRNA sequence. More preferably, the small sequence RNA of the sequence structure mutation can produce m 6 A on the new mRNA complementary to the small RNA after the mutation. More preferably, the small RNA mutation at the mutation site and the motif m 6 A matched pair. More preferably, the mutated small molecule RNA is as shown in any of SEQ ID No. 29-32.
  • the miRNA modulator is capable of increasing or decreasing the level of endogenous miRNA.
  • miRNA modulators capable of increasing the level of endogenous miRNA include, but are not limited to, mimics that overexpress miRNA, enzymes involved in miRNA production, genes of enzymes involved in miRNA production, expression vectors of genes comprising enzymes involved in miRNA production, or Host cells, or any other exogenously designed small RNA that increases the level of miRNA expression.
  • the enzyme involved in miRNA production is the miRNA producing enzyme Dicer.
  • miRNA modulators capable of reducing the level of endogenous miRNA include, but are not limited to, miRNA inhibitors or any of the exogenously designed small molecule RNAs that reduce the level of miRNA expression. More preferably, the miRNA inhibitor is an expression vector or host cell of a gene related to miRNA degradation, a gene related to miRNA degradation, or a gene containing an enzyme related to miRNA degradation.
  • the small molecule RNA is an siRNA of a miRNA production-related enzyme. More preferably, the miRNA The related enzyme that is produced is the miRNA-producing enzyme Dicer. More preferably, the siRNA of the miRNA generating enzyme Dicer is as shown in any of SEQ ID No. 1-6.
  • the agent is a pharmaceutical composition. More preferably, the pharmaceutical composition is for the treatment of cancer and several brain diseases such as autism, Alzheimer's disease and schizophrenia.
  • the invention also provides the use of a reagent as described above for modulating m 6 A modification levels or m 6 A modification mediated functions.
  • the present invention also provides a use of the above agents functions in the regulation of the formulation prepared m 6 A regulation or modification levels m 6 A modified mediated in.
  • the invention also provides a method of modulating m 6 A modification level or m 6 A modification mediated function, which comprises modulating m 6 A modification with the above reagents.
  • the m 6 A modification mediated functions include, but are not limited to, regulation of cell fate regulation, biological function of the organism, or modulation of disease treatment. More preferably, the m 6 A modification mediated functions include important biological processes such as circadian clocks involved in m 6 A modification, meiosis, and proliferation of embryonic stem cells; cancer and several brain diseases such as autism, Alzheimer's disease and schizophrenia. More preferably, the m 6 A modification mediated function comprises cell reprogramming. More preferably, the method is carried out in vitro. More preferably, the method is not a method of treatment.
  • the present invention also provides a modulator 6 A m applied in the regulation of cell reprogramming.
  • the m 6 A modulator is an m 6 A inhibitor or promoter. More preferably, the m 6 A inhibitor is cycloleucine or other methyltransferase inhibitor acting on the methyl donor S-adenosylmethionine (SAM), such as 3-deaza Glycosides, siRNAs of methyltransferases, and the like. More preferably, the siRNA of the methyltransferase is as shown in any of SEQ ID No. 41-43.
  • the m 6 A promoter is an m 6 A methyltransferase.
  • the present invention also provides a method of modulating cell reprogramming, wherein the method is modulated using the above m 6 A modulator.
  • the present invention also provides an application of the m-adjusting agent 6 A formulation is prepared in the regulation of cell reprogramming.
  • the present invention proposes to identify cell type-specific and previously unreported m 6 A by analyzing the distribution profile of m 6 A modification in multiple pluripotent stem cells and differentiated cells. Modified features.
  • the m 6 A modification site was found to be a potential miRNA target region. Further studies showed that miRNA is involved in the regulation of the abundance of m 6 A and the modification of target-specific abundance in both mouse and human cells.
  • the present inventors have also found m 6 A modified cell change affects reprogramming efficiency.
  • the present invention provides a novel method for modulating RNA m 6 A modification using miRNA.
  • the method of the present invention has important application value for the first time in the treatment of diseases caused by disorder of m 6 A level.
  • Figure 1 is a characteristic of a total of m 6 A modifications in the four cell lines of Example 1.
  • A has m 6 A modified gene enriched biological pathways stably expressed in four cell lines.
  • B Distribution of m 6 A modified regions on transcripts stably expressed in cell lines and having a consistently modified map. Each black line indicates that there is a m 6 A modification in the corresponding region.
  • TcSS transcription start site region
  • 5' UTR 5' untranslated region
  • CDS coding region
  • TsTS translation termination site region
  • 3' UTR 3' untranslated region.
  • FIG 2 is a m 2 Example 6 A modified embodiment is a potential site of miRNA target region; Proportion and control area (A) of the predicted miRNA mouse cells may be targeted by m 6 A modified region. '***' stands for Fisher's exact test p ⁇ 2.2e-16. (B) Proportion of miRNA-targeted m 6 A modified regions expressed in HeLa cells and ratio of control regions. '***' stands for Fisher's exact test p ⁇ 2.2e-16.
  • NSC cells miRNA Dicer enzyme mRNA knockdown of m 6 A modified abundance (FIG. 3A) using small interfering RNA (siRNA). Knockdown of Dicer in human HeLa cells reduced m 6 A modification abundance at the cellular level (Fig. 3B); '**' stands for Student's t-test p ⁇ 0.01;'***' stands for Student's t-test p ⁇ 0.001.
  • FIG. 4 is a representation of the miRNA-producing enzyme Dicer in Example 4, and the m 6 A abundance was detected by the dot-blot method.
  • Overexpression of Dicer plasmid using improved cellular level m 6 A modified abundance (FIG. 4A).
  • Overexpression of D icer in human HeLa cells increased m 6 A modification abundance at the cellular level (Fig. 4B); '***' represents Student's t-test p ⁇ 0.001.
  • Figure 5 is a diagram showing the overexpression of miR-668-3p, miR-1981-5p, miR-1224-5p, miR-330-5p and miR-455-3p for its target by m 6 A-QPCR in Example 5.
  • '*' stands for Student's t-test p ⁇ 0.05;
  • ***' stands for Student's t-test p ⁇ 0.001.
  • Figure 6 is a diagram showing the knockdown of miR-668-3p, miR-1981-5p, miR-484, miR-330-5p and miR-455-3p for its target site by m 6 A-QPCR in Example 6.
  • miR-668-3p KIF1B miR-1981-5p ,: TAF5L miR-484,: miR-330-5p NFE2L1,: and miR-455-3p
  • TCF4 Effect of m 6 a modified PIGT abundance.
  • '*' stands for Student's t-test p ⁇ 0.05
  • '**' stands for Student's t-test p ⁇ 0.01
  • '***' stands for Student's t-test p ⁇ 0.001.
  • Figure 7 is a diagram showing the overexpression of miR-330-5p-mutant, miR-668-3p-mutant, miR-1981-5p-mutant and miR-1224-5p by the method of m 6 A-QPCR in Example 7.
  • - mutant its newly generated target site miR-330-5p-mutant: FBXO21, miR-668-3p-mutant: TATAG1, miR-1981-5p-mutant: FAM129B and miR-1224-5p - Mutant: The effect of the m 6 A modification abundance of DDX6.
  • '**' stands for Student's t-test p ⁇ 0.01.
  • Figure 8 is a graph showing AP staining (A) and number of clones (B) of the effect of m 6 A methylase inhibitor in Example 8 on iPS efficiency, and '***' represents Student's t-test p ⁇ 0.001.
  • Figure 9 is a graph showing the effect of knockdown of m 6 A methyltransferase on iPS efficiency in Example 9 on AP staining (A) and the number of clones (B), and '**' on Student's t-test p ⁇ 0.01.
  • ESC embryonic stem cells
  • i PSC induced pluripotent stem cells
  • NSC neural stem cells
  • SC testicular support cells
  • the medium for SC cells was formulated into 450 ml DMEM, 50 ml FBS, and 5 ml 100 x streptomycin.
  • NSC medium was added to E2 and bFGF (concentration: 20 ng/ml) in N2B27 medium.
  • Mouse ES cells and iPS cell culture medium were DMEM containing 20% fetal bovine serum (FBS, Gibco), and 1000 U of LIF was added ( Leukemia inhibitory factor, Chemicon), 2 mM glutamine (Sigma), 1 mM sodium pyruvate (Sigma), and 0.1 mM ⁇ -mercaptoethanol (Sigma), 0.1 mM non- Non-essential amino acid (Gibco) and the like.
  • PCR model used in the following examples was a Stratagene Mx 3000P real-time PCR instrument purchased from Jitai.
  • the reagents used in the following examples are analytical grade reagents and are commercially available from conventional sources.
  • RNA is isolated from the cells and is enriched for high purity and high integrity mRNA. The mRNA is then interrupted into fragments of approximately 100 nucleotides in size and then precipitated with ethanol for later use. A portion of the fragmented mRNA was used to construct a transcriptome sequencing library. Transcriptome library construction and sequencing were performed according to standard protocols provided by Illumia. Another part of the mRNA fragments comprising fragments are used to enrich m 6 A and transcriptome sequencing using the same method comprises m 6 A mRNA enriched sequenced.
  • transcriptome sequencing data as a control, we identified distributed over a 7,000-8,000 33,000-43,000 genes m 6 A enriched regions in each cell line m 6 A-Seq data.
  • the transcript is first divided into five regions, the transcription initiation region (TcSS), the 5' untranslated region (5'UTR), the protein coding region (CDS), the translation termination region (TsTS), and the 3' untranslated region (3). 'UTR), and the m 6 A modification map of each gene in 4 samples was plotted according to whether each region contained m6A modification.
  • Mouse mature miRNA sequences were downloaded from the database miRBase (version number 20) and aligned with the m 6 A modified region enriched motif.
  • the previously reported m 6 A modification motif is the RRACH motif.
  • m 6 A seed region and the reverse complement of a miRNA motifs were screened.
  • To assess whether the ratio of m 6 A motifs that match the miRNA seed region is a random event we generated 500 sets of mock sequences for each cell line and used the same criteria to screen for motifs complementary to the miRNA seed region and calculate Its proportion.
  • To systematically compare the relationship between miRNA and m 6 A modified regions we used the tool miRanda to align mature miRNA sequences to m 6 A modified regions.
  • To investigate whether the targeting relationship between miRNA and m 6 A modified regions is conserved in humans we analyzed m 6 A modified regions and miRNAs in HeLa cells using published m 6 A-Seq data from human HeLa cells. Relationship.
  • NSC and HeLa cells were cultured normally. After 24 h of inoculation, Lipofecamine RNAi Max (Invitrogen, 13778150) and RFect siRNA Transfection were used respectively when the cell fusion degree was 50%.
  • the reagent (Bio-Tran) reagent was transfected, the transfected Dicer siRNA was a mixture of three siRNAs, and the sequence of three mouse Dicer siRNAs was:
  • the sequence of the three human Dicer siRNAs is:
  • each siRNA was 60 nM.
  • the cells were collected 24 hours after transfection, and total RNA was extracted by TRIzol method.
  • mRNA was extracted from the mRNA purification kit (Ambion, 61006). The prepared mRNA was transferred to a nylon membrane, incubated with rabbit anti-m 6 A antibody (1:1000) (Synaptic Systems, 202003) at 4 ° C overnight, and the secondary antibody HRP-conjugated Goat anti-rabbit IgG was incubated (DakoCytomation).
  • small interfering RNA was used to knock down the expression of miRNA-producing enzyme Dicer, and the abundance of m 6 A was detected by dot-blot method.
  • the results showed that knocking down Dicer can reduce m 6 A abundance accordingly (Fig. 3A left), the gray-scale scan and statistical analysis of the results of dot-blot, the decrease of m 6 A abundance after knocking down Dicer, set the control group to 1, and the decrease of m 6 A abundance after knocking down Dicer is 0.528 There was a significant difference from the control group (Fig. 3A right).
  • NSC and HeLa cells were cultured normally. After 24 h of inoculation, Lipofecamine 2000 (Invitrogen, 11668019) and polyethylenimine (Polysciences, 24765) reagents and pCI-Myc-Dicer plasmid (mouse transcript number; NM_148948.2; human transcript number: NM_030621.4) was transfected together, cells were collected 24 h after transfection, total RNA was extracted by TRIzol method, and used. mRNA was extracted from the mRNA purification kit (Ambion, 61006).
  • the prepared mRNA was transferred to a nylon membrane, incubated with rabbit anti-m 6 A antibody (1:1000) (Synaptic Systems, 202003) at 4 ° C overnight, and the secondary antibody HRP-conjugated Goat anti-rabbit IgG was incubated (DakoCytomation). , p0448) (1:5000) After incubation for 30 min at room temperature, add 1 ml of the exposure solution (GE, RPC2232) to 1 ml of normal temperature for 1 min, then expose and photograph. The signal intensity of dot-blot was quantified using Gel-Pro analyzer software (Media Cybernetics).
  • NSC cells were cultured normally. After 24 h of inoculation, miR-668-3p, miR-1981-5p, miR-1224-5p, miR-330-5p were transfected with Lipofecamine RNAi Max (Invitrogen, 13778150) reagent at 50% cell fusion. And miR-455-3p, the final concentration of each miRNA was 20 nM. The sequences used were all synthesized from Genepharma. Cells were harvested 24 h after transfection. Extraction of total RNA using the TRIzol method, using The mRNA purification kit (Ambion, 61006) will extract mRNA.
  • RNA Fragmentation Reagents (Ambion, AM8740) reagent was interrupted at 94 ° C for 30 s into fragments of approximately 300 nt size.
  • the m 6 A antibody (Synaptic Systems, 202003), which was twice the amount of RNA, was incubated for 2 h at 4 ° C in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4). The mixture was incubated with 50 ⁇ l Protein A (Sigma, P9424) for 4 h at 4 °C.
  • RNA bound to the beads was eluted with 0.5 mg/ml m 6 A (BERRY & ASSOCIATES, PR 3732), and then RNA was extracted with TRIzol (Invitrogen, 15596-018).
  • the enriched m 6 A binding RNA was reverse transcribed by MMLV enzyme (Promega), and the m 6 A modification abundance of its target region was detected by Real-Time Quantitative PCR (qRT-PCR).
  • the sequence of miR-668-3p used is:
  • the sequence of miR-1981-5p used is:
  • the sequence of miR-1224-5p used is:
  • the sequence of miR-330-5p used is:
  • the sequence of miR-455-3p used is:
  • the upstream primer is: CCTTCTACCGTTTCGAGGC (SEQ ID No. 12);
  • the downstream primer is: TGCAATGATCCAACTCCAGA (SEQ ID No. 13);
  • the upstream primer is: TTGGCATCTGCTGGTGAG (SEQ ID No. 14);
  • the downstream primer is: CCATGGAGGCAGAAGCA (SEQ ID No. 15);
  • the upstream primer is: AAGGACCCAAGTCCTCAGC (SEQ ID No. 16);
  • the downstream primer is: GGCCTGACTTGGCATGA (SEQ ID No. 17);
  • the upstream primer is: TGCAACTTGAGGGACGACT (SEQ ID No. 18);
  • the downstream primer is: AGTGTGGGAGGATTGCCA (SEQ ID No. 19);
  • the upstream primer is: AGCGGTACGTGAGTGGCTA (SEQ ID No. 20);
  • the downstream primer was: CACGACATCCAGCAGCA (SEQ ID No. 21).
  • the m 6 A-qRT-PCR results showed changes in the m 6 A level of the target site overexpressing the miRNA.
  • the targeted m 6 A modified regions of the detected miR-668-3p, miR-1981-5p, miR-1224-5p, miR-330-5p and miR-455-3p were mapped to miR-668-3p:KIF1B, respectively.
  • overexpression of the miRNA can correspondingly increase the m 6 A abundance of the corresponding target region.
  • We set the control group to 1.
  • the m 6 A of the corresponding regions of miR-668-3p, miR-1981-5p, miR-1224-5p, miR-330-5p and miR-455-3p increased to 7.587, 5.847, respectively. 2.793, 4.857 and 4.407, and the difference was statistically significant.
  • RNAi Max Lipofecamine RNAi Max (Invitrogen, 13778150) reagent after 24 h of inoculation.
  • the miR-330-5p inhibitor and the miR-455-3p inhibitor have a final concentration of 100 nM per miRNA inhibitor.
  • the sequences used were designed to be synthesized from Genepharma. Cells were harvested 24 h after transfection. Extraction of total RNA using the TRIzol method, using The mRNA purification kit (Ambion, 61006) will raise the mRNA.
  • RNA Fragmentation Reagents (Ambion, AM8740) reagent at 94 ° C for 30 s.
  • the m 6 A antibody (Synaptic Systems, 202003), which was twice the amount of RNA, was incubated for 2 h at 4 ° C in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4). The mixture was incubated with 50 ⁇ l Protein A (Sigma, P9424) for 4 h at 4 °C.
  • RNA bound to the beads was eluted with 0.5 mg/ml m 6 A (BERRY & ASSOCIATES, PR 3732), and then RNA was extracted with TRIzol (Invitrogen, 15596-018).
  • the enriched m 6 A binding RNA was reverse transcribed by MMLV enzyme (Promega), and the m 6 A modification abundance of its target region was detected by Real-Time Quantitative PCR (qRT-PCR).
  • the sequence of the miR-668-3p inhibitor used is:
  • the sequence of the miR-1981-5p inhibitor used was:
  • the sequence of the miR-484 inhibitor used is:
  • the sequence of the miR-330-5p inhibitor used is:
  • the sequence of the miR-455-3p inhibitor used is:
  • Amplification primers for amplifying miR-668-3p target site KIF1B, miR-1981-5p target site TAF5L, miR-330-5p target site TCF4, miR-455-3p target site PIGT are as described in Example 5. Show.
  • the upstream primer is: TCGGCGACAGGAGAGAA (SEQ ID No. 27);
  • the downstream primer was: TGTTAGGTCCAGGCCCA (SEQ ID No. 28).
  • the m 6 A-qRT-PCR results showed a change in m 6 A levels that inhibited miRNA expression at the corresponding target site.
  • the targeted m 6 A modified regions of the detected miR-668-3p, miR-1981-5p, miR-484, miR-330-5p and miR-455-3p were mapped to miR-668-3p:KIF1B, miR, respectively.
  • miR-484 NFE2L1
  • miR-330-5p TCF4
  • miR-455-3p transcript of PIGT.
  • Inhibition of miRNA expression compared to the control group can correspondingly reduce the m 6 A abundance of the corresponding target region.
  • We set the control group to 1.
  • the m 6 A of the corresponding regions of miR-668-3p, miR-1981-5p, miR-484, miR-330-5p and miR-455-3p were reduced to 0.171, 0.214, 0.606, 0.619, respectively. And 0.601, and the difference is statistically significant.
  • RNAi Max Lipofecamine RNAi Max (Invitrogen, 13778150) reagent after 24 h of cell inoculation.
  • the design principle of the mutant is mutation miR-330-5p, miR-668-5p, miR-1981-5p and miR-1224-5p seed region (5'2- The 3 nucleotides of 8 nt) are targeted to a new m 6 A modified region different from the original miRNA. The final concentration of each miRNA was 20 nM. The sequences used were all synthesized from Genepharma.
  • RNA Fragmentation Reagents (Ambion, AM8740) reagent was interrupted at 94 ° C for 30 s into fragments of approximately 300 nt size.
  • the m 6 A antibody (Synaptic Systems, 202003), which was twice the amount of RNA, was incubated for 2 h at 4 ° C in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4).
  • RNA bound to the beads was eluted with 0.5 mg/ml m 6 A (BERRY & ASSOCIATES, PR 3732), and then RNA was extracted with TRIzol (Invitrogen, 15596-018).
  • MMLV enzyme Promega
  • m 6 A modification abundance of its target region was detected by Real-Time Quantitative PCR (qRT-PCR).
  • the sequence of the miR-330-5p mutant used was:
  • the sequence of the miR-668-3p mutant used was:
  • the sequence of the miR-1981-5p mutant used was:
  • the sequence of the miR-1224-5p mutant used is:
  • the upstream primer is: TTACGGGAAGCGGAGCA (SEQ ID No. 33);
  • the downstream primer is: GCATCAGGCAGAAGCCA (SEQ ID No. 34);
  • the upstream primer is: CCTCTCCCAACAGGCAA (SEQ ID No. 35);
  • the downstream primer is: ACGCTGGACTCTGAGCTTG (SEQ ID No. 36);
  • the upstream primer is: TGTGGGAAAGGTGGCTG (SEQ ID No. 37);
  • the downstream primer is: AGCCCACAGAAAACGGG (SEQ ID No. 38);
  • the upstream primer is: TGCCAACCTTGGACTGC (SEQ ID No. 39);
  • the downstream primer was: TCCAAGGCACCCCTCA (SEQ ID No. 40).
  • the m 6 A-qRT-PCR results showed a change in the level of newly generated target site m 6 A corresponding to the overexpressed miRNA mutant.
  • the targeted m 6 A modified regions of the detected miR-330-5p-mutant, miR-668-5p-mutant, miR-1981-5p-mutant and miR-1224-5p-mutant were localized at miR, respectively -330-5p-mutant: FBXO21, miR-668-3p-mutant: TAGAP1, miR-1981-5p-mutant: FAM129B and miR-1224-5p-mutant: transcript of DDX6.
  • miRNA overexpression mutants corresponding increase in the target area m 6 A newly generated abundance.
  • MEF cells About 1 ⁇ 10 4 MEF cells were inoculated, and 4 transcription factors Oct4, Sox2, Klf4 and c-Myc were transfected, and iPS cells were induced by KOSR system.
  • m 6 A inhibitor cycloleucine In order to detect the effect of m 6 A inhibitor cycloleucine on cell reprogramming, The MEF cells of the experimental group were treated with the m 6 A inhibitor cycloleucine daily until the 10th day of induction, while the control cells were treated with DMSO (dimethyl sulfoxide). After 15 days of induction, alkaline phosphatase staining was used to detect reprogramming efficiency, and induced reprogrammed cell clones were counted and statistically analyzed.
  • DMSO dimethyl sulfoxide
  • siRNAs of METTL3 were transfected with Lipofecamine RNAi Max (Invitrogen, 13778150) reagent for 3 days. The final concentration of each siRNA was 60 nM, which was transfected 4 times. The control group was transfected with nonsense siRNA.
  • the sequences of the three siRNAs are:
  • siRNA The sequence of meaningless siRNA is:
  • the sequences used were all synthesized from Genepharma. After 15 days of induction, alkaline phosphatase staining was used to detect reprogramming efficiency, and induced reprogrammed cell clones were counted and statistically analyzed. The results showed that METTL3 knockdown significantly reduced the number of iPS clone formation compared to the control group, we set the control group to 1, and the experimental group to 0.214 (Fig. 9A), and was statistically significant (Fig. 9B). The results indicate that knocking down METTL3 with siRNA can significantly reduce iPS efficiency during iPS induction.

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Abstract

L'invention concerne un procédé pour la régulation, par un miARN, du niveau de modification de la N6-méthyladénosine (m6A) sur des molécules d'ARN et d'autres applications associées correspondantes. Par l'augmentation ou la diminution du miARN, le niveau de modification de m6A peut être augmenté ou diminué de manière correspondante et la fonction de modification de la médiation de m6A est en outre modulée. L'invention concerne également un procédé de modulation pour influencer la reprogrammation cellulaire par la modulation de m6A et un modulateur utilisé dans ce procédé.
PCT/CN2015/071582 2015-01-26 2015-01-26 Procédé de régulation, par un miarn, du niveau de modification de m6a et ses applications Ceased WO2016119113A1 (fr)

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CN113717279A (zh) * 2021-07-28 2021-11-30 武汉爱博泰克生物科技有限公司 m6A重组兔单克隆抗体及制备方法
WO2022007890A1 (fr) * 2020-07-09 2022-01-13 Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences Compositions et procédés d'inhibition de ythdf1
CN116555162A (zh) * 2023-05-06 2023-08-08 郑州大学第一附属医院 基于piRNA与m6A甲基化在高糖损伤肾小管上皮细胞的调控应用
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WO2022007890A1 (fr) * 2020-07-09 2022-01-13 Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences Compositions et procédés d'inhibition de ythdf1
CN113717279A (zh) * 2021-07-28 2021-11-30 武汉爱博泰克生物科技有限公司 m6A重组兔单克隆抗体及制备方法
WO2024039764A3 (fr) * 2022-08-17 2024-04-18 Ohio State Innovation Foundation Analyse épitranscriptomique de gliome
CN116555162A (zh) * 2023-05-06 2023-08-08 郑州大学第一附属医院 基于piRNA与m6A甲基化在高糖损伤肾小管上皮细胞的调控应用
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