WO2016119113A1 - Method for mirna to regulate modification level of m6a and applications thereof - Google Patents

Abstract

Disclosed are a method for miRNA to regulate the modification level of N6-methyladenosine, m⁶A on RNA molecules, and related further applications thereof. By increasing or decreasing miRNA, the modification level of m⁶A can be increased or decreased correspondingly, and the mediation modifying function of m⁶A is further modulated. Also disclosed is a modulation method for affecting cell reprogramming by m⁶A modulation and a modulator used therein.

Description

Regulation method of miRNA on m6A modification level and its application Technical field

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.

Background technique

The post-transcriptional modification of RNA lays a chemical foundation for the diversification of RNA function. The 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. The m ⁶ A modification can be removed by RNA demethylase, and the two demethylases that have been identified are FTO and ALKBH5, respectively. YTH domain protein (YTHDF1-3) is a recently discovered family of proteins binding to m ⁶ A and ssRNA, wherein the binding capacity and m ⁶ A YTHDF2 strongest. Human YTHDF2 "reader" protein identified by m ⁶ A selective regulation of mRNA degradation.

Although 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. 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. The plasticity and dynamics of m ⁶ A modification on ribonucleic acid make it a new epigenetic regulatory marker and participate in important biological processes such as circadian clock, meiosis and proliferation of embryonic stem cells (Fustin, JM, et al.; Okamura, H.; 2013. RNA-methylation-dependent RNA processing controls the speed of the circadian clock. Cell 155, 793-806.; Schwartz, S., et al.; High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation Program in yeast meiosis.Cell 155,1409-1421.;Geula,S.,et al.;2015.m ⁶ A mRNA methylation facilitats resolution of naive pluripotency towards differentiation.Science.), but its function and regulation mechanism is very The extent is unknown.

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. 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. Previous reports have suggested that 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.

Summary of the invention

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.

On the one hand, mitogen sequencing of mouse embryonic stem cells, induced pluripotent stem cells, neural stem cells and testicular support cells, m ⁶ A enrichment sequencing (m ⁶ A-Seq), and systematic analysis of sequencing data. 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.

We then systematically examined the relationship between the m ⁶ A modified region and the miRNA. The results indicate that the expressed miRNA can target most of the m ⁶ A modified regions (75%). And mouse cell lines similar to those found, HeLa cells, 75% of the area of m ⁶ A modification is the expression of miRNA target site.

On the other hand, in NSC and human HeLa cells, 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.

In another aspect, in NSC cells, 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.

In another aspect, m ⁶ A. generated on NSC cells, the mutated RNA can be small molecules with mutations in the new small RNA complementary to mRNA

In still another aspect, 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.

Therefore, the present invention provides the following technical solutions:

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.

Preferably, the modifying agent is capable of increasing or decreasing the level of m ⁶ A.

Preferably, 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.

Preferably, the miRNA modulator is capable of increasing or decreasing the level of endogenous miRNA.

Preferably, 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.

More preferably, the enzyme involved in miRNA production is the miRNA producing enzyme Dicer.

Preferably, 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.

More preferably, 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.

Preferably, 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.

Preferably, 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.

Preferably, 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 technical solution of the present invention, from the technical level: 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. From the application level: by manipulating the expression of miRNA, it can be achieved: 1) the change of m ⁶ A overall modification level; 2) the change of miRNA specific m ⁶ A site modification level; 3) the change of small molecule RNA sequence can generate new m ⁶ A modification; 4) regulation of important biological processes such as circadian clock, meiosis and proliferation of embryonic stem cells involved in m ⁶ A modification; 5) regulation of m ⁶ A by miRNA can be used as cancer and autism, Alzheimer's Therapeutic targets for diseases such as schizophrenia and obesity. 6) In summary, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

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.

3 in Example 3. 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.

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. Effect of m ⁶ a modified PIGT abundance: site of miR-668-3p: KIF1B, miR- 1981-5p: TAF5L, miR-1224-5p: SSRP1, miR-330-5p: TCF4 and miR-455-3p . '*' 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.

detailed description

It is understood that the specific embodiments described herein are shown by way of example, and are not intended to limit the invention. The main features of the present invention can be applied to various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to recognize that many equivalents can be used in the specific steps described herein, using only routine experimentation. These equivalents are considered to be within the scope of the invention and are covered by the claims.

Unless otherwise specified, embryonic stem cells (ESC), induced pluripotent stem cells (i PSC), neural stem cells (NSC), and testicular support cells (SC) in the following examples were purchased from the Institute of Zoology, Chinese Academy of Sciences.

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 β-mercaptoethanol (Sigma), 0.1 mM non- Non-essential amino acid (Gibco) and the like.

Unless otherwise indicated, the PCR model used in the following examples was a Stratagene Mx 3000P real-time PCR instrument purchased from Jitai.

Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from conventional sources.

Example 1

Mouse embryonic stem cells, induced pluripotent stem cells, neural stem cells and Sertoli cells transcriptome sequencing, m ⁶ A-rich sequence (m ⁶ A-Seq), and m ⁶ A modification site verification. Total 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. In 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. To examine the distribution of m ⁶ A modifications on transcripts stably expressed in four cell lines, we used Shannon entropy-based methods to identify genes stably expressed in all samples and whether these genes have a consistent m ⁶ A modification pattern. 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.

We identified 8,558 genes stably expressed in all samples. Among them, 3,880 transcripts have m ⁶ A modifications in all cells tested, and 2,489 transcripts share at least one m ⁶ A modified region. The genes encoding these 3,880 transcripts tend to be involved in biological processes associated with cell cycle regulation, transcriptional regulation, RNA processing, and epigenetic modification (Fig. 1A). We subsequently investigated whether 3,880 stably expressed transcripts with modifications in all samples had consistent m ⁶ A modification patterns in the four cell lines. Of all the cell types tested, approximately 50% of the transcripts had stable m ⁶ A modifications in the coding and translation termination regions, while the ratio of transcription initiation region to 5' untranslated region was only 5%. Of the 4 cell lines tested, only 437 (11% of 3,880) transcripts shared a consistent m ⁶ A modification pattern, of which 325 transcripts shared at least 1 m ⁶ A modified region (Fig. 1B).

Example 2

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. Under conditions allowing a mismatch, 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.

We systematically examined the relationship between m ⁶ A modified regions and miRNAs. By aligning the sequences of mouse mature miRNAs collected by miRBase to the identified m ⁶ A modified regions, the results indicated that the expressed miRNAs can target most of the m ⁶ A modified regions (75%), which is significantly higher than Control area (Fisher's exact test, p < 2.2e-16) (Fig. 2A). In view of the fact that we only tested the regularity of m ⁶ A modification in mouse cells in the experiment, in order to study whether the targeting relationship between miRNA and m ⁶ A modified regions is conserved in humans, we have already used human HeLa cells. Published m ⁶ A-Seq data. Similar to the findings in mouse cell lines, 75% of the m ⁶ A modified regions in HeLa cells are the target sites for expressed miRNAs, which is significantly higher than the control region (Fisher's exact test, p<2.2e- 16) (Fig. 2B).

Example 3

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:

GAGCGCCGAUCUCUAAUUA (SEQ ID No. 1);

GGGAAAGAGACUGUUAAAU (SEQ ID No. 2);

GGUGCUCCCAGUAAUCAAA (SEQ ID No. 3);

The sequence of the three human Dicer siRNAs is:

UGCUUGAAGCAGCUCUGGA (SEQ ID No. 4);

AAGGGCACCCAUCUCUAAU (SEQ ID No. 5);

GCCAAGGAAAUCAGCUAAA (SEQ ID No. 6).

mRNA purification kit(Ambion,61006)提取mRNA。将准备好的mRNA转移到尼龙膜上，用兔抗m⁶A抗体(1:1000)(Synaptic Systems,202003)4℃孵育过夜，洗膜后孵育二抗HRP-conjugated Goat anti-rabbit IgG(DakoCytomation,p0448)(1:5000)室温孵育30min后加上曝光液(A，B液等量混合)(GE Healthcare,RPN2232)1ml常温孵育1min后进行曝光并拍照。dot-blot的信号强度使用Gel-Pro analyzer软件(Media Cybernetics)进行定量统计。The final concentration of 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). , 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).

In NSC cells, small interfering RNA (siRNA) 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). In human HeLa cells, the expression of Di ^{6 was} knocked down by small interfering RNA (siRNA), and the abundance of m ⁶ A was detected by dot-blot method. The results showed that knocking down Dicer can reduce the abundance of m ⁶ A (Fig. 3B). Left), the gray-scale scanning and statistical analysis of the results of dot-blot, the decrease of m ⁶ A abundance after knocking down Dicer, the control group was set to 1, and the decrease of m ⁶ A abundance after knocking down Dicer was 0.131. and the control group were significantly different (the right in FIG. 3B), indicating that regulation m ⁶ a modified Dicer overall level is conserved in humans and rodents.

Example 4

mRNA purification kit(Ambion,61006)提取mRNA。将准备好的mRNA转移到尼龙膜上，用兔抗m⁶A抗体(1:1000)(Synaptic Systems,202003)4℃孵育过夜，洗膜后孵育二抗HRP-conjugated Goat anti-rabbit IgG (DakoCytomation,p0448)(1:5000)室温孵育30min后加上曝光液(A，B液等量混合)(GE Healthcare,RPN2232)1ml常温孵育1min后进行曝光并拍照。dot-blot的信号强度使用Gel-Pro analyzer软件(Media Cybernetics)进行定量统计。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).

In NSC cells, the expression of Dicer was increased by plasmid overexpression, and the abundance of m ⁶ A was detected by dot-blot method. The results showed that overexpression of Dicer could increase the abundance of m ⁶ A (Fig. 4A left), for dot- The results of the blot were scanned by gray scale and statistical analysis. The over-expression of Dicer increased the abundance of m ⁶ A, and the control group was 1. The over-expression of Dicer increased the abundance of m ⁶ A to 4.461, which was significantly different from the control group. (Figure 4A right). In human HeLa cells, the expression of Dicer was increased by plasmid overexpression, and the abundance of m ⁶ A was detected by dot-blot method. The results showed that overexpression of Dicer could increase the abundance of m ⁶ A (Fig. 4B left). The results of dot-blot were scanned by gray scale and statistical analysis. The over-expression of Dicer increased the abundance of m ⁶ A, and the control group was set to 1. The over-expression of Dicer increased the abundance of m ⁶ A to 8.668, compared with the control group. significant differences (the right in FIG. 4B), indicating that regulation m ⁶ a modified Dicer overall level in humans and rodents are conserved.

Example 5

mRNA purification kit(Ambion,61006)将提取mRNA。利RNA Fragmentation Reagents(Ambion,AM8740)试剂94℃，30s将mRNA打断成约300nt大小的片段。利用2倍于RNA量的m⁶A抗体(Synaptic Systems,202003)在IPP缓冲液中(150mM NaCl,0.1％NP-40,10mM Tris-HCl,pH 7.4)4℃孵育2h。再将混合物和50μl Protein A(Sigma,P9424)4℃继续孵育2h。清洗三遍，用0.5mg/ml m⁶A(BERRY&ASSOCIATES,PR 3732)洗脱结合在珠子上RNA，后用TRIzol(Invitrogen,15596-018)提取RNA。富集的m⁶A结合RNA经过MMLV酶(Promega)反转录后，利用Real-Time Quantitative PCR(qRT-PCR)检测其靶区域的m⁶A修饰丰度。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 μl Protein A (Sigma, P9424) for 4 h at 4 °C. After washing three times, 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:

UGUCACUCGGCUCGGCCCACUACC (SEQ ID No. 7);

The sequence of miR-1981-5p used is:

GUAAAGGCUGGGCUUAGACGUGGC (SEQ ID No. 8);

The sequence of miR-1224-5p used is:

GUGAGGACUGGGGAGGUGGAG (SEQ ID No. 9);

The sequence of miR-330-5p used is:

UCUCUGGGCCUGUGUCUUAGGC (SEQ ID No. 10);

The sequence of miR-455-3p used is:

GCAGUCCACGGGCAUAUACAC (SEQ ID No. 11);

Amplification of miR-668-3p target site KIF1B

The upstream primer is: CCTTCTACCGTTTCGAGGC (SEQ ID No. 12);

The downstream primer is: TGCAATGATCCAACTCCAGA (SEQ ID No. 13);

Amplification of miR-1981-5p target site TAF5L

The upstream primer is: TTGGCATCTGCTGGTGAG (SEQ ID No. 14);

The downstream primer is: CCATGGAGGCAGAAGCA (SEQ ID No. 15);

Amplification of miR-1224-5p target site SSRP1

The upstream primer is: AAGGACCCAAGTCCTCAGC (SEQ ID No. 16);

The downstream primer is: GGCCTGACTTGGCATGA (SEQ ID No. 17);

Amplification of miR-330-5p target site TCF4

The upstream primer is: TGCAACTTGAGGGACGACT (SEQ ID No. 18);

The downstream primer is: AGTGTGGGAGGATTGCCA (SEQ ID No. 19);

Amplification of miR-455-3p target site PIGT

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. , miR-1981-5p: TAF5L, miR-1224-5p: SSRP1, miR-330-5p: TCF4 and miR-455-3p: transcript of PIGT. Compared to the control group, 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.

Example 6

mRNA purification kit(Ambion,61006)将提mRNA。利用RNA Fragmentation Reagents(Ambion,AM8740)试剂94℃，30s将mRNA打断成约300nt大小的片段。利用2倍于RNA量的m⁶A抗体(Synaptic Systems,202003)在IPP缓冲液中(150mM NaCl,0.1％NP-40,10mM Tris-HCl,pH 7.4)4℃ 孵育2h。再将混合物和50μl Protein A(Sigma,P9424)4℃继续孵育2h。清洗三遍，用0.5mg/ml m⁶A(BERRY&ASSOCIATES,PR 3732)洗脱结合在珠子上的RNA，后用TRIzol(Invitrogen,15596-018)提取RNA。富集的m⁶A结合RNA经过MMLV酶(Promega)反转录后，利用Real-Time Quantitative PCR(qRT-PCR)检测其靶区域的m⁶A修饰丰度。Normally cultured NSC cells, transfected with miR-668-3p inhibitor, miR-1981-5p inhibitor, miR-484 inhibitor, and 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. The mRNA was disrupted into fragments of about 300 nt size using 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 μl Protein A (Sigma, P9424) for 4 h at 4 °C. After washing three times, 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:

GGUAGUGGGCCGAGCCGAGUGACA (SEQ ID No. 22);

The sequence of the miR-1981-5p inhibitor used was:

GCCACGUCUAAGCCCAGCCUUUAC (SEQ ID No. 23);

The sequence of the miR-484 inhibitor used is:

AUCGGGAGGGGACUGAGCCUGA (SEQ ID No. 24);

The sequence of the miR-330-5p inhibitor used is:

GCCUAAGACACAGGCCCAGAGA (SEQ ID No. 25);

The sequence of the miR-455-3p inhibitor used is:

GUGUAUAUGCCCGUGGACUGC (SEQ ID No. 26);

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.

Amplification of miR-484 target site NFE2L1

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. -1981-5p: TAF5L, miR-484: NFE2L1, miR-330-5p: TCF4 and 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.

Example 7

mRNA purification kit(Ambion,61006)将提取mRNA。利RNA Fragmentat ion Reagents(Ambion,AM8740)试剂94℃，30s将mRNA打断成约300nt大小的片段。利用2倍于RNA量的m⁶A抗体(Synaptic Systems,202003)在IPP缓冲液中(150mM NaCl,0.1％NP-40,10mM Tris-HCl,pH 7.4)4℃孵育2h。再将混合物和50μl Protein A(Sigma,P9424)4℃继续孵育2h。清洗三遍，用0.5mg/ml m⁶A(BERRY&ASSOCIATES,PR 3732)洗脱结合在珠子上RNA，后用TRIzol(Invitrogen,15596-018)提取RNA。富集的m⁶A结合RNA经过MMLV酶(Promega)反转录后，利用Real-Time Quantitative PCR(qRT-PCR)检测其靶区域的m⁶A修饰丰度。Normally cultured NSC cells, transfected with miR-330-5p-mutant, miR-668-5p-mutant, miR-1981-5p with Lipofecamine RNAi Max (Invitrogen, 13778150) reagent after 24 h of cell inoculation. - Mutant and miR-1224-5p-mutant, 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. 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 μl Protein A (Sigma, P9424) for 4 h at 4 °C. After washing three times, 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-330-5p mutant used was:

UGUCAGCGCCUGUGUCUUAGGC (SEQ ID No. 29);

The sequence of the miR-668-3p mutant used was:

UGACUCACGGCUCGGCCCACUACC (SEQ ID No. 30);

The sequence of the miR-1981-5p mutant used was:

GAAAACGGUGGGCUUAGACGUGGC (SEQ ID No. 31);

The sequence of the miR-1224-5p mutant used is:

GAGAGCAGUGGGGAGGUGGAG (SEQ ID No. 32);

Amplification of the miR-330-5p-mutant target site FBXO21

The upstream primer is: TTACGGGAAGCGGAGCA (SEQ ID No. 33);

The downstream primer is: GCATCAGGCAGAAGCCA (SEQ ID No. 34);

Amplification of the miR-668-5p-mutant target site TATAG1

The upstream primer is: CCTCTCCCAACAGGCAA (SEQ ID No. 35);

The downstream primer is: ACGCTGGACTCTGAGCTTG (SEQ ID No. 36);

Amplification of the miR-1981-5p-mutant target site FAM129B

The upstream primer is: TGTGGGAAAGGTGGCTG (SEQ ID No. 37);

The downstream primer is: AGCCCACAGAAAACGGG (SEQ ID No. 38);

Amplification of miR-1224-5p-mutant target site DDX6

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. Compared with the control group, miRNA overexpression mutants corresponding increase in the target area m ⁶ A newly generated abundance. We set a control group of 1, miR-330-5p-mutant, miR-668-5p-mutant, miR-1981-5p-mutant and miR-1224-5p-mutant new targeted m ⁶ A The m ⁶ A of the modified region was increased to 1.494, 1.407, 1.142, and 1.290, respectively, and the difference was statistically significant.

Example 8

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. 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. The results showed that compared with the control group, the m ⁶ A inhibitor cycloleucine treatment group significantly reduced the number of iPS clone formation, we set the control group to 1, and the experimental group to 0.513 (Fig. 8A), and statistics Meaning (Figure 8B). The results indicate that m ⁶ A inhibitors can significantly reduce iPS efficiency during iPS induction.

Example 9

Inoculation of about 1x10 ⁴ MEF cells, transfection of 4 transcription factors Oct4, Sox2, Klf4 and c-Myc, induction of iPS cells by KOSR system, in order to detect the effect of knockdown of methylation transferase METTL3 on cell reprogramming, Three 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:

GGAGAUCCUAGAGCUAUUA (SEQ ID No. 41);

CUGCACUUCAGACGAAUUA (SEQ ID No. 42);

GCUACCGUAUGGGACAUUA (SEQ ID No. 43).

The sequence of meaningless siRNA is:

UAGAACGUCUAGGUAUCCC (SEQ ID No. 44).

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.

Claims (10)

An agent for regulating the level of m ⁶ A modification on an RNA molecule, characterized in that the reagent comprises a miRNA, a miRNA modulator or exogenously introduced into a small molecule RNA similar to a miRNA; preferably, the reagent can increase or decrease m ⁶ A level of modification. The reagent according to claim 1, wherein said small RNA is a small molecule RNA having an exogenous design paired with an m ⁶ A motif; more preferably, said small RNA is relative to an endogenous source Small molecule RNA with structural mutations in the sex mi RNA sequence. The agent of claim 1 wherein said miRNA modulator is capable of increasing or decreasing the level of endogenous miRNA. Preferably, miRNA modulators capable of increasing the level of endogenous miRNA include mimics overexpressing miRNA, enzymes related to miRNA production, genes encoding miRNA production-related enzymes, expression vectors or host cells of genes comprising miRNA-producing enzymes, Or any other exogenously designed small RNA that increases the level of miRNA expression; preferably, a miRNA modulator that reduces the level of endogenous miRNA, including but not limited to a miRNA inhibitor or any exogenous design, can be reduced a small molecule RNA of a miRNA expression level, more preferably, the miRNA inhibitor is an expression vector or a host cell of a gene related to miRNA degradation, a gene related to miRNA degradation, or a gene including an enzyme related to miRNA degradation, Small RNAs are siRNAs that are involved in miRNA production. The agent according to any one of claims 1 to 3, wherein the agent is a pharmaceutical composition. Use of an agent according to any of claims 1-3 for modulating m ⁶ A modification levels or m ⁶ A modification mediated functions. A reagent according to any one of 1-3 in the preparation of the regulation or modification levels m m ⁶ A Formulation ⁶ A modified regulation mediated functions in the claims. A method of modulating m ⁶ A modification level or m ⁶ A modification mediated function, comprising modulating m ⁶ A modification with an agent according to any of claims 1-3. One kind of m ⁶ A modifier Application reprogrammed cells was adjusted. Application of a modifier m ⁶ A formulation is prepared in the regulation of cell reprogramming. A cell reprogramming adjusting method characterized by comprising adjustment of the reprogrammed cells m ⁶ A modifier.

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