KR101310063B1 - Artificial microrna for increasing expression of cell adhesion molecule by knock-down of utx or jmjd3 gene expression - Google Patents

Abstract

The present invention provides an artificial miRNA (amiRNA) that inhibits expression of UTX or JMJD3 by binding to mRNA transcribed from a gene encoding UTX or from a gene encoding JMJD3 using RNA interference technology. .

Description

ARTIFICIAL MICRORNA FOR INCREASING EXPRESSION OF CELL ADHESION MOLECULE BY KNOCK-DOWN OF UTX OR JMJD3 GENE EXPRESSION}

The present invention relates to artificial microRNAs that inhibit expression of UTX protein or JMJD3 protein and increase expression of cell adhesion molecule (CAM). More specifically, the present invention increases the expression of cell adhesion molecules while binding to mRNA transcribed from a gene encoding UTX or mRNA transcribed from a gene encoding JMJD3 and inhibiting expression of UTX or JMJD3. It is about an artificial micro RNA to play a role.

Epigenetic changes do not change the gene sequence, but include methylation of DNA, histone modifications, nonhistone modifications, and short RNA production. These changes regulate gene activity and expression.

The N-terminal region of the histones undergoes various modifications depending on the environment, and as a result, the structure of chromatin is changed, and as a result, the expression of a specific gene is affected. The best known histone modification is acetylation, which is dynamically regulated by histone acetylase and deacetylase. If histone acetylation activates gene expression, histone methylation affects both the transcriptional activity and inactivity of the gene.

Histone methylation refers to the methylation of lysine and arginine residues in histone proteins, with each lysine undergoing three different forms of methylation, and arginine also undergoes three different forms of methylation. Histone methylation involves the activation or inactivation of the relevant gene, depending on which residue of the histone protein is methylated. In general, methylation of lysine present in H3K9, H3K27 and H4K20 inactivates genes, and methylation of lysine present in H3K4, H3K36 and H3K79 promotes gene activity.

Transcription inhibition by H3K27-methyl mark regulates the expression of transcription of line-specific genes in lineage-committed progenitor cells (Mikkelsen et al., Nature. 2007; 448 (7153): 553-60). H3K27me3 plays an important role in the pluripotentcy of ES (embryonic stem) cells, plasticity during embryonic development, polycomb-mediated gene silencing and X chromosome inactivation (Schuettengruber et al. ., Cell. 2007; 128 (4): 735-45; Boyer et al., Nature. 2006; 441 (7091): 349-53; Sparmann and van Lohuizen, Nat Rev Cancer. 2006; 6 (11): 846 -56; Lee et al., Cell. 2006; 125 (2): 301-13; Trojer et al., Cell. 2006; 125 (2): 213-7). Neuronal specific genes are activated with at least a 100-fold reduction in H3K27 methylation while ES cells differentiate into neural precursors.

H3K27 is methylated by Polycomb compelx (PRC2) proteins and H3K27me inhibits gene expression at developmental stages. This phenomenon occurs when three methylation of the 27th lysine of the histone protein (H3K27me3), the chromatin is tangled, preventing the gene expression. H3K27me3 plays an important role in developmental fate determination and cell growth regulation (Sparmann and van Lohuizen, Nat Rev Cancer. 2006; 6 (11): 846-56). Recently, JmjC-domain proteins UTX and JMJD3, which remove the methyl mark of H3K27me, have been reported (Agger et al., Nature. 2007; 449 (7163): 731-4; De Santa et. al., Cell. 2007; 130 (6): 1083-94.Epub 2007 Sep 6; Lan et al., Nature. 2007; 449 (7163): 689-94 and Lee et al., Science.2007; 318 ( 5849): 447-50.).

UTX (ubiquitously transcribed tetratricopeptide repeat (X chromosome) protein is on the X chromosome and consists of six N-terminal tetratricopeptied repeat (TPR) domains and a C-terminal JmjC domain (Hong et al., Proc Natl Acad Sci). US A. 2007; 104 (47): 18439-44.). UTX genes are expressed throughout mouse and human genes beyond X chromosome inactivation (Greenfield et al., Hum Mol Genet. 1998; 7 (4): 737-42.). UTX, along with UTY (ubiquitously transcribed tetratricopeptide repeat, Y chromosome) and JMJD3, belongs to the JmjC domain-containing protein subfamily (Klose et al., Nat Rev Genet. 2006; 7 (9): 715-27.). UTY is 88% homologous to the Y-link homolog of UTX. JMJD does not have a TPR domain but has a JmjC domain that is homologous to UTX and UTY.

Growth, guidance, and stabilization of axons and dendrites or neurites during neuronal differentiation may be attributed to extracellular guiding cues and intratracelluar signaling events. It occurs sequentially in time by complex interactions (Hansen et al., Signaling mechanisms of neurite outgrowth induced by the cell adhesion molecules NCAM and N-Cadherin 2008 Cell. Mol. Life. Sci. 65 3809-3821). Cell surface molecules such as Cadherins, integrin, selectins, and Ig (immunoglobulin) of cell adhesion molecules (CAMs) react to activate proteins in neurons. Activated proteins, such as cell signaling, gene expression and the regulation of the cytoskeleton, occur inside the cell, leading to neurite guidance and elongatin. The extracellular domain of the cell adhesion molecule is adhisin by trans interacting with other cells or the matrix outside the cell. Generally, cell adhesion molecules are transmemebrane proteins and are composed of extracellular domains that play an important role in adsorption and intracellular domains that interact with the cytoskeleton. Thus, it acts as a direct link between the external growth and guidance cues of the cell and the scaffold inside the cell, thereby affecting cell morphology and growth.

The present inventors continue to study the effects of the expression of UTX and Jmjd3, which demethylates H3K27 methylation using RNA interference technology, amiRNA technology, and then on the expression of cell adhesion molecules during neuronal differentiation. The present invention has been reached.

 It is an object of the present invention to provide an artificial miRNA (amiRNA) that inhibits expression of UTX or JMJD3 by binding to mRNA transcribed from a gene encoding UTX or from a gene encoding JMJD3 using RNA interference technology. It is for.

Another object of the present invention is to provide an artificial miRNA (amiRNA) that increases the expression of cell adhesion molecules.

Still another object of the present invention is to provide a gene encoding an amiRNA that inhibits the expression of UTX or JMJD3, an expression vector comprising the gene, and a cell transformed with the expression vector.

The present invention provides artificial miRNA (amiRNA) that inhibits expression of UTX or JMJD3. The amiRNA may be selected from the group consisting of SEQ ID NOs: 1 to 8.

In the present invention, the amiRNA binds to mRNA transcribed from a gene encoding UTX or mRNA transcribed from a gene encoding JMJD3 to inhibit expression of UTX or JMJD3 to increase expression of cell adhesion molecules. Can be.

The cell adhesion molecule may be selected from the group consisting of NRP1, ATP2C1, SIRPA, FREM3, FPRL1, SPON1, PCDHB10, PXN, CLEC4M, PCDHA5, NCAM2, ADAM12, PCDH12, CDK5R1, HES5, MYF5, POSTN, ACTN1 and CD93. .

The expression of "UTX or JMJD3" means that the UTX or JMJD3 protein is formed through the genetic information transfer process of the UTX or JMJD3 gene.

In the present invention, "artificial miRNA (amiRNA)" is a micro RNA (miRNA) designed to specifically act on a target gene, and miRNA when recombinant DNA is injected into a cell that can express a desired "artificial miRNA". RNA is expressed by the expression / action mechanism of. Artificial miRNAs serve to inhibit the expression of target genes.

In the present invention, "specific" means to specifically select a target in a cell, or to specifically bind or react with the target. The present invention provides UTX or JMJD3 gene (eg mRNA) specific amiRNAs.

The UTX or JMJD3 gene sequence can be included as long as it is a known sequence of human and animal. Specifically, it includes, but is not limited to, the known sequences of GenBank access numbers NM_021140.2 (UTX) and NM_001080424.1 (JMJD3).

In addition, cell adhesion molecules selected from the group consisting of NRP1, ATP2C1, SIRPA, FREM3, FPRL1, SPON1, PCDHB10, PXN, CLEC4M, PCDHA5, NCAM2, ADAM12, PCDH12, CDK5R1, HES5, MYF5, POSTN, ACTN1 and CD93 The sequence can include any known sequence of humans and animals. Specifically, but not limited to known sequences, such as GenBank (GenBank) Access Number in Table 1 below.

[Table 1]

The amiRNA may be selected from the group consisting of SEQ ID NOs: 1-8. Specifically, SEQ ID NOs: 1 to 4 are amiRNAs that knock down expression of UTX, and SEQ ID NOs: 5 to 8 are amiRNAs that inhibit expression of Jmjd3. In the present invention, the same as each sequence number, amiRNA of SEQ ID NO: 1 "amiRNA1", amiRNA of SEQ ID NO: 2 will be expressed as "amiRNA2".

 The amiRNA has a small hairpin structure similar to the form of miRNA produced in the body.

The present invention also provides a gene encoding the amiRNA. The gene may be DNA selected from the group consisting of SEQ ID NOs: 9-16. Specifically, SEQ ID NOs: 9 to 12 are genes encoding amiRNA for UTX, and SEQ ID NOs: 13 to 16 are genes encoding amiRNA for Jmjd3.

 The method for preparing the amiRNA is not particularly limited, and examples thereof include a method of chemically synthesizing an RNA oligonucleotide, a method of synthesizing a small RNA using in vitro transcription, and the like.

The present invention also provides a recombinant expression vector comprising the gene encoding the amiRNA.

A gene encoding (encoding) the nucleotide sequence of the recombinant amiRNA may be synthesized and inserted into an expression vector.

The amiRNA expression vector can be transformed or infected into cells so that amiRNAs of the present invention can be expressed temporarily or permanently in the cells. The recombinant expression vector of the present invention may be constructed by recombinant DNA methods known in the art.

The expression vector may be selected from the group consisting of plasmids, lentiviral vectors, retroviral vectors, adenovirus vectors known in the art, used for replication and expression of mammalian cells or other target cell types. Especially preferred are viral vectors in the present invention. The reason for this is that viral vectors have high efficiency of nucleic acid delivery in vivo, and therefore, the effect is expected to be high when applied to RNAi.

In the present invention, it is particularly preferable to use a lentivirus. Lentiviruses are a retroviral vector that can transduce even non-dividing cells, and the injected gene can be expressed for several months or longer (Kafri T, et al., Nat Genet 1997; 17: 314-317. ), Has an advantage in maintaining RNAi for a long time.

The present invention also provides a transformed cell prepared by introducing the expression vector into a host cell.

The expression vector into which the gene encoding the amiRNA sequence is inserted can be introduced into a host cell by methods well known to those skilled in the art. Introduction methods include, but are not limited to, electroporation and lipofection, and methods known in the art may be selected.

Cells that can be used in the present invention may be selected from the group consisting of E. coli, yeast, heart cells, lymphocytes, liver cells, vascular endothelial cells, spleen cells, cancer cells, animal cell lines such as CHO, Cos7, NIH3T3, insect cells, Although not limited, all possible cells can be used as host cells.

As in the present invention, when amiRNA is delivered into a cell using a viral vector, the stability is much higher than that of amiRNA directly injected into the cell. The intracellular delivery method of the siRNA or shRNA mainly used until now is a method of directly injecting into the cell from outside the cell, there is a disadvantage that the cell is easily modified. The present invention used a viral vector system to overcome these shortcomings and ensure stability in cell delivery of amiRNA.

In addition, the conventional synthetic siRNA, synthetic shRNA, and antisense oligonucleotides have a short half-life, so that the effect of inhibiting gene expression is maintained for only 2-3 days. Therefore, there is a problem that a large amount of synthetic siRNA (dsRNA, etc.) must be repeatedly injected in order for the synthetic siRNA to have a continuous effect in the cell.

In order to overcome this drawback, the present invention transformed or infected the amiRNA expression vector into the cell so that the amiRNA of the present invention could be expressed temporarily or permanently in the cell. Such amiRNAs transformed or infected into cells may have a lasting effect in the cells.

The amiRNA of the present invention has an effect of increasing the expression of cell adhesion molecules by inhibiting the expression of UTX or JMJD3. Through the amiRNA of the present invention will be able to specifically understand the gene regulation mechanism of cell adhesion molecules.

1 and 2 show the design of amiRNAs according to the invention.
Figure 3 shows a vector map capable of expressing amiRNA according to an embodiment of the present invention.
Figure 4 shows the gene expression patterns in UTX amiRNA infected cells and Jmdj3 amiRNA infected cells.
5 shows the expression pattern of NRP1 gene during neuronal differentiation of UTX amiRNA infected cells and Jmjd3 amiRNA infected cells.
Figure 6 shows the expression pattern of NCAM2 gene during neuronal differentiation of UTX amiRNA infected cells and Jmjd3 amiRNA infected cells.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications can be made within the scope and spirit of the present invention. It is obvious to the skilled person, and it is natural that such variations and modifications fall within the scope of the appended claims.

Example  1: materials and methods

1-1: Human Embryonic Carcinoma Cells NCCIT  Culture and Differentiation into Neurons

Human embryonic carcinoma cells were distributed by the US ATCC. Human embryonic carcinoma cells were cultured using an incubator with 37 ° C. and 5% CO 2 in growth medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin and 100 μg / ml streptomycin in RPMI 1640 medium. It was.

To differentiate human embryonic carcinoma cells into neurons, the medium of human embryonic carcinoma cells was removed and treated with 0.25% trypsin-EDTA (Invitrogen ™, Carlsbad, Calif.) For 5 minutes at 37 ° C. After the reaction, the same amount of growth medium was added to inhibit the trypsin activity. After centrifuging the cell solution for 5 minutes at 1,000 rpm, remove the supernatant to remove the supernatant, collect only the cells, add the growth medium, transfer 4 X 106 ~ 5 X 106 cells to the microbial culture dish 37 ℃, 5% The cells were cultured in an incubator supplying CO 2. After 24 hours of incubation, 10 μM of retinoic acid was added and incubated for one week. After one week of incubation, the cells were transferred to a T75 flask and incubated for one week with the addition of 10 μM of RA. After one week of incubation, the cells were separated into single cells by treatment with trypsin-EDTA, and these cells were transferred to new T75 plates and incubated for two days. After incubation, trypsin-EDTA was used to collect the cells and divide 7.5 × 10 6 cells into mitotic inhibitors (1 μM 1-6-D-arabinofuransylcytosine, 10 μM 2′-deoxy-5-fluorouridine, 10 μM 1-β-D-ribofuranosyluracil) was added and incubated for one week. Cells cultured for one week were treated with trypsin-EDTA to cover 1 to 5 X 10 ⁴ cells with polys-L-lysine (Sigma-Aldrich, St. Louis, MO) coated slips (Nunc, Roskilde, Denmark). The cells were aliquoted into 24 well plates (Corning, Inc. New York, NY) and differentiated into neurons.

1-2: Immunocytochemistry

After differentiating human embryonic carcinoma cells into neurons, the cells were fixed by removing medium, washing with PBS, and soaking in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 1 hour at room temperature. Fixed cells were 0.1% BSA (bovine serum albumin, InvitrogenTM, Carlsbad, CA) containing 10% horse serum (InvitrogenTM, Carlsbad, CA) and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) Soak in / PBS solution and left for 1 hour. The cells were then reacted at room temperature for 1 hour in 0.1% BSA / PBS solution containing the antibody. Antibody beta tubulin III (Tu-20, Abcam, Cambridge, UK) was diluted 1: 250, and antibody glial fibrillary acidic protein (GFAP, Dakocytomation, Glostrup, Denmark) was diluted 1: 1000. The antibody Tryptophan hydroxylase (TPH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted 1: 250. After the reaction, the cells were washed three times with 0.1% BSA / PBS solution, and then reacted at room temperature for 1 hour in 0.1% BSA / PBS solution containing the secondary antibody. After washing three times with 0.1% BSA / PBS solution, a mounting solution (Vectashield, Vector Laboratories, Peterborough, UK) containing DAPI (4'-6-diamidino-2-phenylindole), which stains the nuclei on the coverslip, was Drops were dropped and fixed on the slide glass, and observed using a phase contrast microscope (Olympus, Hertfordshire, UK).

1-3: amiRNA  Design Lentivirus  synthesis

As shown in Figures 1 to 3, sequences targeting Utx (NM_021140.2) and Jmjd3 (NM_001080424.1) were designed to have a stem-loop structure using the BLOCK-iT RNAi designer. Invitrogen's Gateway system was used to clone amiRNAs targeting Utx or Jmid3. The designed amiRNA sequences were synthesized into complementary two-stranded DNA, hybridized to double-stranded DNA, and inserted into pLenti 6.2-GW / EmGFP-miR vectors. Negative control vectors were cloned using the pcDNA6.2-GW / EmGFP-miR-neg control plasmid (Invitrogen). The amiRNA sequence inserted into the pLenti 6.2-GW / EmGFP-miR vector was recombined with the pDON221 vector (BP reaction). After performing the BP reaction, the pLenti6.4 R4R2 / V5-DEST vector and the pENTR 5 ′ promoter clone vector were subjected to LR reaction to insert an amiRNA sequence into the final lentiviral vector. The BP reaction and the LR reaction are based on INVITROGEN's manual. 3 μg of the lentiviral vector prepared and 9 μg of ViraPower packaging mix were mixed and reacted at room temperature for 20 minutes to make a DNA-lipopeptamine 2000 complex, which was added to 293FT cells and incubated in a CO ₂ incubator for 1 day. . After incubation, lipofectamine was removed and incubated for 48-72 hours in DMEM medium (10% FBS, 1X PS) containing sodium pyruvate. After 48 hours of culture, the medium containing the lentiviral was harvested and centrifuged to obtain the virus from the supernatant from which the cells were removed. The resulting virus was infected with NIH-3T3 cells and observed for 10 days using an antibiotic (blasticidin S 5 μg / ml) to observe the fluorescence of the virus-infected cells, stained with crystal violet to determine the titer of the lentiviral. .

1-4: On human embryonic cells Lentivirus  infection

Human embryonic carcinoma cells were dispensed into 24 well plates with 1 × 10 ⁵ cells and cultured for 1 day. After incubation, the medium was removed and the lentiviral with each amiRNA sequence was added and then infected for 12 hours. After infection, the lentiviral solution was removed and RPMI-1640 (10% FBS, 1X PS) was added and incubated for 48 hours. After incubation, blasticidine S (5 ug / ml) was added to select only cells resistant to antibiotics.

1-5: Gene chip  analysis

Total RNA extraction from human embryonic carcinoma cells was isolated by a method provided using Trizol (Invitrogen ™, Carlsbad, CA). 8 μg of total RNA was added to 50 μM T7-oligo primer and 2 μl of the primer, and reacted at 70 ° C. for 10 minutes, and then left on ice for 2 minutes. After reacting the reaction solution with 1-strand cDNA buffer, 10 mM DTT, 500 μM dNTP mix for 2 minutes at 42 ° C., 200 units / μl of Superscript II Reverse transcriptase; 1 μl of Invitrogen ™, Carlsbad, CA) was added and reacted at 42 ° C. for 1 hour. After the reaction was completed, it was left on ice for 2 minutes, and a second-strand buffer, 200 μM dNTP mix, 10 unit DNA ligase, 40 unit DNA polymerase I, and 2 unit RNase H were added, followed by 2 hours at 16 ° C. Reacted. After the reaction, 10 unit T4 DNA polymerase was added and reacted at 16 ° C. for 5 minutes to stop the reaction by adding 10 μl of 0.5 M EDTA. A cRNA was synthesized by adding a reaction buffer, ribonucleotide, DTT, RNase inhibitor, and T7 RNA polymerase to the synthesized cDNA, and mixing lightly every 30 minutes for 5 hours at 37 ° C. To the synthesized cRNA was added 50 pM Oligo B2, hybridization control, 0.1 mg / ml Heringsump DNA, 0.5 mg / ml acetylated BSA and 2X hybridization buffer and denatured at 99 ° C. for 5 minutes. Denatured cRNA was injected into the chip (Mouse Genome 430A 2.0 GeneChips), and then hybridization reaction was performed at 45 ° C. for 16 hours. After 16 h of the hybridization reaction, the reaction solution was removed and washed with a wash buffer. Streptavidin-Phycoerythrin (MES staining buffer, 2 mg / ml activated BSA, 1 mg / ml steptavidin-phycoerythrin) and antibody (MES staining buffer, 2 mg / ml activated BSA, 10 mg) for staining After staining with / ml goth IgG, 3 μg / ml biotin-labeled solution, the washing process was automatically performed using a Fluidics station (Affymetrix, Inc. Santa Clara, Calif.) Scanning was performed using a laser confocal scanner (Affymetrix, Inc. Santa Clara, Calif.). Scanned images were analyzed using GeneChip® Operating Software (GCOS) / microarray suite version 5.0 (MAS 5.0, Affymetrix, Inc. Santa Clara, Calif.). Statistical algorithms of MAS 5.0 were used to calculate signal intensity, probe set detection, probe set (gene expression) change, and log ratio of the signal.

1-6: Real time Reverse transcription Polymerase chain reaction  ( real - time RT - PCR )

Real-time reverse transcription polymerase chain reaction was performed with total RNA extracted from human embryonic carcinoma cells. The polymerase chain reaction solution using SYBR ^® green was subjected to real-time polymerase chain reaction with ABI 7500 (Applied Biosystems, Carlsbad, CA) using SYBR ^® Premix Ex Taq ™ II (Takara BIO, Shiga, Japan). Each primer was added to the reaction solution so as to have a final concentration of 2 pmol and a final concentration of 0.01 μg cDNA. DNA denaturation was repeated 40 times for 30 seconds at 95 ° C, annealing for 30 seconds at 60 ° C, and synthesis for 30 seconds at 72 ° C. All amplification and detection was performed in 96 well-plates covered with optical caps (Applied Biosystems, Carlsbad, Calif.). The product of each cycle was measured for increased amounts using the properties of SYBR ^® green binding to two strands of DNA. The polymerase chain reaction was denatured at 95 ° C. for 1 minute, annealed at 60 ° C., and then increased at 2 ° C. per minute from 60 ° C. to 95 ° C. for 1 minute (Lyondagger et al., 1998). All experiments were repeated three more times and normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH). The results were analyzed using a program provided by Applied Biosystems (Carlsbad, CA). The ΔC _T values obtained from the experiments were determined using a 2- ^{ΔΔ CT} method (Livak and Schmittgen, 2001). CT values of the genes and GAPDH were obtained from NC amiRNA-infected cells (NC), UTX amiRNA-infected cells (UTX kd), and Jmjd3 amiRNA-infected cells (Jmjd3 kd). Subtracted to obtain the ΔC _T value. To obtain ΔΔC _T value resulting from the ΔC _T value was out of the amiRNA UTX-infected cells and infected cells Jmjd3 amiRNA ΔC _T value for the gene obtained ΔC _T value for each gene in the human embryonic carcinoma cells obtained from. The ΔΔC _T value obtained was calculated by ^subtracting 2 ^{−ΔΔ CT} to determine the increase or decrease of gene expression in UTX amiRNA infected cells and Jmjd3 amiRNA infected cells. If the final calculated value is higher than '1', the expression is increased and if it is lower than '1', the expression is decreased. After the differentiation into neurons, the expression patterns of the genes were analyzed in NC amiRNA infected cells (NC), UTX amiRNA infected cells (UTX kd) and Jmjd3 amiRNA infected cells (Jmjd3 kd).

1-7: Protein Isolation

Human embryonic carcinoma cells were removed from the medium and washed twice with PBS. The supernatant obtained by adding 500 μl of RIPA buffer to the washed cells was spun for 15 minutes on an orbital shaker and centrifuged at 4 ° C., 13,000 rpm for 15 minutes, and transferred to a new 1.5 ml tube. The protein obtained was stored at −70 ° C. until protein quantification and Western blot analysis.

1-8: Protein Quantitation

Proteins obtained from human embryonic carcinoma cells were quantified. Protein analysis kit (Pierce) was prepared by diluting in distilled water in a ratio of 1: 5 (working solution). BSA standards were prepared at 1, 0.5, 0.1, 0.05, 0.01, 0 mg / ml. 10 μl of standard was added to a 96 well plate and 200 μl of working solution was added. The isolated protein was diluted 10-fold and put into 10 wells of 96 well-plates, and 200 μl of working solution was added. The prepared standard and the protein to be quantified were left at room temperature for 5 minutes. Absorbance was measured at a wavelength of 595 nm using an ELISA reader and protein concentration was measured using a standard curve.

Example  2: Human Embryonic Carcinoma  To the cell amiRNA Gene knock-down using

Sequences targeting Utx or Jmjd3 were designed to have a stem-loop structure using the BLOCK-iT RNAi designer. UTX amiRNA, Jmjd3, and amiRN cloning were performed using Invitrogen's BLOCK-iT ™ HiPerform ™ expression system to synthesize lentiviral. Negative control (NC) amiRNA was cloned into the BLOCK-iT ™ HiPerform ™ expression system using the pcDNA6.2-GW / EmGFP-miR-neg control plasmid (Invitrogen) to synthesize a lentiviral. The obtained lentivirus was infected with human embryonic carcinoma cells to obtain stable UTX amiRNA infected cells, Jmjd3 amiRNA infected cells and NC amiRNA infected cells. Total RNA of the obtained cells was extracted and subjected to real-time reverse transcriptase chain reaction to analyze the expression patterns of UTX, Jmjd3, Ezh2, and Suz12 genes. Primers used in real-time reverse transcription polymerase chain reaction are shown in Table 2 below. Ezh2 and Suz12 are proteins included in the histone methyltransferase complex and are not altered by decreased UTX or Jmjd3 gene expression. Ezh2 and Suz12 performed real-time PCR together to confirm that UTX or Jmjd3 was specifically reduced.

[Table 2]

The results are shown in Fig. 4, UTX amiRNA-infected cells showed 40% decrease in UTX gene expression compared to NC amiRNA-infected cells, but the expression of Jmjd3 and Ezh2 were almost unchanged, and the expression of Suz12 was slightly decreased. In Jmjd3 amiRNA-infected cells, the expression of Jmjd3 gene was reduced by 50% compared to NC amiRNA-infected cells, but the expression of UTX, Ezh2, and Suz12 was almost unchanged.

Example  3: UTX amiRNA  Infected cells and Jmjd3 amiRNA  Cell adhesion molecule of infected cell ( Cell  Adhesion Molecule : CAM ) Expression

TriRNA was extracted from UTX amiRNA infected cells, Jmjd3 amiRNA infected cells, and NC amiRNA infected cells, and two-week-differentiated cells, respectively. The reaction was performed to analyze the gene expression patterns. As a result of a simple functional analysis, UTX amiRNA infected cells increased / decreased expression of 918 genes more than two times compared to NC amiRNA infected cells. Among these altered genes, 397 genes, including genes involved in protein biosynthesis (GO: 6412, p-value: 0.0126), such as RPS19, AGT7, RPS10, MRPS12, ABO, RPLP2, FAU, UPF1, TLR2, etc. More than twice the expression was increased. In addition, 521 genes more than doubled, including genes involved in the establishment of protein localization (GO: 45184, p-value: 0.014) such as STEAP3, SRP54, BET1, YWHAZ, SSR1, and ARF4 genes. Decreased.

Jmjd3 amiRNA infected cells increased / decreased expression more than 2 times in total of 1308 genes compared to NC amiRNA infected cells. Among these altered genes, 694 genes more than doubled in expression, including genes involved in cell physiological processes (GO: 50875, p-value: 0.00243) such as SCIN, NFYC, KIF5A, GMDS, MRP21 and RINB genes. Increased. In addition, 614 genes, including genes involved in ion transport (GO: 6811, p-value: 0.0281), such as ATP13A2, NALCN, KCTD9, COL3A1, MRS2L, and GABRG2 genes, reduced expression by more than two-fold.

Two weeks after differentiation, UTX amiRNA infected cells increased / decreased expression more than two-fold by a total of 964 genes compared to NC amiRNA cells two weeks after differentiation. Among the changed genes, 568 genes more than doubled in expression, including genes involved in biological process regulation (GO: 50789, p-value: 0.00552) such as PQBP1, BRWD1, SLC12A4, NRP1, LGALS12, and DMRTA2 genes. . In addition, 396 genes reduced expression more than two-fold, including genes involved in fat biosynthesis (GO: 8610, p-value: 0.0324) such as PIK4CB, HSD3B7, IDI1, PTGES3, PLCE1, and PECR genes.

Two weeks after the differentiation, the Jmjd3 amiRNA-infected cells increased / decreased expression more than two-fold in total of 677 genes compared to NC amiRNA cells after two weeks of differentiation. Among the changed genes, 396 genes more than doubled in expression, including genes related to cell development (GO: 48468, p-value: 0.0136) such as NRP1, MYH11, CEP290, NME5, DAZL, and GNAQ genes. In addition, 280 genes, including genes related to cell biosynthesis (GO: 44249, p-value: 0.00523), such as TRASL2, HSD3B7, SYTL1, RPS9, EIF2S2, and RPL39 genes, reduced expression by more than two-fold.

As a result of analyzing the obtained microarray data by function, genes such as NRP1, ATP2C1, and SIRPA, cell adhesion molecules, increased significantly after 2 weeks of differentiation. The results are shown in Table 3 below.

[Table 3]

Expression of NRP1 and NCAM genes involved in the growth, induction and stabilization of axons and dendrites during neuronal differentiation of these cell adhesion molecules was analyzed using real-time RT PCR. It was. The primer used at this time is as Table 4 below. Other primers are the same as in Table 2 above.

[Table 4]

As shown in FIG. 5, NRP1 gene increased 1.6-fold in Jmjd3 amiRNA-infected cells (NCCIT of FIG. 5), and increased in both UTX amiRNA-infected cells and Jmjd3 amiRNA-infected cells. In UTX amiRNA infected cells, expression was increased even after 2 weeks of differentiation, indicating that neuronal differentiation and adhesion increased more than NC amiRNA infected cells. In the case of the NCAM gene, the expression of UTX amiRNA infected cells also increased in undifferentiated cells, and even after two weeks of differentiation, the amount of UTX amiRNA infected cells increased more than the NC amiRNA infected cells (FIG. 6).

Attach an electronic file to a sequence list

Claims (7)

An artificial miRNA (amiRNA) selected from the group consisting of SEQ ID NOs: 1 to 4, which binds to mRNA transcribed from a gene encoding UTX and inhibits expression of UTX to increase expression of cell adhesion molecules.
The method of claim 1, wherein the cell adhesion molecule is NRP1, ATP2C1, SIRPA, FREM3, FPRL1, SPON1, PCDHB10, PXN, CLEC4M, PCDHA5, NCAM2, ADAM12, PCDH12, CDK5R1, HES5, MYF5, POSTN, ACTN1 and CD93 Artificial miRNA, which is selected from the group.
The gene encoding the amiRNA of claim 1.
The gene of claim 3, wherein the gene is DNA selected from the group consisting of SEQ ID NOs: 9-12.
Recombinant expression vector comprising a gene encoding the amiRNA of claim 1.
The recombinant expression vector of claim 5, wherein the expression vector is selected from the group consisting of lentiviral vectors, retroviral vectors, and adenovirus vectors.
A cell transformed with the recombinant expression vector of claim 5.

Images