KR101304992B1 - Artificial microrna for increasing expression of cell adhesion molecule by knock-down of 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 micro-RNA that increases the expression of cell adhesion molecules by inhibiting the expression of JMJD3 gene {ARTIFICIAL MICRORNA FOR INCREASING EXPRESSION OF CELL ADHESION MOLECULE BY KNOCK-DOWN OF JMJD3 GENE EXPRESSION}

The present invention relates to artificial microRNAs that increase the expression of cell adhesion molecules (CAM) by inhibiting the expression of UTX protein or JMJD3 protein. More specifically, the present invention inhibits the expression of UTX or JMJD3 by binding to mRNA transcribed from the gene encoding UTX or mRNA transcribed from the gene encoding JMJD3, while increasing the expression of cell adhesion molecules. It is about artificial micro RNAs that play a role in letting go.

Epigenetic changes do not change the gene sequence, but include DNA methylation, histone modification, non-histone modification, and short RNA generation. And by this change, the activity and expression of the gene is regulated.

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

Histone methylation refers to methylation at the lysine and arginine residues of histone proteins. Each lysine undergoes three different types of methylation, and arginine also undergoes three different types of methylation. In histone methylation, the related gene is activated or inactivated 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.

Inhibition of transcription by the H3K27-methyl mark regulates the expression of transcription of lineage-specific (LS) genes in lineage-committed (LC) 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 embryogenesis, 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). During the differentiation of ES cells into neural precursors, neuronal-specific genes are activated with at least a 100-fold decrease in H3K27 methylation.

H3K27 is methylated by Polycomb compelx (PRC2) proteins, and H3K27me inhibits gene expression in the developmental stage. This phenomenon is caused by three methylation of the 27th lysine of the histone protein (H3K27me3), chromatin is entangled and gene expression is prevented. H3K27me3 plays an important role in determining developmental fate and regulating cell growth (Sparmann and van Lohuizen, Nat Rev Cancer. 2006;6(11):846-56). Recently, UTX and JMJD3, JmjC-domain proteins, which are H3K27-specific demethylases that remove the methyl mark of H3K27me, have been reported (Agger et al., Nature. 2007;449(7163):731-4; De Santa et al. 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 located on the X chromosome and is composed of six tetratricopeptied repeat (TPR) domains at the N-terminus and JmjC domains at the C-terminus (Hong et al., Proc Natl Acad Sci). US A. 2007;104(47):18439-44.). The UTX gene escapes from X chromosome inactivation and is expressed in both mouse and human genes (Greenfield et al., Hum Mol Genet. 1998;7(4):737-42.). UTX belongs to the JmjC domain-containing protein subfamily along with UTY (ubiquitously transcribed tetratricopeptide repeat, Y chromosome) and JMJD3 (Klose et al., Nat Rev Genet. 2006;7(9):715-27.). UTY is the Y-link homolog of UTX and has 88% homology. 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 (dendrites or neurites) during neuronal differentiation are the mainstays of extracellular guiding cues and intracelluar signaling events. It occurs sequentially in a timely manner 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 the cell adhesion molecule (CAM) react to activate proteins in neurons. By activated proteins, things such as cell signaling, gene expression, and cytoskeleton regulation occur inside the cell, leading to neurite guidance and elongatin. The extracellular domain of the cell adhesion molecule is adsorbed by cross-reacting with other cells or with the matrix outside the cell. In general, a cell adhesion molecule is a transmemebrane protein, and is composed of an extracellular domain that plays an important role in adsorption and an intracellular domain that interacts with the cytoskeleton. Therefore, it acts as a direct link between the external growth and guiding cues of the cell and the scaffold inside the cell, affecting the cell morphology and growth.

The inventors of the present invention continued to study the effect on the expression of cell adhesion molecules during neuronal differentiation after reducing the expression of UTX and Jmjd3, which demethylated H3K27 methylation using RNA interference technology, that is, amiRNA technology. The present invention has been reached.

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

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

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 containing the gene, and a cell transformed with the expression vector.

The present invention provides an artificial miRNA (amiRNA) that inhibits the 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 inhibits the expression of UTX or JMJD3 by binding to mRNA transcribed from the gene encoding UTX or the mRNA transcribed from the gene encoding JMJD3, thereby increasing the expression of cell adhesion molecules. I can.

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" refers to the formation of UTX or JMJD3 protein through the process of transmitting the genetic information 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 when a DNA capable of expressing a desired "artificial miRNA" is recombined and injected into a cell, miRNA It is an RNA expressed by an expression/action mechanism of. Artificial miRNA plays a role in inhibiting the expression of the target gene.

In the present invention, "specific" refers to specifically selecting a target within a cell, or specifically binding or reacting with a target. The present invention provides a UTX or JMJD3 gene (eg, mRNA) specific amiRNA.

The UTX or JMJD3 gene sequence may be included as long as it is a known human and animal sequence. Specifically, GenBank accession numbers NM_021140.2 (UTX) and NM_001080424.1 (JMJD3) include, but are not limited to, known sequences.

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 may include any known sequence of humans and animals. Specifically, it includes known sequences such as GenBank access numbers in Table 1 below, but is not necessarily limited thereto.

[Table 1]

The amiRNA may be selected from the group consisting of SEQ ID NOs: 1 to 8. Specifically, SEQ ID NOs: 1 to 4 are amiRNAs that knock-down the expression of UTX, and SEQ ID NOs: 5 to 8 are amiRNAs that inhibit the expression of Jmjd3. Hereinafter, in the present invention, the amiRNA of SEQ ID NO: 1 is denoted as “amiRNA1” and the amiRNA of SEQ ID NO: 2 is denoted as “amiRNA2” in the same manner as each of the sequence numbers.

The amiRNA has a small hairpin structure similar to that of miRNA made in the body.

In addition, the present invention provides a gene encoding the amiRNA. The gene may be a DNA selected from the group consisting of SEQ ID NOs: 9 to 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 of preparing the amiRNA is not particularly limited, but examples thereof include a method of chemically synthesizing RNA oligonucleotides, a method of synthesizing small RNA using in vitro transcription, and the like.

In addition, the present invention provides a recombinant expression vector comprising a gene encoding the amiRNA.

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

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

The expression vector may be used by selecting from the group consisting of plasmids, lentiviral vectors, retroviral vectors, and adenovirus vectors known in the art, which are used for replication and expression of mammalian cells or other target cell types. Viral vectors are particularly preferred in the present invention. The reason for this is that viral vectors are highly efficient in delivering nucleic acids in vivo, so it is thought that their effect will be high when applied to RNAi.

It is most preferable to use a lentivirus in particular in the present invention. Lentivirus is a kind of retroviral vector that can be transduced even in 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 advantageous advantage in maintaining RNAi for a long time.

In addition, the present invention 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 may be introduced into a host cell by a method well known to those skilled in the art. Introduction methods include electroporation and lipofection, but are not limited thereto, 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, and insect cells. It is not limited and 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 better than that of directly injecting amiRNA into the cell. The method of intracellular delivery of siRNA or shRNA, which has been mainly used so far, is a method of injecting directly into the cell from the outside of the cell, and this method has the disadvantage that cells are easily transformed. The present invention used a viral vector system to overcome these shortcomings and secure stability in cell delivery of amiRNA.

In addition, 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 to 3 days. Therefore, there is a problem in that a large amount of synthetic siRNA (dsRNA, etc.) must be repeatedly injected in order for the synthetic siRNA or the like to exhibit a lasting effect in the cell.

In order to overcome this drawback, the present invention transforms or infects the amiRNA expression vector into cells so that the amiRNA of the present invention can be expressed temporarily or permanently in cells. The amiRNA transformed or infected into a cell in this way can exhibit a lasting effect in the cell.

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

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

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to the engineer, and it is natural that such modifications and modifications fall within 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 purchased from ATCC in the United States. Human embryonic carcinoma cells are cultured in RPMI 1640 medium in a growth medium containing 10% FBS (fetal bovine serum), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C, using an incubator supplying 5% CO2. I did.

In order to differentiate human embryonic carcinoma cells into neurons, the medium of human embryonic carcinoma cells was removed, and 0.25% trypsin-EDTA (InvitrogenTM, Carlsbad, CA) was treated and reacted at 37° C. for 5 minutes. After the reaction, the same amount of growth medium was added to inhibit trypsin activity. After centrifuging the cell solution at 1,000 rpm for 5 minutes, remove the supernatant to remove trypsin, collect only cells, add growth medium, and transfer 4 X 106 ~ 5 X 106 cells to a microbial culture dish at 37℃, 5% It was cultured in an incubator supplying CO2. After 24 hours of incubation, 10 μM of retinoic acid was added and incubated for a week. After incubation for a week, the cells were transferred to a T75 flask, and 10 μM of RA was added and cultured for a week. After incubation for a week, the cells were separated into single cells by treatment with trypsin-EDTA, and these cells were transferred to a new T75 plate and cultured for 2 days. After incubation, cells were collected by treatment with trypsin-EDTA, divided into 7.5 X 106 cells, and mitotic inhibitors (1 μM 1-6-D-arabinofuransylcytosine, 10 μM 2′-deoxy-5-fluorouridine, in 10 cm culture dishes). 10 μM 1-β-D-ribofuranosyluracil) was added and incubated for a week. Cells cultured for one week were treated with trypsin-EDTA, and 1-5 X 10 ⁴ cells were coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO) and covered slips (Nunc, Roskilde, Denmark) Was dispensed into a 24-well plate (Corning, Inc. New York, NY) into which the cells were placed and differentiated into neurons.

1-2: Immunocytochemistry

After differentiating human embryonic carcinoma cells into neurons, the cells were fixed by removing the medium, washing with PBS, and immersing 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). /PBS solution was soaked and left for 1 hour. Thereafter, the cells were reacted at room temperature in a 0.1% BSA/PBS solution containing an antibody for 1 hour. The antibody beta tubulin III (Tu-20, Abcam, Cambridge, UK) was diluted 1:250, and the antibody glial fibrillary acidic protein (GFAP, Dakocytomation, Glostrup, Denmark) was diluted 1:1000. And the antibody Tryptophan hydroxylase (TPH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted 1:250. The cells after the reaction were washed three times with a 0.1% BSA/PBS solution, and then reacted at room temperature for 1 hour in a 0.1% BSA/PBS solution containing a secondary antibody. After washing three times with 0.1% BSA/PBS solution again, a mounting solution (Vectashield, Vector Laboratories, Peterborough, UK) containing DAPI (4'-6-diamidino-2-phenylindole) that stains the nuclei on the coverslip was added. Drops were dropped and fixed on a slide glass, and observed using a phase contrast microscope (Olympus, Hertfordshire, UK).

1-3: amiRNA Department of Design Lentivirus synthesis

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. In order to clone amiRNA targeting Utx or Jmid3, Invitrogen's Gateway system was used. The designed amiRNA sequence was synthesized as a complementary two-stranded DNA, and hybridized to form a double-stranded DNA, and then inserted into the pLenti 6.2-GW/EmGFP-miR vector. The negative control vector was 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 subjected to a recombination reaction (BP reaction) with the pDON221 vector. After performing the BP reaction, the pLenti6.4 R4R2/V5-DEST vector and the pENTR 5'promoter clone vector were subjected to LR reaction, and the amiRNA sequence was inserted into the final lentiviral vector. The BP reaction and LR reaction are according to INVITROGEN's manual. 3 μg of the prepared lentiviral vector and 9 μg of ViraPower packaging mix were mixed and reacted at room temperature for 20 minutes to create a DNA-Lipofectamine 2000 complex, which was added to 293FT cells and incubated for 1 day in a _{CO 2 incubator.} . After incubation, lipofectamine was removed, and cultured for 48 to 72 hours in DMEM medium (10% FBS, 1X PS) containing sodium pyruvate. After 48 hours of culture, the medium containing the lentivirus was harvested and centrifuged to obtain a virus from the supernatant from which the cells were removed. The obtained virus was infected with NIH-3T3 cells, and the fluorescence of the virus-infected cells was observed using an antibiotic (blasticidin S 5 μg/ml) for 10 days, and stained with crystal violet to determine the titer of the lentivirus. .

1-4: In human embryonic cells Lentivirus infection

Human embryonic carcinoma cells were aliquoted into a 24-well plate with 1 ^{×10 5 cells and cultured for 1 day.} After incubation, the medium was removed and lentivirus having each amiRNA sequence was added, followed by infection for 12 hours. After infection, the lentivirus solution was removed, 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 the method provided using Trizol (InvitrogenTM, Carlsbad, CA). 50 μM T7-oligo primer 2 μl was added to 8 μg of total RNA, reacted at 70° C. for 10 minutes, and then left on ice for 2 minutes. A first-strand cDNA buffer, 10 mM DTT, and 500 μM dNTP mix were added to the reaction solution and reacted at 42° C. for 2 minutes, and then 200 unit/μl of Superscript II reverse transcriptase; InvitrogenTM, Carlsbad, CA) was added and reacted at 42° C. for 1 hour. After the reaction is over, let stand on ice for 2 minutes, add 2-strand buffer, 200 μM dNTP mix, 10 unit DNA ligase, 40 unit DNA polymerase I, and 2 units RNase H, at 16° C. for 2 hours Reacted. After the reaction was over, 10 unit T4 DNA polymerase was added and reacted at 16° C. for 5 minutes, and 10 μl of 0.5 M EDTA was added to stop the reaction. A reaction buffer, ribonucleotide, DTT, RNase inhibitor, and T7 RNA polymerase were added to the synthesized cDNA, and cRNA was synthesized by gently mixing every 30 minutes while reacting at 37° C. for 5 hours. 50 pM Oligo B2, hybridization control, 0.1 mg/ml Herring Sperm DNA, 0.5 mg/ml acetylated BSA and 2X hybridization buffer were added to the synthesized cRNA, followed by denaturation at 99° C. for 5 minutes. After injecting the denatured cRNA into a chip (Mouse Genome 430A 2.0 GeneChips), a hybridization reaction was performed at 45° C. for 16 hours. After 16 hours of the hybridization reaction, the reaction solution was removed and washed with a washing buffer. For staining, streptavidin-phycoerythrin (MES staining buffer, 2 mg/ml activated BSA, 1 mg/ml steptavidin-phycoerythrin) and antibodies (MES staining buffer, 2 mg/ml activated BSA, 10 mg) /ml of goat IgG, 3 μg/ml of biotin-labeled antibody) after staining with fluids station (Affymetrix, Inc.Santa Clara, Calif.) Scanning was performed using a laser confocal scanner (Affymetrix, Inc. Santa Clara, CA). The scanned images were analyzed using GeneChip® orewriting software (GCOS)/microarray suite version 5.0 (MAS 5.0, Affymetrix, Inc. Santa Clara, CA). Using the statistical algorithm of MAS 5.0, the signal intensity, probe set detection, probe set change [probe set (gene expression) change], and the log ratio of the signal were calculated.

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. Polymerase chain reaction solution using SYBR ^® green was performed in 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 to obtain a final concentration of 2 pmol and a final concentration of 0.01 μg of cDNA. DNA denaturation was repeated 40 times at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and synthesis at 72°C for 30 seconds. All amplification and detection were performed in 96 well-plates covered with optical caps (Applied Biosystems, Carlsbad, CA). The amount of increase in the product of each cycle was measured using the property of ^{SYBR ®} Green to bind to the double stranded DNA. After polymerase chain reaction, the melting curve was analyzed by denaturing at 95° C. for 1 minute, annealing at 60° C., increasing by 2° C. per minute from 60° C. to 95° C. (Lyondagger et al., 1998). All experiments were repeated three or more times, and standardized with glyceraldehydes 3-phosphate dehydrogenase (GAPDH). The results were analyzed using a program provided by Applied Biosystems (Carlsbad, CA). _{The ΔC T} value obtained from the experiment was a 2- ^{ΔΔ CT} method (Livak and Schmittgen, 2001). After obtaining CT values of each gene and GAPDH in NC amiRNA-infected cells (NC), UTX amiRNA-infected cells (UTX kd), and Jmjd3 amiRNA-infected cells (Jmjd3 kd), the CT values of GAPDH were calculated from the CT values of each gene. 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 resulting ΔΔC _T value was ^{calculated by putting it in 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 increases, and if it is lower than '1', the expression decreases. In addition, the expression patterns of each gene were analyzed in NC amiRNA-infected cells (NC), UTX amiRNA-infected cells (UTX kd), and Jmjd3 amiRNA-infected cells (Jmjd3 kd) after differentiation into neurons.

1-7: protein separation

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

1-8: protein quantification

Proteins obtained from human embryonic carcinoma cells were quantified. Protein analysis kit (Pierce) was prepared by diluting in distilled water at a ratio of 1:5 (working solution). BSA standards were prepared at 1, 0.5, 0.1, 0.05, 0.01, and 0 mg/ml. After 10 µl of the standard was added to a 96-well plate, 200 µl of a working solution was added thereto. The separated protein was diluted 10 times, added 10 µl each to a 96 well-plate, and 200 µl each of the 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, and lentiviruses were synthesized. The 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 lentivirus. 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 transcription polymerase chain reaction to analyze the expression patterns of UTX, Jmjd3, Ezh2, and Suz12 genes. The primers used in the real-time reverse transcription polymerase chain reaction are shown in Table 2 below. Ezh2 and Suz12 are proteins contained in the histone methyltransferase complex, and are not changed by the decrease of UTX or Jmjd3 gene expression. Real-time PCR was performed together with Ezh2 and Suz12 to confirm that UTX or Jmjd3 decreased specifically.

[Table 2]

The results are shown in FIG. 4. 4, UTX amiRNA-infected cells had a 40% decrease in UTX gene expression compared to NC amiRNA-infected cells, but the expression of Jmjd3 and Ezh2 was almost unchanged, and the expression of Suz12 was reduced by a small amount. In Jmjd3 amiRNA-infected cells, the expression of Jmjd3 gene was decreased 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 Expression of Cell Adhesion Molecule (CAM) in amiRNA- infected cells

Total RNA was extracted from UTX amiRNA-infected cells, Jmjd3 amiRNA-infected cells, NC amiRNA-infected cells, and two-week differentiated cells, respectively, using Trizol, and then hybridized to NimbleGen Human whole genome 12-plex arrays by labeling Cy3 using an aRNA labeling kit. The reaction was performed to analyze the gene expression pattern. As a result of simple functional analysis, UTX amiRNA-infected cells increased/decreased the expression of a total of 918 genes more than twice that of NC amiRNA-infected cells. Among these altered genes, 397 genes including genes related to protein biosynthesis (GO:6412, p-value:0.0126) such as RPS19, AGT7, RPS10, MRPS12, ABO, RPLP2, FAU, UPF1, and TLR2 genes are 2 Over-fold expression was increased. In addition, 521 genes, including genes related to the establishment of protein localization (GO:45184, p-value:0.014), such as STEAP3, SRP54, BET1, YWHAZ, SSR1, and ARF4 genes, are more than double expressed. Decreased.

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

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

Two weeks after differentiation, Jmjd3 amiRNA-infected cells had a two-fold increase/decrease in expression of a total of 677 genes compared to NC amiRNA cells two weeks after differentiation. Among the changed genes, 396 genes, including genes related to cell development (GO:48468, p-value:0.0136), such as NRP1, MYH11, CEP290, NME5, DAZL, and GNAQ genes, were more than doubled in expression. 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, decreased more than double their expression.

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

[Table 3]

Analysis of the expression patterns of NRP1 and NCAM genes, which are related to the growth, induction, and stabilization of axons and dendrites during neuronal differentiation among these cell adhesion molecules, using real-time RT PCR. I did. The primers used at this time are shown in Table 4 below. Other primers are the same as in Table 2 above.

[Table 4]

As shown in Figure 5, the expression of the NRP1 gene was increased by 1.6-fold in Jmjd3 amiRNA-infected cells in non-differentiated cells (NCCIT in FIG. 5). In UTX amiRNA-infected cells, expression increased even after 2 weeks of differentiation, indicating that neuronal differentiation and adhesion were higher than those of NC amiRNA-infected cells. In the case of the NCAM gene, the expression of UTX amiRNA-infected cells was increased even in non-differentiated cells, and it can be seen that even after 2 weeks of differentiation, the amount of UTX amiRNA-infected cells increased more than that of the NC amiRNA-infected cells (FIG. 6).

Attach electronic file of sequence list

Claims (7)

An artificial miRNA (amiRNA) selected from the group consisting of SEQ ID NOs: 5 to 8 which bind to mRNA transcribed from a gene encoding JMJD3 and inhibit the expression of JMJD3 to increase the 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: 13-16.
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