KR102095298B1 - Reagent composition for inhibiting Ninj1 comprising microRNA - Google Patents

Abstract

The present invention relates to a reagent composition for inhibiting Ninj1 and a method for inhibiting Ninj1 containing micro RNA showing an inhibitory effect of Ninj1 (Ninjurin-1) as an active ingredient, more specifically miR125a, miR145, miR184, miR206 and miR761 micro RNA As it is confirmed that the expression of cytokines is inhibited by inhibiting Ninj1 expression in phagocytic and inflammatory animal models, a composition containing the micro RNA as an active ingredient may be provided as a reagent composition for inhibiting Ninj1 (Ninjurin-1).

Description

Reagent composition for inhibiting Ninj1 comprising microRNA}

The present invention relates to a reagent composition and a method for inhibiting Ninj1 containing NiRNA 1 (Ninjurin-1) inhibitory effect containing NiRNA as an active ingredient.

Human Ninj1 (Ninjurin-1) is located on the chromosome 9q22 and consists of 152 amino acids. The mouse Ninj-1 is located on chromosome 13 and consists of 152 amino acids. Two transmembrane domains can be predicted in the Ninj-1 amino acid sequence. And through experiments, it is known that Ninj-1 is a protein located on the cell membrane (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). From this fact, it can be assumed that the N-terminal region of Ninj-1 extends out of the cell.

Ninj-1 is known to bind between allogeneic proteins, and its expression rate increases in an inflammatory environment or animal model of multiple sclerosis and is mainly involved in the mobility of cancer cells and immune cells and transendothelial migration. Is known.

Ninj-1 is expressed in various tissues. In the RNA stage, the heart, brain, placenta, lung, liver, skeletal muscle (sk.Muscle), kidney (kidney) ), Interest (pancreas), spleen, thymus, prostate, testis, ovary, small intestine (sm.int.), Colon, blood, It has been reported to be expressed in the adrenal gland and Dorsal Root Ganglia (DRG) and in the liver, kidney, thymus, uterus, adrenal gland, retina and dorsal roots at the protein level. .

As described above, as it is confirmed that Ninj-1 is expressed in a wide range of tissues to induce various diseases, drugs or treatment methods capable of exhibiting an effect of inhibiting Ninj-1 are related diseases induced by Ninj-1, including inflammation. It can be a new treatment strategy to prevent or treat them.

Republic of Korea Patent Publication No. 10-2010-0065953 (2010.06.17. Public)

As described above, the present invention provides a composition containing micro RNA as an active ingredient capable of effectively inhibiting Ninj-1, which is expressed in a wide range of tissues and induces various diseases, to study Ninj-1 related signaling processes and Ninj It is intended to provide a reagent composition for inhibiting Ninj-1 for the study of therapeutic or therapeutic methods of related diseases induced by -1.

The present invention provides a reagent composition for inhibiting Ninj1 (Ninjurin-1), which contains micro RNA as an active ingredient and is selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761.

The present invention provides a method for inhibiting Ninj1 in vitro, comprising the step of treating micro RNA selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761 to cells isolated from individuals other than humans. do.

In addition, it provides a method for inhibiting Ninj1 in vivo, including the step of treating micro RNA selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761 in inflammatory animal models other than humans.

According to the present invention, as miR125a, miR145, miR184, miR206 and miR761 micro RNAs are found to inhibit expression of cytokines by inhibiting Ninj1 expression in macrophage and inflammatory animal models, containing the micro RNA as an active ingredient The composition may be provided as a reagent composition for inhibiting Ninj1 (Ninjurin-1), and a method for inhibiting Ninj1 (Ninjurin-1) using the micro RNA may be provided.

1 is a result of miRNA PCR analysis performed to identify micro RNAs expected to exhibit Ninj1 expression control effects.
Figure 2 is a result of confirming the Ninj1 expression control effect of micro RNA, Figure 2A is a result of confirming the inhibitory effect of Ninj1 expression of miR125a-5p, miR145, miR184, miR206 and miR761 in Raw 264.74 cells, Figure 2B is miRNA mimic 125a- This is the result of confirming the concentration-dependent Ninj1 expression inhibitory effect of 5p.
Figure 3 is a result confirming the effect of micro RNA on the expression of the Ninj1 sub-cytokine-related gene, Figure 3A is a LPS-treated Raw 264.74 cell group and LPS and miRNA mimic 125a-5p in the cell group treated with Ninj1 sub-cytokine related As a result of confirming the expression of the gene, FIG. 3B is a result of confirming the expression of the Ninj1 sub-cytokine related gene in the cell group overexpressing Ninj1 and the cell group treated with miRNA mimic 125a-5p in cells overexpressing Ninj1.
Figure 4 is a result of confirming the effect of inhibiting macrophage activity of micro RNA, LPS-treated bone marrow-derived macrophages and Raw 264.74 cells are miRNA mimic 125a-5p treated and the results confirming the adhesion of macrophages.
5 is a result of confirming the inflammation inhibitory effect of micro RNA in a mouse animal model in which inflammation is induced by LPS, and FIG. 5A is a PCR analysis result confirming Ninj1 expression level by miRNA mimic 125a-5p treatment in the inflammatory model, FIG. 5B Is a Western blot analysis result confirming the level of Ninj1 expression by miRNA mimic 125a-5p treatment in the inflammatory model.
6 is a fluorescent staining result confirming the expression level of Ninj1 in the eye tissue of the mouse animal model induced inflammation by LPS.
7 is a result of confirming the effect of improving the retinal damage of micro RNA in the experimental group treated with 20 μM miR mimic 125a-5p in the diabetic retinopathy animal model and the untreated group, and FIG. 7A confirmed the level of Ninj1 expression in the eyes of each experimental group PCR analysis results, Fig. 7B is a PCR analysis result confirming the level of tubulin expression in the eyes of each experimental group, Fig. 7C is a Western blot analysis result confirming the expression level of Ninj1 and tubulin in the eyes of each experimental group.
FIG. 8A shows the results of staining FICT-dextran and diabetic whole of the retina, HCL and GCL, INL and ONL, and tissue staining of Ninj1 and F4 / 80. As a result, FIG. 8B is a result of confirming the total thickness of the retina and the thicknesses of GCL, INL and ONL.

Hereinafter, the present invention will be described in more detail.

The present invention can provide a reagent composition for inhibiting Ninj1 (Ninjurin-1), which contains micro RNA as an active ingredient and is selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761. .

The micro RNA can inhibit the activity of macrophages by inhibiting Ninj1 expression or activity in macrophages in vitro.

The micro RNA can inhibit Ninj1 expression or activity in vivo in an inflammatory animal model.

According to an embodiment of the present invention, miR125a, miR145, miR184, miR206 and miR761 micro RNAs, which are expected to influence the regulation of expression of Ninj1, actually confirm the effect of Ninj1 and cytokine-related gene expression, Raw 264.7 As a result of transfecting the cells with scramble RNA as a control and miRNA mimic for the micro RNA, and processing LPS to confirm the effect on Ninj1 expression, both miRNA mimics identified as in FIG. 2A significantly reduced Ninj1 expression, Among them, as the effect of miRNA 125a-5p mimic was found to be the best, PCR analysis was performed by processing the miRNA 125a-5p mimic by concentration.

As a result, it was confirmed that the expression of Ninj1 is decreased depending on the concentration of miRNA 125a-5p mimic as shown in FIG. 2B. In addition, as a result of confirming that miRNA 125a-5p mimic can influence the expression regulation of Ninj1 sub-cytokine-related genes, as shown in FIG. 3, in the LPS-treated group and the Ninj1 overexpression group, most of the low-cytokine-related genes increased, and miRNA 125a In the -5p mimic treatment group, it was confirmed that all the expressions of the lower cytokines were reduced.

From the above results, it was confirmed that miRNA 125a-5p regulates the expression of the lower cytokine-related gene by controlling the expression of Ninj1.

Accordingly, the present invention can provide a method of inhibiting Ninj1 in vitro, comprising the step of processing micro RNA on cells isolated from individuals other than humans.

In more detail, the cells may be macrophages, and the micro RNA may be selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761.

In addition, the present invention can provide a method of inhibiting Ninj1 in vivo, including the step of processing micro RNA in an inflammatory animal model other than a human.

In more detail, the micro RNA may be selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental example  1> Cell culture

Raw 264.7 cells were purchased from ATCC and maintained at 5% CO2 at 37 ° C.

Raw 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (Invitrogen, Carlsbad, CA).

< Experimental example  2> Cytokine analysis

RayBio mouse cytokine antibody array C1000 (RayBiotech, Inc.) was applied to serum-free conditioned media (SFCM) according to the manufacturer's instructions. Briefly, the membrane was incubated overnight with 1 ml of SFCM, then washed and re-incubated with biotin-bound anti-cytokine.

Then, the membrane was visualized by incubating with HRP-coupled streptavidin secondary antibody and chemiluminescence (FUSION-SL4, Vilber) was detected.

< Experimental example  3> Micro RNA real-time quantitative PCR  analysis

According to the manufacturer's instructions using Hybrid-RTM micro RNA isolation kit (GeneAll ^® ), miRNA was purified from incised eye or cultured cells, and using miScript reverse trnascription kit (Qiagen Inc.) according to the manufacturer's instructions. , Isolated 200ng miRNAs were synthesized.

Thereafter, real-time PCR was performed with the target miRNA pre-armor using miScript SYBR green PCR kit (Qiagen Inc.). 200 ng of cDNA was diluted to 20 ng for miRNA PCR.

MiRNAs were quantified with U6 short RNA provided as a standardized control.

The base sequence of the miRNA primer is shown in Table 1 below.

The heat cycle conditions were 15 cycles at 95 ° C, followed by 40 cycles at 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 70 ° C for 30 seconds. Cycle thresholds were calculated using automatic baselines and 0.1 thresholds for in vivo and in vitro samples.

miRNA accession number Primer sequence miR-1a MI0000139 3'-TGGAATGTAAAGAAGTATGTAT-5 ' miR-206-3p MIMAT0000239 3'-TGGAATGTAAGGAAGTGTGTGG-5 ' miR-184-3p MIMAT0000213 3'-TGGACGGAGAACTGATAAGGGT-5 ' miR-34a MI0000584 3'-TGGCAGTGTCTTAGCTGGTTGT-5 ' miR-145a-5p MIMAT0000157 3'-GTCCAGTTTTCCCAGGAATCCCT-5 ' miR-214 MI0000698 3'-ACAGCAGGCACAGACAGGCAGT-5 ' miR-761 MI0004306 3'-GCAGCAGGGTGAAACTGACACA-5 ' miR-125a-5p MIMAT0000135 3'-TCCCTGAGACCCTTTAACCTGTGA-5 ' miR-378 MI0000795 3'-ACTGGACTTGGAGTCAGAAGG-5 ' miR-449c MI0004645 3'-AGGCAGTGCATTGCTAGCTGG-5 ' miR-338-3p MIMAT0000582 3'-TCCAGCATCAGTGATTTTGTTG-5 '

< Experimental example  4> miRNA  mimic transfection

For overexpression or inhibition of miRNAs, miRNA mimics (miR-125a-5p, # MSY0000135; miR-145a-5p, # MSY0000157; miR-184-3p, # MSY0000213; miR-761, # MSY0003893; miR-206-3p , # MSY0000239, Qiagen, Valencia, CA, USA) was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

< Experimental example  5> Western Blot

For Western blot, samples were lysed using lysis buffer with 40 mM Tris-cl (pH 7.4), 10 mM EDTA, 120 mM Nacl, 0.1% NP-40, 1 mM PMSF, 2 μg / ml leupeptin. The lysate was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane.

The membrane was blocked with a phosphate buffer solution containing 0.5% Tween 20 and 5% skim milk powder (TBS; 10 mM Tris-Cl pH 7.4) and incubated overnight at 4 ° C with the primary specific antibody included in the blocking solution. Washed and incubated with secondary antibody for 1 hour at room temperature. Each protein band was detected by chemiluminescence (FUSION-SL4, Vilber).

< Experimental example  6> In the bone marrow Derived  Macrophages ( BMDM ) detach

Macrophages derived from bone marrow were isolated from the femur and tibia of 10-week-old C57BL / 6 tm1a / tm1a mice and 10-week-old 10-Week-old C57BL / 6 wild type mice.

The isolated bone marrow-derived macrophages were cultured for 4 hours in RPMI 1640 medium containing 10% FBS and 1% antibiotic, and then for bone marrow-derived macrophage differentiation, 20 ng / ml macrophage colony stimulating factor; cat no. Peprotech 315-02) was added to the medium and cultured for 7 days.

< Experimental example  7> Attachment analysis

The day before cell inoculation, 5 μg / ml fibronectin (cat no. Gibco 33010-018), 10 μg / ml collagen type I (cat no.BD 354249), 10 μg / ml laminin, a component of ECMs, in each well of a 96-well plate (cat no. sc-29012) and coated with 5 μg / ml gelatin (cat no. sigma G1890) for 24 hours at 4 ° C. and washed with washing buffer (0.1% BSA in medium) for 2 hours the next day.

A blocking buffer (0.5% BSA in medium) was added to each well, incubated at 37 ° C for 60 minutes, washed once again with a washing buffer, and a 96 well plate was maintained at 4 ° C.

Counted at 2 × 10 ⁴ cells per well, dispensed into each well, washed cells with wash buffer and fixed with 4% PFA for 10 min.

Cells were attached to the bottom, stained with 5 mg / ml crystal violet and dried for one day. The next day, 100 μl of 1% SDS was added to each well and incubated for 30 minutes at room temperature, followed by ELISA analysis at 550 nm.

< Experimental example  8> Animal experiment

One. With endotoxin  Production of induced inflammatory animal models

7-week-old C57BL / 6 wild-type mice were injected intraperitoneally 3 times over 4 days with 6.6 μg LPS (330 μg / kg of body weight), and 20 μM miRNA mimic was intravenously injected into the eye using Lipofectamine 2000.

Euthanized 24 hours after the last LPS treatment.

2. To streptozotocin (STZ)  Production of induced diabetes animal models

In the diabetic mouse model, a high dose of streptozotocin (STZ; Sigma, S0130) was induced by a method in which the pancreatic beta cells were toxic. A diabetes model induced by streptozotocin was developed using 8-week-old male C57BL / 6 mice and streptozotocin-sensitive ICR mice.

All animals were fed only water without food from 4 hours before STZ treatment.

STZ dissolved in 50 mM citrate buffer (pH4.5) was injected into the C57BL / 6 mice intraperitoneally at a dose of 180 mg / kg, and STZ dissolved in 50 mM citrate buffer (pH4.5) in ICR mice. Was injected intraperitoneally in a single dose of 100 mg / kg.

In addition, the same dose of 50 mM citrate buffer (pH4.5) was injected intraperitoneally as a control. Food was fed normally after the abdominal injection was complete.

At this time, 10% sucrose was supplied instead of water for 3 days to prevent mortality caused by fatal hypoglycemia, and on day 4, only mice with fasting blood glucose of 250 mg / dL were selected and used in the experiment.

3. miRNA  mimic Inside the eyeball  injection

10 weeks after diabetes, lipofectamine added with 20 μM miR mimic 125a-5p was injected into the eyeballs of ICR mice.

< Experimental example  9> Fluorescein ( FITC ) -Dextran perfusion

25 mg / ml FITC-dextran (70 kDa) was perfused through the tail vein and retinal plane specimen fixation was confirmed under a fluorescence microscope.

< Example  1> Ninj1's  Regulating expression miRNA  Confirm

PCR analysis was performed to confirm the difference in expression of miRNA in 5-day-old ocular (A) and LPS-stimulated Raw 264.7 cells (B), which are the highest periods of Ninjurin1 expression.

As a result, miR125a, miR145, miR184, miR206, and miR761, which consistently showed expression differences in both conditions, as shown in FIG. 1, may be proposed as Ninj1 high regulatory factors.

< Example  2> miRNA  mimic Ninj1  Confirmation of inhibitory effect and effect on cytokine-related gene expression

In order to confirm the effect of the previously identified micro RNA on the expression of Ninj1 and cytokine-related genes, raw 264.7 cells were transfected with scramble RNA as a control and miRNA mimic for the micro RNA, and then treated with LPS to express Ninj1. The effect was confirmed.

As a result, all of the identified miRNA mimics as shown in FIG. 2A significantly reduced Ninj1 expression, and among them, it was confirmed that the effect of miRNA 125a-5p mimic was the best, PCR was performed by processing the miRNA 125a-5p mimic by concentration. Analysis was performed.

As a result, it was confirmed that the expression of Ninj1 is decreased depending on the concentration of miRNA 125a-5p mimic as shown in FIG. 2B.

In addition, it was confirmed that miRNA 125a-5p mimic can influence the expression regulation of Ninj1 sub-cytokine related genes.

As a result, as shown in Figure 3, the LPS-treated group and the Ninj1 overexpression group showed that most of the lower cytokine-related genes increased, and the miRNA 125a-5p mimic treatment group showed that the expression of the lower cytokine was decreased.

From the above results, it was confirmed that miRNA 125a-5p regulates the expression of the lower cytokine-related gene by controlling the expression of Ninj1.

< Example  3> miRNA  Confirm the effect of inhibiting macrophage activity

In order to confirm the effect of miRNA on macrophages by using one of the main functions of macrophages, the attachment assay was performed to confirm changes in macrophage adhesion by miRNA mimic.

The day before the experiment, the 96-well plate was coated with ECM components such as collagen, fibronectin, lamin, and gelatin, and the degree of attachment of macrophage (BMDM) and Raw 264.7 cells derived from bone marrow was confirmed.

As a result, as shown in FIG. 4, the adhesion ability was significantly increased in both cells treated with LPS, but the decrease in adhesion was significantly confirmed in the cell group treated with miRNA mimic 125a.

From the above results, it was confirmed that miRNA mimic 125a shows an effect of reducing the adhesion capacity of macrophages.

< Example  4> In the inflammatory model miRNA  Confirmation of anti-inflammatory effect

To confirm the in vivo effect of miRNA, an inflammatory mouse model stimulated by LPS was used. The model was confirmed by performing real-time PCR and Western blot analysis by injecting miRNA into the vitreous cavity after injecting LPS daily into the intraperitoneal injection for 4 days, then injecting miRNA into the vitreous cavity.

As a result, as shown in FIG. 5, Ninj1 was significantly increased in the LPS group, while expression was decreased in the miRNA mimic 125a-treated group.

In addition, as a result of the Ninj1 fluorescence staining after fabricating the OCT block with ocular tissues, as shown in FIG. 6, it was confirmed that Ninj1 with increased expression in the LPS group was decreased in the miRNA mimic 125a treatment group.

From the above results, it was confirmed that miRNA mimic 125a showed an inhibitory effect of Ninj1 in an LPS-induced inflammatory mouse model.

< Example  5> In diabetic retinopathy model miRNA  Check effect

Like the brain, the blood-retinal barrier (BRB), which is a blood-retinal barrier, is present in the eye. When diabetic retinal disease develops, the barrier is damaged and the influx of foreign substances into the tissue increases and a severe inflammatory reaction occurs. In this process, macrophages are known to mediate inflammation and weaken the barrier.

Accordingly, in order to confirm that miRNA regulating the expression of Ninj1 can alleviate the damage, after administering dextran tagged with fluorescent material FITC, the degree of BRB damage was confirmed by observing it in a flat mount.

In addition, in order to confirm the expression of Ninj1, real-time PCR and Western blot were performed, and finally, H & E staining and Ninj1 and F4 / 80 were tissue stained.

As a result, as shown in Table 2, compared to the normal control group, the body weight of the mouse was significantly decreased in the diabetic group and hyperglycemia was maintained, and it was confirmed that diabetes was induced as the HbA1c increased to 8%. And as a result of performing Western blot, as shown in FIGS. 7A to 7C, Ninj1 increased significantly in the diabetic group (DM), and a significant decrease was observed in the miRNA mimic treatment group.

On the other hand, after injecting FITC dextran by intravenous injection, the eyeballs were extracted and unfolded with a flat mount and confirmed by a fluorescence microscope. Referring to FIGS. 8A and 8B, in the diabetic group, the barrier was damaged and leakage of dextran was confirmed. On the other hand, it was confirmed that the leakage was significantly reduced in the miRNA mimic treatment group compared to the diabetic group.

In addition, in the result of staining the eye tissues with H & E, it was confirmed that the total thickness of the retina and the thickness of GCL, INL and ONL were decreased in the diabetic group and significantly increased in the miRNA mimic treatment group, and finally Ninj1 and F4 / 80. As a result of tissue staining, Ninj1 expression and the number of macrophages increased in the diabetic group, but the number of macrophages decreased in the miRNA mimic treatment group.

From the results, it was confirmed that miRNA mimic modulates Ninj1 in an animal model in which diabetes is induced, and improves Ninj1-mediated diabetic retinopathy.

The specific parts of the present invention have been described in detail above, and it is obvious to those skilled in the art that this specific technology is only a preferred embodiment, whereby the scope of the present invention is not limited. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

It contains a micro RNA as an active ingredient, the micro RNA is a reagent composition for inhibiting Ninj1 (Ninjurin-1) expression, characterized in that it is selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761. The method according to claim 1, wherein the miR125a is in vitro (in vitro) Ninj1 expression inhibitory reagent composition for inhibiting the activity of macrophages by inhibiting Ninj1 expression or activity in macrophages. The method according to claim 1, wherein the miR125a is a reagent composition for inhibiting Ninj1 expression, characterized in that to inhibit Ninj1 expression or activity in vivo in an inflammatory animal model. A method for inhibiting Ninj1 expression in vitro, comprising the step of treating micro RNA selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761 to cells isolated from individuals other than humans. A method of inhibiting Ninj1 expression in vivo, comprising the step of treating micro RNA selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761 in an inflammatory animal model other than humans.

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