KR20200008788A - Reagent composition for inhibiting Ninj1 comprising microRNA - Google Patents

Abstract

The present invention relates to a reagent composition for inhibiting Ninj1 comprising microRNA having an inhibitory effect on Ninjurin-1 (Ninj1) as an active ingredient and an inhibitory method. More specifically, it is confirmed that miR125a, miR145, miR184, miR206, and miR761 microRNA inhibit the expression of Ninj1 in phagocytes and inflammatory animal models and thus inhibit the expression of cytokines. Therefore, the composition comprising the microRNA as an active ingredient may be provided as a reagent composition for inhibiting Ninjurin-1(Ninj1).

Description

Reagent composition for inhibiting Ninj1 comprising microRNA containing micro RNA as an active ingredient

The present invention relates to a reagent composition for inhibiting Ninj1 containing microRNA having an inhibitory effect on Ninj1 (Ninjurin-1) as an active ingredient, and a method for inhibiting the inhibition.

Human Ninj1 (Ninjurin-1) is located on chromosome 9q22 and consists of 152 amino acids. Mouse Ninj-1 is located on chromosome 13 and consists of 152 amino acids. Two transmembrane domains can be predicted from the Ninj-1 amino acid sequence. Experiments have shown that Ninj-1 is a protein located in cell membranes (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). This fact suggests that the N-terminal region of Ninj-1 may extend out of the cell.

Ninj-1 is known to bind to homologous proteins, and its expression rate is increased in inflammatory environments or in animal models of multiple sclerosis, and is mainly involved in the motility and transendothelial migration of cancer cells and immune cells. Known.

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

As Ninj-1 is found to be expressed in a wide range of tissues to induce a variety of diseases, drugs or treatments that may have an effect of inhibiting Ninj-1 include related diseases induced by Ninj-1, including inflammation. It may be a new treatment strategy to prevent or treat them.

Republic of Korea Patent Publication No. 10-2010-0065953 (Published June 17, 2010)

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, thereby studying Ninj-1 related signaling processes and Ninj The present invention provides a reagent composition for inhibiting Ninj-1 for the study of a therapeutic agent or method for treating related diseases induced by -1.

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

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

In addition, the present invention provides a method of inhibiting Ninj1 in vivo, including treating microRNAs selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761 in an inflammatory animal model other than human.

According to the present invention, as miR125a, miR145, miR184, miR206 and miR761 micro RNA was found to inhibit Ninj1 expression in macrophages and inflammatory animal models to inhibit the expression of cytokines, 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 microRNA may be provided.

1 is a miRNA PCR analysis performed to identify the micro RNA expected to exhibit a Ninj1 expression regulatory effect.
2 is a result of confirming the control effect of Ninj1 expression of micro RNA, Figure 2A is a result 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- The result of confirming the concentration dependent Ninj1 expression inhibitory effect of 5p.
FIG. 3 shows the effect of microRNA on the expression of Ninj1 subcytokine related genes. FIG. 3A is related to Ninj1 subcytokine in LPS-treated Raw 264.74 cell group and LPS- and miRNA mimic 125a-5p cell group. 3B shows the results of confirming the expression of the Ninj1 lower cytokine related genes in the cell group overexpressing Ninj1 and the cell group treated with miRNA mimic 125a-5p in the cells overexpressing Ninj1.
4 is a result of confirming the effect of inhibiting the macrophage activity of the microRNA, the result of treatment of miRNA mimic 125a-5p to LPS-treated bone marrow-derived macrophages and Raw 264.74 cells, respectively, and confirmed the adhesion of macrophages.
5 is a result of confirming the inflammation inhibitory effect of the micro RNA in the mouse animal model induced inflammation by LPS, Figure 5A is a PCR analysis result confirming the Ninj1 expression level by miRNA mimic 125a-5p treatment in the inflammation model, Figure 5B Is a Western blot analysis confirming the Ninj1 expression level by miRNA mimic 125a-5p treatment in the inflammation model.
6 is a fluorescence staining result confirming the Ninj1 expression level 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 the micro RNA in the experimental group and the 20 group treated with 20 μM miR mimic 125a-5p diabetic retinopathy animal model, Figure 7A confirms the Ninj1 expression level in the eye of each experimental group 7B is a PCR analysis result confirming the tubulin expression level in the eye of each experimental group, Figure 7C is a Western blot analysis result confirming the Ninj1 and tubulin expression level in the eye of each experimental group.
Figure 8A shows the results of FICT-dextran staining confirming diabetic barrier damage to the degree of dextran leakage in the eyes of each experimental group, H & E staining of the entire retina, GCL, INL and ONL, and tissue staining of Ninj1 and F4 / 80. 8B is a result of checking 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 contains micro RNA as an active ingredient, the micro RNA can provide a reagent composition for inhibiting Ninj1 (Ninjurin-1), characterized in that selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761. .

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

The microRNA may inhibit Ninj1 expression or activity in vivo in an inflammatory animal model.

According to an embodiment of the present invention, in order to confirm the effect of miR125a, miR145, miR184, miR206 and miR761 microRNAs expected to affect the expression regulation of Ninj1 actually on the expression of Ninj1 and cytokine related genes, Raw 264.7 As a result of transfecting miRNA mimic to the microRNA and the scramble RNA as a control group, LPS treatment confirmed the effect on Ninj1 expression. As shown in FIG. 2A, both miRNA mimics significantly reduced Ninj1 expression. Among them, the miRNA 125a-5p mimic was found to have the best effect, and the miRNA 125a-5p mimic was treated by concentration to perform PCR analysis.

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

From the above results, it was confirmed that miRNA 125a-5p regulates the expression of Ninj1, thereby regulating the expression of its lower cytokine related genes.

Accordingly, the present invention can provide a method for inhibiting Ninj1 in vitro, including treating microRNAs with cells isolated from individuals other than humans.

In more detail, the cell may be a macrophage, and the microRNA may be selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761.

In addition, the present invention can provide a method for inhibiting Ninj1 in vivo, including the step of processing micro RNA in an inflammatory animal model other than 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 understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain 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% CO 2, 37 ° C.

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

< Experimental Example  2> Cytokine Analysis

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

The membrane was then visualized by incubating with HRP-bound streptavidin secondary antibody and detecting chemiluminescence (FUSION-SL4, Vilber).

< Experimental Example  3> Micro RNA Real Time Quantitative PCR  analysis

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

Thereafter, real-time PCR was performed with a target miRNA primer using a 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 served as a standardized control.

Base sequences of miRNA primers are shown in Table 1 below.

Heat cycle conditions were followed by 40 cycles of 15 seconds at 95 ° C., 15 seconds at 94 ° C., 30 seconds at 55 ° C., and 30 seconds at 70 ° C. Cycle thresholds were calculated using automatic baseline 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) were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

< Experimental Example  5> Weston Blot

For western blots, samples were dissolved using lysis buffer containing 40 mM Tris-cl (pH 7.4), 10 mM EDTA, 120 mM Nacl, 0.1% NP-40, 1 mM PMSF, 2 μg / ml leupeptin. Lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane.

The membrane was blocked with phosphate buffer solution (TBS; 10 mM Tris-Cl pH 7.4) containing 0.5% Tween 20 and 5% skim milk powder and incubated overnight at 4 ° C. with a 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  Macrophage ( BMDM ) Separation

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

< Experimental Example  7> Attachment Analysis

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

Blocking buffer (0.5% BSA in medium) was added to each well and incubated at 37 ° C. for 60 minutes, then washed again with a wash buffer and maintained at 96 ° C. in a 96 well plate.

Count wells at 2 × 10 ⁴ cells per well, dispense into each well, wash cells with wash buffer and fix for 10 min with 4% PFA.

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

< Experimental Example  8> Animal Experiment

One. With endotoxin  Induced Inflammation Animal Model

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

Euthanasia was performed 24 hours after the last LPS treatment.

2. With streptozotocin (STZ)  Induced Diabetes Animal Model

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

Four hours before STZ treatment, all animals were fed with no water and only water.

The experimental group of C57BL / 6 mice was intraperitoneally injected with 180 mg / kg of STZ dissolved in 50 mM citrate buffer (pH4.5), and IZ mice were dissolved in 50 mM citrate buffer (pH4.5). Was injected intraperitoneally in a single dose of 100 mg / kg.

In addition, the control group was injected intraperitoneally with the same dose of 50 mM citrate buffer (pH 4.5). Food was normally supplied after the abdominal injection was completed.

At this time, 10% sucrose was supplied instead of water for 3 days to prevent death due to fatal hypoglycemia, and on day 4, only mice having fasting glucose of 250 mg / dL were used for the experiment.

3. miRNA  mimic Intraocular  injection

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

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

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

< Example  1> Ninj1's  Regulating expression miRNA  Confirm

PCR analysis was performed to identify the difference in miRNA expression in 5-day-old eyes (A) and LPS-stimulated Raw 264.7 cells (B).

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

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

To confirm the effect of the microRNAs identified above on the expression of Ninj1 and cytokine related genes, raw 264.7 cells were transfected with control scramble RNA and miRNA mimic for the microRNA, and then treated with LPS for Ninj1 expression. The effect was confirmed.

As a result, all miRNA mimics identified as shown in FIG. 2A significantly reduced Ninj1 expression, and among them, miRNA 125a-5p mimics were found to have the best effect, and the miRNA 125a-5p mimics were treated by concentration to PCR. The analysis was performed.

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

In addition, we confirmed whether miRNA 125a-5p mimic can influence the expression regulation of Ninj1 subcytokine related genes.

As a result, as shown in FIG. 3, the LPS-treated group and the Ninj1 over-expressed group mostly increased the lower cytokine-related genes, and the miRNA 125a-5p mimic treated group reduced all the expression of the lower cytokine.

From the above results, it was confirmed that miRNA 125a-5p regulates the expression of Ninj1, thereby regulating the expression of its lower cytokine related genes.

< Example  3> miRNA  Inhibition of macrophage activity

In order to confirm the effect of miRNA on macrophages using adhesion, one of the main functions of macrophages, adhesion analysis was performed to confirm the change of macrophage adhesion by miRNA mimic.

On the day before the experiment, 96-well plates were coated with ECM components collagen, fibronectin, lamin and gelatin, and then the degree of adhesion of BMDM-derived raw 264.7 cells to bone marrow was confirmed.

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

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

< Example  4> Inflammation model miRNA  Inflammation inhibitory effect

To confirm the in vivo effects of miRNAs, an inflammatory mouse model stimulated by LPS was used. The model was confirmed by performing intraperitoneal injection of LPS daily for 4 days to induce inflammation, injecting miRNA into the mouse's eye in the vitreous cavity, and then extracting the eyeballs of the inflammatory model mice and performing real-time PCR and western blot analysis.

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

In addition, as a result of Ninj1 fluorescence staining after producing the OCT block in the eye tissue, it was confirmed that Ninj1 increased expression in the LPS group in the miRNA mimic 125a treatment group as shown in FIG.

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

< Example  5> in diabetic retinopathy model miRNA  Check the effect

Like the brain, the eye has a blood-retinal barrier (BRB), which is a blood vessel-tissue barrier. When diabetic retinopathy develops, the barrier is damaged, increasing the inflow of foreign substances into the tissue, and a serious inflammatory reaction occurs. In this process, macrophages are known to mediate inflammation and weaken the barrier.

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

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

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

On the other hand, after injecting FITC dextran by intravenous injection, eyeballs were extracted and unfolded with a flat mount and confirmed by fluorescence microscopy. Referring to FIGS. On the other hand, the miRNA mimic treatment group was confirmed that the leakage was significantly reduced compared to the diabetic group.

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

The results showed that miRNA mimic regulates Ninj1 and improves Ninj1-mediated diabetic retinopathy in diabetic animal models.

Having described the specific part of the present invention in detail, it is apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. 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 micro RNA as an active ingredient, the micro RNA is a reagent composition for inhibiting Ninj1 (Ninjurin-1), characterized in that selected from the group consisting of miR125a, miR145, miR184, miR206 and miR761. The reagent composition for inhibiting Ninj1 of claim 1, wherein the microRNA inhibits the activity of macrophages by inhibiting Ninj1 expression or activity in macrophages in vitro. The reagent composition for inhibiting Ninj1 according to claim 1, wherein the micro RNA inhibits Ninj1 expression or activity in vivo in an inflammatory animal model. A method of inhibiting Ninj1 in vitro comprising treating micronuclear cells 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 in vivo, comprising treating a microRNA selected from the group consisting of miR125a, miR145, miR184, miR206, and miR761 in an inflammatory animal model other than human.

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