Application of miR-1295a in preparation of medicine for treating atopic dermatitis

Abstract

The invention discloses an application of miR-1295a (micro Ribonucleic Acid-1295a) in preparation of a medicine for treating atopic dermatitis. The nucleotide sequence of the miR-1295a is as shown in SEQ ID No: 5. The inventor of the invention finds that the content of miR-1295a in the blood of a patient with atopic dermatitis is remarkably increased in the related research of the atopic dermatitis, and finds that miR-1295a can be combined with the 3 'UTR region of silk polyprotein mRNA and inhibit the expression of the silk polyprotein mRNA, and the content of miR-1295a in the blood of the patient with atopic dermatitis can be used as a molecular marker for diagnosing atopic dermatitis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of miR-1295a in the preparation of drugs for treating atopic dermatitis. Background Art

[0002] Atopic Dermatitis (AD) is a chronic, recurrent, inflammatory skin disease, often accompanied by severe itching, dry skin and eczema-like skin lesions, which seriously affects the quality of life of patients. The symptoms of atopic dermatitis vary with age: in infants, the skin lesions are mostly seen on the cheeks, forehead and scalp, manifested as erythema, papules, papulovesicles, densely packed, easy to exude, erode and crust, with severe itching; in children, the skin lesions mostly occur in the cubital fossa, popliteal fossa, flexor side of the wrist and other parts, showing dark red color, often accompanied by scratch marks and lichenification, and the itching persists; in adolescents and adults, the skin lesions are mostly seen in the cubital fossa, popliteal fossa, neck and other parts, manifested as localized lichenification or generalized dry papules, with severe itching.

[0003] Atopic dermatitis is a disease caused by the combined action of multiple factors, multiple links and multiple pathways. Skin dysfunction has been considered as the basic feature of atopic dermatitis. Skin barrier dysfunction can lead to the infiltration of microorganisms, allergens and other environmental factors, which in turn leads to consequences including inflammation, sensitization of allergens and aggregation of microorganisms. The expression levels of intercellular junction proteins between skin cells, such as filaggrin and occludin, are significantly decreased in the skin of patients with atopic dermatitis, which is considered to be an important cause of skin barrier dysfunction.

[0004] miRNA (MicroRNA) is a class of endogenous small non-coding RNAs, about 19-25 nucleotides in length. The miRNA gene is first transcribed into a longer primary transcript (pri-miRNA), about 300-1000 bases in length. The pri-miRNA is cleaved by Drosha enzyme in the nucleus to form a precursor miRNA (pre-miRNA) with a hairpin structure, about 70-90 bases in length. The pre-miRNA is transported to the cytoplasm with the help of Exportin-5 protein and further cleaved by Dicer enzyme, finally generating a mature miRNA about 20-24 nucleotides in length. The latest research shows that some miRNAs are abnormally expressed in atopic dermatitis, but there is still no consensus on which miRNAs are related to the occurrence and development of atopic dermatitis.

[0005] Therefore, it is necessary to find miRNAs related to the occurrence and development of atopic dermatitis, so as to provide an effective means for the clinical treatment of atopic dermatitis. Summary of the Invention

[0006] The object of the present invention is to provide the use of miR-1295a in the preparation of a drug for treating atopic dermatitis.

[0007] The use of miR-1295a in the preparation of a drug for treating atopic dermatitis, wherein the nucleotide sequence of the miR-1295a is as shown in SEQ ID No: 5.

[0008] The said use includes the preparation of a drug for alleviating atopic dermatitis and preventing the recurrence of atopic dermatitis.

[0009] It includes complexing miR-1295a with a carrier and then compounding it with an anti-inflammatory drug and / or a pharmaceutically acceptable excipient to form a pharmaceutical composition.

[0010] A pharmaceutical composition, wherein the pharmaceutical composition takes miR-1295a as an active ingredient.

[0011] The said pharmaceutical composition contains miR-1295a combined with a carrier and / or a pharmaceutically acceptable excipient.

[0012] The said pharmaceutically acceptable excipients include various excipients, diluents and adjuvants; the excipients themselves are not essential active ingredients and have no excessive toxicity after use; such excipients include but are not limited to: physiological saline, buffer solution, glucose, water, glycerol, ethanol, etc.

[0013] The said carrier is a lentiviral expression vector.

[0014] The said carrier is a liposome or chitosan.

[0015] A marker for detecting atopic dermatitis, wherein the marker is miR-1295a.

[0016] The expression level of the said marker miR-1295a is increased in patients with atopic dermatitis.

[0017] The beneficial effects of the present invention: The inventors of the present invention found in the related research on atopic dermatitis that the content of miR-1295a in the blood of patients with atopic dermatitis is significantly increased. At the same time, it was found that miR-1295a can bind to the 3'UTR region of filaggrin mRNA and inhibit its expression. The content level of miR-1295a in the blood of patients with atopic dermatitis can be used as a molecular marker for diagnosing atopic dermatitis. Description of the Drawings

[0018] Figure 1 It is the content of miR-1295a in the sera of healthy people and patients with atopic dermatitis.

[0019] Figure 2 It is the relative luciferase activity of Group 1 - Group 4.

[0020] Figure 3 To detect the change in FLG content by Q-PCR.

[0021] Figure 4 For the ROC curve of miR-1295a in detecting atopic dermatitis. Detailed implementation manners

[0022] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Example 1 The content of miR-1295a in the blood of patients with atopic dermatitis is higher than that in healthy people

[0024] Collect 60 serum samples each from healthy people and patients with atopic dermatitis. The typical skin lesion characteristic sites included in patients with atopic dermatitis: flexural sites such as the cubital fossa, popliteal fossa, and anterior neck, and it can involve the trunk, limbs, face, and hands; types: mainly chronic dermatitis, manifested as dry and hypertrophic plaques (lichenification), and scattered millet to peanut-sized prurigo-like nodules can be seen in some patients; itching: severe itching is the core symptom, which is aggravated at night, and the skin lesions are often aggravated or secondary infections are caused due to scratching; it is often accompanied by abnormal skin barrier, manifested as xeroderma: dry skin and desquamation all over the body.

[0025] Obtain miR in the blood through a miR extraction kit, and detect and compare the relative content of miR-1295a in the serum by using the Q-PCR method;

[0026] The selected internal reference is: has-miR-16-5p (5’-UAGCAGCACGUAAAUAUUGGCG-3’ SEQ ID NO: 1);

[0027] Internal reference reverse transcription primer: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA-3’ (SEQ ID NO: 2);

[0028] qPCR primer F: 5’-TAGCAGCACGTAAATA-3’ (SEQ ID NO: 3);

[0029] qPCR primer R: 5’-GTGCAGGGTCCGAGGT-3’ (SEQ ID NO: 4).

[0030] has-miR-1295a (5’-UUAGGCCGCAGAUCUGGGUGA-3’, SEQ ID NO: 5);

[0031] Reverse transcription primer: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT GGATACGACTCACCC-3’ (SEQ ID NO: 6);

[0032] qPCR primer F: 5’-TTAGGCCGCAGATCT-3’ (SEQ ID NO: 7);

[0033] qPCR primer R: 5’-GTGCAGGGTCCGAGGT-3’ (SEQ ID NO: 8).

[0034] The results are as Figure 1 shown that the content of miR-1295a in the serum of patients with atopic dermatitis is significantly higher than that in the serum of healthy people.

[0035] Example 2 Dual-luciferase reporter gene assay

[0036] Construct the pmirGLO-FLG-3’-UTR dual-luciferase reporter plasmid, and use the recombinant plasmid pmirGLO-FLG-3’-UTR to detect the regulatory effect of has-mir-1295a on the 3’-UTR region of the FLG gene. Co-transfect the pmirGLO-FLG-3’-UTR plasmid and the has-mir-1295a mimic into 293T cells. Seed exponentially growing cells in a 6-well plate at a density of 5×10 5 cells / well, and incubate overnight in a 37°C, 5% CO2 incubator until the cells are 80% confluent. Then, perform co-transfection of the pmirGLO-FLG-3’-UTR plasmid and the has-mir-1295a mimic in a biosafety cabinet.

[0037] The luciferase activity of each group was measured using a Luciferase kit (Promega) and a firefly luciferase detector (Beahold), and 3 replicate wells were set up for each group. Working solution 1 was used for the detection of firefly luciferase activity (Working solution 1: Solution A (2 mL) + Solution B (40 μL) + Solution C (10 μL)); Working solution 2 was used for the detection of Renilla luciferase activity: Solution D (2 mL) + Solution E (10 μL). Lysis of cells: The culture medium was removed, and the cells were washed once with PBS. 500 μL of lysis buffer was added to each well, and the mixture was shaken at room temperature for 15 min. 20 μL of cell lysate was taken into a 1.5 mL centrifuge tube. Working solution 1 was added, the delay was set to 2 sec, and the luminescence was measured by the luminometer for 10 sec. Working solution 2 was added, the delay was set to 2 sec, and the luminescence was measured by the luminometer for 10 sec for detection. The grouping is as follows:

[0038] Group 1: Wild-type sequence of filaggrin (FLG) + negative control miRNA (miR NC);

[0039] Group 2: Wild-type sequence of filaggrin (FLG) + miR-1295a mimic;

[0040] Group 3: Mutant sequence of filaggrin (FLG) + negative control miRNA (miR NC);

[0041] Group 4: Mutant sequence of filaggrin (FLG) + miR-1295a mimic.

[0042] The experimental results showed that miR-1295a could bind to the 3' UTR region of filaggrin mRNA and inhibit its expression ( Figure 2 ).

[0043] Example 3

[0044] In the keratinocyte cell line HaCaT, miR-1295a mimic (RiboBio, product number: miR10005885-1-5) was transfected, and the change in the content of FLG was detected by Q-PCR;

[0045] The internal reference was GAPDH:

[0046] qPCR primer F: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO: 9);

[0047] qPCR primer R: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO: 10);

[0048] Filaggrin (FLG) primers:

[0049] qPCR primer F: 5’-aggtaccattgcaggagtcc-3’ (SEQ ID NO: 11);

[0050] qPCR primer R: 5’-gtgacgagtgcctgattgtc-3’ (SEQ ID NO: 12).

[0051] The experimental results are as Figure 3 shown. There were significant differences between the miR-1295a mimic group and the two control groups, p < 0.01.

[0052] Example 4

[0053] Serum samples from 80 patients with atopic dermatitis and 80 normal healthy individuals were recollected to verify the diagnostic efficacy of miR-1295a. The expression level of miR-1295a was used as the detection variable to judge the diagnostic result. Analysis method: The ROC of the index was analyzed using MedCalc Statistical Software version.

[0054] The results of the ROC curve showed ( Figure 4 ), AUC = 0.9069, p value < 0.01. The above results indicate that miR-1295a can be used as a diagnostic marker for atopic dermatitis.

[0055] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of miR-1295a in the preparation of a medicament for treating atopic dermatitis, characterized in that, The nucleotide sequence of the miR-1295a is shown as SEQ ID No:

5.

2. The application according to claim 1, characterized in that, The application includes preparing a drug for alleviating atopic dermatitis and preventing the recurrence of atopic dermatitis.

3. The application according to claim 1 or 2, characterized in that, It includes compounding a drug composition by combining miR-1295a with a carrier and then compounding it with an anti-inflammatory drug and / or a pharmaceutically acceptable excipient.

4. A pharmaceutical composition, characterized in that, The drug composition uses miR-1295a as an active ingredient.

5. The pharmaceutical composition according to claim 4, wherein The drug composition contains miR-1295a combined with a carrier and / or a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The carrier is a lentiviral expression vector.

7. The pharmaceutical composition according to claim 6, wherein, The carrier is liposome or chitosan.

8. A biomarker for detecting atopic dermatitis, characterized in that, The biomarker is miR-1295a.

9. The marker for detecting atopic dermatitis according to claim 8, wherein The expression level of the biomarker miR-1295a is increased in patients with atopic dermatitis.

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