Use of miR-21 in preparation of drug for treating intrauterine adhesion and/or thin endometrium

Abstract

Provided is use of miR-21 in preparation of a therapeutic drug or diagnostic reagent for intrauterine adhesion and/or thin endometrium.

Claims

1. A method for treating intrauterine adhesion and/or thin endometrium, comprising increasing an amount of miR-21 in endometrial epithelial cells and/or interstitial cells so as to treat intrauterine adhesion and/or thin endometrium.

2. The method according to claim 1, wherein a therapeutically effective amount of miR-21 is provided to a subject in need of treatment for a disease caused by intrauterine adhesion and/or thin endometrium.

3. The method according to claim 2, wherein the disease is uterine infertility, recurrent abortion, or placenta adhesion and implantation caused by intrauterine adhesion and/or thin endometrium.

4. A method comprising detecting expression of miR-21 in a subject having or suspected of having intrauterine adhesion and/or thin endometrium with a reagent specific for miR-21.

5. A method for detecting intrauterine adhesion and/or thin endometrium, comprising performing the method according to claim 4 to measure whether there is underexpression of miR-21 in endometrial epithelial cells and/or interstitial cells.

6. The method according to claim 4, wherein the reagent is selected from the group consisting of PCR and qPCR primers, Taqman probes, digoxigenin or fluorescent dye-labeled probes, miRNA expression profile chips, and deep sequencing reagents.

7. The method according to claim 6, wherein the reagent comprises a digoxigenin or fluorescent dye-labeled probe that includes SEQ ID NO. 1.

8. The method according to claim 2, wherein the subject has intrauterine adhesion in which there is adhesion between uterine walls of the subject.

9. The method according to claim 2, wherein the subject has thin endometrium in which endometrial thickness is less than 7 mm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the analysis of miR-21 expression in endometrial tissues by in situ hybridization. A: miRNA negative control; B: miRNA positive expression control (miR-16); C: normal human endometrial miR-21 expression; D: endometrial miR-21 expression in a patient with intrauterine adhesion

(2) FIG. 2 is the effect of miR-21 on cell fibrosis-related gene expression.

(3) Panels A-D are the expression of E-cadherin, fibrosis-related genes FN1, a-SMA, and Collagen I, respectively.

(4) FIG. 3 is the effect of miR-21 on Np63-induced endometrial epithelial cell fibrosis.

(5) Panel A shows the immunofluorescence results of E-cadherin; Panel B shows the immunofluorescence results of N-cadherin.

DETAILED DESCRIPTION

Example 1 Expression of miR-21 in Endometrial Tissues of Normal Patients and Patients with Severe Intrauterine Adhesion

(6) 1. Material, Reagent and Device

(7) 1.1 Source of Human Endometrial Tissue

(8) 5 normal endometrial tissues and 5 endometrial tissues of patients with severe intrauterine adhesion syndrome were collected. When the endometrial tissues were obtained, the size of follicles was detected by ultrasound to ensure that acquisition stages for the endometrial tissues were all late proliferation stages. All participants signed written informed consent form and the tests were approved by the Ethics Committee of Nanjing Drum Tower Hospital.

(9) 1.2 Primary Reagent

(10) HASProbe™ MicroRNA in-situ detection probe (Pengekiphen biotechnology, Inc.), hybridization kit (Pengekiphen biotechnology, Inc.), Anti-Digoxigenin-AP (Roche), NBT/BCIP developer (Pengekiphen biotechnology, Inc.), xylene, ethanol, DEPC water, PBS, PFA, proteinase K, acetic anhydride, formamide, TEA, SSC, MAB, and Tween.

(11) 1.3 Primary Instrument

(12) Hybridizer (Thermobrite), shaker, fluorescence microscope (Leica).

(13) 1.4 Primary Method

(14) The expression of miR-21 was detected by adopting a paraffin section in-situ hybridization method. The section was treated twice with xylene, each for 15 min, treated with gradient of alcohol (100% ethanol for 5 min, 100% ethanol for 5 min, 95% ethanol for 3 min, 85% ethanol for 3 min, 70% ethanol for 3 min, and 30% ethanol for 3 min), and then washed with DEPC treated water for 3 min, and washed with 1×PBS for 3 min. After which, the section was treated with 4% PFA on ice for 10 min, and washed twice with 1×PBS, each for 5 min. After soaking with proteinase K at room temperature for 15 min, the section was washed with 1×PBS for 2 min. The section was rinsed with 1×TEA on a shaker for 10 min, and treated with 2×SSC for 5 min. After air drying, prehybridization was performed by incubating the section for 1 h, followed by hybridization overnight. The section was soaked with 1×SSC (containing 50% formamide+0.1% Tween 20) for 15 min, and soaked twice with 0.2×SSC (containing 50% formamide+0.1% Tween 20), each for 10 min. The section was treated twice with 1×MAB (containing 0.1% Tween 20) at room temperature, each for 10 min. The section was blocked with a blocking buffer at 37° C. for 30 min, and then antibody (Anti-DIG-antibody) was added and left overnight at 4° C. After elution, the section was treated with an NBT/BCIP developer, mounted, and then observed under a microscope and photographed.

(15) 2. Result

(16) In the endometrial tissues of patients with severe intrauterine adhesion, the expression of miR-21 in epithelial cells and interstitial cells were all remarkably reduced, in which the reduced expression of miR-21 in the glandular epithelial cells was most remarkable (FIG. 1).

Example 2 Effect of miR-21 on Fibrosis of Endometrial Epithelial Cells

(17) 1. Material, Reagent and Device

(18) 1.1 Primary Reagents

(19) Collagenase (Sigma), hyaluronidase (Sigma), DNAase (Roche), epithelial cell medium (Gibco), serum (Gibco), Trizol (Invitrogen), reverse transcriptase (Takara), qPCR enzyme (Roche).

(20) 1.2 Primary Instrument

(21) Cell incubator, shaker, qPCR instrument (Roche), PCR instrument (ABI)

(22) 1.3 Primary Method

(23) 1.3.1 the Separation and Culture of Primary Endometrial Epithelial Cell

(24) An endometrial tissue was placed in a fresh 60 mm culture dish, sheared until no visible block existed, a digestive juice prepared was added, the mixture was beaten and uniformly mixed, and placed in an incubator at 37° C. for 5 min; the culture dish was taken out, and the digestion condition was observed under a microscope; DNAase was added in a concentration of 4 mg/mL, and digestion was continued for 5 min; the digested tissue was blown and beaten and the tissue suspension was dropwise added onto a 40 μm sieve. The residual tissues in the sieve was transferred to a clean 35 mm culture dish, 2 ml of mixed solution of protease and collagenase was added, fully and uniformly mixed, and placed in an incubator at 37° C. for 5 min; whether the digested glandular tissue had been dispersed into single glands was observed under a microscope, and then an epithelial cell culture solution was added to stop digestion, and the glands were placed in a culture dish for culture.

(25) 1.3.2 RNA Extraction and Real-Time PCR

(26) Total RNA was extracted by a Trizol method. 1 ug of RNA was reversely transcribed to obtain cDNA, which was diluted with RNase-free water in double volume and subjected to fluorescent quantitative PCR detection by an SYBRGreen method with 3 replicate wells per sample. The expression levels of different target genes were counted by using AACT values with GAPDH as an internal reference.

(27) 2. Result

(28) After transfecting the endometrial epithelial cells with miR-21 for 48 h, the mRNA level of E-cadherin was remarkably increased, and the FN1, a-SMA and Collagen I gene expression were remarkably reduced, indicating that miR-21 inhibited the cell fibrosis process.

Example 3 Reversion of Np63-Induced Endometrial Epithelial Cell Fibrosis by miR-21

(29) 1. Material, Reagent, and Device

(30) 1.1 Primary Reagent

(31) E-cadherin antibody (Abcam), N-cadherin antibody (Abcam), fluorescent secondary antibody (Jackson), DAPI-containing mountant (Abcam), PFA, antibody dilution (Gibco), methanol, Tween, and PBS.

(32) 1.2 Primary Instrument

(33) Fluorescence microscope (Leica), shaker

(34) 1.3 Primary Method

(35) The expression of E-cadherin and N-cadherin were detected by a cell immunofluorescence method. After sliding, the endometrial epithelial cells were infected with Np63 adenovirus for 24 h. A cell-loaded slide was then removed after transfecting with miR-21 for 48 h. The cells were washed with 1×PBS for 3 times, each for 5 min, and then fixed with PFA at room temperature for 15 min, washed with 1×PBS for 3 times, each for 5 min. The cells were treated with pre-chilled methanol for 5 min and washed with 1×PBS for 3 times, each for 5 min. After blocking with 2% BSA at room temperature for 1 h, E-cadherin and N-cadherin antibodies were added and left overnight at 4° C. The cells were then washed with PBST for 3 times, each for 5 min, the excessive primary antibody was thoroughly washed, and the water on a surface of the slide was blotted up after the last wash. A fluorescent secondary antibody was added dropwise, the mixture was incubated in an incubator at 37° C. for 30 min in the dark and washed with PBST for 3 times, each for 5 min, the water on the surface of the slide was blotted up after the last wash, DAPI dye containing an anti-quenching agent was dropwise added, observed under a fluorescent microscope and photographed in the dark.

(36) 2. Result

(37) Np63 remarkably inhibited E-cadherin expression and promoted upregulation of N-cadherin expression. However, miR-21 promoted E-cadherin expression and reversed Np 63-induced upregulation of N-cadherin expression. The result showed that miR-21 had the effect of reversing fibrosis, and can be used for the treatment of the diseases caused by intrauterine adhesion and thin endometrium and/or uterine fibrosis, or the preparation of the drug for treating the diseases caused by intrauterine adhesion, thin endometrium and/or uterine fibrosis.