WO2023206742A1 - Mirna molecule mir-206 for regulating and controlling uterine muscle contraction and use thereof - Google Patents

Abstract

Provided are a miRNA molecule miR-206 for regulating and controlling uterine muscle contraction and use thereof, particularly use of miR-206, a precursor of miR-206, or a miR-206 mimic in preparing a drug for preventing and/or treating premature birth.

Description

A kind of miRNA molecule miR-206 that regulates uterine muscle contraction and its application Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a miRNA molecule miR-206 that regulates uterine muscle contraction and its application.

Background technique

Premature birth refers to delivery between 28 and 37 weeks of gestation and is the main cause of adverse perinatal outcomes. Every year, more than 1 million newborns die worldwide due to premature birth and related complications. China's premature birth rate is about 5% to 15%, and more than 1.17 million premature babies are born every year. Neonatal death and severe injuries related to premature birth are serious medical and social problems. The core links of spontaneous preterm birth include premature cervical ripening, decidua activation and uterine contraction caused by various factors. At present, the drugs used in clinical treatment of premature labor are mainly chemical tocolytic inhibitors, such as ritodrine hydrochloride, nifedipine and atosiban. They have their own contraindications and most of them are only used to extend the gestational age for 48 hours to complete the promotion. When the fetal lungs are mature, the above drugs cannot achieve the effect of preventing and treating premature birth. There is an urgent need to find new highly specific biomolecule targets to provide new strategies for the prevention and treatment of premature birth.

MicroRNA (miRNA) is an endogenous short-chain non-coding RNA with a size of 20 to 23 bp. It specifically binds to the 3'-untranslated region (3'-UTR) of the target gene mRNA through the 5' end. and exert post-transcriptional regulatory effects. miRNA can specifically regulate the expression of target genes and plays an important role in the prevention and treatment of diseases. miRNA-based gene therapy can be used as an effective treatment for premature birth.

Contents of the invention

The purpose of the first aspect of the present invention is to provide the application of miR-206 in the preparation of drugs for preventing and/or treating premature birth.

The purpose of the second aspect of the present invention is to provide the use of nucleic acid molecules expressing miR-206 or the precursor of miR-206 in the preparation of drugs for preventing and/or treating premature birth.

The third aspect of the present invention aims to provide expression cassettes, vectors, and cell lines containing miR-206 nucleic acid molecules for use in the preparation of drugs for preventing and/or treating premature birth.

The purpose of the fourth aspect of the present invention is to provide the use of reagents or compositions that target up-regulation of miR-206 in the preparation of drugs for preventing and/or treating premature birth.

The purpose of the fifth aspect of the present invention is to provide the application of miR-206 in preparing GJA1 inhibitors.

The object of the sixth aspect of the present invention is to provide a medicine for treating premature labor or inhibiting uterine muscle contraction.

The purpose of the seventh aspect of the present invention is to provide the application of a reagent for detecting the expression of miR-206 in the preparation of products for diagnosing premature birth.

The purpose of the eighth aspect of the present invention is to provide the application of miR-206 as a target in screening drugs for diagnosis of premature birth.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

The first aspect of the present invention provides the application of miR-206 in the preparation of drugs for preventing and/or treating premature birth;

Application of the precursor of miR-206 in the preparation of drugs to prevent and/or treat premature birth;

Application of miR-206 mimetics in the preparation of drugs for the prevention and/or treatment of premature birth.

Preferably, the nucleotide sequence of miR-206 is 5'-UGGAAUGUAAGGAAGUGUGUGG-3' (SEQ ID No. 1).

Preferably, the miR-206 inhibits the contractile function of uterine smooth muscle.

Through transcriptome sequencing analysis and experimental verification of human uterine muscle cells overexpressing miR-206, the present invention found that miR-206 is lowly expressed in uterine muscle tissue during labor, and overexpression of miR-206 can significantly inhibit uterine smooth muscle in vitro. The expression of the key contraction protein GJA1 thereby reduces the contractile function of uterine smooth muscle cells. Overexpression of miR-206 in premature animal models can effectively reduce the occurrence of premature birth.

Preferably, the miRNA mimics (miRNA mimics/agomir) simulate the endogenous miRNA of the organism and are synthesized using chemical synthesis methods, which can enhance the function of the endogenous miRNA. Specifically, the miR-206 mimic is a small fragment of double-stranded miRNA designed and synthesized for the mature form of miR-206. Its function is the same as that of mature miR-206 in vivo, and it can upregulate the content of corresponding miR-206 in cells. , enhance the function of endogenous miR-206.

Preferably, the miR-206 of the present invention includes functional equivalents of the constitutive nucleic acid molecule, i.e., variants, which exhibit the same functions of the complete miR-206 nucleic acid molecule, which may be obtained by deletion, substitution or insertion of nucleotide residues. mutation.

It is well known to those in the field that in order to ensure the stability of miRNA, protective bases, such as TT, can be added to one or both ends of the miRNA; the bases of the miRNA can also be modified, but without affecting the function of the miRNA. Therefore, those skilled in the art are well aware that the sequence obtained by base modification of miR-206 or by adding bases at both ends without affecting the function of miR-206 is also included in the protection scope of the present invention. That is, the so-called Said miR-206 can be modified by any one or more ribonucleotides in the sequence shown in SEQ ID No.1.

Preferably, the miR-206 is a mature miRNA, precursor miRNA or primary transcript.

Preferably, the nucleic acid molecule of miR-206 can exist in a single-stranded or double-stranded form. Mature miR-206 is mainly in a single-stranded form, while the precursor of miR-206 is partially self-complementary to form a double-stranded form. structure. The nucleic acid molecule of miR-206 can be in the form of RNA, DNA, PNA, or LNA.

Preferably, the drug achieves the purpose of preventing and/or treating premature birth by inhibiting the expression of GJA1.

Preferably, the drug inhibits the expression of GJA1 in cells by increasing the content/expression of miR-206 or its mimic in uterine smooth muscle cells.

Preferably, the method for increasing the content of miR-206 in uterine smooth muscle cells is: using a cell transfection reagent to wrap the nucleotide fragment of miR-206 or its mimic, and transfecting the uterine smooth muscle cells.

Preferably, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, Adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorbent carriers.

Preferably, the drug can be prepared into various dosage forms as needed, including but not limited to solutions, tablets, granules, patches, capsules, liposome-encapsulated RNA, bacterial-borne plasmid expression RNA methods, or viral packaging expression. RNA method, nanomaterial assembly, ointment, suppository.

Preferably, the administration route of the drug is not limited, including but not limited to intraperitoneal injection, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal spray, rectal or vaginal infusion, oral spray, uterine injection, For topical or systemic transdermal application to the skin.

A second aspect of the present invention provides the use of nucleic acid molecules expressing miR-206 or a precursor of miR-206 in the preparation of drugs for preventing and/or treating premature birth.

Preferably, the drug achieves the purpose of preventing and/or treating premature birth by inhibiting the expression of GJA1.

Preferably, the drug inhibits the expression of GJA1 in cells by increasing the content/expression of miR-206 or its mimic in uterine smooth muscle cells.

Preferably, the method for increasing the content of miR-206 in uterine smooth muscle cells is: using a cell transfection reagent to wrap the nucleotide fragment of miR-206 or its mimic, and transfecting the uterine smooth muscle cells.

Preferably, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, Adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorbent carriers.

Preferably, the drug can be prepared into various dosage forms as needed, including but not limited to solutions, tablets, granules, patches, capsules, liposome-encapsulated RNA, bacterial-borne plasmid expression RNA methods, or viral packaging expression. RNA method, nanomaterial assembly, ointment, suppository.

Preferably, the administration route of the drug is not limited, including but not limited to intraperitoneal injection, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal spray, rectal or vaginal infusion, oral spray, uterine injection, For topical or systemic transdermal application to the skin.

The third aspect of the present invention provides the use of expression cassettes, vectors, and cell lines containing miR-206 nucleic acid molecules in the preparation of drugs for preventing and/or treating premature birth.

Preferably, the vector includes a plasmid vector, a viral vector or a eukaryotic expression vector.

Preferably, the plasmid vector may be an optional plasmid and the eukaryotic expression vector does not include propagation material.

Preferably, the viral vector can be any suitable vector, including but not limited to retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) vectors, alphavirus vectors .

Preferably, the eukaryotic expression vector can be any appropriate expression vector, including but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector, pTRE expression vector, or a modified vector based on a known expression vector, such as pBin438, pCAMBIA1301, etc.

Preferably, the drug achieves the purpose of preventing and/or treating premature birth by inhibiting the expression of GJA1.

Preferably, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, Adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorbent carriers.

Preferably, the drug can be prepared into various dosage forms as needed, including but not limited to solutions, tablets, granules, patches, capsules, liposome-encapsulated RNA, bacterial-borne plasmid expression RNA methods, or viral packaging expression. RNA method, nanomaterial assembly, ointment, suppository.

Preferably, the administration route of the drug is not limited, including but not limited to intraperitoneal injection, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal spray, rectal or vaginal infusion, oral spray, uterine injection, For topical or systemic transdermal application to the skin.

The fourth aspect of the present invention provides the use of reagents or compositions that target up-regulation of miR-206 in the preparation of drugs for preventing and/or treating premature birth;

Application of agents or compositions that enhance the activity of miR-206 in the preparation of drugs for preventing and/or treating premature birth.

Preferably, the drug achieves the purpose of preventing and/or treating premature birth by inhibiting the expression of GJA1.

Preferably, the drug inhibits the expression of GJA1 in cells by increasing the content/expression of miR-206 or its mimic in uterine smooth muscle cells.

Preferably, the method for increasing the content of miR-206 in uterine smooth muscle cells is: using a cell transfection reagent to wrap the nucleotide fragment of miR-206 or its mimic, and transfecting the uterine smooth muscle cells.

Preferably, the agent that upregulates miR-206 or enhances the activity of miR-206 can be a protein, an oligonucleotide, or a small molecule compound.

Preferably, the reagent can be a mimic of miR-206, an agonist of miR-206, a precursor of miR-206, or a vector carrying miR-206.

MiR-206 mimics or agonists are designed based on its sequence. MiR-206 agonists are specially labeled and chemically modified double-stranded small RNAs. After the miR-206 mimics or agonists are transferred into the human body, they Can significantly upregulate the expression of miR-206.

Preferably, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, Adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorbent carriers.

Preferably, the drug can be prepared into various dosage forms as needed, including but not limited to solutions, tablets, granules, patches, capsules, liposome-encapsulated RNA, bacterial-borne plasmid expression RNA methods, or viral packaging expression. RNA method, nanomaterial assembly, ointment, suppository.

Preferably, the administration route of the drug is not limited, including but not limited to intraperitoneal injection, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal spray, rectal or vaginal infusion, oral spray, uterine injection, For topical or systemic transdermal application to the skin.

The fifth aspect of the present invention provides the application of miR-206 in preparing GJA1 inhibitors;

Application of the precursor of miR-206 in the preparation of GJA1 inhibitors;

Application of miR-206 mimics in the preparation of GJA1 inhibitors;

Application of reagents targeting up-regulation of miR-206 in the preparation of GJA1 inhibitors;

Application of reagents that target the activity of miR-206 in the preparation of GJA1 inhibitors.

The seventh aspect of the present invention provides a drug for treating premature birth or inhibiting uterine muscle contraction, which contains an agent or composition that targets up-regulating miR-206 or enhancing the activity of miR-206.

Preferably, the agent that upregulates miR-206 or enhances the activity of miR-206 can be a protein, an oligonucleotide, or a small molecule compound.

Preferably, the reagent can be a mimic of miR-206, an agonist of miR-206, a precursor of miR-206, or a vector carrying miR-206.

Preferably, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, Adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorbent carriers.

Preferably, the drug can be prepared into various dosage forms as needed, including but not limited to solutions, tablets, granules, patches, capsules, liposome-encapsulated RNA, bacterial-borne plasmid expression RNA methods, or viral packaging expression. RNA method, nanomaterial assembly, ointment, suppository.

Preferably, the administration route of the drug is not limited, including but not limited to intraperitoneal injection, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal spray, rectal or vaginal infusion, oral spray, uterine injection, For topical or systemic transdermal application to the skin.

A seventh aspect of the present invention provides the use of a reagent for detecting the expression of miR-206 in the preparation of products for diagnosing premature birth.

Preferably, the product includes a chip or a kit.

Preferably, the chip includes a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier.

Further preferably, the oligonucleotide probe includes part or all of the sequence specifically corresponding to miR-206.

Preferably, the kit includes reagents for detecting the expression level of miR-206.

Preferably, the kit also contains a primer set for amplifying miR-206.

Further preferably, the primer set of miR-206 is as follows:

Upstream primer: 5’-GGCGGTGGAATGTAAGGAAG-3’ (SEQ ID No. 4).

Downstream primer: 5’-GGCTGTCGTGGACTGCG-3’ (SEQ ID No. 5).

Preferably, the oligonucleotide probes may also include oligonucleotide probes targeting miRNAs that have been reported in the prior art and can be used to diagnose premature birth. The joint diagnosis of premature birth by detecting multiple miRNA indicators by placing multiple miRNA detection probes on the same chip is also within the scope of the present invention.

Preferably, the reagent further includes primers and/or probes targeting miRNAs that have been reported in the prior art for diagnosing premature birth. The joint diagnosis of premature birth by detecting multiple miRNA indicators by placing multiple miRNA detection primers and/or probes in the same kit is also within the scope of the present invention.

Preferably, the solid phase carrier includes various commonly used materials in the field of gene chips, such as but not limited to nylon membrane, glass slide or silicon modified with active groups (such as aldehyde groups, amino groups, etc.) slides, unmodified glass slides, plastic sheets, etc.

The eighth aspect of the present invention provides the application of miR-206 as a target in screening drugs for diagnosis of premature birth.

The beneficial effects of the present invention are:

The present invention discloses for the first time miR-206 as a therapeutic target for preterm birth. It has not been reported before that miR-206 is related to preterm birth. Overexpression of miR-206 can significantly inhibit the contractility of uterine smooth muscle cells, thereby preventing and treating premature birth. role in reducing the incidence of premature birth and the number of premature babies.

By detecting the expression level of miR-206 in subjects, it can be determined whether the subjects are at risk of premature birth, thereby guiding clinicians to provide prevention or treatment options to the subjects.

Description of the drawings

Figure 1 is a volcano plot of miRNA expression in uterine muscle tissue before labor and labor, and labeled miRNAs are differentially expressed miRNAs.

Figure 2 shows the ROC curves of term pregnant women who are not in labor and term pregnant women who are in labor.

Figure 3 is a statistical graph showing the correlation between miR-206 expression and GJA1 expression in uterine smooth muscle tissue during labor.

Figure 4 is a schematic diagram of the sequence of the luciferase reporter vector GJA1 (upper picture) and a statistical diagram of the dual-luciferase activity detection results (lower picture).

Figure 5 shows the expression of miR-206 and GJA1 in uterine smooth muscle cells in different treatment groups, ** represents p<0.01.

Figure 6 is a schematic diagram of the treatment methods of different treatment groups of CD1 mice.

Figure 7 is a statistical graph of pregnancy duration results of CD1 mice in different treatment groups.

Figure 8 shows fetal photos (upper picture), fetal weight statistics (lower left picture) and length (lower right picture) of CD1 mice in different treatment groups.

Figure 9 is a graph showing GJA1 protein detection of CD1 mice in different treatment groups.

Figure 10 shows the results of the effect of miR-206 on uterine muscle contraction. The upper left picture is the integral statistical diagram under the contraction wave curve, the upper right picture is the contraction amplitude statistical diagram, and the lower picture is the uterine muscle contraction waveform diagram.

Detailed ways

The present invention will now be described in detail with reference to specific embodiments, but is not intended to limit the scope of the present invention.

The materials and reagents used in this example are materials and reagents obtained from commercial sources unless otherwise specified.

In the present invention, the "complementary" mentioned may be completely complementary or partially complementary. The complete complementarity means that the sequences are completely matched and no sticky ends are formed. The partial complementarity means that the sequences are completely matched, but sticky ends are formed.

The RNA in the present invention is microRNA (microRNA, or miRNA), which refers to a single-stranded oligoribonucleotide. Ribonucleotides are long chain molecules formed by condensation of nucleotides through phosphodiester bonds. Ribonucleotide molecules are composed of one base molecule, one ribose sugar molecule, and phosphate.

In the present invention, the "modification" refers to any one or more groups or combinations thereof connected to ribose and/or bases in the RNA molecular chain provided by the present invention or its complementary chain.

Example 1 Expression of miRNA-206 in laboring uterine muscle

1. Collect samples

The inventor collected human uterine muscle tissue from full-term pregnant women who were not in labor and full-term pregnant women who underwent cesarean section at Guangzhou Women and Children's Medical Center. The human uterine muscle tissue was taken from the edge of the uterine muscle incision during cesarean section. , the above samples are samples to exclude pregnancy complications such as gestational diabetes, eclampsia, etc. Among them, the human uterine muscle tissue of full-term pregnant women (10 cases) was used as the control group, and the human uterine muscle tissue of full-term pregnant women (10 cases) was used as a control group. As the observation group, there was no significant difference in the BMI value and age of pregnant women in the control group and the observation group. Informed consent was obtained from each pregnant woman beforehand, and all samples obtained were approved by the organization's ethics committee.

2. Extraction and sequencing of tissue RNA

The collected human uterine muscle tissue was washed and cut into pieces. Total RNA from the human uterine muscle tissue was extracted using Trizol reagent (Invitrogen, Cat. No. 15596018), and human uterine muscle miRNA was purified using PAGE. The obtained miRNA was subjected to high-throughput sequencing and library construction. The sequenced reads were compared by Bowtie2, quantified using featureCounts software, and gene difference analysis was performed using DESeq2 software. The threshold was (p<0.01,|fold change|> 2).

The results are shown in Figure 1. Compared with the non-labor group, uterine muscle miR-206, miR-196a-5p and miRNA-615-3p were significantly down-regulated in the labor group, and miR-206 was the most significantly differentially expressed miRNA, indicating that in the labor group The expression of miR-206 is low in the human uterine muscle tissue of pregnant women at term. The subject's gene sequence was subjected to Receiver Operating Characteristic Curve (ROC) analysis, and the results showed (Figure 2) that miR-206 has high sensitivity and specificity in identifying labor (AUC=0.8333, p=0.0055) .

The nucleotide sequence of miRNA-206 is: 5’-UGGAAUGUAAGGAAGUGUGUGG-3’ (SEQ ID No. 1).

MiRNA mimics mimic the endogenous miRNA of organisms and are synthesized using chemical synthesis methods, which can enhance the function of endogenous miRNA. Specifically, miRNA-206 mimics, which are mimics of miR-206, are small fragments of double-stranded miRNA designed and synthesized for the mature form of miR-206. They have the same effect as mature miR-206 in vivo and can upregulate the corresponding miR-206 in cells. The content of 206 enhances the function of endogenous miR-206. Mimics of miR-206 contain the sequence shown in SEQ ID No. 1 and/or its complementary sequence.

miRNA-206 mimics: positive strand 5’-UGGAAUGUAAGGAAGUGUGUGG-3’ (SEQ ID No. 2), reverse strand 5’-CCACACACUUCCUUACAUUCCA-3’ (SEQ ID No. 3).

Example 2 Effect of mimics of miR-206 on GJA1 expression in uterine smooth muscle cells

1. Primary culture of uterine smooth muscle cells

Collect human uterine smooth muscle tissue from caesarean section, wash it with Hanks solution to remove surface blood stains, and use surgical tweezers to remove adherent connective tissue and other non-cultured tissues; after cleaning again, use a scalpel to cut the tissue into several small pieces , move into a penicillin vial or small beaker, add PBS, use elbow ophthalmic scissors to repeatedly cut the tissue until the tissue becomes a paste, about ^1mm3 in size. After letting it stand for a while, use a pipette to suck off the upper liquid, add appropriate buffer and wash again; use trypsin (the volume ratio of trypsin to tissue block is 1:0.25) to digest and separate the small tissue blocks into cell clusters or dispersed cells. Single cells are collected to facilitate further culture. Collect the cell suspension and use a counting board to count the cells; aliquot them into culture bottles containing DMEM medium containing 10v/v% FBS (cell confluence is about 30%), and place in CO ₂ In the incubator, 5% CO ₂ was used for static culture at 37°C; when the cell confluence reached 80% to 90%, the cells were digested and passaged with 0.25% trypsin to obtain human uterine smooth muscle cells for later use.

2. Prediction and verification of the binding of miRNA-206 to downstream GJA1

The miRNA target gene prediction tool miRanda was used to predict the binding site of miR-206 and GJA1, and analysis of the labor uterine muscle transcriptome revealed that the expression of miRNA-206 was negatively correlated with GJA1 (Figure 3). Further select a fragment of about 150 bp upstream and downstream of the binding site and clone it downstream of the luciferase reporter gene to construct the luciferase reporter vector Luc-GJA1-wild type; use the principle of changing the base arrangement to construct a mutant luciferin Enzyme reporter vector Luc-GJA1-mutant type; wild-type and mutant vectors were co-transfected with miRNA-206 mimics into HEK293 cells, and 36 hours after transfection, a dual-luciferase reporter gene detection system was used to detect Firefly and Renilla luciferin. enzyme activity.

As shown in Figure 4, up-regulation of miR-206 expression can significantly reduce luciferase activity ((mean±SEM) (n=3), **p<0.005), and after mutating the recognition site, miR-206 has no effect on fluorescence. Loss of inhibition of prime enzyme activity.

3. Transfection of miRNA-206 mimics via lentivirus

The sequences of miRNA-206 and negative control NC mimics (5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID No. 10)) were combined with the GV309 vector to construct a lentiviral expression vector, and the miRNA-206 and negative control were combined using lentiviral infection technology. The lentiviral expression vector of NC Mimics was used to infect primary human uterine smooth muscle cells with an MOI (multiplicity of infection) value of 50; human uterine smooth muscle cells not infected with the lentiviral expression vector were used as a blank control group. Three days after the cells were infected with lentivirus, the total RNA of human uterine smooth muscle cells was extracted using the kit RNeasy Plus Mini Kit (ESscience, Cat. No. RN001), and the reverse transcription kit Fast miRNA Reverse Transcription Kit (ESscience, Cat. No. miR002) was used. ), PrimeScript RT Master Mix (TAKARA, Cat. No. RR036A) was used to reverse-transcribe miRNA and mRNA, and TB Green Premix Ex Taq II (TAKARA, Cat. No. RR820A) was used to perform real-time quantitative PCR to detect and calculate miR-206 and GJA1. Express the situation. Among them, the primers used in real-time quantitative PCR of miR-206 and GJA1 are: miR-206-F: 5'-GGCGGTGGAATGTAAGGAAG-3' (SEQ ID No. 4), miR-206-R: 5'-GGCTGTCGTGGACTGCG-3' (SEQ ID No. 5), GJA1-F: 5'-GGTGGACTGTTTCCTCCTCG-3' (SEQ ID No. 6), GJA1-R: 5'-GGAGCAGCCATTGAAATAAGC-3' (SEQ ID No. 7).

The results showed that there was no significant difference in the expression of miR-206 and GJA1 in human uterine smooth muscle cells infected with the negative control lentiviral expression vector and the blank control group not infected with the lentiviral expression vector; while when human uterine smooth muscle cells were After infection with the miRNA-206 mimics lentiviral expression vector, the expression of miR-206 in human uterine smooth muscle cells increased significantly, and the expression of GJA1 decreased significantly (Figure 5).

Example 3 Therapeutic effect of miRNA-206 mimic in premature animal model

In order to obtain the overexpression of miR-206, we used agomir of miR-206. agomir is a specially chemically modified double-stranded small RNA that regulates the biological function of the corresponding target gene by simulating endogenous miR-206. The sense strand base The base is Ome (methoxy) modified, and the antisense chain is modified with Chol (cholesterol). It can exist stably in mice and is easily taken up by cells. The corresponding agomir NC is used as a negative control. The sequence of agomiR-206 is: forward strand 5’-UGGAAUGUAAGGAAGUGUGUGG-3’ (SEQ ID No. 8), reverse strand 5’-chol-CCACACACUUCCUUACAUUCCA-3’ (SEQ ID No. 9).

CD1 mice (6 to 8 weeks old) were used to construct a premature birth model using mifepristone RU486. On the 15th day of pregnancy, RU486 (200 μg/kg/d, mixed with sesame oil to make 100 μL) was subcutaneously injected into the mice. Premature labor occurs within the next 20 hours. The specific steps are as follows: Divide CD1 mice into 7 groups, with 6 mice in each group, which are respectively the blank control group (NP group, mice are not treated with any treatment, normal pregnancy), and the sesame oil treatment group (on the 15th day of pregnancy of CD1 mice). subcutaneous injection of 100 μL sesame oil), RU486 treatment group (subcutaneous injection of RU486 on the 15th day of pregnancy of CD1 mice), agomiR-NC+RU486 co-treatment group (administration of agomiR-NC+RU486 to mice for three consecutive days on the 11th, 12th, and 13th day of pregnancy of CD1 mice). agomiR-NC was injected into the tail vein, 80mg/kg/d, mixed with normal saline to 100uL; RU486 was injected subcutaneously on the 15th day), miR-206 and RU486 were co-treated in group I (i.e., the high-dose miR-206 + RU486 group, in On the 11th, 12th and 13th days of pregnancy of CD1 mice, agomiR-206 was injected into the tail vein of the mice for 3 consecutive days, 80mg/kg/d, mixed with physiological saline to make 100uL; on the 15th day, RU486), miR-206 and RU486 co-treatment group II (i.e., low-dose miR-206 + RU486 group), agomiR-206 was injected into the tail vein of CD1 mice for three consecutive days on the 11th, 12th, and 13th days of pregnancy, 40 mg/kg/d, with normal saline. The configuration is 100uL; RU486 is injected subcutaneously on the 15th day) and the miR-206 treatment group (agomiR-206 is injected into the tail vein of the mice for 3 consecutive days on the 11th, 12th and 13th days of pregnancy of CD1 mice, 80mg/kg/d, Use physiological saline to prepare 100uL) (Figure 6). After treating each mouse according to the above grouping, the pregnancy duration and fetal outcome of each CD1 mouse were observed and counted.

The pregnancy duration and fetal outcome of each CD1 mouse were observed and counted. The results showed that the pregnancy duration of the mice in the miR-206 treatment group was not significantly different from that of the mice in the blank control group; three days of continuous injection of RU486 before the injection of RU486 The dose (80mg/kg/d) of agomiR-206 can significantly extend the pregnancy duration of premature model mice, and can even reach full-term delivery, and increase the fetal survival rate; a low dose (40mg/kg) was injected for three consecutive days before the injection of RU486 /d) agomiR-206 did not significantly extend the pregnancy duration of mice (Figure 7). By weighing and measuring the length of the fetuses of mice in the agomiR-NC+RU486 group, miR-206 low dose+RU486 group, and miR-206 high dose+RU486 group, the results showed that the miR-206 low dose+RU486 group and agomiR- There was no significant difference in fetal weight and length of mice in the NC+RU486 group, while fetal weight and length in the high-dose miR-206+RU486 group were significantly increased (Figure 8).

The uterine muscle strips of the maternal mice in the agomiR-NC+RU486 group, the low-dose miR-206+RU486 group, and the high-dose miR-206+RU486 group were collected, and the uterine muscle strips of each female mouse were detected by Western Blot. GJA1 protein expression, the results showed that agomiR-206 can effectively reduce the expression of GJA1 in the uterine muscle of maternal mice in a dose-dependent manner (Figure 9).

Further use mouse uterine muscle tissue to conduct muscle tension experiments, as follows: take uterine muscle strips from mice in the blank control group and the miR-206 treatment group, remove visible blood vessel tissue and other non-uterine smooth muscle tissue, and cut into 1*0.5*0.5 cm size, hang it in a tissue water bath, in which Krebs equilibrium solution (119mM NaCl, 25mM NaHCO ₃ , 1.2mM KH ₂ PO ₄ , 4.7mM KCl, 1.2mM MgSO ₄ ·H ₂ O, 0.026mM Na) was added in advance ₂ ·EDTA·2H ₂ O, 11.5mM glucose, 2.5mM CaCl ₂ ·H ₂ O), and preheat to 37°C to completely immerse the uterine muscle strips in Krebs balance solution, and turn on the gas mixture (95% O ₂ + 5% CO ₂ ) simulates the hypoxia-reoxygenation state of the laboring uterus, observe and adjust the initial tension value 1g through the Powerlab sensor, and balance contraction for 1 hour. Oxytocin (final concentration: 1 nM) was added to each tissue tank to trigger the contraction of the uterine muscle strip, and this time period was set as T0. Simulate the uterine hypoxia-reoxygenation state during labor: turn off the gas mixture, introduce 100% nitrogen to cause tissue hypoxia for 10 to 15 minutes, then re-introduce the gas mixture for 30 to 45 minutes, and repeat this cycle 3 times. Set the reoxygenation sections of each cycle as T1, T2 and T3 respectively. The signal was received through PowerLab PL3508 throughout the process, and the contraction parameters of the uterine muscle strip were recorded by Labchart Pro V8.0.7. The results showed that compared with CD1 mice that were not treated with agomiR-206, the contraction intensity and contraction amplitude of the uterine muscle strips of CD1 mice treated with agomiR-206 were significantly reduced (p<0.05) (Figure 10).

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various modifications can be made without departing from the purpose of the present invention. Variety. In addition, the embodiments of the present invention and the features in the embodiments may be combined with each other without conflict.

Claims (10)

1. Application of at least one of (1) to (3) in the preparation of drugs for preventing and/or treating premature birth;

   (1)miR-206;

   (2) Precursor of miR-206;

   (3)miR-206 mimics.
2. Application of nucleic acid molecules expressing miR-206 or a precursor of miR-206 in the preparation of drugs for preventing and/or treating premature birth.
3. Application of expression cassettes, vectors, and cell lines containing miR-206 nucleic acid molecules in the preparation of drugs for preventing and/or treating premature birth.
4. (4) or (5) application in the preparation of drugs for the prevention and/or treatment of premature birth;

   (4) Reagents or compositions that target up-regulation of miR-206;

   (5) Agents or compositions that enhance the activity of miR-206.
5. The application according to any one of claims 1 to 4, characterized in that the drug achieves the purpose of preventing and/or treating premature birth by inhibiting the expression of GJA1.
6. Use of at least one of (6) to (10) in preparing a GJA1 inhibitor;

   (6)miR-206;

   (7) Precursor of miR-206;

   (8)miR-206 mimic;

   (9) Reagents that target the upregulation of miR-206;

   (10) Agents that target the activity of miR-206.
7. A medicine for treating premature birth or inhibiting uterine muscle contraction, characterized in that the medicine contains an agent or composition that targets up-regulation of miR-206 or enhances the activity of miR-206.
8. Application of reagents for detecting the expression of miR-206 in the preparation of products for diagnosing premature birth.
9. The application according to claim 8, wherein the product includes a chip or a kit. Preferably, the chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier; The kit preferably includes reagents for detecting the expression level of miR-206; more preferably, the kit also includes a primer set for amplifying miR-206.
10. Application of miR-206 as a target in screening drugs for diagnosis of premature birth.

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