阅读说明：本技术 miR-431作为靶点在制备促进SP表达的药物中的应用 (Application of miR-431 as target point in preparation of medicine for promoting SP expression ) 是由 杨洋 沈艳青 程锐 于 2019-10-08 设计创作，主要内容包括：本发明公开了miR-431作为靶点在制备促进SP表达的药物中的应用。MicroRNA-431作为治疗靶点在制备由肺表面活性蛋白不足所致疾病的药物中的应用。抑制miR-431表达的物质通过直接靶向SMAD4抑制BMP4/活化素/TGF-β信号通路来促进肺表面活性蛋白的表达,从而在制备由肺表面活性蛋白不足所致疾病的药物中的应用。本发明探索了miR-431及其靶基因-SMAD4的功能。数据表明抑制miR-431可通过直接靶向SMAD4抑制BMP4/活化素/TGF-β信号通路来促进SP的表达。该研究提供了miR-431的重要分子和细胞基础,作为胎儿肺发育的潜在标志物和肺发育疾病的潜在治疗靶标之一。(The invention discloses application of miR-431 as a target point in preparation of a medicament for promoting SP expression. Application of MicroRNA-431 as a therapeutic target in preparation of medicines for treating diseases caused by deficiency of lung surfactant protein. The substance for inhibiting miR-431 expression can be used for inhibiting BMP 4/activin/TGF-beta signal pathway by directly targeting SMAD4 to promote the expression of lung surfactant protein, so that the substance can be applied to the preparation of medicines for treating diseases caused by lung surfactant protein deficiency. The invention explores the functions of miR-431 and a target gene SMAD4 thereof. The data indicate that inhibition of miR-431 can promote expression of SP by inhibiting the BMP4/activin/TGF- β signaling pathway by directly targeting SMAD 4. The research provides important molecules and cellular basis of miR-431, and the miR-431 is used as a potential marker of fetal lung development and one of potential therapeutic targets of lung development diseases.)

Application of MicroRNA-431 as a therapeutic target in preparation of medicines for treating diseases caused by deficiency of lung surfactant protein.

2. The use of claim 1, wherein the miR-431 expression inhibitor inhibits BMP 4/activin/TGF-beta signaling pathway through direct targeting of SMAD4 to promote expression of lung surfactant protein, so that the use of the miR-431 expression inhibitor in preparation of medicines for treating diseases caused by lung surfactant protein deficiency is realized.

3. Use according to claim 1 or 2, characterized in that the disease caused by a deficiency in lung surfactant protein is a pulmonary developmental disease, preferably neonatal respiratory distress syndrome.

4. Application of a substance for inhibiting miR-431 expression in preparation of a medicament for promoting lung surfactant protein expression.

5. The use according to claim 3, characterized in that the substance inhibiting miR-431 expression promotes the expression of lung surfactant protein by inhibiting BMP4/activin/TGF- β signaling pathway by directly targeting SMAD 4.

Technical Field

The invention belongs to the field of biological medicines, and relates to application of miR-431 as a target point in preparation of a medicine for promoting SP expression.

Background

Preterm birth rates have generally increased by about one-third (1) over the last 25 years. Most premature infants live in the saccular phase of lung development (26 to 36 gestational weeks), while lung structures are immature, Surfactant Proteins (SPs) are inefficient, fluid absorption is delayed in the lungs and gas exchange is inefficient. Evidence has accumulated over the years that infants born less than 37 weeks have consistently high respiratory morbidity. Lung development is a complex process, accompanied by a series of carefully planned events. Premature delivery disrupts normal lung development, leading to a range of respiratory diseases. Neonatal Respiratory Distress Syndrome (RDS) is a condition of pulmonary insufficiency which begins in the natural process at or shortly after birth and increases in severity within the first 48 hours after birth, a common disease of premature infants. Early/moderate preterm infants (23-33 weeks gestation) have an RDS incidence of about 45%, whereas preterm infants (34-36 weeks gestation) account for about 4% and neonatal infants about 1% (more than 37 weeks gestation)). The main causes of RDS are surfactant protein deficiency and structural immaturity. Therefore, improved understanding of lung development may lead to a desire for better prevention and treatment of respiratory diseases in the future.

SMAD4, a common SMAD of the SMAD family, is a common mediator of the TGF- β signaling pathway. Previous studies reported that in the classical signaling pathway, activated Smad2 and Smad3 form complexes with Smad4, Smad4 is a component common to all TGF- β family members, and such complexes localize trans-nuclear to regulate transcription of target genes. Notably, there is increasing evidence that the SMAD 4-dependent TGF- β signaling pathway plays an important role in the differentiation of endodermal cells and in the inhibition of epithelial-to-mesenchymal transition (EMT), which is critical for pulmonary branch morphogenesis. Bone morphogenetic protein 4 belongs to the TGF-beta superfamily, is a multifunctional peptide and plays an important role in normal lung development.

Micrornas (mirnas) are a class of small, about 22-nucleotide long, non-coding RNAs that inhibit translation of target mrnas, and play important roles in cell proliferation, cell differentiation, and organ development. Several studies have demonstrated that over 100 miRNAs show significant changes in expression during lung development. However, little is known about whether or how they regulate lung development.

Disclosure of Invention

The invention aims to overcome the blank in the prior art and provides application of MicroRNA-431 as a therapeutic target in preparing a medicament for treating diseases caused by deficiency of lung surfactant protein.

The invention also aims to provide application of the miR-431 expression inhibition substance in preparation of a medicament for promoting lung surfactant protein expression.

The purpose of the invention can be realized by the following technical scheme:

application of MicroRNA-431 as a therapeutic target in preparation of medicines for treating diseases caused by deficiency of lung surfactant protein.

The application is preferably that the substance for inhibiting miR-431 expression promotes the expression of the lung surfactant protein by directly targeting SMAD4 to inhibit BMP 4/activin/TGF-beta signal channel, so that the application in preparing the medicine for treating diseases caused by lung surfactant protein deficiency is realized.

The disease caused by the deficiency of the lung surfactant protein is a lung development disease, preferably neonatal respiratory distress syndrome.

Application of a substance for inhibiting miR-431 expression in preparation of a medicament for promoting lung surfactant protein expression.

Preferably, the substance for inhibiting miR-431 expression inhibits BMP 4/activin/TGF-beta signal channel through directly targeting SMAD4 to promote the expression of lung surfactant protein.

Has the advantages that:

the invention explores the functions of miR-431 and a target gene SMAD4 thereof. Our data suggest that inhibition of miR-431 can promote expression of SP by inhibiting the BMP4/activin/TGF- β signaling pathway by directly targeting SMAD 4. In addition, the research provides important molecular and cellular basis of miR-431 as a potential marker of fetal lung development and one of potential therapeutic targets of lung development diseases.

Drawings

FIG. 1 shows the state of cells under light and fluorescence microscopy at 100-fold magnification A549 cells at different concentrations of puromycin, we found that when the cells were completely killed, the optimal puromycin concentration was 1.2. mu.g/ml. Panel B shows representative pictures of four types of lentiviruses (LV-SMAD4+, LV-SMAD4+ -NC, LV-SMAD 4-and LV-SMAD4-NC) infecting the A549 cell line after 72 hours and 96 hours.

FIG. 2 validation of lentiviruses in four types of stable cell lines by RT-PCR (LV-SMAD 4)⁺,LV-SMAD4⁺-NC,LV-SMAD4^-And LV-SMAD4^--NC) SMAD4, BMP4, SP and miR-431. (A) Expression of SMAD4mRNA in a lentiviral cell line; (B) expression of BMP4mRNA in a lentiviral cell strain; (C) expression of SP-A mRNA in lentiviral cell lines; (D) expression of SP-B mRNA in lentiviral cell lines; (E) expression of SP-C mRNA in lentiviral cell lines; (F) expression of miR-431mRNA in a lentiviral cell line. By t-test (^*P<0.05，^**P<0.001，^***P<0.0005，****P<0.0001) test the statistical significance of all real-time PCRs.

FIG. 3 detection of lentiviruses in four types of stable cell lines using Western blotting (LV-SMAD 4)⁺,LV-SMAD4⁺-NC,LV-SMAD4^-And LV-SMAD4^--NC) protein expression of SMAD4, BMP4 and SP. (A) Western blot results of SMAD4, BMP4 and SP (SP-A, SP-B and SP-C) were examined and β -actin was used as an internal control. (B) Relative expression of SMAD4 protein. The data demonstrate that lentiviruses are stable against four cell types (LV-SMAD 4)⁺,LV-SMAD4⁺-NC,LV-SMAD4^-And LV-SMAD4^--NC). (C) Relative expression of BMP4 protein. (D) Relative expression of SP-A protein. (E) Relative expression of SP-B protein. (F) In that respect Relative expression of SP-C protein. All Western blot assays shown were tested for statistical significance by t-test (^*P<0.05，^**P<0.001，^****P<0.0001)。

FIG. 4 relative expression levels of miR-431, BMP4, SPs and SMAD4 in LV-SMAD 4-stable cell lines transfected with miR-431 mimics, inhibitors and corresponding controls (micic-nc and inhibitor-nc) were determined by RT-PCR. (A) Expression of miR-431mRNA in LV-SMAD 4-cells confirmed the transfection efficiency of the mock, inhibitor and corresponding control. (B) BMP4mRNA expression in LV-SMAD 4-cells transfected with miR-431 mimics, inhibitors and corresponding controls. (C) SP-A mRNA expression. (D) SP-B mRNA expression. (E) SP-C mRNA expression. (F) SMAD4mRNA expression. By t-test (^*P<0.05，^**P<0.001，^***P<0.0005，^****P<0.0001) test the statistical significance of all real-time PCRs.

FIG. 5 LV-SMAD4 in transfection of miR-431 mimics, inhibitors and corresponding controls (mimic-nc and inhibitor-nc)^-Expression of BMP4 and SPs (SP-A, SP-B and SP-C) protein levels was verified in stable cell lines. (A) In that respect Western blot results of BMP4 and SP (SP-A, SP-B and SP-C), β -actin was used as an internal control. (B) Relative expression of BMP4 protein. (C) Relative expression of SP-A protein. (D) Relative expression of SP-B protein. (E) Relative expression of SP-C protein. All Western blot analyses shown were tested for statistical significance by t-test (^*P<0.05，^**P<0.001，^***P<0.0005)。

FIG. 6 determination of LV-SMAD4 transfected with miR-431 mimic, inhibitor and corresponding control (mimic-nc and inhibitor-nc) by RT-PCR⁺And stabilizing the relative expression amounts of miR-431, BMP4, SPs and SMAD4 in the cell line. (A) Expression of miR-431mRNA in LV-SMAD4+ cells confirmed the transfection efficiencies of the mock, inhibitor and corresponding control. (B) BMP4mRNA expression. (C) SP-A mRNA expression. (D) SP-B mRNA expression. (E) SP-C mRNA expression. (F) SMAD4mRNA expression. All shown real-time PCRs were tested for statistical significance by t-test (^*P<0.05，^**P<0.001，^***P<0.0005)。

FIG. 7 LV-SMAD4 transfected with miR-431 mimic, inhibitor and corresponding control (mimic-nc and inhibitor-nc) was determined by western blot⁺(iii) determination of BMP4, SMAD4 and SP in the protein level in the stable cell line of (1). (A) Western blot results of SMAD4, BMP4 and SP (SP-A, SP-B and SP-C) were examined and β -actin was used as an internal control. (B) Relative expression of BMP4 protein. (C) Relative expression of SP-A protein. (D) Relative expression of SP-B protein. (E) Relative expression of SP-C protein. (F) Relative expression of SMAD4 protein. All Western blot analyses shown were tested for statistical significance by t-test (^*P<0.05，^**P<0.001，^***P<0.0005)。

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