Use of an expression inhibitor of the resf1 gene in the preparation of a drug for treating organ fibrosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of an expression inhibitor of a RESF1 gene in preparation of a medicine for treating organ fibrosis. The application finds that in fibrotic tissues and TGF-beta 1 induced activated fibroblasts, the expression level of RESF1 is significantly increased, and further in-vivo and in-vitro functional experiments show that after specific knockdown of RESF1 by RNA interference technology, the expression of key markers of myofibroblasts can be significantly down-regulated, which proves that blocking the expression of RESF1 can effectively block the activation and transformation of fibroblasts into myofibroblasts, and then inhibit the migration and contraction ability of fibroblasts, and can significantly reduce the organ fibrosis area and pathological severity in an animal model in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of RESF1 gene expression inhibitors in the preparation of drugs for treating organ fibrosis. Background Technology

[0002] Organ fibrosis is a crucial pathological basis for the progression of many chronic diseases to end-stage organ failure, commonly affecting tissues and organs such as the heart, lungs, liver, kidneys, skin, and blood vessels. The core pathological features of fibrosis include abnormal fibroblast activation, excessive formation of myofibroblasts, excessive extracellular matrix deposition, and tissue remodeling. Long-term, persistent fibrosis can lead to decreased tissue elasticity, organ dysfunction, and even organ failure.

[0003] Fibroblasts play a crucial role in the development and progression of fibrosis. Under the stimulation of injury, inflammation, mechanical stress, or pro-fibrotic factors, resting fibroblasts can be activated and transform into myofibroblasts. Myofibroblasts typically exhibit elevated expression of fibrosis-related molecules such as α-SMA, POSTN, and COL1A1, and possess enhanced migration, contraction, and extracellular matrix secretion capabilities. Among these, the TGF-β signaling pathway is a classic and important pathway for inducing fibroblast activation and the fibrotic process.

[0004] Currently, clinical treatment options for fibrotic diseases are limited. Some anti-fibrotic drugs can be used for specific diseases, such as pirfenidone and nintedanib in idiopathic pulmonary fibrosis, but overall, they still suffer from limited indications, insufficient efficacy, numerous side effects, and difficulty in reversing existing fibrosis. Therefore, finding new fibrosis regulatory targets and developing more targeted therapeutic strategies is an important direction in the current treatment of fibrotic diseases. Existing technologies for treating organ fibrosis also include RNA interference (siRNA) therapies. siRNA technology can exert its therapeutic effect by specifically reducing the expression of target genes. Compared with traditional small molecule drugs, siRNA has advantages such as strong targeting, flexible design, and the ability to target gene targets that are difficult for traditional drugs to act on.

[0005] RESF1, or retroelement silencing factor 1, is a molecule associated with reverse transcription element silencing, chromatin structure regulation, and transcriptional regulation. Public database annotations indicate that RESF1 is predicted to possess histone-binding and histone methyltransferase-binding activities and may participate in the positive regulation of DNA methylation-dependent heterochromatin assembly, suggesting that RESF1 may affect the transcriptional activity of specific genes by regulating chromatin state and epigenetic modifications. RESF1 is predicted to be located in the cell nucleus, and its molecular function is related to transcriptional silencing, chromatin remodeling, and the maintenance of gene expression homeostasis. However, the effects of existing siRNA molecules designed targeting RESF1 on fibroblast activation, myofibroblast transformation, and organ fibrosis remain unclear. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of an inhibitor of the RESF1 gene expression in the preparation of drugs for treating organ fibrosis.

[0007] Application of RESF1 gene expression inhibitors in the preparation of drugs for treating organ fibrosis.

[0008] Preferably, the RESF1 gene includes the human RESF1 gene and its mammalian homologs.

[0009] Preferably, the organ fibrosis is pulmonary fibrosis.

[0010] Preferably, the drug is used to inhibit fibroblast activation.

[0011] Preferably, the drug is used to inhibit the migration and contraction ability of fibroblasts.

[0012] Preferably, the drug is used to reduce the expression of α-SMA, POSTN, and COL1A1.

[0013] Preferably, the drug comprises one or more siRNA, shRNA, antisense oligonucleotide, ribozyme, and gene editing tools targeting the RESF1 gene.

[0014] Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID NO.1~SEQ ID NO.2.

[0015] Preferably, the siRNA has two deoxythymidine residues attached to its 3' end.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovers that RESF1 plays an important role in fibroblast activation and fibrosis. By designing and screening siRNA molecules that target and silence RESF1, the expression of RESF1 can be effectively reduced, the transformation of fibroblasts into myofibroblasts can be inhibited, and the expression of fibrosis markers can be reduced, thereby producing an anti-fibrotic effect.

[0017] This invention proposes that silencing RESF1 can serve as an important target for regulating fibroblast activation and organ fibrosis. It provides a siRNA molecule that targets RESF1, reducing RESF1 expression through RNA interference, thus offering a novel nucleic acid drug candidate molecule for anti-fibrotic therapy.

[0018] RESF1 siRNA can reduce the expression of fibrosis markers such as α-SMA, POSTN, and COL1A1 in fibroblasts and inhibit the transformation of fibroblasts into myofibroblasts. It has the potential for multi-organ fibrosis applications. The siRNA is precisely designed based on the RESF1 mRNA sequence and has strong targeting compared with traditional small molecule drugs. It can be combined with existing delivery technologies such as LNP, liposomes, exosomes or tissue-targeting nanoparticles to improve the feasibility of in vivo application. Attached Figure Description

[0019] Figure 1 To reduce the expression level of RESF1 mRNA in siRESF1.

[0020] Figure 2 The expression level of RESF1 protein was reduced by siRESF1. In the figure, A represents the results of Westren Blot detection and B represents the statistical results.

[0021] Figure 3 siRESF1 knockdown reduced RESF1 and decreased the expression levels of TGFβ1-induced myofibroblast marker genes.

[0022] Figure 4 The expression level of α-SMA induced by TGFβ1 was reduced by siRESF1. In the figure, A represents the immunofluorescence result and B represents the statistical result.

[0023] Figure 5 The scratch assay showed that siRESF1 inhibited the migration ability of fibroblasts induced by TGFβ1. In the figure, A represents the scratch assay result and B represents the statistical result.

[0024] Figure 6 The Tanswell assay showed that siRESF1 inhibited the migration ability of fibroblasts induced by TGFβ1. In this assay, A represents the Tanswell assay results and B represents the statistical results.

[0025] Figure 7The gel shrinkage experiment showed that siRESF1 inhibited the contractile ability of fibroblasts induced by TGFβ1. In the figure, A represents the gel shrinkage experiment results and B represents the statistical results.

[0026] Figure 8 Intratracheal instillation of siRESF1-LNP reduced the severity of bleomycin-induced pulmonary fibrosis in mice. In the figure, A represents staining results and B represents statistical results. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0028] Organ fibrosis is a crucial pathological basis for the progression of many chronic diseases to end-stage organ failure. Its core characteristics include abnormal activation of fibroblasts, their transformation into myofibroblasts, and excessive deposition of extracellular matrix. Transforming growth factor-β1 (TGF-β1) is a classic pro-fibrotic factor that induces fibroblast activation.

[0029] The inventors discovered that the expression level of retroelement silencing factor 1 (RESF1) was significantly increased in fibrotic tissue and TGF-β1-induced activated fibroblasts. Further in vitro and in vivo functional experiments showed that specifically knocking down RESF1 using RNA interference significantly downregulated the expression of key myofibroblast markers (α-SMA, POSTN, COL1A1, etc.). This result confirms that blocking RESF1 expression can effectively prevent the activation and transformation of fibroblasts into myofibroblasts, thereby inhibiting fibroblast migration and contractile ability, and significantly reducing the area and severity of organ fibrosis in animal models.

[0030] Based on the above target identification conclusions, this invention carried out the design and activity verification of an inhibitory nucleic acid drug (siRNA) targeting this target: 1. RESF1 siRNA sequence design The inventors designed a 19-nucleotide siRNA sequence targeting the REF1 mRNA region and modified the siRNA sequence by attaching two deoxythymidine residues (dT) to the 3' end to improve the stability and silencing efficiency of the siRNA molecule. The specific sequence is shown in Table 1 below.

[0031] Table 1: siRNA sequences 2. Validation of RESF1 siRNA efficiency siRESF1 and control siRNA were transfected into the 3T3 fibroblast cell line using liposome transfection. Transfected cells were cultured in high-glucose DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (100 U / mL each) for 48 hours in a cell incubator at 37°C, 5% CO2, and saturated humidity. After culture, cells were collected, and the knockdown efficiency of siRNA on RESF1 was detected by rtPCR and Westren Blot, respectively. The results are shown below. Figure 1 and Figure 2 As shown, siRESF1 can significantly reduce the mRNA and protein expression levels of RESF1.

[0032] 3. RESF1 siRNA inhibits TGFβ1-induced fibroblast activation. siRESF1 and its control sequences were transfected into the 3T3 fibroblast line using liposome transfection technology. After 24 hours of routine culture, the cells were stimulated with solvent and 10 ng / ml TGFβ1. rtPCR was performed 48 hours later. Resf1 and myofibroblast marker genes Postn , Acta2 , Col1a1 The mRNA expression level of the gene and the α-SMA expression level were detected by immunofluorescence. The results are as follows: Figure 3 and Figure 4 As shown, siRESF1 was successfully knocked down. Resf1 mRNA expression levels and reduced Postn , Acta2 , Col1a1 The mRNA expression levels and α-SMA expression levels of the gene suggest that RESF1 siRNA inhibits TGF-β-induced fibroblast activation.

[0033] 4. RESF1 siRNA inhibits the migration and contraction ability of fibroblasts induced by TGFβ1. siRESF1 transfection, TGFβ1 treatment, and fibroblasts were as described above. Cell scratch, Transwell, and gel shrinkage assays were performed on the fibroblasts, and the results are as follows. Figures 5-7 As shown, siRESF1 was found to inhibit the migration and contraction of fibroblasts.

[0034] 5. RESF1 siRNA reduces the severity of bleomycin-induced pulmonary fibrosis in mice.

[0035] Lipid nanoparticles (LNPs) were chosen to encapsulate siRESF1 as an in vivo delivery strategy for animal experiments. During preparation, an ethanol mixture containing ionizable cationic lipids was rapidly mixed with an acidic buffer solution in which siRESF1 was dissolved using microfluidic technology. The mixture was then dialyzed in PBS to neutralize the pH and concentrated, ultimately yielding siRESF1-LNPs with uniform particle size and high encapsulation efficiency.

[0036] In animal experiments, 6-10 week old male C57BL / 6J mice were randomly divided into a control group (Vehicle+siNC-LNP), a model group (BLM+siNC-LNP), and a treatment group (BLM+siRESF1-LNP), with 3 mice in each group. After isoflurane inhalation anesthesia, a pulmonary fibrosis model was established by intratracheal infusion of 5.0 mg / kg bleomycin. On day 7 of model establishment, 2 mg / kg siRESF1-LNP was administered intratracheally (the control and model groups received the same amount of siNC-LNP), once every 3 days, until day 28 after model establishment. Lung tissue samples were then collected for subsequent evaluation.

[0037] The results first confirmed the successful construction of the model: compared with the control group, the model group mice not only experienced weight loss, but also showed significantly upregulated pathological histological features and pro-fibrotic factors (such as α-SMA and collagen), confirming that bleomycin successfully induced severe pulmonary collagen deposition and structural damage. Based on this, lung tissue was fixed, paraffin-embedded, and sectioned, and the degree of pulmonary fibrosis in each group was further examined using Sirius red staining. It was found that compared with the severe lesions in the model group, siRESF1-LNP intratracheal instillation significantly reduced pulmonary collagen deposition, effectively alleviating the area and severity of bleomycin-induced pulmonary fibrosis in mice. Figure 8 .

[0038] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0039] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0040] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of RESF1 gene expression inhibitors in the preparation of drugs for treating organ fibrosis.

2. The application according to claim 1, characterized in that, The RESF1 gene includes the human RESF1 gene and its mammalian homologs.

3. The application according to claim 1, characterized in that, The organ fibrosis is pulmonary fibrosis.

4. The application according to claim 1, characterized in that, The drug is used to inhibit fibroblast activation.

5. The application according to claim 1, characterized in that, The drug is used to inhibit the migration and contraction of fibroblasts.

6. The application according to claim 1, characterized in that, The drug is used to reduce the expression of α-SMA, POSTN, and COL1A1.

7. The application according to claim 1, characterized in that, The drug includes one or more siRNA, shRNA, antisense oligonucleotide, ribozyme, and gene editing tools targeting the RESF1 gene.

8. The application according to claim 7, characterized in that, The nucleotide sequence of the siRNA is shown in SEQ ID NO.1~SEQ ID NO.

2.

9. The application according to claim 8, characterized in that, The siRNA has two deoxythymidine residues attached to its 3' end.

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