Application of KIF20A expression inhibitor in preparation of medicine for treating keloid

Abstract

The invention relates to the field of biological medicines, and discloses application of a KIF20A expression inhibitor in preparation of a medicine for treating keloid. The KIF20A expression inhibitor is a substance capable of reducing mRNA (messenger ribonucleic acid) and / or protein level of KIF20A. The invention provides a novel medicine for treating keloid, the expression of targeted KIF20A can increase the apoptosis of keloid fibroblasts by causing keloid fibroblast G2 / M phase retardation, meanwhile, the migration ability of the keloid fibroblasts is reduced, and the medicine has a treatment effect on the keloid.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, it relates to the use of a KIF20A expression inhibitor in the preparation of a medicament for treating keloids. Background Technology

[0002] Keloids are benign skin tumors caused by excessive proliferation of fibroblasts and abnormal deposition of extracellular matrix during the skin wound healing process. They are characterized by continuous growth, invasion of surrounding normal skin, and a high recurrence rate after surgery. Current treatment methods for keloids generally include intralesional injection of corticosteroids (triamcinolone acetonide), 5-fluorouracil, etc., surgical excision combined with radiotherapy, or laser therapy. While the efficacy is currently acceptable, the treatment options remain relatively limited. The main drawbacks are a high recurrence rate, potential local skin atrophy, and poor patient compliance.

[0003] KIF20A is a member of the kinesin superfamily, encoding a protein closely related to cell division. Recent studies have found that KIF20A expression is upregulated in various cancers, and it can promote tumor progression by promoting tumor cell proliferation, migration, and invasion. Furthermore, some literature indicates that KIF20A expression can be downregulated by upstream FOXM1 stimulation, regulating the expression of cell cycle genes essential for DNA replication and mitosis, such as Cycline B1 and CDK1, ultimately controlling abnormal transcriptional programs during the G2 / M phase of cells.

[0004] However, the role of KIF20A in keloids and its potential as a therapeutic target have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to explore the application of KIF20A expression inhibitors in the preparation of drugs for treating keloids, in order to provide keloid patients with a new and effective treatment method, reduce the recurrence rate, and improve the quality of life of patients.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution: In one aspect, this application provides the use of KIF20A expression inhibitors in the preparation of drugs for treating keloids. Furthermore, the KIF20A expression inhibitor is a substance capable of reducing the mRNA and / or protein levels of KIF20A.

[0007] Furthermore, the KIF20A expression inhibitor includes siRNA, shRNA, ASO, or a CRISPR / dCas9 gene transcription repression system.

[0008] Secondly, this application provides a pharmaceutical composition for treating keloids, the pharmaceutical composition comprising a therapeutically effective amount of a KIF20A expression inhibitor, and a pharmaceutically acceptable carrier or excipient.

[0009] Furthermore, the KIF20A expression inhibitor is a substance capable of reducing the mRNA and / or protein levels of KIF20A.

[0010] Furthermore, the KIF20A expression inhibitor includes siRNA, shRNA, ASO, or a CRISPR / dCas9 gene transcription repression system.

[0011] In summary, this application has the following beneficial effects: This invention provides a novel therapeutic drug for keloids. Targeting the expression of KIF20A can increase apoptosis in keloid fibroblasts by causing G2 / M phase arrest, while reducing the migration ability of keloid fibroblasts, thus achieving a therapeutic effect on keloids. Attached Figure Description

[0012] Figure 1 Figure 1 shows the experimental results of the effect of KIF20A knockdown on the phenotype of keloid fibroblasts (KFs) in this embodiment of the invention. Figures AC and D show the expression and knockdown efficiency of KIF20A in KFs. Figure D shows the changes in cell cycle distribution (G2 / M phase arrest) of KFs after KIF20A knockdown. Figure EI shows the downregulation of CDK1 and Cyclin B1 expression at both the mRNA and protein levels in KFs after KIF20A knockdown. Figure J shows the increased apoptosis rate of KFs after KIF20A knockdown. Figure K shows the decreased migration ability of KFs after KIF20A knockdown. Detailed Implementation

[0013] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0014] Unless otherwise specified, all materials and instruments used in this embodiment are commercially available, and all experimental methods used are existing methods.

[0015] Materials and Methods 1. Primary cell source Both keloid patients and normal controls were from Huashan Hospital, Shanghai. Keloid patients were diagnosed according to the classification and assessment criteria of the 2015 JSW Scar Scale (JSS). Age- and sex-matched healthy Chinese individuals served as normal controls. This study was approved by Fudan University, and all participants signed written informed consent forms. Primary keloid fibroblasts (KFs) and normal human skin fibroblasts (NFs) were isolated and cultured using the tissue block adherence method in DMEM medium containing 10% FBS at 37°C and in a humidified incubator with 5% CO2.

[0016] 2. Intervention of KIF20A expression in keloid fibroblasts 1*10 seed per well in a 6-well plate 5 For keloid fibroblasts, once the cells have reached 30%-50% confluence, prepare the transfection reagent. First, prepare 150 μL opti-men + 9 μL Lipo RNAimax (Invitrogen) and 150 μL opti-men + 3 μL siRNA / NC in each well. After mixing these two solutions and letting them stand for 5 minutes, add them to the culture medium. Incubate at 37°C for 4-6 hours, then replace with complete culture medium and continue culturing. The following are the KIF20A siRNA and NC sequences:

[0017] 3. Cell cycle analysis 1*10 seed per well in a 6-well plate 5 Cells were collected and transfected with KIF20AsiRNA when the cell confluence reached 30%-50%. After 72 hours, the cells were processed further. Subsequently, the cells were digested with trypsin and fixed in ice-cold 70% ethanol overnight at 4°C. The cells were then centrifuged, and the pellet was resuspended in PBS. Cell cycle staining was performed using the protocol provided by the Beyotime Cell Cycle and Apoptosis Analysis Kit. Cell cycle analysis was performed using a flow cytometer (CytoFLEX LX, Beckman Coulter, CA, USA). The data were analyzed using FlowJo software. Statistical methods were used to analyze the results.

[0018] 4. Apoptosis analysis As described above, cells were grown in 6-well plates to 70%-80% confluence. Apoptosis was determined by flow cytometry using the Annexin V-FITC apoptosis detection kit, following the instructions (Lianke Apoptosis Kit). Cells were collected 72 hours after KIF20A siRNA transfection, incubated with 5 μL Annexin V-FITC, then incubated with 10 μL PI at room temperature for 10 min, and resuspended in buffer. These stained cells were then analyzed using flow cytometry with FlowJo software after adjusting compensation.

[0019] 5. Transwell test Primary keloid fibroblasts were seeded into six-well plates at a density of 70%-80% and transfected with siRNA. After 24 hours of treatment, the cells were resuspended in serum-free medium and seeded in Transwell chambers at a cell density of approximately 6*10⁶ cells / well. 4 Cells were collected per well and 15% FBS DMEM was added outside the chamber. The chambers were then cultured for 24 hours. The chambers were collected, rinsed with PBS, and then fixed with paraformaldehyde for 30 minutes. After rinsing with PBS once, the chambers were stained with crystal violet for 15 minutes. The chambers were then washed with PBS again, and the cells inside the chambers were gently wiped away with cotton swabs. After drying, the chambers were photographed and counted under a microscope.

[0020] 6. Statistical Analysis: Statistical analysis was performed using Graph Pad Prism 8.0. At least three independent experiments were conducted for each group. Results are expressed as mean and standard deviation (SD). Differences between groups were assessed using independent samples t-tests or one-way ANOVA. A p-value <0.05 was considered statistically significant.

[0021] Example 1: Application of siRNA targeting KIF20A in inhibiting fibroblast growth in keloids This embodiment analyzes keloid fibroblasts and normal fibroblasts before and after PHS treatment to identify the key target gene: KIF20A.

[0022] To further elucidate the molecular mechanisms of irradiation treatment of keloid fibroblasts, we knocked down the expression level of the key downregulated gene KIF20A in KFs to explore the role of KIF20A in keloid fibroblasts (Figures AC). After interfering with KIF20A expression, KFs exhibited significant G2 / M phase cell cycle arrest (increasing from 4.01% to 39.5%) and a significant decrease in the proportion of G0 / G1 phase (decreasing from 89.0% to 53.2%) (Figure D). Further analysis using qRT-PCR and Western blot revealed that knocking down KIF20A expression in KFs significantly reduced the mRNA and protein expression levels of CDK1 and Cycline B1 (Figure EI). Simultaneously, knocking down KIF20A expression induced apoptosis in KFs, increasing the apoptosis rate to 16%, significantly higher than the empty spin group (Figure J). Knocking down KIF20A expression in KFs also led to a significant decrease in cell migration ability, with the number of migrating cells decreasing from 1800 to 1200 (Figure K).

[0023] This demonstrates that targeted silencing of KIF20A can effectively inhibit the malignant phenotype of keloid fibroblasts, verifying its feasibility as a therapeutic target.

[0024] The beneficial effect of this invention is that it provides a novel therapeutic drug for keloids. Targeting the expression of KIF20A can increase apoptosis of keloid fibroblasts by causing G2 / M phase arrest, while reducing the migration ability of keloid fibroblasts, thus achieving a therapeutic effect on keloids.

[0025] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Application of KIF20A expression inhibitors in the preparation of drugs for treating keloids.

2. The application according to claim 1, characterized in that, The KIF20A expression inhibitor is a substance that can reduce the mRNA and / or protein levels of KIF20A.

3. The application according to claim 2, characterized in that, The KIF20A expression inhibitors include siRNA, shRNA, ASO, or CRISPR / dCas9 gene transcriptional repression systems.

4. A pharmaceutical composition for treating keloid scars, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of a KIF20A expression inhibitor, and a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical composition according to claim 4, characterized in that, The KIF20A expression inhibitor is a substance that can reduce the mRNA and / or protein levels of KIF20A.

6. The pharmaceutical composition according to claim 5, characterized in that, The KIF20A expression inhibitors include siRNA, shRNA, ASO, or CRISPR / dCas9 gene transcriptional repression systems.

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