Use of circ-0007527 as a target in screening drugs for treating peritoneal dialysis-related peritoneal fibrosis

Abstract

The application provides application of circ-0007527 as a target in screening of drugs for treating peritoneal dialysis related peritoneal fibrosis. The application also provides application of an inhibitor of circ-0007527 in preparation of a drug for treating peritoneal dialysis related peritoneal fibrosis. In a mouse peritoneal fibrosis model induced by high glucose peritoneal dialysis solution, circRNA expressed in peritoneal tissues is screened, and after bioinformatics analysis, it is found that the differential expression amount of circ-0007527 in the fibrotic peritoneal tissue is the highest, and the high expression of circ-0007527 in the fibrotic peritoneal tissue is verified by real-time fluorescent quantitative PCR. Further in-vitro cell experiments find that down-regulation of the expression of circ-0007527 can significantly inhibit peritoneal mesothelial cell phenotype transformation, proliferation and apoptosis, and thus reduce peritoneal injury and delay peritoneal fibrosis progression.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmacy, and relates to circular RNA circ-0007527, in particular to application of circ-0007527 as a target in screening of drugs for treating peritoneal dialysis-related peritoneal fibrosis. BACKGROUND

[0002] Chronic kidney disease (CKD) is a global high-incidence disease. According to the first national cross-sectional study of chronic kidney disease, the total prevalence of CKD in China has reached 10.8%, with more than 120 million patients. If the renal function of CKD patients continues to deteriorate, it will further develop into end-stage renal disease (ESRD). In recent years, with the increasing incidence of diabetes and hypertension, the number of ESRD patients in various countries has also been rising, and due to its poor prognosis, long course, and large personal and social burden, ESRD has become a major global public health problem.

[0003] At present, peritoneal dialysis (PD) is one of the renal replacement therapy methods for ESRD patients, and is an ideal and effective treatment method before kidney transplantation. Peritoneal dialysis is to use the patient's own peritoneum to filter solutes, and to inject 1.5% or 2.5% glucose-lactate peritoneal dialysis solution into the patient's abdominal cavity through a catheter, and to use the concentration gradient difference (diffusion of solutes and osmosis of water) on both sides of the peritoneum to remove metabolic products, toxic substances in the body of uremic patients and to correct water and electrolyte imbalance.

[0004] Compared with hemodialysis, peritoneal dialysis has the following advantages: protecting the residual renal function of patients, stable hemodynamics, light economic burden, suitable for home treatment, relatively free time, and high rate of returning to society, etc. Therefore, peritoneal dialysis has a high popularity in clinical practice and is more easily accepted by ESRD patients and their families. However, long-term peritoneal dialysis can cause peritoneal fibrosis, resulting in changes in peritoneal structure and impaired function, leading to ultrafiltration failure, withdrawal from the peritoneal dialysis cohort and then only having the option of hemodialysis or kidney transplantation. Therefore, the research on the mechanism and prevention of peritoneal fibrosis is of great significance for the quality of life and prognosis of clinical PD patients.

[0005] Peritoneal fibrosis is a common complication in patients with peritoneal dialysis, the normal structure of peritoneum is destroyed, peritoneal mesothelial cells are damaged, which affects the ultrafiltration function of peritoneal dialysis, and has a significant impact on the quality of life, treatment effect and prognosis of patients. Non-physiological peritoneal dialysis fluid contains glucose and glucose degradation products (GDPs) and advanced glycation end productions (AGEs) produced during the production and processing and disinfection process, including methylglyoxal, glyoxal and 3-deoxyglucosone, which can directly cause peritoneal mesothelial cell damage and shedding, and is one of the causes of cell phenotype transformation.

[0006] Studies have shown that peritoneal mesothelial cell damage is the initiating factor in the whole process of peritoneal fibrosis, mainly manifested as the destruction of the normal skeleton of peritoneal mesothelial cells, the weakening of cell adhesion and the loss of original cell morphology and epithelial mesenchymal transformation (EMT), increased invasiveness, causing extracellular matrix accumulation and fibrous tissue proliferation, and eventually developing into peritoneal fibrosis. Therefore, peritoneal mesothelial cell phenotype transformation, proliferation and apoptosis are the key regulatory mechanisms of peritoneal fibrosis.

[0007] Circular RNA (circRNA) is a kind of non-coding RNA molecule with closed loop structure, without 5' cap structure and 3' poly(A) structure, mainly located in cytoplasm or stored in exosomes, not affected by RNA exonuclease, more stable and not easy to degrade, which has been proved to exist widely in various eukaryotes. Most circRNAs are circularized by exons, and some circRNAs are lariat structures circularized by introns. CircRNA has characteristics of tissue specificity, disease specificity, time sequence specificity and high stability.

[0008] In recent years, a large number of studies have shown that circRNA is closely related to the growth and development of organisms, stress response, disease occurrence and development, and its application prospects in disease diagnosis markers and targeted therapy are predicted, but its biological function is still largely unknown. Current research has confirmed that circRNA plays an important regulatory role in various tumor diseases and is involved in the fibrosis process of various solid organs, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis and myocardial fibrosis, but the role of circRNA in peritoneal dialysis-related peritoneal fibrosis has not been reported.

[0009] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length, with many different uses in biology. siRNA is known to primarily participate in RNA interference (RNAi) by specifically regulating gene expression. Summary of the Invention

[0010] To address the aforementioned technical problems in the prior art, this invention provides the application of circ-0007527 as a target in screening drugs for the treatment of peritoneal fibrosis associated with peritoneal dialysis. This application aims to solve the technical problem that there are currently no effective drugs for treating peritoneal fibrosis after long-term peritoneal dialysis.

[0011] This invention provides the application of circ-0007527 as a target in screening drugs for the treatment of peritoneal dialysis-related peritoneal fibrosis.

[0012] This invention also provides the use of an inhibitor of circ-0007527 in the preparation of a medicament for treating peritoneal dialysis-related peritoneal fibrosis.

[0013] This invention also provides the use of circ_0007527siRNA in the preparation of a drug for treating peritoneal dialysis-related peritoneal fibrosis, wherein the sense strand of circ_0007527siRNA is 5'-GAACAAGGACCAUGGGUUUTT-3' and the antisense strand is 5'-AAACCCAUGGUCCUUGUUCTT-3'.

[0014] This invention also provides the application of the reagent for detecting circ-0007527 in the preparation of a kit for detecting peritoneal tissue fibrosis.

[0015] This invention utilizes high-throughput sequencing of circRNAs to screen for circRNAs expressed in peritoneal tissue in a high-glucose peritoneal fibrosis model induced by peritoneal dialysis. Bioinformatics analysis revealed that circ-0007527 exhibited the highest differential expression level in fibrotic peritoneal tissue, which was further validated by quantitative real-time PCR. In vitro cell experiments showed that downregulating circ-0007527 expression significantly inhibited peritoneal mesothelial cell phenotypic transformation, proliferation, and apoptosis, thereby alleviating peritoneal damage and delaying the progression of peritoneal fibrosis.

[0016] The circ-0007527 described in this invention is the first discovered and proven effective circular RNA in regulating peritoneal fibrosis, filling a research gap in the relationship between circRNAs and peritoneal dialysis-related peritoneal fibrosis. Specific information about circ-0007527 is as follows: Chromosome number: chr16; circRNA start site: 20656542; circRNA end site: 20657609; Positive and negative strands: +; circRNA full length: 1067; circRNA splice length: 147; circRNA source gene: ENSMUSG00000006998; circRNA splice information: exon: 20656543-20656689.

[0017] Compared with existing technologies, the technological advancements of this invention are significant. The circ-0007527 screened in this invention is the first discovered and proven effective circular RNA in regulating peritoneal fibrosis. Downregulating the expression of circ-0007527 can significantly inhibit peritoneal mesothelial cell phenotypic transformation, proliferation, and apoptosis, thereby reducing peritoneal damage and delaying the progression of peritoneal fibrosis. Therefore, circ-0007527 provides a new therapeutic target and approach for the preparation of drugs to prevent and treat peritoneal fibrosis associated with peritoneal dialysis. Its related inhibitors hold promise for clinical use in preventing and treating peritoneal fibrosis after peritoneal dialysis.

[0018] This invention discloses a circular RNA circ-0007527 and its application in the preparation of drugs for the prevention and treatment of peritoneal dialysis-related peritoneal fibrosis. Studies have shown that downregulating the expression of circ-0007527 can significantly inhibit peritoneal mesothelial cell phenotypic transformation, proliferation, and apoptosis, thereby reducing peritoneal damage and delaying the progression of peritoneal fibrosis. Attached Figure Description

[0019] Figure 1 A mouse peritoneal fibrosis model induced by 4.25% high-glucose peritoneal dialysis fluid was constructed.

[0020] Figure 2 circ-0007527 is highly expressed in fibrotic peritoneal tissue.

[0021] Figure 3 Inhibiting circ-0007527 expression can block peritoneal mesothelial cell phenotypic transformation.

[0022] Figure 4 Inhibiting circ-0007527 expression can downregulate Fibronectin expression and restore E-cadherin expression.

[0023] Figure 5Inhibiting circ-0007527 expression can block peritoneal mesothelial cell proliferation.

[0024] Figure 6 Inhibiting circ-0007527 expression can block peritoneal mesothelial cell apoptosis. Detailed Implementation

[0025] Example 1: Materials and Methods

[0026] 1) Reagents and consumables

[0027] Fibronectin, E-cadherin, p27, Cyclin E, Bax, Cleaved caspase 3, and Bcl-2 antibodies were purchased from Cell Signaling Technology. Collagen I (A2), PCNA, and GAPDH antibodies were purchased from Santa Cruz. α-SMA antibody and other reagents were purchased from Sigma-Aldrich. siRNA was purchased from Shanghai Gemma Gene.

[0028] 2) Mouse peritoneal fibrosis model and experimental grouping

[0029] A mouse peritoneal fibrosis model was established according to methods reported in the literature. All animal experiments were conducted in accordance with the Chinese Regulations on the Management and Use of Laboratory Animals.

[0030] A mouse model of peritoneal fibrosis induced by 4.25% high-glucose peritoneal dialysis fluid for 28 days:

[0031] a. Sham group (n=10): Intraperitoneal injection of the same volume of 0.9% saline for 28 days;

[0032] b. PDF group (n=10): Intraperitoneal injection of 100ml / kg of 4.25% high glucose peritoneal dialysis solution for 28 days.

[0033] 3) Cell culture and processing

[0034] Human peritoneal mesothelial cells (HMrSV5) were cultured in DMEM / Highglucose medium containing 5% fetal bovine serum, 0.5% penicillin, and streptomycin at 37°C, 5% carbon dioxide, and 95% air. Stimulation with TGF-β1 (2 ng / ml) or high-glucose peritoneal dialysis medium (4.25%) for 36 hours was followed by transfection with Con siRNA and circ-0007527 siRNA, respectively. Proteins were extracted after 36 hours and stored at -80°C for molecular biological analysis.

[0035] 4) Circular RNA sequencing

[0036] Differentially expressed proteins among groups were identified, and ClusterProfiler software was used to perform GO functional enrichment analysis, KEGG pathway enrichment analysis, and gene set enrichment analysis (GSEA) on the differentially expressed gene sets. The enrichment expression of related genes in signaling pathways was determined by constructing a co-expression network (WGCNA).

[0037] 5) Qualitative observation of peritoneal morphology and fibrosis

[0038] Masson staining: Tissue is fixed in 4% paraformaldehyde solution, routinely dehydrated and embedded, dewaxed to water, treated with potassium dichromate mordant for 12-18 hours, stained with Weiger's hematoxylin for 5-10 minutes, rinsed briefly with running water, differentiated with 1% hydrochloric acid alcohol, rinsed with running water for several minutes to regain blue color, stained with Ponceau S and acid fuchsin for 5-10 minutes, rinsed quickly with distilled water, treated with phosphomolybdic acid aqueous solution for about 3-5 minutes, without rinsing with water, counterstained directly with aniline blue solution for 2-5 minutes, treated with 1% glacial acetic acid for 1 minute, dehydrated multiple times with 95% alcohol, dehydrated with anhydrous alcohol, cleared with xylene, and mounted with neutral resin.

[0039] 6) Immunoblotting

[0040] Cell samples were mixed with 200 μL of protein lysis buffer and allowed to stand for 20 minutes. Centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit (Sunbio). An 8% separating gel and a 5% stacking gel were then prepared, samples were added, and electrophoresis was performed. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel. The samples were then wet-transferred to a PVDF membrane and washed three times (5 min each) in TBST. A 5% skim milk solution was prepared using TBST, and the membrane was immersed in the solution and incubated at room temperature for 1 hour. The primary antibody was diluted 1:1000 with blocking buffer and incubated with the membrane overnight at 4°C. The membrane was then washed three times (5 min each) with TBST. The HRP-labeled secondary antibody was diluted 1:2000 with blocking buffer and incubated with the membrane for 1.5 hours. The membrane was then washed three times (5 min each) with TBST and exposed to ECL. After photographing, the images were analyzed for grayscale using LabWorks software. Change in relative content = target protein gray level / GAPDH gray level.

[0041] 7) Immunofluorescence staining

[0042] (a) Blocking: Circle the tissue with a hydrophobic pen, prepare blocking solution (PBS + 2% BSA + 0.2% Triton X-100), and block at room temperature for 1 hour. (b) Primary antibody incubation: Discard the blocking solution, add 50 μL of primary antibody dilution solution (prepared with the blocking solution) to each tissue, cut an appropriate size sealing film and cover it on the primary antibody dilution solution to ensure uniform antibody incubation on the slide, and incubate overnight at 4°C. (c) Secondary antibody incubation: After washing the slide four times with PBS, prepare the corresponding fluorescent secondary antibody (1:1000), add 50 μL to each tissue, and incubate at room temperature in the dark for 1 hour. (d) DAPI counterstaining of cell nuclei: Discard the secondary antibody, rinse four times with PBS, and stain with DAPI staining solution for 10 min. (e) Mounting: Add anti-fluorescence quencher, slowly cover the slide from one side of the tissue, avoiding air bubbles, and gently press the slide with your finger to remove excess quencher. Apply nail polish to the gap between the coverslip and the slide, let it dry, take a picture immediately, and store the slide at 4°C after mounting. (f) Data analysis: Observe the slide under a microscope after mounting and analyze the image results.

[0043] 8) Statistical analysis

[0044] SPSS 20.0 software was used to perform statistical analysis on the obtained data. All quantitative data were expressed as mean ± standard deviation, and t-tests and ANOVA were used. P < 0.05 was considered statistically significant.

[0045] Example 2: Construction of a mouse peritoneal fibrosis model induced by 4.25% high-glucose peritoneal dialysis fluid

[0046] The animal model used in this invention is a mouse peritoneal fibrosis model induced by 4.25% high-glucose peritoneal dialysis fluid. Mice were intraperitoneally injected daily with 4.25% high-glucose peritoneal dialysis fluid (100 ml / kg). Peritoneal and serum samples were collected after 28 days for subsequent experimental analysis. Masson staining was performed. Figure 1 A) and Sirius red staining ( Figure 1 C) Thickened subperitoneal areas were observed in the PDF group, suggesting the presence of peritoneal fibrosis lesions. Furthermore, compared to the Sham control group, the percentage of quantitatively positive areas was significantly increased. Figure 1 B and 1D) indicate that we have successfully established a mouse peritoneal fibrosis model, providing the necessary experimental basis for subsequent research.

[0047] Example 3: circ-0007527 was highly expressed in fibrotic peritoneal tissue.

[0048] Based on the aforementioned mouse model, we performed high-throughput sequencing of circular RNA in the Sham and PDF groups, and then conducted a series of bioinformatics analyses. These included using ClusterProfiler software for GO functional enrichment analysis, KEGG pathway enrichment analysis, and gene set enrichment analysis (GSEA) of differentially expressed genes, and constructing a co-expression network (WGCNA) to determine the enrichment expression of relevant genes in signaling pathways. Data analysis revealed that circ-0007527 had the highest differential expression level in fibrotic peritoneal tissue. Figure 2 A). Simultaneously, we further verified using real-time quantitative PCR that circ-0007527 is highly expressed in fibrotic peritoneal tissue. Figure 2 B). This result suggests that circ-0007527 may be involved in the process of peritoneal dialysis-related peritoneal fibrosis.

[0049] Example 4: Inhibiting circ-0007527 expression can block peritoneal mesothelial cell phenotypic transformation.

[0050] Based on the high-throughput sequencing results of circular RNA in Example 3, the full-length gene sequence of the target gene circ-0007527 was determined. The RNA precursor was cleaved by RNase III to obtain a siRNA molecule precursor of appropriate length, which was then introduced into cells to inhibit gene expression.

[0051] The sequence of circ_0007527 siRNA is 5'-GAACAAGGACCAUGGGUUUTT-3' (sense strand) and 5'-AAACCCAUGGUCCUUGUUCTT-3' (antisense strand).

[0052] In further in vitro cell experiments, peritoneal mesothelial cells were stimulated with TGF-β1 (2 ng / ml) for 36 hours and transfected with Con siRNA and circ-0007527 siRNA, respectively, to observe the effect of circ-0007527 on the phenotypic transformation of peritoneal mesothelial cells to EMT. Quantitative real-time PCR results confirmed that TGF-β1 stimulation significantly upregulated the expression level of circ-0007527 in peritoneal mesothelial cells. Figure 3 A).

[0053] Immunoblotting results indicated that inhibiting circ-0007527 expression with specific siRNA downregulated the expression levels of α-SMA, Collagen I, and Fibronectin, and restored the expression of the epithelial cell marker E-cadherin. Figure 3The results (BF) indicate that circ-0007527 is related to cell phenotypic transformation, and inhibiting circ-0007527 expression can block peritoneal mesothelial cell phenotypic transformation.

[0054] Example 5: Inhibiting circ-0007527 expression downregulates Fibronectin expression and restores E-cadherin expression.

[0055] Based on Western blotting, we further detected the expression of Fibronectin and E-cadherin in different treatment groups using immunofluorescence staining. The results also confirmed that the expression level of Fibronectin-positive cells in the circ-0007527 siRNA group was significantly lower than that in the Con siRNA group (…). Figure 4 A and 4B), while the expression level of E-cadherin in positive cells was significantly increased ( Figure 4 (C and 4D) further demonstrate that inhibiting circ-0007527 expression can block the phenotypic transformation of peritoneal mesothelial cells.

[0056] Example 6: Inhibiting circ-0007527 expression can block peritoneal mesothelial cell proliferation.

[0057] In subsequent experiments, we investigated the relationship between circ-0007527 and cell proliferation by detecting several cell proliferation-related marker proteins (p27, PCNA, and Cyclin E). The results showed that TGF-β1 stimulated abnormal proliferation of peritoneal mesothelial cells, and transfection with siRNA to inhibit circ-0007527 expression significantly increased p27 levels, while significantly decreasing the expression levels of PCNA and Cyclin E. Figure 5 AD). Immunofluorescence staining also confirmed that the number of PCNA-positive cells in the circ-0007527 siRNA group was significantly lower than that in the control group (AD). Figure 5 E and 5F). The above results indicate that inhibiting circ-0007527 expression can block peritoneal mesothelial cell proliferation.

[0058] Example 7: Inhibition of circ-0007527 expression can block peritoneal mesothelial cell apoptosis.

[0059] Peritoneal mesothelial cell apoptosis is a key mechanism in the progression of peritoneal injury and peritoneal fibrosis, mainly due to the presence of glucose and its degradation products in the non-biocompatible peritoneal dialysis fluid used by peritoneal dialysis patients. Therefore, in an in vitro study, we used a 4.25% high-glucose peritoneal dialysis fluid to stimulate peritoneal mesothelial cells, thus mimicking this injury process. The results confirmed that in vitro stimulation with the high-glucose peritoneal dialysis fluid induced apoptosis in peritoneal mesothelial cells, manifested by increased levels of pro-apoptotic genes Bax and Cleaved caspase 3, and decreased levels of the anti-apoptotic gene Bcl-2. Inhibition of circ-0007527 expression using siRNA reversed the expression levels of these proteins. Figure 6 The expression of circ-0007527 (AF) inhibited peritoneal mesothelial cell apoptosis. This result indicates that inhibiting circ-0007527 expression can block peritoneal mesothelial cell apoptosis.

Claims

1. The use of an inhibitor of circ-0007527 in the preparation of a drug for treating peritoneal dialysis-related peritoneal fibrosis, wherein the inhibitor of circ-0007527 is circ_0007527siRNA, the sense strand of which is 5'-GAACAAGGACCAUGGGUUUTT-3' and the antisense strand is 5'-AAACCCAUGGUCCUUGUUCTT-3'.

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