A circular non-coding RNA circRNA_013145 and its application

Abstract

The present invention relates to the field of molecular biology, specifically to a circular noncoding RNA (circRNA_013145), its use in the preparation of diagnostic reagents for diabetic erectile dysfunction (DMED), and its use in DMED-related drugs. By exploring the regulatory function of RNA circRNA_013145 on phenotypic transformation of corpus cavernosum smooth muscle cells (CCSMCs) and damage to human umbilical vein endothelial cells (HUVECs), the present invention discovered that circRNA_013145 expression plays a significant role in the development and progression of diabetic erectile dysfunction (DMED). Consequently, the present invention provides a biomarker suitable for the detection and identification of diabetic erectile dysfunction, as well as a target and potential drug for the treatment of DMED.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to circular non-coding RNA circRNA_013145, and particularly relates to the use of a preparation for detecting the expression amount thereof in the preparation of a diagnostic reagent for diabetic erectile dysfunction or the use of the preparation of related drugs. Background Art

[0002] Diabetes mellitus (DM) is one of the most important metabolic diseases affecting human health worldwide, often causing multiple systemic complications. Diabetic erectile dysfunction (DMED) is a common complication of diabetes, characterized by the inability of diabetic patients to achieve or maintain sufficient penile hardness during intercourse for a satisfying sexual experience. Compared with non-diabetic men, the incidence of diabetic erectile dysfunction is approximately 3-5 times higher; ED affects nearly 50-75% of men with DM, significantly reducing their quality of life, self-confidence, and family and social harmony.

[0003] One of the key pathological features of diabetic erectile dysfunction (DMED) is distal vascular damage caused by hyperglycemia. This is characterized by underlying endothelial cell damage, accompanied by chronic inflammation, oxidative stress, cellular senescence or apoptosis, and involves phenotypic transformation of endothelial cells and cavernous smooth muscle cells, neuronal dysfunction, and pathological changes such as leukomalacia and fibrosis. Existing mechanistic research and various treatments have provided a certain understanding of the development and progression of DMED and have been shown to enhance erectile function. However, the disease's pathogenesis is complex and drug efficacy is limited. Further exploration of new treatments and deeper understanding of the underlying mechanisms of DMED treatment are crucial.

[0004] The penis is composed of three cylindrical compartments: the paired corpora cavernosa and corpus spongiosum. Penile erection, or swelling, is a complex hemodynamic process involving relaxation of the corpus cavernosum smooth muscle, increased arterial blood flow, and impaired venous return. Cavernous smooth muscle cells (CCSMCs) are widely accepted as the primary effector cells, comprising 42–50% of the corpus cavernosum cells and playing a key role in the erectile response. Phenotypic conversion refers to the shift of SMCs from a contractile to a synthetic state, accompanied by increased proliferation and migration, decreased expression of α-smooth muscle actin (α-SMA) and desmin, and increased osteopontin levels. This conversion is crucial in vascular disease and erectile dysfunction (ED), and occurs under hypoxic conditions in the pathological changes of cavernous nerve (CN) damage and diabetic ED. The penis is also considered a vascular tissue with unique structural features. Endothelial cells (ECs) constitute the innermost layer of the vasculature and regulate vascular homeostasis. Sustained hyperglycemia significantly promotes oxidative stress, fibrosis, and inflammation, leading to endothelial dysfunction, which contributes to the progression of diabetic ED. As mentioned above, the phenotypic transformation of CCSMCs, the functional impairment caused by EC damage, and the reduction of cell number due to two types of apoptosis are important causes of DMED.

[0005] Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs generated by reverse splicing of pre-mRNAs. They are stable, conserved, and widely expressed in the eukaryotic transcriptome. Studies have shown that circRNAs are associated with a variety of diseases, including diabetes, cardiovascular disease, and cancer. However, to date, studies on the expression and function of circRNAs in DMED are limited. Summary of the Invention

[0006] The present invention aims to provide a method for the use of circRNA_013145, a noncoding circular RNA that is significantly overexpressed in penile tissue of patients with diabetic erectile dysfunction and affects the function of corpus cavernosum smooth muscle cells (CCSMCs) and human umbilical vein endothelial cells (HUVECs). The technical solutions described in this invention provide in-depth understanding of the precise molecular mechanisms linking circRNA_013145 to the progression of diabetic erectile dysfunction (DMED), potentially promoting the development of more effective molecularly targeted therapies and possessing significant clinical significance.

[0007] In a first aspect of the present invention, a preparation for detecting the expression level of circular non-coding RNA circRNA_013145 is provided for use in preparing a reagent for diagnosing or predicting the risk of developing diabetic erectile dysfunction. The nucleotide sequence of the RNA circRNA_013145 is shown in SEQ ID No. 1.

[0008] Optionally, the preparation includes specific primers for detecting circRNA_013145, including a forward primer with a nucleotide sequence as shown in SEQ ID No. 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 3.

[0009] In a second aspect of the present invention, a preparation for detecting the expression level of circular non-coding RNA circRNA_013145 is provided for use in preparing a kit for early screening or auxiliary diagnosis of diabetic erectile dysfunction, wherein the nucleotide sequence of the RNA circRNA_013145 is shown in SEQ ID No. 1.

[0010] In a third aspect, the present invention provides the use of a circular non-coding RNA circRNA_013145 having a nucleotide sequence as shown in SEQ ID No. 1 in the preparation or screening of drugs for treating diabetic erectile dysfunction.

[0011] Optionally, the drug contains the circRNA_013145 expression inhibitor as an active ingredient, and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0012] In a fourth aspect, the present invention provides an inhibitor that can reduce the risk of diabetic erectile dysfunction by inhibiting the expression of circular non-coding RNA circRNA_013145 having a nucleotide sequence as shown in SEQ ID No. 1.

[0013] Optionally, the inhibitor includes an siRNA targeting circRNA_013145, and the sequence of the siRNA includes any one of SEQ ID No. 4 to 6.

[0014] Optionally, the inhibitor comprises siRNA having a sequence as shown in SEQ ID No.4.

[0015] The present invention discloses a circular non-coding RNA circRNA_013145, whose expression level is significantly correlated with diabetic erectile dysfunction (DMED); by exploring the regulatory function of RNA circRNA_013145 on the phenotypic transformation of cavernous smooth muscle cells CCSMCs and vascular endothelial cell damage HUVECs, it was found that circRNA_013145 plays an important role in the occurrence and development of diabetic erectile dysfunction (DMED). That is, the present invention provides a biomarker and therapeutic target for the detection and identification of diabetic erectile dysfunction; by screening this target, patients with diabetic erectile dysfunction can be discovered early, thereby improving the patient's prognosis. In addition, based on the correlation between circRNA_013145 and DMED, the present invention also provides the use of the circRNA in the preparation of a drug for treating DMED diseases, and by inhibiting the expression of the circRNA, the risk of DMED can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To identify differentially expressed circRNAs in penile tissue from DMED rats. (a) Box plot shows the distribution of circRNAs in samples from the DMED and control groups, demonstrating that the two data sets had nearly identical distributions after normalization. (b) Scatter plot shows the changes in circRNA expression between the DMED and control groups. Points outside the green line represent circRNAs with a greater than 2-fold change in logarithmic expression between the two groups. (c) Volcano plot shows the distribution of differentially expressed circRNAs in microarray analysis. (d) Heat map shows the expression profiles of circRNAs in DMED and control tissues, with red bands representing high relative expression and green bands representing low relative expression.

[0017] Figure 2 The figure shows the expression of differentially expressed circRNAs verified by qRT-PCR. The figure shows the level of circRNA_013145 in penile tissue of DMED rats measured by qRT-PCR. Values are expressed as mean ± SD. *P < 0.05, **P < 0.01.

[0018] Figure 3Figure 2 shows the RT-PCR analysis results of circRNA_013145 and its interaction information with the most likely miRNAs. Figure (a) shows the RT-PCR analysis results of circRNA_013145; Figure (b) shows the binding site of circRNA_013145 with rno-miR-185-5p in the 3' untranslated region (UTR). "2D structure" displays the MRE sequence, target miRNA seed type, and 3' mate sequence. "Local AU" displays the AU content 30 nt upstream and downstream of the seed sequence. Red bars represent A / U and high accessibility, while black bars represent G / C and low accessibility seeds. The degree of accessibility is indicated by the height of the bar. "Position" represents the most likely relative MRE position of circRNA_013145 in the linear representation.

[0019] Figure 4 Figure 2. GO, KEGG pathway, and PPI network analysis of miR-1843b-5p target genes. Figures a, b, c, d, and e show the biological processes, molecular functions, cellular components, KEGG pathway enrichment, and PPI networks of miR-1843b-5p target genes, respectively. Nodes represent target proteins, and edges represent interactions between two targets.

[0020] Figure 5 The expression level and cellular distribution of circRNA_013145 in CCSMCs and HUVECs. Panel (a) shows the circularization of Asph exons 2-3 to form circRNA_013145; (b) and (c) indicate that the expression level of circRNA_013145 is higher in CCSMCs and HUVECs induced by high glucose than in cells treated with low glucose; (d) and (e) indicate that circRNA_013145 is resistant to RNase R treatment in CCSMCs and HUVECs, a notable characteristic of circular RNAs; (f) shows the relative expression levels of circRNA_013145 and Asph mRNA at the indicated time points in CCSMCs and HUVECs treated with actinomycin D; (g) shows the relative expression of circRNA_013145 in the nucleus and cytoplasm of CCSMCs and HUVECs measured by RT-PCR; and (h) shows the expression of circRNA_013145 in CCSMCs and HUVECs confirmed by fluorescence in situ hybridization (FISH). Scale bars represent 50 μm. *P<0.05, **P<0.01.

[0021] Figure 6Figure 2: Effects of si-Circ_013145 on the expression level of circRNA_013145 in CCSMCs and HUVECs, and on cell viability. (a) qRT-PCR analysis of circRNA_013145 levels in CCSMCs and HUVECs transfected with si-Circ_013145; (b) CCK-8 assay to determine the effect of silencing circRNA_013145 on cell viability.

[0022] Figure 7 Figure 1 shows the inhibition of inflammation and oxidative stress in CCSMCs and HUVECs by si-CircRNA_013145. Figures (a, b, c, d, e, and f) show the levels of TNF-α, IL-6, MDA, SOD, NO, and cGMP in CCSMCs and HUVECs expressing silencing circRNA_013145, as measured by ELISA. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 for the HG group versus the control group; P < 0.05, ΔΔP < 0.01 for the si-Circ_#1 group versus the si-Circ_NC group.

[0023] Figure 8 Figure 3. Effects of circRNA_013145 knockdown on proliferation, migration, and apoptosis of CCSMCs and HUVECs. A: The effect of circRNA_013145 knockdown on cell proliferation was measured by a colony formation assay; B: The effect of circRNA_013145 knockdown on wound healing was measured by a wound healing assay; C: Flow cytometry analysis of apoptosis in transfected CCSMCs and HUVECs. *P < 0.05, **P < 0.01 for the si-Circ_#1 group and the si-Circ_NC group.

[0024] Figure 9The effects of knockdown of circRNA_013145 on the levels of phenotypic transformation-related proteins and apoptosis-related proteins in CCSMCs and HUVECs were shown in Figure 1. (a) Immunofluorescence staining was used to analyze the protein expression levels of α-SMA, desmin, and osteopotin. The scale bar is 50 μm. (b) Western blot was used to detect the protein levels of p62, PKG, Bcl-2, Beclin-1, Caspase-9, LC3, Bax, and Caspase-3 in CCSMCs. (c) Western blot was used to detect the protein expression levels of p62, PKG, Bcl-2, eNOS, Beclin-1, Caspase-9, Caspase-3, LC3, Bax, VACM-1, and ICAM-1 in HUVECs transfected with circRNA_013145. *P < 0.05, **P < 0.01, compared with the si-Circ_#1 group and the si-Circ_NC group.

[0025] Figure 10 Figure 1 shows some experimental results showing that knockdown of circRNA_013145 improved erectile function, cell phenotypic transformation, and apoptosis in DMED rats. Figure 1 shows the evaluation of erectile function in DMED rats treated with Ad-sh_circRNA_013145; Figure 1 shows representative traces of ICP and MAP for all four groups of rats; Figure 1 shows representative H&E and Masson light micrographs (scale bar = 100 μm) of the corpus cavernosum, with arrows indicating blood vessels; Figure 1 shows the quantification of CD31 (green, scale bar = 100 μm) and the number of blood vessels in the groups by immunofluorescence staining in DMED rats.

[0026] Figure 11Another set of experimental results shows that knockdown of circRNA_013145 improves erectile function, phenotypic transformation, and apoptosis in DMED rats. (a) Representative images of apoptotic cells (green, scale bar = 100 μm) stained by Tunel in the corpus cavernosum of rats treated with sh_circRNA_013145 in all four groups; (b) Real-time PCR analysis assessing the expression levels of circRNA_013145, miR-185-5p, and RhoA in all groups; (c) Representative Western blot results for α-SMA, desensitizer, RhoA, p62, Beclin-1, PKG, eNOS, VACM-1, ICAM-1, osteopontin, LC3, Bax, and Bcl-2 in penile tissue between groups. GAPDH or β-actin was used as loading control. All results are representative of three independent experiments. Data are expressed as mean ± SD. **P<0.01 compared with the control group; ΔP<0.05, ΔΔP<0.01 compared with the DMED and DMED-Ad-NC groups. DETAILED DESCRIPTION

[0027] Example 1 CircRNA differential expression determination and the reliability of circRNA_013145 as a DMED disease detection marker and drug research target

[0028] 1.0 Animals and Samples

[0029] In this study, 70 male Sprague-Dawley rats weighing 172-186 g were used. A diabetic model was initially established by intraperitoneal injection of streptozotocin (STZ). Finally, 40 DMED rats that failed to exhibit an erectile response within 30 minutes were screened using apomorphine (APO, 100 μg / kg). These 40 DMED rats were then randomly divided into four groups: DMED, low-dose, medium-dose, and high-dose LCG (n=10) (for more detailed sample information, see reference PMID: 32896625). After erectile function testing at week 4, penile tissues were collected, frozen in liquid nitrogen, and stored at −80°C for further analysis. Four samples each from the control and DMED groups were selected for circRNA microarray labeling and hybridization. The animal protocol for this study was approved by the Animal Care and Use Committee of Zhejiang Chinese Medical University.

[0030] 1.1 RNA extraction and quality control

[0031] Total RNA was extracted from penile tissue using TRIzol reagent according to the manufacturer's instructions. The concentration of RNA samples was determined by OD260 using a NanoDrop ND-1000 instrument. RNA integrity was assessed by electrophoresis on denaturing agarose gels.

[0032] 1.2 CircRNA microarray labeling and hybridization

[0033] The Arraystar standard protocol provided by Kangcheng Company was used. Briefly, total RNA was digested with RNaseR to remove linear RNA and enrich circular RNA. The enriched circular RNA was then amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar SuperRNA Labeling Kit; Arraystar). The labeled cRNA was then hybridized to the Arraystar Rat circRNA Array (8x15K, Arraystar). After washing the slides, the array was scanned using an Agilent scanner G2505C.

[0034] 1.3 Microarray data collection and analysis

[0035] Scanned images were imported into Agilent Feature Extraction Software (version 11.0.1.1, Agilent Santa Clara, CA, United States) for raw data extraction. Quantile normalization and subsequent data processing were performed using the R software limma package. The statistical significance of differences was conveniently assessed using a t-test. Statistically significant differentially expressed circRNAs between the two groups were identified using volcano plot filtering. CircRNAs with a fold change of 2.0 and a p < 0.05 were selected as significantly differentially expressed. Hierarchical clustering was performed to visualize distinguishable circRNA expression patterns between samples.

[0036] 1.4 Results

[0037] After normalization of circRNA expression profiles, the samples showed high normalization, e.g. Figure 1 As shown in a. Scatter plot Figure 1 b Visualization of the changes in circRNA expression between the DMED group and the control group. A total of 1,288 differentially expressed circRNAs were identified, including 641 upregulated circRNAs and 647 downregulated circRNAs. Volcano plots were used to visualize the differentially expressed circRNAs between the DMED group and the control group, with a significance threshold of p value < 0.05 and an absolute fold change ≥ 2 ( Figure 1 c). Hierarchical cluster analysis further illustrates the different circRNA expression patterns in samples ( Figure 1 d).

[0038] To validate the circRNA microarray results, qRT-PCR analysis was performed on the top five up-regulated and down-regulated circRNAs in penile tissue samples from the control and DMED groups. The relative expression level of circRNA_013145 was significantly up-regulated in DMED rats compared with the control group ( Figure 2 These results support a significant positive correlation between circRNA_013145 and DMED, suggesting that it could serve as a biomarker for the disease and a potential target for drug development.

[0039] Example 2 Enrichment analysis of circRNA_013145 targeted genes and potential pathways for their function

[0040] 2.1 Construction of circRNA-miRNA-mRNA regulatory network

[0041] CircRNAs can crosstalk by competing with common microRNAs, and microRNA response elements (MREs) are the basis of this interaction. Circular RNAs play a vital role in regulating the level of miRNA-mediated gene expression regulation by sequestering miRNAs. To determine the potential function of circRNA_013145, Arraystar's homemade miRNA target prediction software based on TargetScan was used to predict circRNA / microRNA interactions. It was found that circRNA_013145 had a good binding rate with miRNA miR-1843b-5p ( Figure 3 ).

[0042] 2.2 Gene ontology and pathway enrichment analysis

[0043] GO annotation and KEGG pathway analysis were performed by Metascape tool to evaluate the miR-185-5p targeted genes in the circRNA-miRNA-mRNA regulatory network. GO analysis of biological process (BP), molecular function (MF), and cellular component (CC) with p-value less than 0.05 was considered to be significantly enriched by the targeted genes.

[0044] 2.3PPI Network Construction and Analysis

[0045] Based on the genes targeted by miR-185-5p, a PPI network was constructed using the STRING database, with the species restricted to Rattus norvegicus. PPIs with a confidence score ≥0.4 were retained and further imported into Cytoscape for visualization. Furthermore, to identify core genes in the PPI network, the network's topological parameters were analyzed. The degree of a node refers to the number of surrounding genes directly connected to it in the PPI network. Node size was visualized using degree values, and genes with a degree greater than the median degree of all nodes were selected as hub genes for miR-185-5p.

[0046] 2.4 Results

[0047] like Figure 4 As shown, GO analysis revealed enrichment of biological processes such as cellular response to peptides, Ras protein signaling, synaptic organization, and membrane organization; cellular component analysis identified enrichment of synaptic membrane, presynaptic, and leading edge membrane categories. KEGG pathway enrichment analysis showed that miR-1843b-5p target genes were enriched in pathways such as axon guidance, calcium reabsorption regulated by endocrine and other factors, cGMP-PKG signaling, and cAMP signaling. A PPI network of miR-1843b-5p target genes was constructed based on information from the STRING database, revealing 181 nodes and 283 edges. Five central genes were identified within the PPI network, with RhoA having the highest score. These findings suggest that circRNA_013145 may promote the development of DMED through the miR-1843b-5p / RhoA-related signaling pathway.

[0048] Example 3: Effect of Inhibiting CircRNA_013145 Expression in Reversing High Glucose Damage in Cell Models at the Multiple Cell Line Level

[0049] 3.1 Cell culture and transfection

[0050] Primary rat CCSMCs were prepared from the corpus cavernosum of normal male Sprague-Dawley rats. Furthermore, HUVECs were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells from passages 4 to 8 were used in this study. CCSMCs and HUVECs were then divided into three groups: a low glucose group (5 mM glucose); a mannitol group (5 mM glucose + 25 mM mannitol); and a high glucose (HG) group (30 mM glucose) and cultured in DMEM complete medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO₂. Furthermore, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to evaluate the expression of circRNA_013145, miR-185-5p, and RhoA under control or HG conditions.

[0051] 3.2 Cell transfection

[0052] siRNA targeting circRNA_013145 (si-circRNA_013145) was synthesized; miR-185-5p mimics and inhibitors were purchased from RiboBio. CCSMCs and HUVECs were transfected with si-circRNA_013145, si-Circ_negative control (si-Circ_NC), miR-185-5p mimics, miR-185-5p inhibitors, and appropriate NCs using Lipofectamine 3000 transfection reagent.

[0053] The sequence of si-circRNA_013145 is as follows:

[0054] si-Circ_#1:5'-GCAATGAACTGATTCCTTT-3';

[0055] si-Circ_#2: 5'-GCGAAGGACTTCCGTTATA-3';

[0056] si-Circ_#3: 5'-GCTTCTGTTTGTAAGCCTA-3'.

[0057] The above sequences are SEQ ID No. 4-6 in the sequence listing.

[0058] 3.3 qRT-PCR detection

[0059] Total RNA was isolated from penile tissue, CCSMC, and HUVEC using TRIzol reagent. For circRNA and RhoA, total RNA was reverse transcribed into cDNA using Prime Script II First Strand cDNA Synthesis Kit. For miR-185-5p, total RNA was reverse transcribed using TaqMan microRNA Reverse Transcription Kit according to the manufacturer's instructions. SYBR Premix ExTaq TM qRT-PCR was performed on an ABI 7500 system. GAPDH and U6 expression were used as internal controls. -ΔΔCt Methods Relative RNA expression was normalized.

[0060] The primer sequences used in this scheme are shown in the table below, including SEQ ID No. 2-3, SEQ ID No. 7-12.

[0061]

[0062] 3.4 RNaseR and Actinomycin D Treatment

[0063] Total RNA (5 μg) from CCSMCs and HUVECs was incubated with 4 U / mg RNaseR at 37°C for 30 minutes. For actinomycin treatment, 2 μg / ml actinomycin D or DMSO was added to the cells for the indicated times. Then, the RNA expression levels of circRNA_013145 and Asph were detected by qRT-PCR.

[0064] 3.5 Nuclear and cytoplasmic fractionation

[0065] The CCSMC and HUVEC layers were digested into single cells. Then, the cells were resuspended and washed with 1000 μl of PBS and centrifuged at 300 × g for 5 min at 4°C. Next, the nuclear and cytoplasmic fractions were isolated from the cultured CCSMC and HUVEC using NE-PER nuclear and cytoplasmic extraction reagent according to the manufacturer's instructions. The fractionated total RNA was extracted using the QIAamp RNA Mini kit according to the manufacturer's instructions. U6 and GAPDH-treated mRNAs were detected in the isolated RNA as controls for nuclear and cytoplasmic RNA, respectively. The abundance of circRNA_013145 was measured using qRT-PCR.

[0066] 3.6 Fluorescence in situ hybridization (FISH)

[0067] In situ hybridization was performed using probes specific for the circRNA_013145 sequence and miR-185-5p. Briefly, a fluorescein isothiocyanate (FITC) probe was specific for miR-185-5p, and a Cy5-labeled probe was specific for circRNA_013145. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, China). All assay protocols were performed according to the manufacturer's instructions. Finally, images were acquired on a fluorescence inverted microscope.

[0068] 3.7 Cell Counting Kit-8 (CCK-8) Assay

[0069] Cell viability was assessed using the CCK-8 kit and the operation was strictly followed according to the manufacturer's instructions.

[0070] 3.8 Enzyme-linked immunosorbent assay (ELISA)

[0071] Commercial ELISA kits were used to measure the concentrations of inflammatory factors TNF-α and IL-6 in the cell supernatants of HG-treated transfected CCSMC and HUVEC. The concentrations of MDA, SOD, NO, and cGMP were also measured according to the kit instructions.

[0072] 3.9 Colony formation assay

[0073] CCSMCs and HUVECs were transfected with 100 nM si-Circ_#1 and si-Circ_NC and cultured in DMEM containing 10% FBS. 2 Cells were seeded into 6-well plates (100 cells / well) and cultured for 10 days. After incubation, the culture medium was removed, and the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet solution for 30 minutes at room temperature. Colonies were imaged well and counted accurately. All experiments were repeated three times.

[0074] 3.10 Flow cytometry

[0075] The apoptosis rate of CCSMCs and HUVECs was determined using the Annexin V-FITC apoptosis detection kit according to the kit instructions. After incubation, the apoptosis rate was measured using a FACS Calibur flow cytometer.

[0076] 3.11 Wound healing assay

[0077] 5×10 per well 5 Cells were seeded into 6-well plates and incubated at 37°C, 5% CO2 until 100% confluence. A linear scratch was then made on the cell monolayer using a 200 μl pipette tip (denoted as 0 h). Subsequently, the cells were incubated under HG conditions for 24 hours in serum-free and HG medium. After 24 hours of incubation, migration was imaged using an inverted microscope, and the distance at 0 and 24 hours was measured using ImageProPlus 6.0 software.

[0078] 3.12 Immunofluorescence staining

[0079] After fixation in 4% paraformaldehyde for 20 min and washing with PBS, transfected CCSMCs were permeabilized with 0.5% TritonX-100 in PBS for 15 min and blocked with 10% bovine serum albumin (BSA) for 1 hour. The cells were then incubated with primary antibodies against α-SMA (1:200), desmin (1:100), and osteopontin (1:1000) at 4°C overnight, followed by the addition of the corresponding Alexa Fluor 500 antibody. 488-conjugated secondary antibody (1:1000) was used for 1 hour. In addition, cell nuclei were stained with DAPI, and cells were imaged using a microscope as described above.

[0080] 3.13 Luciferase reporter gene assay

[0081] The circRNA_013145 and RhoA fragments containing the miR-185-5p binding site were synthesized by Sangon Biotech Co., Ltd. and named WT-circRNA_013145 or WT-RhoA. Then, the constructs containing wide or mutated circRNA_013145-miR-185-5p and RhoA-miR-185-5p were subcloned into the reporter vector psiCHECK-2 vector. That is, CCSMC and HUVEC (5×10 4 Cells / well were seeded into 24-well plates and then co-transfected with miR-185-5p mimics or miR-185-5pNC and WT-circRNA_013145 / Mut-circRNA_013145 or WT-circRNA_013145. WT-RhoA / Mut-RhoA were transfected using Lipofectamine 3000 according to the manufacturer's instructions for 4 hours, and relative luciferase activity was measured using the dual-luciferase reporter gene assay system.

[0082] 3.14 RNA immunoprecipitation (RIP) assay

[0083] For the RIP assay, CCSMCs and HUVECs were lysed using RIP lysis buffer supplemented with protease and RNase inhibitors. The combination of circRNA_013145, miR-185-5p, and RhoA was examined using the Magna RIP Binding Protein Immunoprecipitation Kit according to the manufacturer's instructions. Briefly, cell lysates were incubated with antibodies against Ago2 or IgG. Finally, the immunoprecipitated RNA was eluted, and the levels of circRNA_013145, miR-185-5p, and RhoA were measured by qPCR.

[0084] 3.15 Western blot analysis

[0085] Transfected CCSMC, HUVEC, and penile tissue were lysed in ice-cold RIPA buffer containing protease inhibitors. The protein concentration in the supernatant was measured using a BCA kit. Equal amounts of protein were then separated by SDS-PAGE at different concentrations and transferred to PVDF membranes, blocked with 5% skim milk for 2 h at room temperature, and then incubated with antibodies against [GAPDH (1:3000), β-actin (1:3000), p62 (1:2000), Beclin-1 (1:2000), LC3Ⅰ / Ⅱ (1:2000), Bax (1:3000), Caspase-9 (1:2000), C Aspase-3 (1:2000), Bcl-2 (1:2000), PKG (1:2000), eNOS (1:3000), VACM1 (1:2000), ICAM-1 (1:2000), RhoA (1:2000), α-SMA (1:2000), desmin (1:100), and osteopontin (1:1000) were incubated overnight at 4°C and then incubated with appropriate secondary antibodies for 2 hours at room temperature. After washing, protein bands were detected using BeyoECL Plus reagent. Finally, the intensity was measured using ImageJ software.

[0086] 3.16 Results

[0087] Microarray analysis identified circRNA 013145 as the most differentially expressed circRNA, originating from exon 2 to exon 3 of the aspartate β-hydroxylase (Asph) gene. Figure 5 a). qPCR results confirmed that circRNA 013145 was significantly upregulated in CCSMCs and HUVECs induced by high glucose (HG) ( Figure 5 b, c). To confirm the circular nature of circRNA_013145, RNaseR treatment was performed, demonstrating that circRNA_013145 was resistant to RNaseR digestion, whereas the linear AsphmRNA was degraded ( Figure 5 d, e). In addition, actinomycin D assay showed that circRNA_013145 exhibited higher stability than its linear mRNA counterpart in CCSMC and HUVEC ( Figure 5 f). Subcellular localization analysis by qRT-PCR and FISH showed that circRNA_013145 was mainly localized in the cytoplasm, confirming its cytoplasmic nature ( Figure 5 g, h). These results collectively indicate that circRNA_013145 is a stable cytoplasmic circRNA in CCSMCs and HUVECs.

[0088] Transfection with siRNA targeting circRNA_013145 knocked down circRNA_013145 in HG-treated CCSMCs and HUVECs ( Figure 6 a). CCK-8 assay showed ( Figure 6 b), silencing circRNA_013145 significantly improved cell viability in HG-treated CCSMCs and HUVECs. Figure 7 As shown, circRNA_013145 knockdown in HG-treated CCSMCs and HUVECs resulted in a significant decrease in TNF-α, IL-6, and MDA levels, while SOD, NO, and cGMP levels increased, indicating reduced inflammation and oxidative stress. These results suggest that circRNA_013145 knockdown improves cell viability, inflammation, and oxidative stress in HG-treated CCSMCs and HUVECs. In addition, colony formation assays showed that ( Figure 8 A), circRNA_013145 knockdown reduced the number of cell colonies in CCSMCs while promoting cell proliferation in HUVECs. Wound healing assays showed that circRNA_013145 knockdown inhibited the migration of CCSMCs but enhanced the migration of HUVECs ( Figure 8 B).

[0089] Flow cytometry analysis showed that knockdown of circRNA_013145 significantly reduced apoptosis in CCSMCs and HUVECs ( Figure 8 C). In addition, IF and WB analysis were used to evaluate the expression levels of phenotypic transformation-related proteins and apoptosis-related proteins, such as Figure 9 As shown in Figure 5, inhibition of circRNA_013145 can improve the phenotypic transformation and HG-induced apoptosis of CCSMCs.

[0090] The above research results show that inhibiting the expression of circRNA_013145 in high glucose-induced CCSMC and HUVEC cell line models in vitro can increase cell viability, reduce cell apoptosis, reduce the expression of oxidative stress and inflammatory factors, and improve the dysfunction of smooth muscle cells and endothelial cells, which makes the inhibitor of circRNA_013145 a drug for diabetic erectile dysfunction.

[0091] Example 4 Animal level verification of the potential role of circRNA_0130145 inhibitors in the treatment of DMED

[0092] 4.1 Functional evaluation of circRNA_013145 knockdown in vivo

[0093] Sixty 6-week-old SD male rats (weighing 200±20 g) were purchased from Shanghai Slake Experimental Animal Co., Ltd. (same as above). Mating tests showed that all rats had normal sexual function. The experimental design was approved by the Animal Care and Use Committee of Zhejiang Chinese Medical University (No. ZSLL-2021-164; Zhejiang, China). The feeding and DMED model preparation methods were the same as before (see literature: PMID: 32896625 for details). Finally, 35 DMED rats were screened for subsequent experiments and randomly divided into DMED group (n=11), DMED-Ad-NC group (n=12) and DMED-Ad-sh_circRNA_013145 (n=12) group. Recombinant adenovirus Ad-circRNA_013145shRNA (titer, 5.6×10 9 puf / ml) and NC adenovirus Ad-null (titer, 6.4×10 9 puf / ml) was purchased from Shanghai Jima Pharmaceutical Co., Ltd. Rats in the DMED, DMED-Ad-NC, and DMED-Ad-sh_circRNA_013145 groups were injected with saline (20 μL), Ad-NC (20 μL), and circRNA_013145 shRNA (20 μL) into the corpus cavernosum every other day for 4 weeks. Rats in the control group were treated with saline alone for 4 weeks. Erectile function and pathological changes in adenovirus-induced DMED rats were evaluated. After 4 weeks of treatment, APO was injected into the neck skin of the rats, and then the rats were observed for half an hour. The number of erections, including penis swelling, glans engorgement, foreskin retraction, and ejaculation in DMED rats was measured. If none occurred, the rat's erection latency was recorded for 30 minutes. In addition, all rats underwent cavernous manometry testing using a four-channel acquisition system to measure maximum intracavernous pressure (maxICP) and mean arterial pressure (MAP). Furthermore, cavernous cell apoptosis and vascular number were determined using TUNEL staining and fluorescent staining, respectively. Afterwards, qRT-PCR and WB were performed to measure the levels of target genes and proteins.

[0094] 4.2 Statistical analysis

[0095] Results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 9.0 and SPSS 22.0. Differences between two or more groups were compared using a two-tailed Student's t-test or one-way analysis of variance with Tukey's post hoc test. A p-value < 0.05 was considered statistically significant.

[0096] 4.3 Results

[0097] Compared with the DMED and Ad-NC groups, rats treated with Ad-sh_circRNA_013145 showed higher erection frequency, erection duration, and shortened latency ( Figure 10 a). maxICP and MAP measurements confirmed that Ad-sh_circRNA_013145 treatment partially restored the erectile function of DMED rats ( Figure 10 b). Histological analysis of penile tissues showed that vascular density and expression of CD31 markers of angiogenesis were increased in DMED rats treated with Ad-sh_circRNA_013145 compared with DMED rats treated with Ad-NC ( Figure 10 c, d). Ad-sh_circRNA_013145 treatment resulted in a decrease in collagen fiber content and partial restoration of smooth muscle content in the corpus cavernosum. TUNEL assay results showed that cell apoptosis was reduced in DMED rats treated with Ad-sh_circRNA_013145 ( Figure 11 a). qRT-PCR and WB analysis further showed that Ad-sh_circRNA_013145 treatment reduced the expression of circRNA_013145 in penile tissue, increased the expression of miR-185-5p, and reduced the expression of RhoA ( Figure 11 b, c). Changes in protein expression of phenotypic transformation markers and apoptosis-related proteins were consistent with those observed in vitro. These findings suggest that circRNA_013145 inhibition can alleviate cavernous cell phenotypic transformation and apoptosis in DMED organisms.

[0098] The technical solutions described in Examples 1-4 above measure the differential expression of circRNA_013145 by microarray labeling and hybridization techniques; bioinformatics analysis is performed on the target genes of RNA circRNA_013145; experiments on multiple cell types demonstrate that RNA circRNA_013145 is associated with enhanced cellular inflammation and oxidative stress levels and the promotion of apoptosis; animal experiments further demonstrate that inhibitors of circRNA_013145 can alleviate phenotypic transformation and apoptosis of cavernous cells in DMED organisms, reduce collagen fiber content in the cavernous body and restore some smooth muscle content, and increase vascular density and angiogenesis. From the above schemes and corresponding experimental results, it can be seen that the circRNA_013145 described in the present invention is significantly associated with DMED, and circRNA_013145 can be used as a molecular marker for detecting the risk of DMED, and accordingly, a detection reagent or kit for detecting DMED can be formed; further, it can also be used as a basis to prepare inhibitors that can inhibit its expression, forming drugs that can treat or prevent DMED.

[0099] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will appreciate that various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention without departing from the spirit and scope of the present invention, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An inhibitor, characterized in that It is an siRNA targeting circRNA_013145, and the sequence of the siRNA is shown as SEQ ID No.4.

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