Identification method of circSS18 R-loop in synovial sarcoma and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an identification method of circSS18R-loop in synovial sarcoma and application of the identification method. Comprising the following steps: identifying circSS18R-loop formed by circular RNA circ0108126 of synovial sarcoma, wherein the nucleotide sequence of the circSS18R-loop is as shown in SEQ ID NO: 1. According to the invention, the method comprises the following steps: CUTamp; the TAG is combined with a qPCR (quantitative polymerase chain reaction) technology to identify the existence of circSS18R-loop in a synovial sarcoma cell line HS-SY-II for the first time; it is found for the first time that the circular circ0108126 can promote proliferation, migration and invasion of synovial sarcoma cells and inhibit apoptosis, and deeper cognition and a new thought are brought to a molecular mechanism for regulating synovial sarcoma through circular RNA.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an identification method and application of circSS18 R-loop in synovial sarcoma. Background Art

[0002] Synovial sarcoma (SS) is a relatively rare malignant tumor, accounting for 5-10% of all soft tissue sarcomas. It predominantly occurs in adolescents and young adults and has an aggressive behavior and high metastatic potential. Synovial sarcoma can arise in any anatomical site but is most common in the extremities, particularly around large joints. The cellular origin of SS remains unclear, but some suggest a multipotent mesenchymal stem cell or neurogenic origin. It presents in three distinct histological morphologies: monophasic, biphasic, or poorly differentiated. The prognosis for patients with SS varies depending on factors such as tumor size, stage at diagnosis, and the presence of metastases. Synovial sarcoma in adults has a high metastatic potential, with 50% of adult patients developing metastatic disease. The 5-year overall survival rate for adults with metastatic SS is 10%, while that for those with localized SS is 76%.

[0003] The hallmark genetic feature of synovial sarcoma is the presence of a chromosomal translocation, t(X;18)(p11;q11), which leads to the formation of the fusion gene SS18-SSX. This fusion gene is a core driver of synovial sarcoma, promoting tumorigenesis and affecting cell function. Currently, there are three fusion gene subtypes of synovial sarcoma: SS18-SSX1, SS18-SSX2, and SS18-SSX4. Approximately two-thirds of synovial sarcomas manifest as the SS18-SSX1 subtype, one-third as the SS18-SSX2 subtype, and the SS18-SSX4 subtype is rare. The SS18-SSX fusion protein produced by the SS18-SSX fusion gene interferes with the normal gene expression regulatory mechanism, affecting chromatin remodeling and gene expression patterns, which are crucial for cell differentiation and proliferation. The oncogenic fusion protein SS18-SSX promotes tumorigenesis and progression primarily through pathways such as BAF complex dysfunction, imbalanced activity of the polycomb repressive complex 2, and activation of stem cell-associated tumor genes. Furthermore, it regulates gene transcription through transcription factors, co-effectors, and histone deacetylases. Therefore, the SS18-SSX fusion gene is not only a diagnostic marker for synovial sarcoma but also a potential candidate target for its biological therapy.

[0004] Circular RNAs (circRNAs), a type of noncoding RNA (ncRNA), are more stably expressed and less susceptible to degradation than linear RNAs. They possess the functions of miRNA sponges, RNA-binding proteins, translation, splicing, and transcription. In addition to regulating host gene transcription by binding to RNA polymerase II and recruiting proteins, circRNAs can also form R-loops (R-loops) to target transcriptional regulatory regions of host genes. For example, in breast cancer, circSMARCA5 can increase the splicing efficiency of homologous exon-deficient mRNAs by forming R-loops, thereby terminating transcription and host gene expression. In pulmonary arterial hypertension, circLrch3 binds to specific DNA sequences within the host gene Lrch3 promoter to form a circR-loop, promoting chromatin activation and DNA demethylation, leading to enhanced Lrch3 transcriptional activity and apoptosis in pulmonary artery smooth muscle cells. Furthermore, compared with linear RNAs, circRNAs hybridize significantly more efficiently with cognate DNA, suggesting that circR-loops have a broader and more potent regulatory potential. Furthermore, given the widespread distribution of circRNAs and R-loops, circR-loops may regulate downstream gene expression through multiple mechanisms. While limited research exists on the relationship between circR-loops and human tumorigenesis, existing studies have shown that circRNAs can bind to parental genes to generate circR-loops, regulating their transcription. Most reported circRNAs are located in the cytoplasm, primarily promoting tumor progression through the ceRNA mechanism of miRNA adsorption or interactions with RNA-binding proteins. However, the functions of circRNAs in the nucleus remain poorly understood. Studies have shown that circRNAs can bind to chromatin-modifying enzymes, regulating gene expression at the epigenetic level and participating in tumor invasion and metastasis. CircMRPS35 recruits KAT7 to the SS18 / 3a promoter, inducing H4K5 acetylation and thereby promoting SS18 / 3a transcription and inhibiting the invasion and metastasis of gastric cancer cells. However, there is currently no evidence that circRNAs can participate in histone acetylation modification, thereby regulating the expression of specific target genes and affecting the invasion and metastasis of synovial sarcoma. Summary of the Invention

[0005] In order to make up for the deficiencies of the existing technology, the present invention provides a method for identifying circSS18 R-loop in synovial sarcoma and its application, providing a new strategy for the diagnosis and treatment of synovial sarcoma.

[0006] The present invention provides a method for identifying circSS18 R-loop in synovial sarcoma, comprising the following steps: identifying circSS18 R-loop formed by synovial sarcoma circular RNA circ_0108126, wherein the nucleotide sequence of the circSS18 R-loop is shown in SEQ ID NO: 1;

[0007] The kit includes RNA circ_0108126 reagent, RNA circ_0108126 primer pair, RNA circ_0108126 probe, and primer pair for the circSS18 R-loop site. The circSS18 R-loop was confirmed to be a circR-loop by treating the CUT&TAG assay products with RNase R and RNase H, and using the S9.6 antibody.

[0008] The RNA circ_0108126 primer pair includes an upstream primer JF1 and a downstream primer JR1. The nucleotide sequence of the upstream primer JF1 is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer JR1 is shown in SEQ ID NO: 3.

[0009] The nucleotide sequence of the RNA circ_0108126 probe is shown in SEQ ID NO: 4;

[0010] The primer pair for the circSS18 R-loop site includes an upstream primer F1 and a downstream primer R1. The nucleotide sequence of the upstream primer F1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO: 6.

[0011] The present invention provides a lentiviral vector for silencing the RNA circ_0108126, and the nucleotide sequence of the lentiviral vector is shown in SEQ ID NO:7.

[0012] The present invention provides a lentiviral vector that overexpresses the RNA circ_0108126, and the nucleotide sequence of the lentiviral vector is shown in SEQ ID NO:8.

[0013] The present invention provides a cell comprising a lentiviral vector for silencing the RNA circ_0108126 or a lentiviral vector for overexpressing the RNA circ_0108126.

[0014] The present invention provides a use of the RNA circ_0108126 or circSS18 R-loop in preparing a drug for treating synovial sarcoma.

[0015] The present invention provides a use of the above-mentioned lentiviral vector in preparing a medicine for treating synovial sarcoma.

[0016] The present invention provides an application of the above cells in preparing a medicine for treating synovial sarcoma.

[0017] In summary, the present invention has the following advantages:

[0018] The present invention, using DRIP-seq technology, discovered for the first time an R-loop site in the SS18 gene region in synovial sarcoma cells. Specific primers were designed to identify whether circular RNAs were correctly circularized, and the expression and localization of the synovial sarcoma circular RNA circ_0108126 in the test samples were analyzed and detected, confirming the stability of the synovial sarcoma circular RNA circ_0108126. The present invention, using CUT&TAG combined with qPCR technology, identified for the first time the presence of the circSS18 R-loop in the synovial sarcoma cell line HS-SY-II. Furthermore, by constructing silencing and overexpression vectors for the synovial sarcoma circular RNA circ_0108126, the present invention stably silenced and overexpressed circ_0108126. After transfection into synovial sarcoma HS-SY-II cells, the present invention discovered for the first time that the circular circ_0108126 could promote synovial sarcoma cell proliferation, migration, and invasion, and inhibit cell apoptosis. This provides a deeper understanding and new insights into the molecular mechanism of circular RNA regulation of synovial sarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is a schematic diagram of the structure of circ_0108126;

[0021] Figure 2 The pie chart shows the distribution of 70044 R-loops peaks in HS-SY-II cells in gene coding regions, intergenic regions, terminator regions, and promoter regions.

[0022] Figure 3 The distribution diagram of DRIP product reads in the gene start region (TSS) and end region (TES);

[0023] Figure 4 This is the location map of R-loop on chromosome;

[0024] Figure 5 The distribution percentage of R-loops peaks in different gene regions and the distribution of fragment lengths;

[0025] Figure 6 is the length distribution diagram of DRIP products;

[0026] Figure 7 The DRIP-seq analysis shows the R-loop site distribution in the SS18 gene region (chr18:23,594,217-23,673,183) in synovial sarcoma cells HS-SY-II.

[0027] Figure 8 RT-PCR and Sanger sequencing images;

[0028] Figure 9 The distribution of circSS18 in HS-SY-II cells was detected by RNA FISH;

[0029] Figure 10 The distribution statistics of circSS18 in HS-SY-II cells were detected by qPCR analysis of cytoplasmic-nuclear separation;

[0030] Figure 11 Electropherogram of circSS18 circularization for gDNA-cDNA amplification experiment verification;

[0031] Figure 12 Statistical diagram of circSS18 stability validation for RNase R experiments;

[0032] Figure 13 Statistical graph for the validation of circSS18 stability by actinomycin D experiment;

[0033] Figure 14 Set up statistical graphs for CUT&Tag-qPCR results after RNase H and RNase R digestion.

[0034] Figure 15 This is a schematic diagram of the lentiviral plasmid for silencing circSS18;

[0035] Figure 16 This is a statistical diagram of circSS18 silencing efficiency in HS-SY-II cells detected by qRT-PCR;

[0036] Figure 17 Fluorescence imaging shows the transfection efficiency of circSS18 silencing plasmid in HS-SY-II cells;

[0037] Figure 18 Schematic diagram of the lentiviral plasmid overexpressing circSS18;

[0038] Figure 19 This is a statistical diagram of the overexpression efficiency of circSS18 in HS-SY-II cells detected by qRT-PCR;

[0039] Figure 20 Fluorescence imaging shows the transfection efficiency of circSS18 overexpression plasmid in HS-SY-II cells;

[0040] Figure 21 The results and statistical graph of the plate cloning experiment show that the proliferation ability of circ_0108126 is reduced after silencing;

[0041] Figure 22 The results and statistical graphs of the invasion and migration assay show that silencing circ_0108126 reduces the invasion and migration abilities.

[0042] Figure 23 This is a statistical diagram of the reduction in proliferation ability after silencing circ 0108126 in the CCK8 experiment;

[0043] Figure 24 The experimental results and statistical graphs of TUNEL assay to detect the increased apoptosis level after silencing circ_0108126;

[0044] Figure 25 These are the experimental results and statistical graphs of the EDU experiment to detect the reduced proliferation level after silencing circ_0108126.

[0045] Figure 26 The results and statistical graph of the plate cloning experiment show that the proliferation ability is increased after overexpression of circ_0108126;

[0046] Figure 27 Invasion and migration assay results and statistical graphs showing the increased invasion and migration abilities after overexpression of circ_0108126.

[0047] Figure 28 This is a statistical chart of the experimental results of the cck8 experiment to detect the increased proliferation ability after overexpression of circ_0108126;

[0048] Figure 29 The experimental results and statistical graphs of TUNEL assay to detect the reduction of apoptosis level after overexpression of circ_0108126;

[0049] Figure 30 These are the experimental results and statistical graphs of the EDU experiment detecting the increased proliferation level after overexpression of circ_0108126. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0051] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0052] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] Example

[0054] 1. Screening of circSS18 R-loop in synovial sarcoma cells

[0055] This example first analyzed the R-loops formed in synovial sarcoma cells (HS-SY-II) by DRIP combined with high-throughput sequencing. The S9.6 antibody specifically binds to the DNA: RNA hybrid chain in the R-loop structure. The fragments forming R-loops in the whole genome of RH30 cells were immunoprecipitated by DRIP experiment, and the DNA fragments in the RNA: DNA hybrid chain were extracted to build a library and sequenced. 70,044 R-loops were formed in the whole genome of HS-SY-II cells, 45.15% of which were distributed in gene coding regions, 47.08% were located in intergenic regions, 3.65% were located in terminator regions, and 4.12% were located in promoter regions. The 70,044 R-loops peaks were distributed on 23 chromosomes (such as Figure 2-Figure 6 ), the DRIP-seq data were further analyzed to determine whether there were R-loops near the SS18 gene on chromosome 18. IGB software compared the R-loops signal of DRIP-seq with the position of the SS18 gene (chr18:23,594,217-23,673,183). The results showed that there was one R-loops signal near the SS18 gene (as shown in Table 1 and Figure 7 shown).

[0056] Table 1 SS18-related R-loops sites in HS-SY-II cells

[0057]

[0058] To further explore circRNAs in synovial sarcoma, we screened for SS18-related circRNAs using two circRNA databases, circBase and circinteractome. A total of 23 circRNAs were screened (as shown in Table 2):

[0059] Table 2SS18-related circRNAs in circBase and circinteractome databases

[0060]

[0061]

[0062] By comparing the HS-SY-II fusion gene-related R-loops sites screened by DRIP-seq in synovial sarcoma cells with the chromosomal position of circSS18 in the circBase and circinteractome databases, we jointly screened circRNAs that may form circR-loops and jointly analyzed SS18-related CircR-loops in synovial sarcoma (as shown in Table 3). We screened out a circSS18R-loop region that may form near the SS18 gene, namely chr18:23652787-23653309 (as shown in Table 3). Figure 7 shown).

[0063] Table 3 Circ SS18 R-loops in synovial sarcoma cells

[0064]

[0065]

[0066] Validation of the circRNA that forms the circSS18 R-loop in synovial sarcoma

[0067] We have previously explored the enrichment of a large number of R-loops in synovial sarcoma cells. We further explored the potential formation of circR-loops in synovial sarcoma cells related to the SS18 gene. We further analyzed circSS18, which was screened from the circRNA database. By designing primers for its junction site, we extracted total RNA from HS-SY-II cells using the Trizol method and amplified it by one-step RT-PCR. We successfully verified circSS18 (i.e., circ_0108126, whose structure is shown in Figure 2) in HS-SY-II cells. Figure 1 The presence of the junction site (as shown) was successfully confirmed by Sanger sequencing. Figure 8 Probes were designed based on the junction site of circ_0108126, and RNA FISH was performed to detect the distribution of the circRNA in HS-SY-II cells. 18s rRNA was used as a cytoplasmic positive control, and U6 was used as a nuclear positive control. Cytoplasmic and nuclear separation experiments were also performed. The results showed that circ_0108126 was distributed in both the cytoplasm and the nucleus (as shown in Figure 2). Figure 9-10 shown).

[0068] Table 4 PCR primer sequence list

[0069]

[0070] Table 5 Probe sequence information

[0071]

[0072] Further experiments were conducted to investigate the circular structure and stability of circSS18, and its circular structure was successfully verified by gDNA-cDNA (e.g. Figure 11 ), the restriction exonuclease RNase R was used to degrade the linear RNA and then perform qPCR to successfully verify the circular structure of circSS18 (e.g. Figure 12 HS-SY-II cells were treated with actinomycin D, and RNA was extracted at 4h, 8h, and 12h, respectively. qPCR amplification of circSS18 and SS18 was performed to detect their expression. The results showed that circSS18 was not easily degraded and was more stable than SS18 (as shown in Figure 2). Figure 13 shown).

[0073] Identification of circSS18 R-loop in synovial sarcoma cells

[0074] There are natural structural differences between R-loop and circR-loop. The restriction endonuclease RNase H can specifically degrade DNA:RNA hybrid chains, while RNase R only degrades R-loop by degrading linear RNA and has no effect on circR-loops. In the CUT&Tag experiment, the DNA:RNA hybrid chain was immunoprecipitated by S9.6 antibody, and then the DNA single strand in the hybrid chain was extracted. During the experiment, RNase H and RNase R treatment groups were added. Primers of the related SS18 gene were designed based on the circSS18 R-loop sequence. qPCR was performed on the CUT&Tag products respectively. After RNase H treatment, the expression of SS18 decreased significantly, confirming that R-loops structure does exist near SS18 DNA; while after RNase R treatment and digestion of linear RNA, the expression of SS18 was less affected, indicating that circSS18 R-loops were formed in this region, successfully confirming that circR-loops can be formed in this fragment region of the SS18 gene (such as Figure 14 shown).

[0075] Table 6 Protein Antibody Information

[0076]

[0077] CircSS18 R-loop in synovial sarcoma cells promotes malignant biological behavior in vitro

[0078] Based on the above verification and identification of circSS18 R-loop, in order to further confirm that circSS18 can form a circR-loop structure and explore the effect of circSS18 R-loop on the SS18 gene, this example selected the human-derived cell line positive for the SS18-SSX1 fusion gene: synovial sarcoma cell line HS-SY-II, and constructed stable cell lines with silencing and overexpression of circSS18, respectively.

[0079] circSS18-related lentiviral vectors were purchased from Suzhou Jima Gene Chemical Technology Co., Ltd. The circSS18 silencing lentiviral vector (LV3-H1-circ_0108126) and its control lentiviral vector (LV3-H1-NC) carried GFP fluorescent protein and puro-resistance gene, while the circSS18 overexpressing lentiviral vector (LV5-EF-1a-circ_0108126) and its control lentiviral vector (LV5-EF-1a-NC) carried GFP fluorescent protein and puro-resistance gene.

[0080] Synovial sarcoma HS-SY-II cells in logarithmic growth phase with good growth status were digested, counted and plated, and incubated in a 37°C incubator with 5% CO2 for 24 hours until the cell confluence reached about 50%-60%, and then virus infection was performed; each lentiviral vector was injected into the cell at 1×10 8 TU / mL, MOI = 10 was added to the corresponding wells of synovial sarcoma cells HS-SY-II, followed by addition of 40uL HirianGA and 40uL HirianGP infection reagents, and cultured in a 37°C, 5% CO2 incubator; 48 hours after transfection, the cell state was observed under a microscope, and the cell culture medium containing the virus solution was replaced with a complete culture medium containing 10% fetal bovine serum; 72 hours after lentiviral infection of synovial sarcoma cells HS-SY-II, puromycin (Puromycin; 10μg / mL) was added to the DMEM complete culture medium for selection, and the culture medium containing puromycin was changed every two days. The cells were observed under a microscope before each culture medium change / cell passaging until the cell growth was stable and almost no dead cells appeared. The puromycin selection concentration was reduced by half to approximately 5μg / ml, and the cells were cultured at this drug concentration for one week. After the fluorescence transfection efficiency of the constructed single silenced and overexpressed cells reached 90%, the expression difference of circSS18 between the experimental group and the control group was detected by qRT-PCR technology, confirming that the stable transfection cell line was successfully constructed (e.g. Figure 15-20 shown).

[0081] The successfully constructed stable cell line overexpressing circSS18 was digested and counted, and CCK8, plate cloning, EDU-555, one-step TUNEL and Transwell assays were performed to analyze the effect of circSS18 (circ_0108126) on synovial sarcoma HS-SY-II cells. The results showed that after silencing circ_0108126, the proliferation rate of synovial sarcoma cells was reduced, the number of invasive and migrating cells was significantly reduced, and the cell apoptosis rate was increased (such as Figure 21-25 ); while synovial sarcoma cells overexpressing circ_0002164 and circ_0108126 had accelerated proliferation, significantly increased the number of invasive and migrating cells, and decreased the apoptosis rate (as shown in Figure 26-30 In vitro functional experiments showed that circ_0108126 could promote the proliferation, invasion, and migration of synovial sarcoma and inhibit its apoptosis.

[0082] In summary, the present invention provides methods for identifying the circSS18 R-loop formed by circ_0108126 in synovial sarcoma and its applications. This method can be used to prepare monoclonal or polyclonal antibodies targeting this site and to develop anti-tumor drugs for the treatment of synovial sarcoma, demonstrating its significant clinical application value.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying circSS18 R-loop in synovial sarcoma, characterized in that: The method comprises the following steps: identifying a circSS18 R-loop formed by a synovial sarcoma circular RNA circ_0108126, wherein the nucleotide sequence of the circSS18 R-loop is shown in SEQ ID NO: 1; The kit includes RNA circ_0108126 reagent, RNA circ_0108126 primer pair, RNA circ_0108126 probe, and primer pair for the circSS18 R-loop site. The circSS18 R-loop was confirmed to be a circR-loop by treating the CUT&TAG assay products with RNase R and RNase H, and using the S9.6 antibody. The RNA circ_0108126 primer pair includes an upstream primer JF1 and a downstream primer JR1. The nucleotide sequence of the upstream primer JF1 is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer JR1 is shown in SEQ ID NO:

3. The nucleotide sequence of the RNA circ_0108126 probe is shown in SEQ ID NO: 4; The primer pair for the circSS18 R-loop site includes an upstream primer F1 and a downstream primer R1. The nucleotide sequence of the upstream primer F1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO:

6.

2. A lentiviral vector for silencing the RNAcirc_0108126 of claim 1, characterized in that: The nucleotide sequence of the lentiviral vector is shown in SEQ ID NO:

7.

3. A lentiviral vector overexpressing the RNAcirc_0108126 of claim 1, characterized in that: The nucleotide sequence of the lentiviral vector is shown in SEQ ID NO:

8.

4. A cell, characterized in that Comprising the lentiviral vector according to claim 2 or 3.

5. Use of the RNA circ_0108126 or circSS18 R-loop according to claim 1 in the preparation of a medicament for treating synovial sarcoma.

6. Use of the lentiviral vector according to claim 2 or 3 in the preparation of a drug for treating synovial sarcoma.

7. Use of the cell according to claim 4 in the preparation of a medicament for treating synovial sarcoma.

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