A protein translating circular RNA and its application

Abstract

This invention provides a protein translated from circular RNA and its applications in the biomedical field. The protein is translated from circular RNA circFOXP1. The protein has a length of 231 amino acids and contains a specific peptide sequence at its C-terminus. This peptide sequence is FPCQWL and exhibits dual localization in the cytoplasm and cell membrane. It directly binds to the non-receptor tyrosine kinase Src on the cell membrane and drives the translocation of Src from the cell membrane to the cytoplasm, thereby activating STAT3. The amino acid sequence of the protein translated from circular RNA is shown in SEQ ID NO.2. Its expression is significantly increased in lung cancer tissues, and it significantly promotes the proliferation and invasion of lung cancer cells and tumor growth in vivo. It effectively inhibits the malignant biological behavior of lung cancer under in vitro and in vivo conditions, and can be applied to the diagnosis, treatment, and preparation of anticancer drugs for lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a protein for translating circular RNA and its applications. Background Art

[0002] In the Human Genome Project, protein-coding genes were annotated for approximately 2.5% of the human genome, while the remaining transcripts were considered non-coding RNAs, including circular RNAs (circRNAs). Circular RNAs (circRNAs) are a class of non-coding RNAs that lack a 5' cap and a 3' poly(A) tail, and are covalently linked in a closed circular structure. Classical theory holds that eukaryotic mRNA-mediated protein translation requires a 5' m7G cap and a 3' poly-A tail; however, circular RNAs, lacking both a 5' m7G cap and a 3' poly-A tail due to their covalently closed circular structure, are considered to lack translational function.

[0003] In existing technologies, research based on high-throughput ribosomal sequencing technology has shown that circular RNA can be transmitted independently of m 7 Special translation elements in the G-hat structure, such as the internal ribosome entry site IRES element or m 6 A-regulated IRES elements translate proteins.

[0004] On the other hand, lung cancer, as one of the leading causes of cancer-related morbidity and mortality worldwide, seriously endangers human health. In my country, lung cancer is on the rise. Lacking specific, stable, and effective diagnostic biomarkers and novel therapeutic targets, most patients are diagnosed at an advanced stage, resulting in poor prognosis. Currently, the main treatments for lung cancer are surgical resection, radiotherapy and chemotherapy, targeted drug therapy, and immunotherapy. Surgical resection is primarily for early-stage patients, while drug therapy suffers from instability and a tendency to develop drug resistance.

[0005] Circular RNAs (RNAs) have a closed circular structure, making them difficult to digest by the ribonuclease RNase R. Compared to linear RNAs, circular RNAs exhibit stable expression. A master's thesis published by Jiangsu University in June 2021 used only 32 lung adenocarcinoma patient samples and only preliminarily assessed the diagnostic role of low expression of hsa_circ_0001320 in peripheral blood of lung adenocarcinoma patients; the conclusions still need to be verified. However, there are currently no reports on the applications of circular RNAs, especially proteins translated from circFOXP1 (circBaseID: hsa_circ_0001320), in the diagnosis, treatment, or preparation of antitumor drugs for lung cancer. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a protein for cyclic RNA translation and its application.

[0007] To achieve the above objectives, the present invention provides a protein translated from circular RNA, which is translated from circular RNA circFOXP1 protein; the protein translated from circular RNA has a length of 231 amino acids and contains a specific peptide sequence at its C-terminus; the peptide sequence is FPCQWL.

[0008] Preferably, the amino acid sequence of the protein translated from the circular RNA is shown in SEQ ID NO.2.

[0009] Preferably, the protein translated from the circular RNA has dual localization in the cytoplasm and cell membrane, directly binding to the non-receptor tyrosine kinase Src in the cell membrane and driving the non-receptor tyrosine kinase Src to translocate from the cell membrane to the cytoplasm, thereby activating STAT3, rather than the bypass activation pathway that relies on JAK2 to activate STAT3, which is different from the traditional JAK2-mediated STAT3 regulatory pathway.

[0010] Preferably, the circular RNA circFOXP1 is formed by back splicing exons 8-12 of the FOXP1 gene, and the sequence contains 692 nucleotides; the nucleotide sequence of the circular RNA circFOXP1 is shown in SEQ ID NO.1; the circular RNA circFOXP1 is translated into a protein (protein FOXP1-231) via an IRES-dependent process.

[0011] Preferably, the circular RNA circFOXP1 nucleotide sequence includes an internal ribosome entry site (IRES) element (543-680 bp), start and stop codons, and an open reading frame across the circularization site.

[0012] As one of the objectives of the invention, the present invention also provides the application of the above-mentioned circular RNA-translated protein in diagnostic and / or therapeutic drugs for lung cancer; the circular RNA-translated protein promotes the proliferation, migration and invasion of lung cancer cells and exhibits high expression on the cell membrane and cytoplasm of the lung cancer cells; especially the expression is significantly increased in lung cancer and intermediate-to-advanced (III+IV) patient tissues.

[0013] Preferably, the lung cancer cells include any one of H292, A549, H23, and H299.

[0014] As one of the objectives of the invention, the present invention also provides a reagent for detecting the expression level of proteins translated from the above-mentioned circular RNA, which can detect the expression level of proteins translated from circular RNA.

[0015] As one of the objectives of the invention, the present invention also provides a kit for diagnosing lung cancer, comprising at least a reagent capable of detecting the protein expression level of the aforementioned circular RNA translation.

[0016] As one of the objectives of the invention, the present invention also provides the application of the proteins translated from the above-mentioned circular RNA as biomarkers or targets in the preparation of drugs for diagnosing and / or treating lung cancer.

[0017] As one of the objectives of the invention, the present invention also provides an antitumor drug, comprising at least the above-mentioned protein translated from circular RNA and reagents for its expression level, as well as a pharmaceutically suitable carrier.

[0018] The aforementioned antitumor drugs include, but are not limited to, drug formulations containing one or more acceptable carriers, diluents, or excipients, such as liposomes, nanoparticles, powders, granules, tablets, etc.; and the antitumor drugs described herein are suitable for administration via any appropriate route of administration, such as injection or infusion (including subcutaneous, intradermal, intramuscular, intra-articular, intragastric, intrasternal, intrathecal, intralesional, intravenous, or intradermal routes), as well as oral, rectal, nasal, local (including buccal, sublingual, or transdermal), vaginal, or parenteral administration routes.

[0019] The nucleotide sequence of the circular RNA circFOXP1 (circBase ID: hsa_circ_0001320) is shown in SEQ ID NO.1, and the amino acid sequence of the protein translated from the circular RNA, namely the translated protein FOXP1-231, is shown in SEQ ID NO.2. The expression of protein FOXP1-231 is significantly increased in lung cancer tissues, and it can significantly promote the proliferation, migration, and invasion of lung cancer cells and promote tumor growth in vivo. It effectively inhibits the malignant biological behavior of lung cancer under in vitro and in vivo conditions, and can be applied to the diagnosis, treatment, and preparation of anticancer drugs for lung cancer.

[0020] The human circular RNA circFOXP1 (circBase ID: hsa_circ_0001320, sequence shown in SEQ ID NO.1) provided by this invention participates in the occurrence and development of lung cancer. As shown in the technical solution provided by this invention, the circular RNA hsa_circ_0001320 has the ability to translate proteins, translating a 231-amino acid protein (named protein FOXP1-231) via an internal ribosome entry site (IRES) element-dependent mechanism. Unlike the full-length FOXP1 protein, protein FOXP1-231 has a unique C-terminal sequence (FPCQWL) and dual localization in the cytoplasm and cell membrane. Protein FOXP1-231 is significantly increased in lung cancer and intermediate-to-advanced lung cancer tissues, indicating its endogenous presence and ability as a diagnostic biomarker for lung cancer. FOXP1-231 can significantly promote the proliferation and migration of lung cancer cells in vitro and tumor growth in vivo, indicating that FOXP1-231 can serve as a novel target for treating lung cancer progression. Importantly, this invention not only reveals a novel, non-classical protein synthesis pattern of FOXP1-231 derived from circular RNA, but also unveils a novel oncogenic mechanism of FOXP1-231: activation of STAT3 via a JAK2-independent bypass pathway, distinct from the traditional JAK2-mediated STAT3 regulatory pathway. Furthermore, the protein FOXP1-231, translated from the circular RNA hsa_circ_0001320, exhibits dual localization across the cell membrane and cytoplasm, directly binding to the non-receptor tyrosine kinase Src at the cell membrane and driving its translocation to the cytoplasm, thereby activating STAT3 via a JAK2-independent bypass pathway. This provides new insights and directions for the prevention and treatment of lung cancer and other tumors. Based on the above analysis, the protein generated from the translation of circular RNA can serve as a novel drug target for the prevention and treatment of lung cancer, and can be stably expressed, thus overcoming the drug resistance problem caused by target loss and expanding new avenues for the preparation of anti-tumor drugs.

[0021] The beneficial technical effects obtained by this invention are as follows:

[0022] 1. This invention constructs a flag-tagged vector system that stably expresses the circular RNA circFOXP1 (circBase ID: hsa_circ_0001320) and prepares a specific antibody targeting the unique C-terminal peptide (FPCQWL) of the novel protein FOXP1-231. Simultaneously, silver staining combined with Western blotting and mass spectrometry analysis verifies that the circular RNA circFOXP1 translates into the novel protein FOXP1-231. The novel protein FOXP1-231 translated from the circular RNA circFOXP1 can serve as a novel biomarker and target for the preparation of drugs for the diagnosis and / or treatment of lung cancer, as well as for the progression of lung cancer.

[0023] 2. The protein FOXP 1-231 provided by the technical solution of this invention can directly bind to the enzyme activity domain of the non-receptor tyrosine kinase Src, promoting the translocation of Src from the cell membrane to the cytoplasm. As a scaffold protein, it promotes the activation of downstream molecules by inducing STAT3 tyrosine phosphorylation through Src binding, which is different from the traditional JAK2-mediated STAT3 regulatory pathway. Based on this, it is significantly different from proteins derived from linear mRNA, thus FOXP 1-231, a protein derived from the translation of stably expressed circular RNA, is expected to overcome the drug resistance problem induced by target loss, providing a new approach for the preparation of antitumor drugs.

[0024] 3. The protein product translated from the circular RNA-hsa_circ_0001320 provided by the technical solution of this invention can be used in the preparation of products for diagnosing lung cancer, products for diagnosing the malignancy of lung cancer, and anticancer drugs. Attached Figure Description

[0025] Figure 1A This is a schematic diagram of the reverse circularization of circular RNA hsa_circ_0001320 in Embodiment 1 of the present invention.

[0026] Figures 1B-1D This is a diagram showing the results of the identification of the reverse circularization site of circular RNA hsa_circ_0001320 using Sanger-seq experiments and polymerization and divergence primer experiments (A549 and H23 cells) in Example 1 of this invention.

[0027] Figures 2A-2E This is a graph illustrating the analysis of the endogenous expression and translational capacity of the circular RNA hsa_circ_0001320 in Example 2 of the present invention.

[0028] Figures 3A-3D This is a diagram illustrating the results of Example 3 of the present invention, which demonstrates that the circular RNA hsa_circ_0001320 is translated into a new protein FOXP1-231 with a length of 231 amino acids via an IRES-dependent manner.

[0029] Figures 4A-4C This is an analysis diagram showing the results of Example 4 of the present invention, which confirms that the protein FOXP1-231 translated from the circular RNA hsa_circ_0001320 is located in the cell membrane and cytoplasm.

[0030] Figures 5A-5B This is a graph showing the expression results of FOXP1-231, the protein translated from the circular RNA hsa_circ_0001320 in lung cancer cells and tissues, in Example 5 of this invention.

[0031] Figures 6A-6FThis is a graph showing the effect of FOXP1-231, the protein translated from the circular RNA hsa_circ_0001320 in Example 6 of the present invention, on the proliferation, migration, and invasion abilities of lung cancer cells.

[0032] Figures 7A-7B This is a graph showing the effect of FOXP1-231, the protein translated from the circular RNA hsa_circ_0001320 in Example 7 of the present invention, on tumor growth. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0034] This invention constructs a flag-tagged vector system that stably expresses the circular RNA circFOXP1 (circBase ID: hsa_circ_0001320) and prepares a specific antibody targeting the unique C-terminal peptide (sequence FPCQWL) of the novel protein FOXP1-231. Simultaneously, silver staining combined with Western blotting and mass spectrometry analysis was used to verify that the circular RNA circFOXP1 translates into a novel protein, FOXP1-231.

[0035] The FOXP1-231 protein sequence contains 231 amino acids, a unique C-terminal peptide sequence (FPCQWL), and unique dual localization to the cell membrane and cytoplasm. This FOXP1-231 sequence is distinct from all previously reported FOXP1 proteins and isoforms. Furthermore, the expression of the FOXP1-231 protein is significantly increased in lung cancer and advanced (III+IV) patient tissues, and this molecule significantly promotes the proliferation, migration, and invasion of lung cancer cells, especially A549 and H23.

[0036] In particular, the protein FOXP1-231 can also promote tumor growth in vivo. Experimental results show that FOXP1-231, translated from the circular RNA circFOXP1, can serve as a novel biomarker for diagnosing lung cancer and its progression, and a new target for the preparation of anticancer drugs. Furthermore, it indicates that FOXP1-231 possesses a unique pro-cancer mechanism: FOXP1-231 directly binds to the enzymatic active domain of the non-receptor tyrosine kinase Src, promoting the translocation of Src from the cell membrane to the cytoplasm. As a scaffold protein, it further promotes Src binding, inducing STAT3 tyrosine phosphorylation to activate downstream molecules. This mechanism suggests that it differs from proteins derived from linear mRNA. The novel protein FOXP1-231, derived from stably expressed circular RNA, holds promise for overcoming drug resistance induced by target loss, providing a new pathway for the preparation of antitumor drugs.

[0037] Furthermore, the present invention provides a reagent for detecting the expression level of circular RNA circFOXP1 (circBaseID: hsa_circ_0001320) in lung cancer and control tissues, for use in the preparation of diagnostic kits for lung cancer.

[0038] This invention provides a kit for detecting the expression level of protein FOXP1-231 for the diagnosis of lung cancer. The amino acid sequence of FOXP1-231 is shown in SEQ ID NO.2.

[0039] Specifically, the above kit can detect the expression level of protein FOXP1-231 in lung cancer and control tissues. Based on the increased expression level of protein FOXP1-231 in lung cancer tissues, lung cancer can be diagnosed and treated. The amino acid sequence of protein FOXP1-231 is shown in SEQ ID NO.2.

[0040] Furthermore, the reagents provided by this invention for detecting the expression level of protein FOXP1-231 can be used in the preparation of anti-lung cancer drugs.

[0041] Preferably, the present invention provides a pharmaceutical composition comprising at least a reagent for detecting the expression level of protein FOXP1-231 and a pharmaceutically suitable carrier.

[0042] Furthermore, the present invention can provide any one or more reagents of the above-mentioned pharmaceutical composition for the preparation of anti-lung cancer drugs, including inhibiting lung cancer cell proliferation, migration and lung cancer progression.

[0043] The present invention relates to the preparation of antitumor drugs, which is not limited to pharmaceutical preparations containing one or more acceptable carriers, diluents or excipients (such as liposomes, nanoparticles, powders, granules, tablets, etc.); the pharmaceutical preparations involved are suitable for application via any appropriate route of administration, such as injection or infusion (including subcutaneous, intradermal, intramuscular, intra-articular, intragastric, intrasternal, intrathecal, intralesional, intravenous or intradermal), and oral, rectal, nasal, local (including buccal, sublingual or transdermal), vaginal or parenteral routes of administration.

[0044] Furthermore, the aforementioned antitumor drugs are preferably used as biomarkers for diagnosing lung cancer and as targeted drugs for treating lung cancer progression.

[0045] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0046] Cell lines and cell culture: The lung cancer cell lines A549, H23, H292, and H1299, and the normal control cell lines Beas-2B and HEK-293T were all purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. All cell lines underwent STR identification. Cells were cultured in complete medium containing 10% fetal bovine serum (GIBCO, NY, USA) and DMEM (GIBCO, NY, USA) medium and 1×10⁻⁶ ppm. 5 U / L penicillin + streptomycin (GIBCO, NY, USA); culture environment: 37℃, 5% CO2.

[0047] Clinical Samples: This case was reviewed by the Clinical Trial Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (YFY2018-KL023-01) and patients signed informed consent forms. A total of 50 patients with non-small cell lung cancer, pathologically diagnosed and treated at the Department of Thoracic Surgery, Affiliated Hospital of Xuzhou Medical University between 2016 and 2020, were included. Tumor tissue and adjacent normal tissue (adjacent normal tissue: tissue >5 cm from the tumor tissue) samples were collected from surgically treated patients and rapidly placed in 1 ml of 4°C RNA later to prevent RNA degradation during freezing. The samples were then flash-frozen in liquid nitrogen and stored at -80°C.

[0048] Unless otherwise specified, all reagents used in this invention are commercially available.

[0049] Example 1

[0050] This embodiment first verified the cyclic properties of hsa_circ_0001320.

[0051] The circular RNA hsa_circ_0001320, located at chr3:71064699-71102924, is formed by backsplicing exons 8-12 of the FOXP1 gene and contains 692 nucleotides, as shown in SEQ ID NO.1. Figure 1A We designed a primer pair specifically for recognizing the circular RNA hsa_circ_0001320, including an upstream primer (SEQ ID NO.3) and a downstream primer (SEQ ID NO.4), and detected its expression using qRT-PCR. Sanger sequencing confirmed its reverse circularization site (…). Figure 1B Simultaneously, agarose gel electrophoresis experiments were performed on cDNA and gDNA in A549 and H23 cells using polymerization and diffusion primers.

[0052] The nucleotide sequence of the circular RNA circFOXP1 (circBase ID: hsa_circ_0001320) is shown in SEQ ID NO.1.

[0053] The experimental scheme of this embodiment includes:

[0054] RNA extraction and real-time quantitative polymerase chain reaction (Real-time PCR) were performed. All primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 1. Total RNA was extracted using Trizol... TM LS (Nanjing Novizan Biotechnology Co., Ltd.). Reverse transcription was performed using the Primer Script reverse transcription kit. TMLS (Invitogen, Carlsbad, USA). The real-time quantitative PCR kit is from TB Green. TM Premix Ex Taq TM 11 (TaKaRa, Dalian, China), detected using a Light Cycler 96 real-time quantitative PCR system (Bio-Rad, California, USA). RNA relative level = 2. ΔΔC Method t. The operating procedure follows the instructions in the product manual.

[0055] Table 1 Primer sequences

[0056]

[0057] Agarose gel electrophoresis and Sanger sequencing experiments

[0058] The extracted PCR samples were subjected to electrophoresis on a 1% agarose gel containing nucleic acid dyes. First, 6× DNA loading buffer (TransGen Biotech, Beijing, China) was added to the sample, and then diluted with double-distilled water to 1× DNA loading buffer. Next, electrophoresis buffer was added to the electrophoresis tank (Bio-Rad, Califomia, USA), and the gel was placed in the tank. 20 μL of sample was loaded, and the instrument was set to 100V for 80 minutes. After electrophoresis, the gel was removed, photographed, and the target band was cut with a clean blade for Sanger sequencing.

[0059] Sanger sequencing results are as follows Figure 1B As shown, the characteristic backsplicing site of the circular RNA hsa_circ_0001320 is "TCTAA". TTCCC". And... Figure 1C The results showed that the expected band was detected in the cDNA lane of A549 cells, but no band was detected in the gDNA lane. Figure 1D As shown, the same results were obtained in H23 cells.

[0060] Therefore, the above results confirm the circularization site of the circular hsa_circ_0001320.

[0061] Example 2

[0062] This embodiment verifies the endogenous expression and translational ability of the circular RNA hsa_circ_0001320.

[0063] The specific nucleic acid probe kit for circular RNA hsa_circ_0001320 (Ribo™ Fluorescent InSitu Hybridization Kit, R11060.7) was purchased from Guangzhou Ribo Biotechnology Co., Ltd.

[0064] Pre-treat glass slides in 24-well plates with UV sterilization for 2 hours; then seed cells at approximately 40% density; after 24 hours, take the pre-treated cells, wash with PBS, fix with paraformaldehyde at room temperature for 10 minutes; wash three times with PBS; then add pre-cooled PBS containing 0.5% Triton X-100 to permeate the cells, incubate at 4°C for 5 minutes; wash three times with PBS; add 400 μL of pre-hybridization solution to each well, block at 37°C for 30 minutes, and preheat the hybridization solution at 37°C.

[0065] After blocking, discard the prehybridization solution and add 200 μL of hybridization solution containing 5 μL of probe. Incubate overnight at 37°C in the dark. The next day, discard the hybridization solution and wash the cells for 5 min with hybridization wash I (4×SSC buffer, 0.1% Tween-20, preheated to 42°C); then wash the cells for 5 min with hybridization wash II (2×SSC, preheated to 42°C); and then wash the cells for 5 min with hybridization wash III (1×SSC buffer, preheated to 42°C). Stain with 1×DAPI at room temperature for 10 min. After washing with PBS, remove the slide, fix it on a glass slide, photograph it, and store it in the dark.

[0066] Sucrose density gradient centrifugation experiment

[0067] Prepare sucrose solutions with different concentration gradients (50%, 40%, 30%, 20%, 10%), and add 2.2 mL of the above sucrose solutions to ultracentrifuge tubes in sequence, and incubate at 4°C overnight to form a uniform concentration gradient liquid in the tubes.

[0068] Add 10 μL (100 μg / mL) of cyclohexane (CHX) to 10 mL of DMEM medium and incubate at 37°C for 10 min. Discard the medium, wash the cells three times with pre-chilled PBS containing CHX, collect the cells in a 1.5 mL EP tube using trypsin, wash the cells again with PBS containing CHX, centrifuge at 500g, and discard the PBS. Then, add 1 mL of PEB solution (PEB solution pre-added with 10 μL CHX, 10 μL protease inhibitor, and 5 μL RNase inhibitor) to the tube, incubate the cells on ice for 10 min, gently inverting the EP tube every 2 min; centrifuge at 12000g at 4°C for 10 min, and collect the supernatant cell lysis buffer; then add the same volume of cell lysis buffer to the top of the ultracentrifuge tube to ensure complete equilibration. Centrifuge at 190000g at 4°C for 90 min. After centrifugation, slowly aspirate the liquid from top to bottom, approximately 1 mL per aliquot. RNA was extracted from each sample separately, and then diluted with 12 μL LEPC water. RNA content was measured, and RNA fraction peaks were plotted. Finally, the remaining RNA fractions were reverse transcribed, and the content of circRNA in each fraction was detected by qRT-PCR to analyze their expression levels.

[0069] See Figure 2A The results showed that, compared with matched adjacent normal control tissues (tissues > 5 cm from tumor tissue), the expression of circular RNA hsa_circ_0001320 was significantly increased in lung cancer patient tissues.

[0070] See Figure 2B The results showed that hsa_circ_0001320 was significantly increased in cytoplasm expression.

[0071] See Figure 2C As shown in the figure, RNA-FISH experiments revealed that hsa_circ_0001320 was mainly located in the cytoplasm.

[0072] See Figure 2D As shown, the sucrose density gradient centrifugation experiment proved that hsa_circ_0001320 can bind to polyribosomes, while puromycin inhibits its binding to ribosomes.

[0073] Figure 2E This is a schematic diagram of the translation potential analysis of the circular RNA hsa_circ_0001320. The translation potential analysis of the circular RNA shows that the nucleotide sequence of hsa_circ_0001320 contains an internal ribosomal entry site (IRES) element (543-680 bp), start and stop codons, and an open reading frame (ORF) that crosses the circularization site, which can encode a new protein product with a length of 231 amino acids.

[0074] The above results indicate that hsa_circ_0001320 is a circular RNA molecule that is highly expressed in lung cancer and has translational capabilities.

[0075] Example 3

[0076] In this embodiment, a new protein product “FOXP1-231” with a length of 231 amino acids was translated using the circular RNA hsa_circ_0001320 in an IRES-dependent manner.

[0077] The amino acid sequence of protein FOXP1-231 is shown in SEQ ID NO.2:

[0078]

[0079] Guangzhou Gise Biotechnology Co., Ltd. ordered three plasmids (catalog number WR|44) fused with the 3×Flag tag [circFOXP1, ATG-mutant (ATG mutated to TTG), FOXP1-231] to transfect A549 and H23 cells. Cells were harvested after 72 hours and proteins were extracted.

[0080] The overexpression of circFOXP1 and the ATG mutant plasmid sequence are shown in SEQ ID NO.7:

[0081]

[0082] The sequence of the FOXP1-231 overexpression plasmid is shown in SEQ ID NO.8:

[0083]

[0084]

[0085] Immunoblotting and an antibody designed specifically for the FOXP1-231 sequence (anti-FOXP1-231, Ibotek (Wuhan) Biotechnology Co., Ltd., WG-06120) and a Flag antibody (PTG, Wuhan, 66008-4-Ig) from Wuhan Sanying Biotechnology Co., Ltd. were used to detect FOXP1-231 protein expression, further identifying the peptide fragment of FOXP1-231 translated from the circular RNA hsa_circ_0001320. Silver staining and mass spectrometry analyses were also performed. A dual-luciferase assay was used to analyze whether the circular RNA hsa_circ_0001320 was translated into protein via IRES.

[0086] The experimental procedure is as follows:

[0087] Western blot assay

[0088] 1) Glue preparation: Take a clean glass plate, fix it, pour in the prepared lower layer glue until it is about 2 cm from the top. After about 30 minutes, when the lower layer glue has completely solidified, add the upper layer glue, insert a comb to absorb air bubbles, and after about 30 minutes, remove the comb.

[0089] 2) Sample loading: Add each group of protein samples and markers to the wells. Perform electrophoresis at 80V, and adjust the voltage to 120V according to the separation of protein samples until the target protein is separated. Cut the PVDF membrane according to the molecular weight of the target protein and the size of the gel, and transfer the membrane at a constant current of 4℃.

[0090] 3) Blocking and Antibody Incubation: After transfer, place the membrane in freshly prepared blocking buffer (5% skim milk) and block on a shaker at room temperature for 2-3 hours. Wash three times with TBST buffer, and incubate with the target antibody solution overnight at 4°C on a shaker. The next day, wash three times with TBST solution, and incubate with the matched secondary antibody at room temperature for 2 hours. Wash three times with TBST solution.

[0091] The next day, wash the sample three times with TBST solution on a shaker for three minutes each time. Incubate the corresponding secondary antibody at room temperature for 2 hours. Prepare the exposure solution for the ECL chemiluminescence substrate kit at a 1:1 ratio. Expose the sample using a chemiluminescence instrument and save the image.

[0092] Sample preparation for silver staining experiment and mass spectrometry detection

[0093] First, load the protein marker and the protein sample to be mass spectrographed, and perform SDS-PAGE electrophoresis (method as in Example 1). Then, immerse the gel in 100 mL of fixative and fix it on a shaker at room temperature for 40 min. Next, wash with 200 mL of ultrapure water for 10 min, discard the water, add 100 mL of silver staining sensitization solution, and shake at room temperature for 2 min. Wash with ultrapure water for 1 min, repeating twice. Discard the water, add 100 mL of silver staining sensitization solution, shake at room temperature for 10 min, discard the water, and wash with ultrapure water for 1–1.5 min. Use 100 mL of silver staining developing solution and shake at room temperature for 3–10 min. Stop the process when a clear band appears, and add 100 mL of stop solution to finish the color development.

[0094] Sample preparation for mass spectrometry detection: Use a clean blade to cut the target silver-stained protein band at 25-40 kDa and store it in ddH2O for mass spectrometry analysis.

[0095] Dual-luciferase activity assay

[0096] IRES wild-type sequence plasmid (543-680bp retained), IRES mutant 1 (543-611bp retained), mutant 2 (611-680bp retained), and control plasmid were constructed from nucleotides 543-680bp of SEQ ID NO.1 (hsa_circ_0001320). All of these plasmids were obtained from Guangzhou Gise Biotechnology Co., Ltd. The above four groups are the IRES wild-type group, mutant group 1, mutant group 2, and control group, respectively.

[0097] The wild-type IRES plasmid sequence is shown in SEQ ID NO. 9.

[0098]

[0099] The sequence of IRES mutant 1 is shown in SEQ ID NO. 10.

[0100]

[0101] The sequence of IRES mutant 2 is shown in SEQ ID NO. 11.

[0102]

[0103] The above dual-luciferase plasmids were transfected into HEK-293T cells. After 48 hours, the cells were harvested and the culture medium was discarded. Approximately 500 μL of reporter gene cell lysis buffer was added, and the cells were lysed at room temperature for 5–10 min; centrifuged at 12000g for 5 min; the supernatant was collected for later use. Next, 20 μL of the sample was taken for dual-luciferase detection. 100 μL of firefly luciferase detection reagent was pipetted and mixed well before measuring the fluorescence intensity. Subsequently, 100 μL of Renilla luciferase detection working solution was added, and the fluorescence intensity was measured. Using Renilla luciferase as an internal control, the IRES activity of different groups was analyzed based on the ratio of firefly luciferase fluorescence to Renilla luciferase fluorescence.

[0104] The results are as follows Figure 3A As shown, in A549 and H23 cells, expression of the novel protein FOXP1-231 could be detected in both the circular RNA hsa_circ_0001320 lane and the FOXP1-231 lane; after the ATG start codon mutation of the circular RNA hsa_circ_0001320 sequence, the novel protein FOXP1-231 was almost undetectable.

[0105] Figure 3B The silver staining experiment results showed that after overexpression of hsa_circ_0001320, the expression of protein FOXP1-231 was significantly increased.

[0106] Figure 3CMass spectrometry analysis confirmed the unique peptide sequence “FPCQWL” of protein FOXP1-231.

[0107] Figure 3D The results of the dual-luciferase reporter assay showed that, compared with the control group, the fluorescence activity of the wild-type IRES group of circular RNA hsa_circ_0001320 was significantly increased, while the activity of the two IRES sequence mutant groups was significantly decreased.

[0108] The above results indicate that the circular RNA hsa_circ_0001320 is translated into a new protein, FOXP1-231, via an IRES-dependent process.

[0109] Example 4

[0110] This embodiment analyzes the subcellular localization of the protein FOXP1-231.

[0111] See Figure 4A A structural comparison diagram of protein FOXP1-231 and the full-length protein FOXP1 is shown. Analysis of the structures revealed that the N-terminal amino acid sequence (101-325) of FOXP1-231 is identical to that of the full-length FOXP1 (677aa). However, FOXP1-231 possesses a unique C-terminal peptide segment, "FPCQWL". Therefore, this invention transfects cells with the plasmid hsa_circ_0001320 and uses immunofluorescence combined with Flag antibody to analyze the subcellular localization of FOXP1-231.

[0112] In addition, H23 cells were transfected with FOXP1-231-3×Flag, and cell membrane and cytoplasmic protein components were extracted. The protein expression of FOXP1-231 in each component was detected by Western blotting.

[0113] The specific experimental plan is as follows:

[0114] Immunofluorescence assay:

[0115] The hsa_circ_0001320 plasmid was transfected into A549 cells in 24-well plates. After 24 hours, the culture medium was discarded, and the cells were washed with PBS for 5 minutes, repeated three times. Cells were then fixed with 4% paraformaldehyde at room temperature for 10 minutes, followed by washing with PBS for 5 minutes, repeated three times. Blocking with 5% BSA for 2 hours was performed, and the blocking solution was discarded. Diluted Flag antibody (Wuhan Sanying Biotechnology Co., Ltd., 66008-4-Ig) was added and incubated overnight at 4°C. Cells were washed with PBS for 5 minutes, repeated three times. Finally, diluted secondary antibody RFP-Booster Alexa was added under light-protected conditions. Cells were incubated with 568 (Wuhan Sanying Biotechnology Co., Ltd., rb2AF568) for 1 hour (room temperature). Cells were then washed with PBS for 5 minutes, repeated three times. Cells were stained with 1×DAPI solution in a dark room for 10 minutes at room temperature. The slide was then removed, and the film was fixed onto a glass slide using a suitable fixative. Images were acquired using a laser scanning confocal microscope.

[0116] See Figure 4B As shown, FOXP1-231 is located in the cell membrane and / or cytoplasm.

[0117] See Figure 4C As shown, the immunoblotting experiment after cell membrane / cytoplasm separation demonstrated that FOXP1-231 was increased in both the cell membrane and cytoplasm.

[0118] The results above indicate that FOXP1-231 is a novel protein molecule with unique dual localization in the cell membrane and cytoplasm, demonstrating that FOXP1-231 has a nuclear localization distinct from the full-length FOXP1 protein.

[0119] Example 5

[0120] This embodiment validates the expression of protein FOXP1-231 in lung cancer cells and tissues.

[0121] Immunoblotting combined with FOXP1-231 antibody [anti-FOXP1-231, Ibotek (Wuhan) Biotechnology Co., Ltd., WG-06120] was used as a control group to detect the protein expression of FOXP1-231 in various lung cancer cell lines (H292, A549, H23, H299). Adjacent normal tissue was used as a control group, and the expression of FOXP1-231 in the tissues was compared with that in lung cancer tissue. Simultaneously, the protein expression of FOXP1-231 in lung cancer tissues from early-stage (I+II) and intermediate-to-late-stage (III+IV) patients was compared. The immunoblotting procedure was the same as in Example 3.

[0122] See Figure 5A and Figure 5B As shown in the figure, the expression of protein FOXP1-231 was significantly increased in lung cancer cells and tissues; specifically, Figure 5A In comparison with the control group (Beas-2B cells), FOXP1-231 expression was significantly increased in various lung cancer cell lines (H292, A549, H23, H299).

[0123] Figure 5BIn comparison with the control group in adjacent normal tissue, FOXP1-231 expression was significantly increased in lung cancer patients and patients with intermediate and advanced (III+IV) lung cancer. *** indicates P < 0.001, * indicates P < 0.05.

[0124] Example 6

[0125] This study investigated the effects of FOXP1-231, a protein translated from circular RNA hsa_circ_0001320, on the proliferation, migration, and invasion abilities of lung cancer cells. Changes in cell proliferation were analyzed using the CCK8 assay, and changes in cell migration and invasion abilities were analyzed using cell migration and invasion assays.

[0126] The specific experimental plan includes:

[0127] Four plasmids were constructed using the above method: control (control group), circFOXP1-3×Flag, ATG-mut-3×Flag mutant plasmid, and FOXP1-231-3×Flag. A549 and H23 cells were transfected with these plasmids and the cells were harvested 48 hours later.

[0128] The expression of circular RNA was detected by PCR, using conventional methods available in the field.

[0129] The results are as shown in 6A and Figure 6B As shown in the figure, the PCR results demonstrate that overexpression and mutation of circular RNA can significantly increase circular RNA expression, but do not affect the level of linear mRNA.

[0130] CCK8 assay: The effects of overexpression of circFOXP1, circFOXP1-ATG mutation, and FOXP1-231 on the proliferation of A549 and H23 cells were detected. 4000 A549 or H23 cells were seeded into each well of a 96-well plate, and the volume was increased to 200 μL / well with complete culture medium. Five replicates were set for each sample group. CCK8 mixture (CCK8:complete culture medium = 1:10) was prepared at 0, 24, 48, and 72 hours, and 10 μL of CCK8 mixture (APExBIO, Houston, USA) was added to each well. The plates were incubated at 37°C for 2 hours, and the absorbance (OD 450 nm) was read using a multi-plate reader (Bio-Tek, Minnesota, USA).

[0131] The results are as follows Figures 6C-6D As shown, compared with the control group, hsa_circ_0001320 and FOXP1-231 significantly improved the proliferation ability of lung cancer cells, while the ATG mutation group did not show a significant increase.

[0132] The results showed that the protein FOXP1-231 translated from the circular RNA hsa_circ_0001320 significantly promoted the proliferation of lung cancer cells, while ATG mutation significantly inhibited cell proliferation.

[0133] Cell migration and invasion assays: This invention uses cell migration and invasion assays to detect the effects of overexpression of circFOXP1, circFOXP1-ATG mutation, and FOXP1-231 on the migration and invasion of lung cancer A549 and H23 cells. A clean Transwell chamber (8.0 μm pore size, Corning Costar, Kennebunk USA) was placed in a 24-well plate, and 800 μL of culture medium containing 15% serum was added along the wall of the 24-well plate to the bottom of the Transwell chamber. 2 × 10⁶ cells were then added. 4 Cells were cultured at a density of 100 μL. 200 μL of the well-mixed cell suspension was added to a Transwell chamber and incubated at 37°C for 24 h. The 24-well plate was then removed, the culture medium discarded, and 800 μL of pre-chilled methanol was added to the lower chamber of the Transwell chamber. Cells were fixed at 25°C for 20 min. The methanol was discarded, and the cells were washed twice with PBS. Crystal violet staining solution was added, and after 30 min, the chamber was inverted and allowed to air dry at room temperature before being photographed under a microscope.

[0134] The results are as follows Figures 6E-6F As shown, compared with the control group, circular RNA hsa_circ_0001320 and FOXP1-231 significantly enhanced the invasive ability of lung cancer cells, while the ATG mutation in the start codon of circular RNA hsa_circ_0001320 significantly inhibited the invasive ability of cells.

[0135] The results showed that the protein FOXP1-231, translated from the circular RNA hsa_circ_0001320, promotes the migration and invasion of lung cancer cells, while ATG mutation significantly inhibits cell invasion. In the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0136] In summary, the protein FOXP1-231 translated from the circular RNA hsa_circ_0001320 is a novel target for promoting lung cancer cell proliferation and invasion.

[0137] Example 7

[0138] This invention uses a nude mouse subcutaneous tumor model to demonstrate the effect of FOXP1-231, translated from the circular RNA hsa_circ_0001320, on tumor growth.

[0139] The experimental procedure is as follows:

[0140] Subcutaneous tumor formation experiment in nude mice: Groups: FOXP1-231 and control group, stable transfection knockdown of hsa_circ_0001320 (circ 0001320-shl) and control group (n=6).

[0141] H23 cells stably transfected with the above plasmid were constructed. 6-7 week old BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected, and 1×10⁻⁶ H23 cell suspensions were collected. 7 / 200μL (cell suspension: matrix gel = 4:1). Inject subcutaneously using a 1 mL syringe. One week later, record the weight and tumor volume of the nude mice every other day; collect the subcutaneous tumors 19-22 days later; subcutaneous tumor volume is calculated as (length × width) 2 ) / 2. All animal experiments were approved by the Animal Experiment Ethics Committee of Xuzhou Medical University and complied with the "Guidelines for the Care and Use of Laboratory Animals".

[0142] See Figures 7A-7B This is a graph showing the relationship between the protein FOXP1-231, translated from the circular RNA hsa_circ_0001320, and tumor growth. Specifically, as shown... Figure 7A As shown, the protein FOXP1-231 promotes tumor growth. Compared with the control group, the FOXP1 overexpression group showed accelerated tumor growth, with a significant increase in tumor weight and volume. Figure 7B As shown, tumors in the circ_0001320 knockdown group (circ_0001320-sh-1) exhibited slow growth, with significantly reduced tumor weight and volume, indicating that circFOXP1 knockdown inhibits tumor growth. Simultaneously, the co-transfected FOXP1-231 group showed accelerated tumor growth, with significantly increased tumor weight and volume, demonstrating that FOXP1-231 reversed the inhibitory effect of circFOXP1 knockdown on tumor growth. In the figure, * indicates P < 0.05, and ** indicates P < 0.01.

[0143] The above results indicate that the novel protein FOXP1-231, translated from the circular RNA hsa_circ_0001320, can promote tumor growth in vivo.

[0144] The above examples demonstrate that the circular RNA hsa_circ_0001320 possesses the ability to translate proteins, specifically a 231-amino acid protein (named protein FOXP1-231) via an internal ribosome entry site (IRES) element-dependent translation. The novel protein FOXP1-231 showed significantly increased expression in lung cancer and intermediate-to-advanced lung cancer tissues, indicating its endogenous presence and potential as a diagnostic biomarker for lung cancer. Furthermore, FOXP1-231 significantly promoted the proliferation and migration of lung cancer cells in vitro and tumor growth in vivo, suggesting that FOXP1-231 is a novel therapeutic target for lung cancer progression.

[0145] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. The use of a protein translated from a circular RNA as a biomarker or target in the preparation of drugs for the diagnosis and / or treatment of non-small cell lung cancer; wherein the protein translated from the circular RNA is translated from circular RNA circFOXP1; wherein the protein translated from the circular RNA has a length of 231 amino acids and contains a specific peptide sequence at its C-terminus; wherein the peptide sequence is FPCQWL; The proteins translated from the circular RNA have dual localization in the cytoplasm and cell membrane; The protein translated from the circular RNA directly binds to the non-receptor tyrosine kinase Src at the cell membrane and drives the non-receptor tyrosine kinase to translocate from the cell membrane to the cytoplasm, thereby activating STAT3. The proteins translated from the circular RNA showed high expression in the cell membrane and cytoplasm of non-small cell lung cancer cells; The proteins translated from the circular RNA promote the proliferation and migration of non-small cell lung cancer cells in vitro and tumor growth in vivo. The amino acid sequence of the protein translated from the circular RNA is shown in SEQ ID NO.

2.

2. The use of a reagent for detecting protein expression levels in the translation of circular RNA in the preparation of a diagnostic kit for non-small cell lung cancer, characterized in that, The amino acid sequence of the protein translated from the circular RNA is shown in SEQ ID NO.2.

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