Small molecule RNA tRF-5023b and application thereof in preparation of anti-non-small cell lung cancer drugs
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG NEW TIME PHARMA CO LTD
- Filing Date
- 2026-03-13
- Publication Date
- 2026-06-23
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule RNA tRF-5023b and its application in the preparation of drugs for treating non-small cell lung cancer. Background Technology
[0002] Non-small cell lung cancer (NSCLC), the main pathological type of lung cancer, accounts for approximately 80%-85% of all lung cancer cases. Its high mortality rate is closely related to its late-stage diagnosis rate, with about 46% of patients already diagnosed at stage IIIB / IV. Although targeted therapy and immune checkpoint inhibitors have significantly improved the prognosis of some patients, many challenges remain in clinical practice: targeted therapy relies on specific driver gene mutations, limiting its applicability to certain populations, and secondary drug resistance is difficult to avoid; immunotherapy has a response rate of less than 20% in patients with negative PD-L1 expression or low tumor mutational burden (TMB), and activation of alternative immunosuppressive pathways such as FGL1 / LAG-3 further leads to treatment escape. Furthermore, immune-related adverse events (irAEs) such as pneumonia and colitis also limit long-term use. Therefore, developing novel treatment strategies that can overcome drug resistance, broaden the applicable population, and have manageable toxicity is urgently needed.
[0003] Nucleic acid drugs, as a third-generation innovative therapy, achieve precise regulation through direct intervention in gene expression, demonstrating unique advantages in the field of cancer treatment. Their core value lies in their ability to target "undruggable" proteins that are difficult to target with traditional small molecule and antibody drugs, and they boast short design cycles and efficient candidate molecule screening. However, the application of existing nucleic acid drugs in solid tumors, especially NSCLC, still faces significant bottlenecks: low delivery system efficiency leads to insufficient targeting to lung tissue, and nuclease degradation and immunosuppressive barriers in the tumor microenvironment (TME) further limit drug efficacy. Although more than 20 nucleic acid drugs have been approved globally, nucleic acid therapies for NSCLC are still in the exploratory stage, urgently requiring breakthroughs in both delivery technology and target selection limitations.
[0004] Small RNA fragments (tRFs) derived from tRNA are a class of non-coding RNAs approximately 14-30 nt in length. Their mechanisms of regulating tumor progression are multidimensional: they can silence target messenger RNAs (mRNAs) via AGO2-mediated silencing, similar to small interfering RNAs (siRNAs); they can also act as competitive endogenous RNAs (ceRNAs) to adsorb microRNAs (miRNAs), relieving their inhibition of tumor suppressor genes; simultaneously, they can directly interfere with the formation of the translation initiation complex or consume the mature tRNA pool, forcing tumor cells into a state of metabolic stress. In NSCLC, some tRFs have been shown to affect chemosensitivity or metastasis, but the target networks of the vast majority of tRFs and their potential to intervene in immune checkpoints remain unresolved. Furthermore, how to improve the stability and targeting of tRFs through engineering remains an unsolved technical challenge. Therefore, in-depth elucidation of the functional mechanisms of specific tRFs in NSCLC and the development of innovative tRF-based drugs will not only provide new ideas for overcoming existing therapeutic limitations but will also promote the clinical translation of nucleic acid drugs in the field of solid tumors. Summary of the Invention
[0005] This invention addresses the shortcomings of existing technologies by providing an application of the small molecule RNA tRF-5023b in the preparation of drugs for treating non-small cell lung cancer.
[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:
[0007] A small RNA tRF-5023b, the nucleotide sequence of which is shown in SEQ ID No. 1, which is: GUCAGGAUGGCCGAGCGGUCUAA.
[0008] This invention provides the application of the small molecule RNA tRF-5023b in the preparation of drugs for treating lung cancer.
[0009] Specifically, the lung cancer is either non-small cell lung cancer or small cell lung cancer.
[0010] More specifically, the lung cancer in question is non-small cell lung cancer.
[0011] Specifically, the small RNA tRF-5023b can inhibit the proliferation of non-small cell lung cancer cells.
[0012] Specifically, the small RNA tRF-5023b specifically binds to the PD-L1 protein.
[0013] Specifically, the small RNA tRF-5023b binds to FGL1 mRNA through complementary base pairing.
[0014] Specifically, the small RNA tRF-5023b inhibits the expression of PD-L1 and FGL1 proteins.
[0015] The present invention provides a drug for treating non-small cell lung cancer, the drug comprising a nucleic acid, a biologically active functional fragment or variant of tRF-5023b; the nucleotide sequence of tRF-5023b is shown in SEQ ID No. 1.
[0016] Preferably, the drug for treating non-small cell lung cancer also includes a pharmaceutically acceptable carrier or excipient. Beneficial effects
[0017] The validation experiments at both the cellular and organoid levels of this invention have demonstrated that the small RNA tRF-5023b can effectively inhibit the growth of non-small cell lung cancer. It can be used to prepare drugs for the treatment of non-small cell lung cancer, providing new methods and ideas for the clinical treatment and development of non-small cell lung cancer.
[0018] This invention marks the first discovery of the use of the small molecule RNA tRF-5023b in the preparation of drugs for treating non-small cell lung cancer. This small molecule RNA drug offers advantages such as simple preparation methods, readily available raw materials, the ability to achieve engineered, large-scale, and standardized preparation and production, and the need for small RNA doses for treatment. Compared to traditional drugs, small molecule RNA drugs typically exert their effects at the gene or its expression level, exhibiting higher specificity and targeting. Furthermore, small molecule RNA drugs possess high activity, short half-life, fewer side effects, high safety, low cost, sufficient control strategies for aseptic / microbial / impurity control, well-developed preparation processes and quality systems, and long-lasting therapeutic effects. In addition, compared to other drugs, the design method for small molecule RNA drugs is simple; corresponding intervention RNA molecules can be designed based on the pathogenic gene sequence, saving target screening time. It can target any gene, predict off-target effects, reduce disease treatment risks, and make precision medicine possible.
[0019] The small RNA tRF-5023b can effectively inhibit the expression of PDL1 and FGL1 proteins. Specifically, by downregulating the expression levels of PDL1 and FGL1 proteins, the small RNA tRF-5023b can block the PD-1 / PD-L1 and FGL1 / LAG3 immune checkpoint pathways, restore the anti-tumor activity of cytotoxic T cells, and thus enhance the body's immune response to non-small cell lung cancer.
[0020] This invention, through RNA immunoprecipitation (RIP) combined with high-throughput sequencing, identifies for the first time that tRF-5023b specifically binds to the PD-L1 protein, and clarifies its effects on the proliferation of lung cancer cells and organoids using CCK8 and other techniques. Western blotting and quantitative RT-PCR confirm that tRF-5023b regulates PD-L1 expression and simultaneously inhibits FGL1 protein expression by binding to FGL1 mRNA through base pairing. This invention reveals the mechanism by which tRF-5023b enhances anti-tumor immune responses by dually inhibiting PD-L1 and FGL1, blocking the PD-1 / PD-L1 and FGL1 / LAG-3 immune checkpoint pathways. Based on these findings, this invention provides a novel application of tRF-5023b in the preparation of single-agent or combined immune checkpoint inhibitor drugs for non-small cell lung cancer (NSCLC), offering new strategies and candidate molecules for precision treatment of NSCLC. Attached Figure Description
[0021] Figure 1 The graph shows the expression levels of tRF-5023b after transfection of the small RNA tRF-5023b mimic of this invention into lung cancer cell lines.
[0022] Figure 2 This is a graph showing the effect of overexpression of the small RNA tRF-5023b of this invention on the malignant phenotype of lung cancer cells;
[0023] Figure 3 Figure 1 shows the effect of overexpression of the small RNA tRF-5023b on the growth of lung cancer organoids. Note: Figure A is organoid LUAD 1, Figure B is organoid LUAD 2, and Figure C is organoid LUAD 3.
[0024] Figure 4 Figure A shows the effect of overexpression of the small RNA tRF-5023b on the expression of PDL1 and FGL1 proteins. Note: Figure A shows the effect on PDL1 protein expression, and Figure B shows the effect on FGL1 protein expression.
[0025] Figure 5 This diagram illustrates the interaction between the small RNA tRF-5023b of this invention and the PDL1 protein. Detailed Implementation
[0026] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto. Unless otherwise specified, the reagents and materials involved in the embodiments are all commercially available products.
[0027] The nucleotide sequence of the small RNA tRF-5023b of this invention is: GUCAGGAUGGCCGAGCGGUCUAA. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to artificially synthesize the small RNA tRF-5023b mimic using chemical methods according to the above specific nucleotide sequence.
[0028] The experimental organoids were all obtained from patients who were pathologically diagnosed with non-small cell lung cancer between July and August 2025, and were approved by the Ethics Committee of Liaoning Cancer Hospital.
[0029] Example 1
[0030] The specific implementation process for constructing a lung cancer cell line overexpressing the small RNA tRF-5023b is as follows:
[0031] (1) Transfection: Cells were evenly seeded in 6-well plates. After the cells adhered, they were infected with Lipofectamine 2000 (Invitrogen, USA) and tRF-5023b mimic (Sangon Biotech).
[0032] (2) RNA extraction: 24 hours after transfection, add 500 μL Trizol (a reagent for total RNA extraction) (Baori Biotechnology Co., Ltd.), and collect the RNA into a 1.5 mL centrifuge tube after cell lysis; add 100 μL chloroform analog (Beyotime Biotechnology), shake vigorously, and let stand at room temperature for 10 minutes; centrifuge at 12000 rpm at 4℃ for 15 minutes, and transfer the colorless upper aqueous phase containing total RNA to a new tube, add an equal volume of isopropanol, mix by inversion, and let stand at room temperature for 10 minutes. Centrifuge at 12000 rpm at 4℃ for 10 minutes, discard the supernatant; add 0.5 mL of 75% ethanol and gently invert twice to wash. Centrifuge at 12000 rpm at 4℃ for 10 minutes, discard the supernatant, and after the residual liquid in the tube has dried, add 30-40 μL of DEPC water (Beijing Solarbio Science & Technology Co., Ltd.) to dissolve; use a NanoDrop ND-1000 nucleic acid quantification instrument to quantify the purity and concentration of the extracted RNA.
[0033] (3) cDNA synthesis: cDNA was synthesized from total RNA by reverse transcription using the miDETECT A Track miRNAqRT-PCR Starter Kit;
[0034] (4) Real-time quantitative PCR: Specific primers were designed based on the tRF-5023b and U6 nucleic acid sequences (primers were synthesized by Ribot Biotech and technical support was provided). qPCR reactions were performed using a qPCR kit (Ribot Biotech miDETECT A Track miRNA qRT-PCR Starter Kit). The reaction system is shown in the table below:
[0035] Table 1: qPCR reaction system
[0036] After mixing the above components thoroughly, a qPCR reaction was performed using a 3-step method.
[0037] Table 2: qPCR reaction procedure
[0038] Determine the characteristics of the reaction based on the dissolution curve, according to Formula 2. -△△Ct The relative expression levels of tRF were calculated, and the statistical results are shown in [the table below]. Figure 1 The NC group was transfected with disordered RNA as a negative control, and the tRF-5023b group was transfected with tRF-5023b mimic.
[0039] Depend on Figure 1 It can be seen that after transfection with the tRF-5023b mimic, tRF-5023b was significantly upregulated in the cell line, indicating that tRF-5023b overexpression was successful.
[0040] Example 2
[0041] The effect of tRF-5023b overexpression on the malignant phenotype of lung cancer cells was investigated, and the specific implementation process is as follows:
[0042] Cell proliferation assay using CCK8 assay: The tRF-5023b overexpressing cell line from Example 1 was digested with trypsin, centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended. Cells were counted using a cell counting chamber, and 1000 cells / well were seeded into 96-well plates. After cell attachment, CCK8 reagent (referred to as day 0) was added at a ratio of 1:10, i.e., 10 μL of CCK8 reagent was added to 100 μL of culture medium. After incubation for 1 hour, the absorbance at 450 nm was measured using a microplate reader. The above operation was repeated on days 1, 2, 3, and 4. The results are shown in the table below. Figure 2 The NC group was transfected with disordered RNA as a negative control, and the tRF-5023b group was transfected with tRF-5023b mimics.
[0043] Depend on Figure 2It can be seen that overexpression of tRF-5023b significantly inhibited the proliferation of A549 cells (lung adenocarcinoma cells in non-small cell lung cancer), thus indicating that tRF-5023b has the ability to inhibit the proliferation of lung adenocarcinoma cells.
[0044] Example 3
[0045] The specific procedure for determining the proliferative capacity of lung cancer organoids after overexpression of tRF-5023b is as follows:
[0046] (1) Organoid transfection: The constructed organoids were plated in 96-well plates and infected with tRF-5023b mimics using Lipofectamine 2000 transfection reagent.
[0047] (2) Observation of organoid proliferation capacity: The growth of organoids was observed regularly and photographed for record-keeping. The results are shown in the table below. Figure 3 In this study, the NC group was transfected with disordered RNA as a negative control, and the tRF-5023b group was transfected with tRF-5023b mimicry. This invention provides the application of tRF-5023b in inhibiting the proliferation of lung adenocarcinoma organoids. (Through...) Figure 3 Three parallel experiments (Figure A: LUAD 1, Figure B: LUAD 2, Figure C: LUAD 3) confirmed that, compared with the NC group transfected with the disordered negative control sequence, the experimental groups transfected with tRF-5023b mimics exhibited a significant phenotype of proliferation inhibition on day 20 of culture. Specifically, the organoid volume in each experimental group did not increase significantly, and growth tended to stagnate; while the organoids in each NC group showed vigorous proliferation under the same culture conditions, forming significantly larger organoid clusters. These results indicate that tRF-5023b can effectively inhibit the proliferative activity of lung adenocarcinoma organoids, and it has a clear application prospect in the preparation of anti-lung adenocarcinoma drugs.
[0048] Depend on Figure 3 It can be seen that overexpression of tRF-5023b significantly inhibits the proliferation of organoids. Therefore, tRF-5023b has the ability to inhibit organoid proliferation.
[0049] Example 4
[0050] The effects of tRF-5023b on the expression of PDL1 and FGL1 proteins were investigated, and the specific implementation process is as follows:
[0051] (1) Construction of tRF-5023b overexpression cell line: The cells were evenly seeded in a 6cm dish. After the cells adhered, they were infected with tRF-5023b mimic (Sangon Biotech) using Lipofectamine 2000.
[0052] (2) Protein extraction: 48 hours after transfection, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. The cells were scraped off with a cell scraper and collected into a 1.5 mL centrifuge tube. After centrifugation at 800 rpm for 5 minutes, the supernatant was removed. Total protein was extracted from the cell samples using Western blotting with a mixture of 1% protease inhibitors (Beyotime Biotechnology, China) and IP cell lysis buffer (Beyotime Biotechnology, China).
[0053] (3) Sample preparation: Protein concentration was measured using the BCA protein concentration assay kit (Beyotime Biotechnology, China). A protein solution with a concentration of 1 μg / μL was prepared using ultrapure water and SDS-PAGE protein loading buffer (Beyotime Biotechnology, China) and boiled for 5 minutes.
[0054] (4) Immunoblotting of proteins: After SDS-PAGE gel electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA); after incubation with blocking buffer (Yamei Biotechnology, China) at room temperature for 15 minutes, washed with TBST buffer (Seville Biotechnology, China), and then hybridized overnight at 4°C with primary antibody, washed three times with TBST buffer for 10 minutes each time, and then incubated with secondary antibody (Wuhan Sanying Biotechnology, China, SA00001-2) at room temperature for 1 hour, washed three times with TBST buffer for 10 minutes each time; the protein bands were visualized using ECL chemiluminescence substrate (Shanghai Tianneng, China); the primary antibodies used in this study were β-Actin (Wuhan Sanying Biotechnology, China, 20536-1-AP), PDL1 (Wuhan Sanying Biotechnology, China, 17952-1-AP), and FGL1 (Wuhan Sanying Biotechnology, China, 16000-1-AP). The results are shown in the table below. Figure 4 The NC group was transfected with disordered RNA as a negative control, and the tRF-5023b group was transfected with tRF-5023b mimic.
[0055] Depend on Figure 4 It can be seen that overexpression of tRF-5023b significantly reduced the levels of PDL1 and FGL1 proteins, indicating that tRF-5023b has the ability to inhibit the expression of PDL1 and FGL1 proteins.
[0056] PD1 is a ligand of PDL1. The binding of the receptor and ligand can promote tumor immune escape. Therefore, inhibiting PDL1 expression can be regarded as blocking the PD1 / PDL1 pathway. Similarly, LAG3 is a ligand of FGL1. Inhibiting FGL1 expression can be regarded as blocking the FGL1 / LAG3 pathway.
[0057] Example 5
[0058] The interaction between tRF-5023b and PDL1 is described in the following details:
[0059] (1) Cell lysis: Western lysate and IP cell lysis buffer containing protease inhibitor (Beyotime Biotechnology, China) and ribonuclease inhibitor (Beyotime Biotechnology, China) were added to the cell pellet and incubated overnight at 4°C;
[0060] (2) Preparation of magnetic beads: Measure 50 μL of magnetic beads (Selleck, China, B23202) into a centrifuge tube, add 1 mL of TBS buffer, mix well, and then aspirate the supernatant on a magnetic rack. Repeat twice. Add another 50 μL of TBS buffer and place on ice for later use.
[0061] (3) Immunoprecipitation: Mix cell lysate supernatant with IgG antibody (Wuhan Sanying Biotechnology, China, 98136-1-RR) or PDL1 antibody (Wuhan Sanying Biotechnology, China, 17952-1-AP), incubate at 4°C for 4 hours by rotation, then add magnetic beads and incubate at 4°C for 4 hours by rotation. Place on a magnetic rack and discard the supernatant. Wash the magnetic beads 6 times with 500 μL of TBS buffer. Add 1 mL of Trizol (Baori Biotechnology Co., Ltd.) to the magnetic bead precipitate mixture.
[0062] (4) RNA extraction, cDNA synthesis, and real-time quantitative PCR were performed as described in Example 1. Statistical results are shown in […]. Figure 5 The IgG group served as an isotype control to exclude interference from nonspecific binding. This group used nonspecific immunoglobulin G from the same species as the experimental group, which effectively distinguished between antibody-specific binding and background adsorption. Results showed that the tRF-5023b enrichment level in the PDL1 group was significantly higher than that in the IgG control group.
[0063] Depend on Figure 5 It is known that tRF-5023b can interact with the PDL1 protein.
[0064] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
Claims
1. A small RNA molecule tRF-5023b, characterized in that, The nucleotide sequence of the small RNA tRF-5023b is shown in SEQ ID No.
1.
2. The use of the small molecule RNA tRF-5023b as described in claim 1 in the preparation of a drug for treating lung cancer.
3. The application according to claim 2, characterized in that, The lung cancer referred to is either non-small cell lung cancer or small cell lung cancer.
4. The application according to claim 2, characterized in that, The lung cancer in question is non-small cell lung cancer.
5. The application according to claim 2, characterized in that, The small RNA tRF-5023b can inhibit the proliferation of non-small cell lung cancer cells.
6. The application according to claim 2, characterized in that, The small RNA tRF-5023b specifically binds to the PD-L1 protein.
7. The application according to claim 2, characterized in that, The small RNA tRF-5023b binds to FGL1 mRNA through complementary base pairing.
8. The application according to claim 2, characterized in that, The small RNA tRF-5023b inhibits the expression of PD-L1 and FGL1 proteins.
9. A drug for treating non-small cell lung cancer, characterized in that, The drug for treating non-small cell lung cancer includes the nucleic acid, bioactive functional fragment, or variant shown in tRF-5023b; the nucleotide sequence of tRF-5023b is shown in SEQ ID No.
1.
10. The medicament for treating non-small cell lung cancer according to claim 9, characterized in that, The drugs for treating non-small cell lung cancer also include pharmaceutically acceptable carriers or excipients.