tRFs associated with non-small cell lung cancer and uses thereof

Abstract

The application belongs to the technical field of molecular diagnosis, and particularly relates to a tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application thereof. The tRF has a significantly up-regulated expression level in NSCLC cancer cells, patient tumor tissues and plasma. The high expression of the tRF in cancer tissues is significantly correlated with the poor prognosis of NSCLC patients. Therefore, a detection reagent of the tRF can be prepared into a kit for diagnosing and / or evaluating the prognosis of NSCLC. Further research results show that the tRF has a significant ability to promote the proliferation, migration activity and glycolysis metabolism enhancement of NSCLC cells. Knocking down the expression of the tRF can inhibit the growth of NSCLC cells in vivo and in vitro. Therefore, an inhibitor of the tRF can be used for preparing a medicine for preventing and / or treating NSCLC, and has an anticancer value.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a tRF associated with non-small cell lung cancer and its applications. Background Technology

[0002] Lung cancer is one of the most common and deadliest malignant tumors in the world; non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, posing a significant threat to human health. NSCLC often presents with no typical symptoms in its early stages, making it easily overlooked by patients. Its early diagnosis rate is extremely low, only 15%, and it is often inoperable, relying on radiotherapy, chemotherapy, and targeted drug therapy to prolong life. Long-term survival rates are low, causing immense suffering for patients and their families. Although significant progress has been made in lung cancer diagnosis and treatment techniques in recent years, the mortality rate remains high, urgently requiring the development of new prevention and treatment strategies and drugs. Elucidating the pathogenesis of lung cancer and implementing effective pathological interventions is undoubtedly the best approach. Therefore, actively exploring new methods for early diagnosis of lung cancer is of significant clinical importance in improving the early diagnosis rate and patient prognosis.

[0003] tRNA-derived fragments (tRFs) are a new class of small non-coding RNAs (sncRNAs) discovered in recent years with the development of high-throughput sequencing technology. They are produced by specific nucleases such as Dicer and angiopoietin through specific cleavage of mature tRNA or its precursor tRNA at different sites. tRFs mainly include subclasses such as tRF-5, tRF-3, tRF-1, 5' tiRNA, and 3' tiRNA. Recent studies have shown that tRFs are differentially expressed in various tumors and influence tumor proliferation, invasion, or metastasis by regulating gene expression and silencing, modulating cell proliferation and apoptosis, and regulating translation. They hold promise as biomarkers for diagnosis and prognosis of cancer or as targets for precision medicine. Summary of the Invention

[0004] The purpose of this application is to provide a tRF associated with non-small cell lung cancer (NSCLC) and its application, aiming to solve the technical problem of providing molecular markers associated with NSCLC.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, this application provides a tRF associated with NSCLC, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] Secondly, this application provides the use of a reagent for detecting tRF expression in the preparation of a kit for diagnosing and / or assessing the prognosis of NSCLC; wherein the nucleotide sequence of the tRF is shown in SEQ ID No. 1.

[0008] In some embodiments, the reagent includes primer pairs for amplifying the tRF, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0009] In some embodiments, the kit includes RNA extraction reagents, RNA reverse transcription reagents, and PCR amplification reagents; and / or,

[0010] The kit includes a negative control as shown in SEQ ID No. 4.

[0011] Thirdly, this application provides a kit for diagnosing and / or prognostically assessing NSCLC, the kit containing reagents for detecting tRF expression, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0012] In some embodiments, the reagent includes primer pairs for amplifying the tRF, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0013] In some embodiments, the kit includes RNA extraction reagents, RNA reverse transcription reagents, and PCR amplification reagents; and / or,

[0014] The kit includes a negative control as shown in SEQ ID No. 4.

[0015] Fourthly, this application provides the use of an inhibitor that inhibits tRF expression in the preparation of a medicament for the prevention and / or treatment of NSCLC; wherein the nucleotide sequence of the tRF is as shown in SEQ ID No. 1.

[0016] Fifthly, this application provides a medicament for the prevention and / or treatment of NSCLC, the medicament containing an inhibitor that inhibits tRF expression, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0017] In some embodiments, the nucleotide sequence of the inhibitor is as shown in SEQ ID No. 5; and / or, the medicament further includes a pharmaceutically acceptable carrier.

[0018] This application identifies a tRF (nucleotide sequence shown in SEQ ID No. 1) associated with NSCLC characteristics through experimental analysis, thus providing a new target for personalized precision treatment of NSCLC. The expression level of this tRF was significantly upregulated in NSCLC cancer cells, patient tumor tissue, and plasma. High expression of this tRF in cancer tissue was significantly associated with poor prognosis in NSCLC patients. Therefore, the detection reagent for this tRF can be used to prepare kits for the diagnosis and / or prognostic assessment of NSCLC. Further research results indicate that this tRF has a significant ability to promote NSCLC cell proliferation, migration activity, and enhanced glycolytic metabolism. Knockdown of tRF expression can inhibit the growth of NSCLC cells in vivo and in vitro. Therefore, inhibitors of this tRF can be used to prepare drugs for the prevention and / or treatment of NSCLC, possessing anti-cancer value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the difference in expression of AS-tDR-008272 in NSCLC cancer tissue and adjacent normal tissue in the embodiments of this application;

[0021] Figure 2 This is a graph showing the difference in expression of AS-tDR-008272 in different NSCLC cell lines (A549, H358) and normal human lung epithelial cells (BEAS-2B) in the embodiments of this application.

[0022] Figure 3 This is a diagram showing the results of an experiment in which AS-tDR-008272 overexpression promoted the proliferation of NSCLC cells (A549) in the embodiments of this application;

[0023] Figure 4 This is a diagram showing the experimental results of AS-tDR-008272 overexpression promoting NSCLC cell (H358) proliferation in the embodiments of this application;

[0024] Figure 5 This is a diagram showing the results of an experiment in this application demonstrating the inhibition of NSCLC cell (A549) proliferation by knocking down AS-tDR-008272 expression.

[0025] Figure 6 This is a diagram showing the results of an experiment in this application demonstrating the inhibition of NSCLC cell (H358) proliferation by knocking down AS-tDR-008272 expression.

[0026] Figure 7 This is a diagram showing the results of an experiment in which AS-tDR-008272 overexpression promoted the migration of NSCLC cells (A549) in an embodiment of this application.

[0027] Figure 8 This is a diagram showing the results of an experiment in which AS-tDR-008272 overexpression promoted the migration of NSCLC (H358) cells in an embodiment of this application.

[0028] Figure 9 This is a diagram showing the results of AS-tDR-008272 overexpression promoting NSCLC cell (A549 and H358) invasion in the embodiments of this application;

[0029] Figure 10 This is a diagram showing the results of knocking down AS-tDR-008272 expression to inhibit the invasive ability of NSCLC cells (A549 and H358) in this embodiment;

[0030] Figure 11 This is a diagram showing the results of AS-tDR-008272 overexpression promoting lactate and pyruvate production in NSCLC cells (A549 and H358) in the embodiments of this application.

[0031] Figure 12 This is a diagram showing the experimental results of the AS-tDR-008272 expression inhibitor inhibiting the growth of NSCLC xenografts in nude mice, where (A) is the change in tumor volume, (B) is the change in tumor weight, and (C) is the change in tumor appearance.

[0032] Figure 13 This is a graph showing the experimental results of the correlation between the expression level of AS-tDR-008272 in NSCLC cancer tissue and pathological stage and poor prognosis in the embodiments of this application. Detailed Implementation

[0033] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0034] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0035] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0036] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0037] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0038] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0039] In-depth exploration of the molecular mechanisms of NSCLC is expected to uncover effective biomarkers and new therapeutic targets, which are crucial for the early diagnosis, treatment, and prognosis of the disease, and are also urgent issues that need to be addressed in the prevention and treatment of NSCLC. While significant progress has been made in the study of the molecular mechanisms by which tRFs regulate tumorigenesis and development, little is known about the mechanisms by which tRFs act on NSCLC and their target genes. The potential therapeutic effects of tRFs on NSCLC require further investigation.

[0040] Glucose metabolism reprogramming is one of the most representative metabolic characteristics in tumors. Tumor cells possess a unique energy metabolism pattern; even under aerobic conditions, they do not utilize mitochondrial oxidative phosphorylation for energy production, exhibiting active aerobic glycolysis, known as the Warburg effect (aerobic glycolysis). Tumor cells provide themselves with substances and energy through aerobic glycolysis to meet their rapid growth needs. Studies have shown that various metabolic enzymes and signaling molecules involved in aerobic glycolysis play important roles in tumorigenesis and development, and are considered important targets for the diagnosis and treatment of malignant tumors.

[0041] Based on this, embodiments of this application have discovered a tRF related to NSCLC and its application, the specific technical solution of which is as follows.

[0042] In a first aspect, embodiments of this application provide a tRF related to NSCLC, the tRF being named AS-tDR-008272, and the nucleotide sequence of the tRF being shown in SEQ ID No. 1.

[0043] SEQ ID No. 1: 5'-GAAGCGGGUGCUCUUAUUUUU-3'.

[0044] For this sequence, a primer for qRT-PCR amplification of AS-tDR-008272 is provided, the sequence of which is shown in SEQ ID No. 2 and SEQ ID No. 3.

[0045] SEQ ID No.2(Forward):5'-CAGAAGCGGGGTGCTCTT-3';

[0046] SEQ ID No. 3 (Reverse): 5'-AGTGCAGGGTCCGAGGTATT-3'.

[0047] The aforementioned primers can detect the expression level of AS-tDR-008272 in this application, providing a foundation for further research on the function of AS-tDR-008272 in NSCLC and the development of drugs targeting AS-tDR-008272. Because the tRF provided in this application's embodiments has significant pro-cancer activity, it provides a new target for personalized precision treatment of NSCLC. This tRF can be used to prepare kits for diagnosing and / or prognostically assessing NSCLC.

[0048] Secondly, embodiments of this application also provide the application of a reagent for detecting tRF expression in the preparation of a kit for diagnosing and / or prognostically assessing NSCLC; wherein the nucleotide sequence of tRF is shown in SEQ ID No. 1. The kit may further include primers for qRT-PCR amplification of AS-tDR-008272, the sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3; it may further include RNA extraction reagents, RNA reverse transcription reagents, and PCR amplification reagents; the RNA extraction reagents are used to extract RNA from samples of tumor tissue from the patient to be tested, the RNA reverse transcription reagents are used to reverse transcribe the extracted RNA into cDNA, and the PCR amplification reagents are used to amplify its expression for quantitative analysis, such as some real-time quantitative PCR reagents; the above reagents can be commonly used in this technical field and can be obtained through conventional techniques or the market.

[0049] Furthermore, the kit also includes a negative control as shown in SEQ ID No. 4. The negative control better reflects the relative expression level of AS-tDR-008272 detected by the kit. In this embodiment, the negative control in the kit is represented by AS-tDR-008272-NC or NC-AS-tDR-008272, with the following sequence:

[0050] SEQ ID No. 4: 5'-GAGUUGCUUUAUCGUCAUGGU-3'.

[0051] Thirdly, embodiments of this application also provide a kit for diagnosing and / or prognostically assessing NSCLC, the kit containing reagents for detecting tRF expression, the nucleotide sequence of tRF being shown in SEQ ID No. 1. The kit may also include the above-mentioned components.

[0052] Fourthly, this application provides the use of an inhibitor of tRF expression in the preparation of a medicament for the prevention and / or treatment of NSCLC; wherein the nucleotide sequence of tRF is shown in SEQ ID No. 1. Because tRF has a significant ability to promote the tumor activity of NSCLC cells, its inhibitor can be used to prepare a medicament for the prevention and / or treatment of NSCLC.

[0053] Specifically, an inhibitor that suppresses the expression of tRF as shown in SEQ ID No. 1, wherein the inhibitor is a single-stranded RNA as shown in SEQ ID No. 5. This inhibitor inhibits the proliferation of NSCLC cells by suppressing the expression of tRF as shown in SEQ ID No. 1, and thus has anti-cancer application value.

[0054] Specifically, the inhibitor, namely AS-tDR-008272-Inhibitor (which can be referred to as Inhibitor in this application embodiment), has the following specific sequence:

[0055] SEQ ID No. 5: 5'-AAGAAUUCUACCACUGAACCACCAAUGC-3'.

[0056] Accordingly, the negative control of the above inhibitor is named AS-tDR-008272inhibitor-NC (which can be abbreviated as Inhibitor-NC in this application), and its specific sequence is as follows:

[0057] SEQ ID NO: 6: 5'-CACAUACAGAACGGCCAUCAAAAUUCUC-3'.

[0058] Finally, embodiments of this application also provide a medicament for the prevention and / or treatment of NSCLC, the medicament containing an inhibitor that can inhibit the expression of tRF as shown in SEQ ID No. 1 and a pharmaceutically acceptable carrier.

[0059] Specifically, the inhibitor is a single strand of RNA as shown in SEQ ID NO:5.

[0060] Specifically, the pharmaceutical products provided in this application are compositions comprising an inhibitor of AS-tDR-008272, and / or other pharmaceutical classes compatible with the inhibitor, as well as pharmaceutically acceptable carriers and / or excipients.

[0061] In one embodiment of this application, small RNA sequencing technology was used to analyze and compare the differences in plasma tRF expression before and after surgery in non-small cell lung cancer (NSCLC) patients, and quantitative PCR was used to verify the sequencing results. The analysis showed that AS-tDR-008272 was highly expressed in the plasma of NSCLC patients before surgery, and the expression level of AS-tDR-008272 in plasma decreased significantly after surgery. This suggests that AS-tDR-008272 is highly correlated with the presence of NSCLC tumor tissue.

[0062] In one embodiment of this application, the expression differences of AS-tDR-008272 between NSCLC cancer tissue / adjacent tissue and NSCLC cancer cells / normal bronchial epithelial cells were analyzed using RNA fluorescent probes and quantitative PCR technology, respectively. The results showed that the expression levels of AS-tDR-008272 in NSCLC cancer tissue and in NSCLC cells were significantly higher than those in adjacent tissue and normal bronchial epithelial cells, respectively. The detection results demonstrate that AS-tDR-008272 is highly expressed in NSCLC. Further analysis revealed that high expression of AS-tDR-008272 in NSCLC cancer tissue was significantly correlated with poor patient prognosis, indicating that AS-tDR-008272 levels can predict the prognosis of NSCLC patients.

[0063] In one embodiment of this application, AS-tDR-008272 was transfected into H358 and A549 cell lines, respectively. It was found that overexpression of AS-tDR-008272 significantly promoted the proliferation and migration of NSCLC cells. The results indicate that AS-tDR-008272 has a significant ability to promote NSCLC cell tumor growth, and AS-tDR-008272 is a newly discovered tRNA fragment that promotes tumor growth.

[0064] In one embodiment of this application, an inhibitor (as shown in SEQ ID No. 5, i.e., interfering RNA) targeting AS-tDR-008272 was synthesized. After transfecting the inhibitor into H358 and A549 cell lines, it was found that knocking down the expression of AS-tDR-008272 could significantly inhibit the proliferation and migration of various NSCLC cells.

[0065] In one embodiment of this application, overexpression of AS-tDR-008272 promotes enhanced glycolytic metabolism in A549 and H358 cells, manifested as increased production of glycolytic end-products lactate and pyruvate. Knockdown of AS-tDR-008272 expression inhibits glycolytic metabolism in NSCLC cells. The results indicate that AS-tDR-008272 promotes cancer cell proliferation and migration by enhancing glycolytic metabolism in NSCLC.

[0066] In one embodiment of this application, a nude mouse xenograft model was constructed by injecting a stable strain of A549 cells subcutaneously into nude mice. The mice were then treated with AS-tDR-008272-related inhibitors and Iinhibitor-NC, respectively. The final experimental results demonstrated that AS-tDR-008272-Inhibitor of this embodiment of the application can significantly inhibit the growth of NSCLC tumors in vivo.

[0067] This application has undergone multiple experiments, and some of the experimental results are presented here for reference to further describe the application in detail. The following is a detailed description in conjunction with specific embodiments.

[0068] Example 1: Differential expression analysis of AS-tDR-008272 in NSCLC

[0069] 1. RNA extraction from cancer tissue

[0070] (1) Take out the NSCLC patient cancer tissue specimen frozen at -80℃ from the freezer, place it on ice, cut about 0.1g of tissue with surgical scissors and put it into a mortar, add liquid nitrogen and grind until it is powdered, then add 1mL Trizol reagent for lysis and transfer to 1.5mL EP tube, and store it in a freezer at -80℃ for later use.

[0071] (2) Thaw the Trizol reagent lysed sample in the EP tube of step (1) above, add 200uL of chloroform, shake for 30 seconds, let stand at room temperature for 10 minutes, and then centrifuge. The centrifugation conditions are: 4℃, 12000 rpm, centrifuge for 20 minutes.

[0072] (3) After centrifugation in step (2), take 400uL of supernatant into a new EP tube, add 600uL of isopropanol, invert 10 times, place at room temperature for 10 minutes, and then centrifuge. The centrifugation conditions are: 4℃, 12000 rpm, centrifuge for 10 minutes.

[0073] (4) After centrifugation in step (3) above, discard the supernatant, add 1 mL of 75% ethanol to wash the lower RNA layer, and centrifuge at 4°C, 7500 rpm for 5 minutes. After centrifugation, remove the supernatant, invert and dry for 5 minutes, add 40 μL LDEPC water (i.e., ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure) to obtain the RNA solution.

[0074] 2. Reverse transcription PCR (RT-PCR)

[0075] Using a reverse transcription kit (RR047A, Takara), after mixing the system according to Table 1, incubate at 37°C for 5 min to eliminate gDNA. Then, perform reverse transcription according to the system in Table 2. The reverse transcription program for the PCR instrument is shown in Table 3. After the reverse transcription program is completed, store the samples at -20°C.

[0076] Table 1

[0077]

[0078]

[0079] Table 2

[0080] reagents Dosage Table 1 Reaction solution of the system 10ul PrimeScript RT Enzyme MixI 1ul RT Primer Mix / Specific Primer 1ul 5xPrimeScript Buffer2 4ul <![CDATA[RNase Free dH2O]]> 4ul Total 20ul

[0081] Table 3

[0082] Step sequence temperature time Step 1 37℃ 15min Step 2 85℃ 5s Step 3 4℃ -

[0083] 3. Real-time quantitative PCR (qRT-PCR)

[0084] (1) Use the Takara kit RR820A for detection. Each sample is made in 3 replicates. Select the internal reference gene U6 according to the target gene. Use the corresponding amplification primers and amplification reagents to prepare the real-time PCR system shown in Table 4. The real-time PCR instrument is operated according to the following program: first 95℃ for 10 minutes, then 40 cycles: 95℃ for 15 seconds; 60℃ for 15 seconds; 72℃ for 30 seconds); and finally 65℃ for 6 seconds.

[0085] Table 4

[0086]

[0087]

[0088] (2) After the real-time quantitative PCR is completed, calculate according to the following formula:

[0089] ΔCt=Ct 目的基因 -Ct 内参基因 ;

[0090] ΔΔCt=ΔCt 实验组 -ΔCt 对照组 ;

[0091] Target gene quantity = 2 -ΔΔCt .

[0092] Detection of AS-tDR-008272 expression in NSCLC cancer tissues and adjacent normal tissues: The expression of AS-tDR-008272 in cancer tissues and adjacent normal tissues was detected using the above-described qRT-PCR procedure and lung cancer tissue microarray. The results are as follows: Figure 1 As shown, Adjacent (n=80) represents 80 adjacent normal tissue samples, I+II (n=53) represents 53 lung cancer tissue specimens with pathological stages I-II, and III (n=27) represents 27 lung cancer tissue specimens with pathological stage III. The results show that the expression level of AS-tDR-008272 in cancer tissues is significantly higher than that in adjacent normal tissues, and the expression level of AS-tDR-008272 is closely related to the development of NSCLC.

[0093] Detection of AS-tDR-008272 expression in NSCLC cells: The expression of AS-tDR-008272 was detected in A549 cell line, H358 cell line, and normal bronchial epithelial cell line (BEAS-2B) using the above qRT-PCR procedure. The results are as follows: Figure 2 As shown in the figure; the results indicate that the expression levels of AS-tDR-008272 in A549 and H358 cells were significantly higher than those in normal bronchial epithelial cells.

[0094] Example 2: High expression of AS-tDR-008272 promotes NSCLC cell proliferation and migration.

[0095] 1. Cell transfection

[0096] In the embodiments of this application, the cells are arranged at 1.5 x 10 5 Seed each cell in a 6-well plate, and transfection can be performed when the cell density reaches 70%-80% after 24 hours.

[0097] The synthesized tRFs (AS-tDR-008272 and AS-tDR-008272-NC) were dissolved in DEPC water to a final concentration of 20 μM. The seeded cells were removed, the supernatant was aspirated, and the cells were washed twice with PBS. After cell adhesion, 1.75 mL of DMEM complete medium (high glucose / low glucose) was added. According to the Lipo3000 instructions, AS-tDR-008272 and its negative control AS-tDR-008272-NC were transfected into the cells using the Lipo3000 transfection reagent. After standing for 20 min, the cells were incubated in a 37°C, 5% CO2 cell culture incubator. After 24 h, the cells were collected for downstream experimental detection.

[0098] 2. Cell proliferation

[0099] Following the steps described above, AS-tDR-008272 and its negative control were transfected into NSCLC A549 and H358 cells. After 24 hours, the cells were digested and centrifuged; after counting, the cell density was adjusted to 2.5 x 10⁻⁶ cells. 4 Cell proliferation assays were performed on 96-well plates labeled with cells / mL for six time points: day 0, day 1, day 2, day 3, day 4, and day 5. 5000 cells were seeded in each well, with five replicates. Cells were incubated for 4 hours before assays were performed, with the time point designated D0. Data were recorded every 24 hours, designated D1, D2, D3, D4, and D5. Cell proliferation was measured according to the CCK-8 assay kit instructions, with 20 experimental wells and 5 control wells per plate. The cell plates at the corresponding time points were removed, and the old culture medium in the wells was aspirated using a 200 μL pipette tip. 110 μL of assay buffer was added to each well, prepared using a high-glucose / low-glucose complete culture medium and CCK-8 reagent at a volume ratio of 10:1. Cells were incubated for 2 hours. OD was measured at 450 nm using a microplate reader. Cell proliferation results are shown below. Figures 3-4 As shown in the figure, the results indicate that, compared with the negative control (NC-AS-tDR-008272 in the figure), overexpression of AS-tDR-008272 (OE-AS-tDR-008272 in the figure) significantly promoted the proliferation of A549 and H358 cell lines.

[0100] The inhibitor that reduces AS-tDR-008272 expression, inhibitor (SEQ ID No. 5), and its negative control, inhibitor-NC (SEQ ID No. 6), were transfected into A549 and H358 cells, respectively. The results of the cell proliferation inhibition experiment are as follows: Figure 5-6 As shown, the results indicate that knockdown of AS-tDR-008272 expression significantly inhibits the proliferation of A549 and H358 cell lines.

[0101] 3. Effects of AS-tDR-008272 overexpression or underexpression on NSCLC cell invasion and migration

[0102] Following the transfection steps described above, AS-tDR-008272 and a negative control, along with the AS-tDR-008272 expression inhibitor Inhibitor and a negative control Inhibitor-NC, were transfected into A549 and H358 cells, respectively. Twenty-four hours later, the effect of AS-tDR-008272 on cell invasion was detected using a Transwell assay. 1 × 10⁻⁶ cells were collected from each well 24 hours after transfection. 5 Cells were seeded in the upper chamber of a Transwell containing 300 μL of 10% fetal bovine serum (FBS) medium, and the lower chamber contained 600 μL of 10% FBS medium. Cells were fixed in 4% paraformaldehyde solution for 20 min, stained with 0.1% crystal violet for 20 min, and photographed in five fields (top, bottom, left, center, and right) under an inverted microscope. The cells were then counted using ImageJ. Results are shown below. Figures 7-8 As shown, compared with the negative control (NC in the figure), AS-tDR-008272 overexpression (TRF in the figure) promoted the invasion of A549 and H358 cells; AS-tDR-008272 knockdown inhibited the invasion of NSCLC cells.

[0103] The effect of AS-tDR-008272 on cell migration was detected using a cell scratch assay: Transfected NSCLC cells were seeded in 6-well plates (5 × 10⁶ cells per well). 3 Cells were cultured at 37°C and 5% CO2 until confluence was >90%. A single scratch was made on the cell monolayer using the tip of a 200 μL pipette. Exfoliated cells were washed away with PBS, and the cells were imaged using an optical microscope at 0 h and 24 h.

[0104] Scratch healing rate = (0h scratch width - 24h scratch width) / 0h scratch width

[0105] The results are as follows Figure 9-10 As shown, overexpression of AS-tDR-008272 promotes the migration of A549 and H358 cells; knockdown of AS-tDR-008272 inhibits the migration of NSCLC cells.

[0106] Example 3: Effects of overexpression or knockdown of AS-tDR-008272 on glycolytic metabolism in NSCLC

[0107] Following the cell transfection procedure in Example 2, AS-tDR-008272 and the negative control AS-tDR-008272-NC, the AS-tDR-008272 expression inhibitor, and the negative control Inhibitor-NC were transfected into A549 and H358 cells, respectively. After 24 hours, the cell culture supernatant was collected, and the levels of lactate (L-lactate) and pyruvate (pyruvate) were measured using a lactate assay kit (coefficient of variation CV 1.5%, recovery assay X = 101%) and a pyruvate assay kit (coefficient of variation CV 1.7%, recovery assay X = 96%) from Nanjing Jiancheng Biotechnology Co., Ltd., respectively. The experimental steps are as follows:

[0108] (1) Lactic acid detection

[0109] Prepare the enzyme working solution: Mix the enzyme stock solution and enzyme dilution solution at a volume ratio of 1:100.

[0110] Preparation of the colorimetric solution: Pour Reagent No. 4 powder into Reagent No. 3, shake and vortex to dissolve the powder completely in the solution, and mix thoroughly. (The enzyme stock solution, enzyme diluent, Reagent No. 4 powder, and Reagent No. 3 mentioned above are all existing reagents in the lactate detection kit.)

[0111] Table 5

[0112]

[0113]

[0114] Prepare the system according to Table 5, place it in a water bath at 37℃ for 10 minutes, add 250uL of sample to each well of the 96-well plate (corresponding to the sample in the blank tube, standard tube, and test tube), and detect the absorbance at OD530nm using a multi-functional microplate reader.

[0115] Formula for calculating lactic acid:

[0116] The absolute OD (measured OD value - blank OD value) ranges from 0.05 to 0.35.

[0117] (2) Detection of pyruvate

[0118] Table 6

[0119]

[0120] Prepare the system according to Table 6 (reagents 2 and 3 in the table are available in the pyruvate assay kit), let it stand for 5 minutes, and measure the absorbance value of the sample at 505 nm.

[0121]

[0122] The results are as follows Figure 11 As shown, overexpression of AS-tDR-008272 promotes enhanced glycolytic metabolism in NSCLC cells, while knockdown of AS-tDR-008272 inhibits the level of glycolytic metabolism in NSCLC cells.

[0123] Example 4: AS-tDR-008272 inhibitor inhibits the growth of NSCLC xenografts in mice.

[0124] 1. Establish a mouse xenograft model

[0125] SPF-grade BALB / c nude mice (5-6 weeks old) were purchased from the Guangdong Provincial Medical Laboratory Animal Center. Animal experiments were conducted in an SPF-grade laboratory animal facility at a room temperature of 23-27°C and a relative humidity of 50%-60%. Mice were allowed free access to food and water under natural diurnal light and shadow conditions. Experiments began after one week of acclimatization. A549 cells in the logarithmic growth phase were used, and the cell density was adjusted to 3 × 10⁻⁶ cells with physiological saline. 6 Cells / 100μL, ice bath. Disinfect the axillary skin of nude mice with povidone-iodine, thoroughly shake the cell suspension, and inoculate into the axilla (100μL / mouse). Two weeks later, white, hard lumps appeared subcutaneously in the axilla of the nude mice, growing to 100mm. 3 If the model does not shrink or disappear, the modeling is considered successful.

[0126] 2. Treatment with the AS-tDR-008272 inhibitor.

[0127] When the transplanted tumor grows to 100 mm 3 Subsequently, the nude mice with tumors were randomly divided into three groups: a control group, an inhibitor group, and an inhibitor-NC group, with six mice in each group. Each group received a local injection of equal volumes of PBS, inhibitor (5 nmol), and inhibitor-NC (5 nmol), respectively, every three days. The control group and the model group received equal volumes of sterile water for injection. The administration was continued for two weeks. During the administration period, the length and short diameter of the tumors in the nude mice were measured every three days, and the tumor volume was calculated using the formula V = (length × width²) / 2 (where length is the longest diameter of the tumor and width is the shortest diameter). After the administration was completed, the nude mice were euthanized by cervical dislocation, the tumors were dissected on ice, photographed, and weighed to calculate the tumor weight inhibition rate.

[0128] Animal experiment results such as Figure 12 As shown, A is a graph depicting changes in tumor volume (Note: d11 represents the tumor volume when it grows to 100 mm). 3(Above, i.e., day 11 after subcutaneous cell injection), B is a graph showing changes in tumor weight, and C is a graph showing the appearance of the tumor (Note: This is a graph of tumors removed from animals after the experiment, with animals divided into three groups of six animals each); As can be seen from the graph, the volume and weight of the transplanted tumors in the AS-tDR-008272 inhibitor group (Inhibitor group) were significantly lower than those in the negative control group (Inhibitor-NC group) and the blank group (Control group), indicating that knocking down AS-tDR-008272 expression can inhibit the growth of NSCLC tumors in vivo.

[0129] Example 5: The expression level of AS-tDR-008272 in NSCLC patient cancer tissues was significantly correlated with glycolytic metabolism and prognosis.

[0130] A probe targeting the AS-tDR-008272 sequence (SEQ ID NO: 7: 5'-AAAAATAAGAGCACCCGCTTC-3') was synthesized, and the expression level of AS-tDR-008272 in an NSCLC tissue microarray (HLugA180Su04, Shanghai Xinchao) was detected using fluorescence in situ hybridization (FISH). Figure 13 As shown, the results indicate that the expression level of AS-tDR-008272 in cancerous tissues was significantly higher than that in adjacent normal tissues, and the expression level of AS-tDR-008272 was higher in specimens with higher pathological stages (Stage I, II, III), indicating that the expression level of AS-tDR-008272 is closely related to the severity of NSCLC (see [reference]). Figure 13 (A in the text). Furthermore, high expression of AS-tDR-008272 in cancerous tissue was significantly associated with poor prognosis in NSCLC patients (hazard ratio HR = 1.98, P = 0.04, see section A). Figure 13 (B) Tissue microarray analysis results demonstrated that the expression level of AS-tDR-008272 was associated with the severity of NSCLC in patients.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a reagent for quantitatively detecting tRF expression levels in the manufacture of a kit for the diagnosis and / or prognostic evaluation of non-small cell lung cancer; wherein, The nucleotide sequence of the tRF is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The reagent includes a primer pair for amplifying the tRF, and the nucleotide sequences of the primer pair are shown as SEQ ID No. 2 and SEQ ID No.

3.

3. Use according to claim 1 or 2, characterized in that, The kit includes RNA extraction reagents, RNA reverse transcription reagents, and PCR amplification reagents; and / or, The kit includes a negative control shown as SEQ ID No. 4.

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