Introduction

Lung cancer (LC) is a profound and pressing global health challenge, consistently ranking as the leading malignancy in terms of both incidence and mortality worldwide. The non-small cell lung carcinoma (NSCLC) subtype constitutes a significant majority of LC diagnoses, approximately 80%, underscoring its prevalence and the imperative for ongoing research and clinical advancements [1, 2]. NSCLC is further classified into large cell carcinoma, adenocarcinoma, and squamous cell carcinoma based on pathological types. Notably, the incidence of lung adenocarcinoma (LUAD) has been rising steadily, now representing the largest proportion of LC cases [3, 4]. Recent advancements in LC treatments, particularly the introduction and use of targeted therapy drugs, have significantly reduced LC mortality, with the two-year survival rate improving from 34 to 42% [1, 5]. Despite these advances, LUAD remains a major life-threatening condition. The primary challenges in LUAD management are invasion and metastasis, which contribute to high relapse rates, poor prognosis, and low long-term survival rates. Factors influencing the recurrence rate of LUAD include initial cancer staging, treatment modalities, patient age, and overall health. Various studies report recurrence rates ranging from 10% to over 50%. According to the American Cancer Society, the five-year survival rate for NSCLC is approximately 25%, with variations in LUAD survival rates depending on multiple factors. [6,7,8] Thus, identifying novel biomarkers and therapeutic targets for LUAD is crucial to enhancing early detection, improving prognosis, and developing superior clinical interventions.

A novel class of micro non-coding RNAs, tRNA-derived fragments (tRFs), has garnered significant attention. These tRFs are small non-coding RNA fragments, 14–30 nucleotides in length, generated from the precise cleavage of precursor and mature tRNA by Dicer1 nuclease, particularly under conditions of oxygen deficiency and oxidative stress. There are five main subtypes of tRFs, classified based on their origin and original location: tRF-1, tRF-3, tRF-5, i-tRF, and tRF-2 [9, 10]. Initially regarded as non-functional degradation products of tRNA, recent studies suggest that tRFs may play functional roles in various molecular biological processes, including gene silencing, RNA processing, protein biosynthesis, and carcinogenesis [11, 12]. Some tRFs are implicated in cell proliferation, migration, and invasion, and are involved in various human diseases such as cancer, neurodegenerative disorders, viral infections, and metabolic diseases [13,14,15].

Our research group has identified 34 subtypes of abnormally expressed tRNA-derived fragments and tRNA-derived stress-induced small RNAs in lung adenocarcinoma tissues compared to adjacent non-cancerous tissues, with 20 subtypes upregulated and 14 subtypes downregulated [16]. RT-qPCR results indicate that tRF-Val-CAC-010 expression is significantly upregulated in LUAD [16]. Bioinformatic analyses suggest that tRF-Val-CAC-010 may regulate disease progression in LUAD, as both tRF-Val-CAC-010 and its target gene are upregulated in lung alveolus development. Furthermore, tRF-Val-CAC-010 is associated with the Gli2 protein in regulating the “Hedgehog signaling pathway,“ [16] with downregulation of this pathway linked to developmental abnormalities and cancer [17]. To elucidate the specific molecular mechanisms of tRF-Val-CAC-010 in LUAD, we investigated its expression and function in LUAD cells both in vitro and in vivo.

Materials and methods

Cell culture

Normal human lung epithelial cells (BEAS-2B) and LUAD cells (A549, PC9) were obtained from Procell Life Science & Technology Co., Ltd., Wuhan, China. The cell lines used in this study were verified using short tandem repeat (STR) profiling to ensure the absence of contamination from exogenous cell lines. The BEAS-2B, A549, and PC9 cell lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco, Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, USA) and 1% penicillin-streptomycin (Gibco, Life Technologies, USA). All cell cultures were maintained under normoxic conditions in a CO2 incubator set at 37 °C, 21% O2, and 5% CO2 (Thermo Fisher Scientific, Rockford, IL, USA).

Cell transfection

Cells were seeded into a 24-well plate at an appropriate ratio for culture, and transfection was initiated when the cell density reached 70–80%. The tRF-Val-CAC-010 inhibitor, inhibitor NC, mimic, and mimic NC were procured from Ribo Biotechnology Co., Ltd. (Guangzhou, China) (Table 1). The inhibitor is a specially modified chemosynthetic miRNA inhibitor designed to inhibit the function of mature miRNA molecules through specific binding. The mimic is a chemically synthesized miRNA mimic that simulates high miRNA expression in cells. Following the protocol provided with the riboFECT™ CP Transfection Kit (166T, Ribo Biotechnology Co., Ltd., Guangzhou, China), BEAS-2B, A549, and PC9 cells underwent transient transfection and were cultured for 48 h, with the medium being replaced by complete medium 24 h post-transfection.

Table 1 tRF-Val-CAC-010 inhibitor, mimic and their negative control sequences
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Experiment grouping:

  1. 1)

    BEAS-2B cells

  2. 2)

    BEAS-2B cells + mimic NC

  3. 3)

    BEAS-2B cells + mimic

  4. 4)

    A549 cells

  5. 5)

    A549 cells + inhibitor NC

  6. 6)

    A549 cells + inhibitor

  7. 7)

    PC9 cells

  8. 8)

    PC9 cells + inhibitor NC

  9. 9)

    PC9 cells + inhibitor

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells in each experimental group using the Trizol method. One milliliter of RNAiso Plus (Thermo Fisher Scientific, Rockford, IL, USA) was added to the cell surface, mixed thoroughly, and left for 5 minutes to ensure complete cell lysis. The RNA was then extracted into 1.5 mL RNase-free Eppendorf (EP) tubes using chloroform and isopropyl alcohol. The RNA was demethylated using the rtStar™ tRF&tiRNA Pretreatment Kit (Arraystar, Rockville, USA). Subsequently, cDNA was synthesized through reverse transcription using the rtStar™ First-Strand cDNA Synthesis Kit (Arraystar, Rockville, USA), with specific linkers added to the 3’ and 5’ ends. The tRF primers designed and synthesized, containing the linker sequence, are detailed in Table 2. qRT-PCR amplification was performed using the SYBR Green qPCR Master Mix (ROX) (Arraystar, Rockville, USA) and the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Rockford, IL, USA).

Table 2 Primer sequence of qRT-PCR
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Cell proliferation

Cell proliferation was assessed using the CCK-8 kit (MedChem Express, Monmouth Junction, NJ, USA). A cell suspension (5000 cells/100 µL) was inoculated into a 96-well plate and transfected for 24 h. Following transfection, cells were cultured for 0, 24, 48, 72, and 96 h in an anaerobic incubator. At each time point, 10 µL of CCK-8 solution was added to each well and incubated for 1 h. Absorbance at 450 nm was measured using a microplate reader (BioTek, Vermont, USA).

Transwell invasion and cell scratch assay

For the Transwell invasion assay, 100 µL of a Matrigel matrix mixture (Matrigel matrix: RPMI-1640 medium = 1:7) was added to each chamber, pre-spread in the upper chamber, and incubated at 37 °C for 4 h until gel solidification. Treated cells were collected and suspended in a serum-free medium before being added to the upper chamber. The lower chamber was filled with 600 µL of medium containing 10% FBS to establish a concentration gradient. Cells were incubated for 24 h. Post-incubation, chambers were immersed in 4% neutral paraformaldehyde for 30 min and stained with 1% crystal violet. Cell invasion was observed under a 100x positive microscope.

In the scratch assay, horizontal lines were drawn on the back of a 24-well plate. When cell density reached 80–90%, a 10 µL pipette tip was used to make perpendicular scratches. Detached cells were washed twice with phosphate-buffered saline (PBS), and serum-free medium was added. Cell migration was observed at 0 and 24 h under a 100x positive microscope.

Cell cycle

Treated cell precipitates were collected, washed, and centrifuged with pre-cooled PBS. Cells were then fixed by gentle mixing with 1 mL of pre-cooled 70% ethanol and stored at 4 °C for at least 2 h or overnight. Following centrifugation, cells were washed with pre-cooled PBS and resuspended in propidium iodide (PI) staining solution (25 µL of PI stock solution + 20 µL of RNase A). After incubating at 37 °C for 30 min, flow cytometry (ACEA Biosciences Inc., San Diego, California, USA) was used to detect cell cycle phases at an excitation wavelength of 488 nm (red fluorescence).

Cell apoptosis

Treated cell precipitates were collected, washed with PBS, and centrifuged. Cells were resuspended in 400 µL of binding buffer. The suspension was divided into three tubes: blank control, fluorescein isothiocyanate (FITC) positive control, and propidium iodide (PI) positive control. Five microliters of FITC dye was added to the FITC positive control tube and incubated in the dark for 15 min. Cell apoptosis was analyzed using flow cytometry (ACEA Biosciences Inc., San Diego, California, USA).

Verification of tumorigenicity in vivo

All BALB/c nude mice were randomly divided into three groups, with three mice per group, and injected subcutaneously with 0.1 mL (1.0 × 10^6 cells/mL) A549 cells under the left forelimb armpit to establish a tumor model. Starting from the day of tumor formation, the control group received injections of antagomir NC, and the experimental group received tRF-Val-CAC-010-antagomir, each administered at multiple points within the tumor (5 nmol per injection) every two days. The model group received an equal volume of normal saline. Injections continued for four weeks. Tumor dimensions, including the long diameter and the perpendicular short diameter, were measured every three days, and tumor volume was calculated using the formula V = (long diameter × short diameter2) / 2). A tumor growth curve was then plotted. After five weeks of injections, the mice were sacrificed, and samples, including subcutaneous tumors and tissues from the heart, liver, spleen, lung, kidney, brain, and intestine, were collected. Half of each tumor was ground for RNA extraction to perform reverse transcription PCR for detecting tRF-Val-CAC-010 expression (refer to Sect. Quantitative real-time polymerase chain reaction (qRT-PCR) for detailed steps). The remaining half was processed into paraffin sections for hematoxylin and eosin (HE) staining and immunohistochemical staining, focusing on cell proliferation marker Ki-67 and invasion marker MMP9.

Verification of tumor metastasis ability in vivo

A549 cells, stably transfected with luciferase lentivirus, were divided into three groups. Two groups were further transfected with tRF-Val-CAC-010 inhibitor and inhibitor NC, respectively. BALB/c nude mice (three mice per group, 4 weeks old) were injected with 0.1 mL of cell suspension (1.0 × 106 cells/mL) from the three groups via the lateral tail vein. After eight weeks, cell metastasis was observed using in vivo live imaging. The mice were then sacrificed, and samples from the heart, liver, spleen, lung, kidney, brain, and intestine were collected. These samples were processed into paraffin specimens and stained with HE and immunohistochemical antibodies, including CK7, NapsinA, and TTF-1, to co-label and confirm whether cancer cells in each organ were lung-derived epithelial cells, thereby verifying tumor metastasis.

All animal treatments were conducted humanely, and the protocol was approved by the Ethics Committee of Kunming Medical University.

The nude mice used in this paper are sourced from Spiff (Beijing) Biotechnology Co., LTD., and the license number is SCXK(Beijing)2019-0010.

All experimental procedures are carried out in strict accordance with the “Care and Use of Laboratory Animals of the National Institutes of Health”, “National Health and Medical Research Council of China” and “Animal Ethics Guidelines of the Laboratory Animal Management Office of Yunnan Province of China”. The license number of animal experiment facilities is: SYXK(Yunnan)K2021-003. After the experiment, cervical vertebra dislocation euthanasia was performed on the naked mice. The euthanasia protocol followed as the suggestion in “AVMA Guidelines for the Euthanasia of Animals: 2020 Edition”, and approved by the Experimental Animal Ethics Committee of Yunnan Luoyu Biotechnology Co., LTD. (Approval number: PZ20211133).

Statistical analysis

GraphPad Prism (V8.0, GraphPad Software Inc., La Jolla, CA, USA) was used for data analysis. Data are presented as mean ± standard deviation. The t-test was used to detect the differences in the measured data between the two groups. One-way ANOVA (analysis of variance) was used for the assessment of the statistical significance between groups. P < 0.05 indicated that the difference was statistically significant. Image J software (V1.53, Media Cybernetics, Silver Springs, MD, USA) was used to analyze the cell scratch and invasion assay image data.

Results

Upregulation of tRF-Val-CAC-010 in LUAD

Consistent with our previous studies, the expression level of tRF-Val-CAC-010 was significantly elevated in LUAD tissue. To further confirm these findings, we conducted in vitro experiments using LUAD cell lines. We cultured BEAS-2B, A549, and PC9 cells, extracted their respective RNAs, and employed qRT-PCR to measure tRF-Val-CAC-010 expression levels. The results indicated that tRF-Val-CAC-010 expression was significantly higher in A549 and PC9 cells compared to BEAS-2B cells (Fig. 1). Thus, it is evident that tRF-Val-CAC-010 expression is upregulated in LUAD.

Fig. 1
figure 1

Relative expression levels of tRF-Val-CAC-010 in BEAS-2B, A549, and PC9 Cells detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001

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tRF-Val-CAC-010 promoted LUAD cell proliferation

To investigate the effect of tRF-Val-CAC-010 on LUAD cells, BEAS-2B cells were transfected with a tRF-Val-CAC-010 mimic to overexpress tRF-Val-CAC-010, while A549 and PC9 cells were transfected with a tRF-Val-CAC-010 inhibitor to suppress its expression. qRT-PCR analysis performed 48 h post-transfection showed a significant increase in tRF-Val-CAC-010 expression in BEAS-2B cells transfected with the mimic, reaching 1000 times the expression level of the control group (Fig. 2A). Conversely, the expression level of tRF-Val-CAC-010 in A549 and PC9 cells transfected with the inhibitor was significantly reduced, with expression levels at only 21% and 26% of the control group, respectively (Fig. 2B, C).

Fig. 2
figure 2

In vitro transfection efficiency of tRF-Val-CAC-010; Efficacy of tRF-Val-CAC-010 on the proliferation of LUAD cells. A. BEAS-2B cells were transfected with tRF-Val-CAC-010 mimic or mimic NC and verified by qPCR. B & C. A549 and PC9 cells were transfected with tRF-Val-CAC-010 inhibitor and inhibitor NC and verified by qPCR. D, E, & F. CCK-8 proliferation assay was used to determine the cell proliferation potential. NC: negative control, ns P > 0.05, * P < 0.05, **P < 0.01, ***P < 0.001

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To further explore the role of tRF-Val-CAC-010 in LUAD cell proliferation, we conducted a CCK-8 assay in BEAS-2B, A549, and PC9 cells. The assay results revealed that, compared to the NC and blank groups, the proliferation capacity of BEAS-2B cells in the overexpression group was significantly enhanced (Fig. 2D). In contrast, the proliferation abilities of A549 and PC9 cells in the inhibition group were markedly decreased (Fig. 2E, F). These data suggest that tRF-Val-CAC-010 promotes the proliferation of LUAD cells.

tRF-Val-CAC-010 promoted the invasion and migration of LUAD cells

To investigate the effect of tRF-Val-CAC-010 on the invasion and migration abilities of LUAD cells, we conducted Transwell invasion assays and cell scratch assays. The Transwell invasion assay results indicated that overexpression of tRF-Val-CAC-010 in BEAS-2B cells significantly enhanced their invasion ability compared to the NC and blank groups (Fig. 3A). Conversely, knockdown of tRF-Val-CAC-010 in A549 and PC9 cells significantly reduced their invasion capabilities (Fig. 3B, C). The cell scratch assay results showed that overexpression of tRF-Val-CAC-010 in BEAS-2B cells promoted cell migration, with no significant differences between the NC and blank groups (Fig. 3D). In contrast, knockdown of tRF-Val-CAC-010 expression in A549 and PC9 cells inhibited cell migration, again with no significant differences between the NC and blank groups (Fig. 3E, F). These findings indicate that tRF-Val-CAC-010 plays a crucial role in promoting the invasion and migration of LUAD cells.

Fig. 3
figure 3

Efficacy of tRF-Val-CAC-010 in malignant phenotypes of lung adenocarcinoma cells. A, B, & C. The invasive capabilities of BEAS-2B cells with tRF-Val-CAC-010 overexpression (A), and A549 (B) and PC9 cells (C) with tRF-Val-CAC-010 knockdown, were assessed using the Transwell method. D, E, & F. The migration abilities of BEAS-2B cells with tRF-Val-CAC-010 overexpression (D), and A549 (E) and PC9 cells (F) with tRF-Val-CAC-010 knockdown, were evaluated through the cell scratch assay. with the statistical analysis diagram of detection, results shown on the right. NC: negative control, ns P > 0.05, * P < 0.05, **P < 0.01, ***P < 0.001. 100x magnification of cell image

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Effect of tRF-Val-CAC-010 on the apoptosis of LUAD cells

To determine the regulatory effect of tRF-Val-CAC-010 on the apoptosis of LUAD cells, we used the Apoptosis Assay Kit (7Sea Biotech) for cell staining and flow cytometry (FCM) for analysis. The FCM results showed no statistically significant difference in the apoptosis rate of BEAS-2B cells in the tRF-Val-CAC-010 mimic group compared to the NC and blank groups (Fig. 4A). However, the apoptosis rate of A549 cells in the tRF-Val-CAC-010 inhibitor group was significantly increased compared to the NC and blank groups, with the difference being statistically significant (Fig. 4B). Similarly, the apoptosis rate of PC9 cells in the tRF-Val-CAC-010 inhibitor group was significantly higher compared to the NC and blank groups (Fig. 4C). These data suggest that inhibition of tRF-Val-CAC-010 expression promotes apoptosis in LUAD cells.

Fig. 4
figure 4

Efficacy of tRF-Val-CAC-010 on cell apoptosis. FCM analysis was used on BEAS-2B (A), A549 (B) and PC9 (C) cells after transient transfection to detect cell apoptosis, with the statistical analysis of FCM results shown on the right. NC: negative control, ns P > 0.05, *P < 0.05, **P < 0.01

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Effect of tRF-Val-CAC-010 on the LUAD cell cycle

To examine the effect of tRF-Val-CAC-010 on the cell cycle, we used the Cell Cycle Kit (7Sea Biotech) for cell staining and analyzed the cell cycle phases using flow cytometry. The FCM results for BEAS-2B cells in the tRF-Val-CAC-010 mimic group showed no statistically significant differences in the G1, S, and G2 phases compared to the NC and blank groups (Fig. 5A). To assess the effects of tRF-Val-CAC-010 on the cell cycle of LUAD cells, A549 and PC9 cells were transfected with tRF-Val-CAC-010 inhibitor for 48 h. FCM analysis revealed that, compared to the control group, there were statistically significant differences in the G2 phase in the A549 cell suppression group, but no significant differences in the G1 and S phases (Fig. 5B). For the PC9 cell inhibition group, there were no statistically significant differences in the G1, S, and G2 phases compared to the control group (Fig. 5C). Therefore, it can be concluded that knockdown of tRF-Val-CAC-010 expression promotes the G2 phase in LUAD cells, but has no effect on other phases of the cell division cycle.

Fig. 5
figure 5

Efficacy of tRF-Val-CAC-010 on cell cycle. (A) According to FCM analysis, the overexpression of tRF-Val-CAC-010 had no significant effect on the cell cycle of BEAS-2B; (B) According to FCM analysis, the knockdown of tRF-Val-CAC-010 could promote the G2 phase of A549 cell cycle; (C) According to FCM analysis, the knockdown of tRF-Val-CAC-010 had no significant effect on PC9 cell cycle. The statistical analysis of FCM test results are shown on the right, NC: negative control, ns P > 0.05, *P < 0.05

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tRF-Val-CAC-010 promotes tumor growth in vivo

We investigated the effect of tRF-Val-CAC-010 on tumor growth in nude mice bearing the tumor. RT-PCR results confirmed successful inhibition of tRF-Val-CAC-010 in the tumors of the tRF-antagomir group (Fig. 6A). The tumor volume in the tRF-antagomir group was smaller compared to the model group and the tRF-antagomir-NC group, with no significant difference observed between the latter two groups (Fig. 6B, C). This suggests that tRF-Val-CAC-010 can promote tumor formation in vivo. HE staining showed no tumor cells in any organs, and the immunohistochemical results of the tumors are presented in Fig. 6D.

Fig. 6
figure 6

Tumor growth-promoting effect of tRF-Val-CAC-010 in vivo. (A) RT-PCR results confirmed that tRF-Val-CAC-010 was successfully inhibited (P < 0.05). (B) The tumor volume of tRF-antagomir group was smaller (P < 0.05) than that of model group and tRF-antagomir-NC group, and there was no significant difference between the latter two groups (P > 0.05). (C) Tumor size in vivo and ex vivo. (D) The results of HE staining and immunohistochemical staining of the tumors

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tRF-Val-CAC-010 promotes tumor metastasis in vivo

The effect of tRF-Val-CAC-010 on LUAD cell metastasis in vivo was demonstrated through metastatic tumor experiments in nude mice. The total flux value from in vivo imaging in the tRF-inhibitor group was lower than that in the A549 model group and the tRF-inhibitor-NC group, with no significant difference observed between the latter two groups (Fig. 7A, B). HE staining and immunohistochemistry results are shown in Fig. 7C and D. Table 3 summarizes the presence of metastatic tumor cells in the organs of nude mice across each group. These findings suggest that tRF-Val-CAC-010 promotes tumor cell metastasis in vivo, thereby facilitating tumor metastasis.

Fig. 7
figure 7

Tumor metastatic effect of tRF-Val-CAC-010 in vivo. A & B. The total flux value of in vivo imaging in the tRF-inhibitor group was lower (P < 0.05) than that in the A549 model group and tRF-inhibitor-NC group, and there was no significant difference between the latter two groups (P > 0.05). C & D. The results of HE staining and immunohistochemistry of each organ

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Table 3 The metastasis of tumor cells in the organs of nude mice in each group
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Discussion

LC remains the leading cause of cancer-related deaths worldwide, despite continuous advancements in LC screening, surgical techniques, medical treatments, and radiation oncology [18, 19]. LUAD, the most prevalent primary LC, accounts for approximately 40% of all LC cases [4]. Although surgery is the primary treatment for LUAD, achieving a surgical cure is challenging when cancer cell metastasis occurs [20]. Therefore, identifying stable potential biomarkers to predict the prognosis of patients with LUAD and guide individualized treatment is crucial.

The pathogenesis of cancer is a complex, multifactorial process, wherein cancer cells acquire survival advantage as well as proliferation, invasion, and migration ability [21]. tRF have been associated with various cancers, including lung, colon, breast, ovarian, liver, gastric, and pancreatic cancers [22,23,24,25,26,27,28]. An in-depth study of tRFs involved in LUAD could provide insights into the pathogenesis and disease progression of LUAD, contributing to the development of potential targeted therapies.

Our study highlights the significant role of tRF-Val-CAC-010 in the development of LUAD, demonstrating that its upregulation is closely linked to increased tumor cell proliferation, invasion, and migration. Through both in vitro experiments and in vivo animal models, we have shown that inhibiting tRF-Val-CAC-010 significantly reduces the malignant behavior of tumor cells, including decreasing proliferation rates, diminishing invasiveness and migration, and promoting cell apoptosis. These findings suggest that tRF-Val-CAC-010 may serve as a novel biomarker and therapeutic target, aiding in the creation of targeted therapeutic strategies for LUAD. Our future research will focus on elucidating the specific molecular mechanisms of tRF-Val-CAC-010 and exploring its potential applications in personalized healthcare.

Tumor heterogeneity, characterized by differences in genotype and phenotype among various cells or cell populations within a tumor, significantly impacts treatment response, disease progression, and recurrence patterns in LC. In the context of targeted therapy, tumor heterogeneity can result in varying treatment responses among different subgroups, thereby affecting treatment efficacy and disease prognosis.

Given that tRF-Val-CAC-010 is upregulated in LUAD, it presents a promising target for developing new targeted therapy strategies. However, it is essential to acknowledge that tumor heterogeneity may influence the effectiveness of targeted therapy for tRF-Val-CAC-010. For instance, the dependency on tRF-Val-CAC-010 may vary across different tumor regions or subclonal populations, making targeted therapy effective for certain cell populations but limited for others.

To address this challenge, future research should investigate how tumor heterogeneity affects the function of tRF-Val-CAC-010 and leverage this knowledge to design more effective treatment strategies. Potential approaches include developing combination therapies targeting tRF-Val-CAC-010 along with other molecules to overcome therapeutic resistance induced by tumor heterogeneity. Alternatively, tRF-Val-CAC-010 could be used as a biomarker to guide personalized treatment, enabling the formulation of more precise treatment plans for different patient groups.

Extensive research has revealed that the biological role of tRF might involve multiple mechanisms, including regulating RNA stability, protein translation, and participating in cell function regulation. These mechanisms influence tumor cell growth, apoptosis, invasion, and metastasis [12, 29]. For example, tRF-Leu-CAG was found to be upregulated in NSCLC tissues and cell lines, promoting cell cycle progression and proliferation. tRF may be involved in signaling pathways through AURKA, suggesting its potential as a diagnostic marker and therapeutic target for NSCLC [22]. Cui, et al. found that tRF-Val, a new 3’ tRNA-derived fragment, was significantly upregulated in gastric cancer (GC) tissues and cell lines, and the expression of tRF-Val was positively correlated with tumor size and invasion depth in GC tissues [30]. Functionally, tRF-Val binds to the EEF1A1 protein, facilitating its transport to the nucleus and interaction with MDM2, thereby inhibiting the p53 downstream pathway, promoting proliferation and invasion, and inhibiting apoptosis of GC cells [30]. Similarly, tRF-19-W4PU732S, derived from mature tRNASer AGA, was highly expressed in breast cancer and promoted malignant activities such as cell viability, invasion, migration, epithelial-mesenchymal transition, and cancer stem cell phenotypes by targeting RPL27A and inhibiting apoptosis [24]. Additionally, studies have shown a positive correlation between the expression of Dicer1 ribonuclease and tRF-20-MEJB5Y13 in colon cancer (CRC). Under anaerobic conditions, upregulated Dicer1 promotes CRC cell invasion and migration by regulating tRF-20-MEJB5Y13 [31].

Our study demonstrates that tRF-Val-CAC-010 is derived from the 5’ terminal of mature tRF-Val-CAC-2-1, encompassing 31 bases. qRT-PCR results revealed that tRF-Val-CAC-010 expression is significantly upregulated in both LUAD tissues and cells. To further explore the role of tRF-Val-CAC-010 in LUAD, we designed and synthesized tRF-Val-CAC-010 inhibitors and mimics for transient transfection. These tools allowed us to manipulate tRF-Val-CAC-010 expression in normal lung epithelial cells and LUAD cells, facilitating the observation and evaluation of its effects on LUAD cell function. Data from the CCK8 cell proliferation assay indicated that tRF-Val-CAC-010 promotes LUAD cell proliferation. Transwell invasion and cell scratch assays showed that tRF-Val-CAC-010 enhances LUAD cell invasion and migration. Flow cytometry analysis revealed that inhibiting tRF-Val-CAC-010 expression promotes LUAD cell apoptosis. Therefore, tRF-Val-CAC-010 holds promise as a diagnostic biomarker and therapeutic target for LUAD.

Nevertheless, the potential mechanism of action of tRF-Val-CAC-010 in LUAD remains unclear, and it is uncertain whether tRF-Val-CAC-010 directly interacts with RNAs or proteins to jointly regulate gene expression in cells. Therefore, tRFs present significant research potential and exploitability, and further investigations could elucidate the extent of tRF interactions with genes and molecules, yielding noteworthy biological insights.

The limitations of this study include a limited sample size and single source, the inability to comprehensively analyze the impact of tumor heterogeneity on tRF-Val-CAC-010 function, and the lack of long-term follow-up data to evaluate associations with survival and recurrence rates. Additionally, while in vitro and in vivo models provide preliminary insights, they may not fully replicate the complexity of human LUAD. The specific mechanism of action of tRF-Val-CAC-010 as a therapeutic target still requires further clarification. Future research should address these limitations to enhance the generalizability of our findings and the accuracy of clinical applications.

Conclusion

In conclusion, our study aimed to explore the effect of tRF on the phenotypic function of LUAD cells and its role in tumorigenesis and progression in vivo. Preliminary data indicate that tRF-Val-CAC-010 promotes the proliferation, migration, and invasion of LUAD cells while inhibiting apoptosis. However, no consistent effects of tRF-Val-CAC-010 on the cell cycle were observed in this study, necessitating further investigation. Additionally, tRF-Val-CAC-010 was shown to promote tumor formation and metastasis in vivo. tRFs constitute a large proportion of small RNAs in many tissues, and their primary biological functions are highly correlated with various diseases, including cancer. This suggests that tRFs could serve as novel diagnostic biomarkers for tumors or provide innovative approaches for developing new therapeutic drugs. Consequently, tRF-Val-CAC-010 holds potential as a novel biomarker for regulating tumor metastasis.