The role of tRF-Val-CAC-010 in lung adenocarcinoma: implications for tumorigenesis and metastasis

Abstract

Objective: Transfer RNA-derived fragments (tRFs) are short non-coding RNA (ncRNA) sequences, ranging from 14 to 30 nucleotides, produced through the precise cleavage of precursor and mature tRNAs. While tRFs have been implicated in various diseases, including cancer, their role in lung adenocarcinoma (LUAD) remains underexplored. This study aims to investigate the impact of tRF-Val-CAC-010, a specific tRF molecule, on the phenotype of LUAD cells and its role in tumorigenesis and progression in vivo.

Methods: The expression level of tRF-Val-CAC-010 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Specific inhibitors and mimics of tRF-Val-CAC-010 were synthesized for transient transfection. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8), while cell invasion and migration were evaluated through Transwell invasion and scratch assays. Flow cytometry was utilized to analyze cell cycle and apoptosis. The in vivo effects of tRF-Val-CAC-010 on tumor growth and metastasis were determined through tumor formation and metastasis imaging experiments in nude mice.

Results: The expression level of tRF-Val-CAC-010 was upregulated in A549 and PC9 LUAD cells (P < 0.01). Suppression of tRF-Val-CAC-010 expression resulted in decreased proliferation of A549 and PC9 cells (P < 0.001), reduced invasion and migration of A549 (P < 0.05, P < 0.001) and PC9 cells (P < 0.05, P < 0.01), enhanced apoptosis in both A549 (P < 0.05) and PC9 cells (P < 0.05), and increased G2 phase cell cycle arrest in A549 cells (P < 0.05). In vivo, the tumor formation volume in the tRF-inhibitor group was significantly smaller than that in the model and tRF-NC groups (P < 0.05). The metastatic tumor flux value in the tRF-inhibitor group was also significantly lower than that in the model and tRF-NC groups (P < 0.05).

Conclusion: This study demonstrates that tRF-Val-CAC-010 promotes proliferation, migration, and invasion of LUAD cells and induces apoptosis in vitro, however, its specific effects on the cell cycle require further elucidation. Additionally, tRF-Val-CAC-010 enhances tumor formation and metastasis in vivo. Therefore, tRF-Val-CAC-010 may serve as a novel diagnostic biomarker and potential therapeutic target for LUAD.

Keywords: Cell phenotypes; Lung adenocarcinoma; Tumor growth; Tumor metastasis; tRF; tRF-Val-CAC-010.

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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