Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A–C) Knockdown efficiency of tRF-3009a (A), tRF-3021a (B), and tRF-3030a (C) in U251 cells transfected with the corresponding LNA oligos, measured by qRT–PCR, normalized to miR-21-5p, and expressed relative to NC LNA (set to 1). (D-F) Overexpression efficiency of tRF-3009a (D), tRF-3021a (E), and tRF-3030a (F) in U251 cells transfected with the corresponding synthetic mimics, measured by qRT–PCR, normalized to miR-21-5p, and expressed relative to NC mimic (set to 1). (G) Northern blot analysis of the parental tRNAs corresponding to tRF-3009a (tRNA-Leu), tRF-3021a (tRNA-Ala), and tRF-3030a (tRNA-Tyr) following the indicated tRF-3 knockdowns; U6 snRNA serves as a loading control. Statistics: Bars in (A–F) represent mean ± SD of three technical replicates (for panel B, n = 3 biological replicates). P values were calculated using unpaired two-tailed t tests with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%). ****p < 0.0001.

Article Snippet: Reverse transcription and qPCR for tRFs were performed using the Mir-XTM miRNA qRT-PCR TB Green® Kit (Takara, 638314) following the manufacturer’s protocol, together with PowerTrackTM SYBR Green Master Mix (A46113), the kit universal reverse primer, and custom specific forward primers. hsa-miR-21-5p was used for normalization.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Over Expression, Northern Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A) Representative Transwell images of invasion (top) and migration (bottom) of U251 cells after LNA-mediated knockdown (KD) of tRF-3009a, tRF-3021a, or tRF-3030a compared with negative-control (NC) LNA. (B–C) Quantification of invasion (B) and migration (C) in U251 cells following tRF-3 KD. Cell numbers are normalized to NC LNA (set to 100%). (D) Representative Transwell images of invasion (top) and migration (bottom) of U251 cells after overexpression (OE) of tRF-3009a, tRF-3021a, or tRF-3030a using synthetic mimics compared with NC mimic. (E–F) Quantification of invasion (E) and migration (F) following tRF-3 OE. Cell numbers are normalized to NC mimic (set to 100%). (G) qRT–PCR analysis of EMT-associated transcripts ( CDH2, SNAI2, VIM, TWIST1 ) in U251 cells after tRF-3021a KD versus NC LNA; expression is normalized to GAPDH and shown relative to NC LNA (set to 1). (H) Immunoblot analysis of N-cadherin, SLUG, and TWIST1 in U251 cells after tRF-3021a KD versus NC LNA; GAPDH serves as a loading control (representative of n = 3 independent experiments). (I) U251 proliferation measured by MTT at 24, 48, and 72 h following KD of tRF-3009a, tRF-3021a, or tRF-3030a versus NC LNA. (J–K) MTT assays in normal astrocytes (J) and U87 glioma cells (K) after tRF-3021a KD versus NC LNA at the indicated time points. Data presentation: For quantified panels, data are mean ± SD from n = 3 independent experiments (B–C, E–F, I–K) or n = 3 biological replicates (G). Statistics: (B–C, E–F) one-way ANOVA with Dunnett’s multiple-comparisons test versus the corresponding NC. (G) unpaired two-tailed t tests with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%). (I–K) two-way ANOVA (time × treatment) with Sidak’s multiple-comparisons test comparing tRF-KD to NC LNA at each time point. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Reverse transcription and qPCR for tRFs were performed using the Mir-XTM miRNA qRT-PCR TB Green® Kit (Takara, 638314) following the manufacturer’s protocol, together with PowerTrackTM SYBR Green Master Mix (A46113), the kit universal reverse primer, and custom specific forward primers. hsa-miR-21-5p was used for normalization.

Techniques: Migration, Knockdown, Negative Control, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: tRF-3021a, a tRNA-Ala-TGC derived 3’ fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

doi: 10.64898/2026.01.26.701835

Figure Lengend Snippet: (A) Quantification of Annexin V–positive cells (early + late apoptotic), (B) Puromycin-labeling assay in U251 cells transfected with NC LNA, tRF-3021a LNA, tRF-3021a mimic, or tRF-3021a LNA + tRF-3021a mimic. Cell lysates were immunoblotted with anti-puromycin; GAPDH serves as a loading control. (C) Puromycin-labeling assay in U251 cells following knockdown of miR-7-5p or miR-9-5p (LNA) compared with NC LNA; GAPDH serves as a loading control. (D-E) qRT–PCR confirmation of miR-7-5p (D) and miR-9-5p (E) knockdown relative to the corresponding NC mimic (set to 1). Data presentation: Data are mean ± SD from n = 3 independent experiments for panel A and technical replicates for panel D and E. Panels (B and C) are representative blots. Statistics: (A) multiple unpaired two-tailed t tests with Benjamini–Krieger–Yekutieli false-discovery-rate correction (Q = 1%); p = 0.002702 (NC vs tRF-3021a KD) and p = 0.013265 (KD vs KD + mimic). (D) unpaired two-tailed t test, p = 0.000049. (E) unpaired two-tailed t test, p = 0.000510. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Reverse transcription and qPCR for tRFs were performed using the Mir-XTM miRNA qRT-PCR TB Green® Kit (Takara, 638314) following the manufacturer’s protocol, together with PowerTrackTM SYBR Green Master Mix (A46113), the kit universal reverse primer, and custom specific forward primers. hsa-miR-21-5p was used for normalization.

Techniques: Labeling, Transfection, Control, Knockdown, Quantitative RT-PCR, Two Tailed Test