Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR levels are associated with WHO grades of glioma. (A) APTR gene amplification rate in WHO grade 2–4 glioma samples from TMUGH samples (left panel, n = 153) and TCGA datasets (right panel, n = 603). (B) The expression level of APTR between different grades of glioma from TMUGH samples (left, control = 20) and TCGA datasets (right, control = 105). (C) APTR copy number is positively correlated with its expression level in TMUGH samples (upper panel) and TCGA datasets (lower panel). (D) The pan-cancer atlas depicting APTR expression in multiple tissues. ∗ P < 0.05; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Amplification, Expressing, Control

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR amplification pattern and expression level as a prognostic biomarker for glioma. (A) Kaplan-Meier (KM) analysis of overall survival (OS) time in WHO grade 2–4 gliomas from TMUGH samples (left, n = 153) and LGG + GBM samples from the TCGA dataset (right, n = 603) with samples stratified by APTR amplification status. (B) KM analysis of OS in WHO grade 4 gliomas from TMUGH samples (left, n = 70) and GBM samples from the TCGA dataset (right, n = 130) with samples stratified by APTR amplification status; (C) KM results revealed the OS outcome in WHO grade 2–4 gliomas from TMUGH samples (left, n = 153) and LGG and GBM samples from the TCGA dataset (right, n = 603) with samples stratified by APTR expression level. (D) KM was applied to assess OS in the WHO grade 4 gliomas from TMUGH samples (left, n = 70) and LGG + GBM samples from the TCGA dataset (right, n = 130) with samples stratified by APTR expression level. (E – F) Receiver operating characteristic (ROC) curve to illustrated relationships between OS and APTR amplification status in grade 2–4 gliomas from TMUGH samples (left) and the TCGA LGG + GBM datasets (right). (G – H) An ROC curve was used to plot the correlation between OS and RNA expression level in grade 2–4 gliomas from TMUGH samples (left) and the TCGA LGG + GBM datasets (right).

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Amplification, Expressing, Biomarker Discovery, RNA Expression

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR amplification pattern and expression level as a prognostic biomarker for glioma. (A) The influence of APTR amplification status and RNA expression level on the OS in grade 2–4 gliomas from TMUGH samples (upper) and the TCGA LGG + GBM datasets (lower) displayed using a forest plot. (B) The impact of APTR amplification status and RNA expression levels on the OS in grade 4 gliomas from TMUGH samples (upper) and the TCGA GBM datasets (lower) was visualized using a forest plot.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Amplification, Expressing, Biomarker Discovery, RNA Expression

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR knockdown inhibits the malignant phenotype of GBM cells in vivo and in vitro . (A) qRT‐PCR results confirmed APTR knockdown in U87MG and U251 cells (n = 3). (B) Cell Counting Kit-8 assays assessed GBM cell proliferation after APTR knockdown (n = 3). (C) Representative graphs and statistical results of cell clone formation experiments (n = 3). (D) Wound-healing assays assessed GBM cell migration capacity after APTR knockdown (n = 3). (E) Transwell assays assessed GBM cell invasion ability after APTR knockdown (n = 3). (F) Representative bioluminescent images of xenograft tumors with the corresponding quantitative analyses obtained from BALB/c nude mice in the U87MG control group (Scramble) and shAPTR groups (APTR-sh1 and APTR-sh2) (n = 5). (G) Survival curves for glioma-bearing mice in the control group (Scramble) and APTR knockdown groups (APTR-sh1 and APTR-sh2) under the indicated conditions (n = 5). ∗∗∗∗ P < 0.0001. Data are expressed as the mean ± SD.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, In Vivo, In Vitro, Quantitative RT-PCR, Cell Counting, Migration, Control

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR functions as a sponge for miR-6734-5p. (A) FISH was performed to visualize the intracellular localization of APTR (green), Scramble probe served as negative control, U6 probe served as positive control. (B) Nuclear-cytoplasmic separation of RNA from U87MG and U251 cells was followed by RT-PCR amplification, and the results were detected using agarose gel electrophoresis. (C) Volcano plots showing the differentially expressed microRNA (miRNA) after APTR knockdown (upper). The intersection between the up-regulated miRNAs (n = 22) from miRNA-seq and the miRNAs predicted by miRanda (n = 164) was determined to identify miRNAs that potentially bind to APTR (lower). (D) qRT‐PCR results showing the miRNA expression levels in U87MG and U251 cells with APTR knockdown. (E) Schematic representation of the luciferase reporter plasmids, including one with the APTR interaction miRNA sequence and another without (upper panel). The dual luciferase reporter assay verified the targeting relationship between APTR and miR-6734-5p (lower panel). (F) An Anti-AGO2 RIP assay was performed in U87MG and U251 cells, followed by qRT-PCR to detect the enrichment ability of AGO2 on APTR or miR-6734-5p compared to that of IgG. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗∗ P < 0.0001.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Negative Control, Positive Control, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Knockdown, Quantitative RT-PCR, Expressing, Luciferase, Sequencing, Reporter Assay

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR functions as a sponge for miR-6734-5p to modulate the Wnt signaling pathway. (A) Continuous scatter plot showed differential transcripts after APTR knockdown from RNA-seq. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showing differential transcripts after APTR knockdown. (C) The overlap between downregulated genes identified in RNA-seq (868) and genes predicted by TargetScan (3888) to potentially bind with miR-6734-5p. ( D) Bubble chart showing the Gene Ontology (GO) term enrichment analysis of the overlapped genes (n = 170). (E) The expression levels of TCF7 and LEF1 in gliomas of various grades from the TCGA database. (F – G) IHC staining of TCF7 and LEF1 (F). Protein levels (LI %) of TCF7 and LEF1 in 153 glioma samples and 20 control brain samples (G). Scale bar, 50 μm . (H) The relationship between miR-6734-5p and TCF7/LEF1 expression in GBM tissues analyzed using correlation coefficients. ∗∗ P < 0.01; ∗∗∗∗ P < 0.0001.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Knockdown, RNA Sequencing, Expressing, Immunohistochemistry, Control

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR indirectly regulates miR-6734-5p that directly targets TCF7 and LEF1. (A – B) qRT‐PCR analysis of the relative mRNA levels of TCF7 and LEF1 (A) and western blots showing the protein levels of TCF7, LEF1, MYC, and CCND1 (B) after APTR knockdown. (C) Luciferase activity quantified in GBM cells including control and APTR knockdown groups. (D – E) The relative mRNA expression of TCF7 and LEF1 was detected using qRT-PCR (D), while the protein levels of TCF7, LEF1, MYC, and CCND1 were determined using western blotting after transfection with miR-6734-5p mimics (E). (F) Luciferase activity was assessed in U87MG and U251 cells after transfection with miR-6734-5p mimics and TCF7/LEF1-binding reporter plasmids. (G) Schematic representation of the luciferase reporter plasmids, including TCF7 with (TCF7-WT) and without (TCF7-Mut) the miRNA interaction sequence (upper); luciferase activities were measured following transfection with TCF7-WT or TCF7-MUT and miR-6734-5p mimics or mimic-control in U87MG and U251 cells (lower). (H) Schematic illustration of the LEF1-WT and LEF1-MUT luciferase reporter vectors (upper panel). Relative luciferase activity in U87MG and U251 cells after transfection with the aforementioned plasmids and miR-6734-5p mimics or mimic-control (lower). (I) AGO2-RIP analysis verified the interaction between miR-6734-5p and TCF7/LEF1. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Luciferase, Activity Assay, Control, Expressing, Transfection, Binding Assay, Sequencing

Journal: Non-coding RNA Research

Article Title: LncRNA APTR amplification serves as a potential glioma biomarker and promotes glioma progression via miR-6734-5p/ TCF7/LEF1 axis

doi: 10.1016/j.ncrna.2025.02.007

Figure Lengend Snippet: APTR promotes malignant GBM cell phenotype through APTR/miR-6734-5p/TCF7/LEF1 axis. (A) Western blot results showing the protein levels of TCF7, LEF1, MYC, and CCND1 in U87MG and U251 cells after treatment with miR-6734-5p-specific ASO. (B) Relative luciferase activity measured in GBM cells after co-treatment with miR-6734-5p-specific ASO and TCF7/LEF1-binding reporter plasmids. (C – E) The cell proliferation (C, D) and invasion (E) were determined upon application of miR-6734-5p-specific ASO (n = 3). ( F ) Schematic diagram shows the mechanism underlying the promotion of malignant GBM cell phenotype by APTR. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0. 0001. Data are presented as mean ± SD.

Article Snippet: The APTR copy number was detected using the TaqMan Copy Number Assay kit (assay ID: Hs04995058_cn, Hs04965917_cn, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Western Blot, Luciferase, Activity Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Effects of circPCNX on p21 mRNA and p21 protein. (A–D) AUF1 binding to p21 mRNA (A), p21 mRNA stability (B), p21 mRNA levels (C), and p21 protein levels (D) were assessed after transfection with pcDNA3(EV) or with pcDNA-circPCNX to overexpress circPCNX. (A) AUF1 binding to circPCNX, PCNX mRNA and p21 mRNA was assessed one day after transfection; values were first normalized to the levels of a transcript (GAPDH mRNA) that encodes a housekeeping protein, and afterwards normalized to the RNA levels in the IgG IP. (B) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after transfection of plasmids, changes in the levels of p21 mRNA (C) or p21 protein (D) were assessed by RT-qPCR and western blot analyses, respectively. (E–H) AUF1 binding to p21 mRNA (E), p21 mRNA stability (F), p21 mRNA levels (G), and p21 protein levels (H) were assessed after transfection with Ctrl siRNA or circPCNX siRNA to silence circPCNX. (E) AUF1 binding to circPCNX, to PCNX mRNA and to p21 mRNA was assessed one day after transfection; as above, values were first normalized to GAPDH mRNA, and afterwards normalized to RNAs in the IgG IP. (F) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after siRNA transfections, changes in the levels of p21 mRNA (G) or p21 protein (H) were assessed by RT-qPCR and western blot analyses, respectively. Data in (A–C, E–G) represent the mean values ± SD from three biological replicates. Significance was established using Student's t-test. ** P ≤ 0.01 or *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Transfection, Blocking Assay, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: AUF1 binding to p21 mRNA is attenuated by circPCNX, but not by mutant circPCNX unable to bind AUF1. (A) Twenty-four hours after transfecting HeLa cells with the plasmids shown, the association of p21 mRNA (or ACTB mRNA, in control reactions) with AUF1 was measured by RIP followed by RT-qPCR analysis and represented as ‘fold enrichment’. (B, C) In HeLa cells transfected with the plasmids indicated, the levels of p21 mRNA (B) were assessed 3 days after transfection by RT-qPCR analysis, and the levels of p21 protein (C) were assessed one and three days after transfection by western blot analysis, including GAPDH as loading control. (D) At the times shown following transfection of HeLa cells with the plasmids indicated, cell proliferation in each transfection group was assessed by using BrdU incorporation analysis. Data in (A, B, D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Mutagenesis, Quantitative RT-PCR, Transfection, Western Blot, BrdU Incorporation Assay

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Expression levels of p21 mRNA and p21 protein are attenuated by circPCNX, but not by a mutant circPCNX unable to bind AUF1. (A) Reporter plasmids were created using the backbone of the psiCHECK2 dual luciferase reporter by inserting the p21 3′UTR (gray) with either the full-length wild-type (3′wt) sequence or with a mutation (3′del) in the AUF1-binding site (red). Vectors using the pCMV6 backbone were also created to express p21 protein from mRNAs that had the intact 3′UTR (3′wt) or had a mutation in the AUF1-binding site (3′del). (B) Forty-eight hours after transfecting HeLa cells with either pcDNA3(EV) or pcDNA3-AUF1 or with the siRNAs shown, the reporter plasmids psiCHECK2-p21-3′wt or psiCHECK2-p21-3′del were transfected and 24 h after that, luciferase activities (RL/FL) in each transfection group were assessed. (C) Twenty-four hours after transfecting HeLa cells with the plasmids indicated, the levels of p21 mRNA (with 3′wt or 3′del 3′UTRs) were measured by RT-qPCR analysis and normalized to neo mRNA expressed from the same plasmids; the levels of control ACTB mRNA were quantified in the same reactions; the levels of p21 protein in each transfection group were assessed by western blot analysis. (D) Ctrl or circPCNX siRNAs were transfected along with the plasmids shown (left, p21 3′wt; right, p21 3′del), and the rates of proliferation were assessed by measuring BrdU incorporation at the times indicated. (E) Summary model. Top, when circPCNX accumulates in cells, it can sequester AUF1 away from its target mRNAs, notably p21 mRNA, in turn allowing p21 mRNA stabilization, increased p21 expression, and growth suppression. Bottom, when circPCNX levels decline, or if circPCNX is unable to bind AUF1 (mutated or truncated), then AUF1 associates with p21 mRNA, reducing p21 mRNA stability and p21 levels, and enhancing cell proliferation. Image was created using BioRender. Data in (B–D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Expressing, Mutagenesis, Luciferase, Sequencing, Binding Assay, Transfection, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay

Journal: iScience

Article Title: GPX4-dependent ferroptosis sensitivity is a fitness trade-off for cell enlargement

doi: 10.1016/j.isci.2025.112363

Figure Lengend Snippet: Increased lipid peroxidation in large cells (A) Digital holography images of RSL3 treatment time course for control, doxorubicin-treated, and palbociclib-treated RPE1 cells. (B) Quantification of imaging parameters at 0 and 4 h after RSL3 addition. The blue numbers in the optical volume indicate the increase in cell volume (swelling) between 0- and 4-h time points. (C) Lipid peroxidation in RPE1 cells using C11-bodipy lipid peroxidation sensor and flow cytometry. Probe oxidation results in a shift of the fluorescence emission peak from red (PE-A) to green (FITC-A) channel. Cells treated with palbociclib were treated with or without 1 μM RSL3 and 1 μM Fer-1 as indicated. Data shown are mean ± SD, n = 3. (D and E) (D) Same as (C) but cells were treated with 50 nM doxorubicin. Statistical analysis in (C and D) was ANOVA followed by Tukey’s test. (E) Digital holographic quantification of cell areas and phase shifts for single GPX4 KO cells treated with (blue line) or without (red line) doxorubicin for 3 days after which they were imaged for 6 h in the presence of ferroptosis inhibitor Fer-1. (F) Same treatment as in (E), but Fer-1 was washed out immediately before imaging. Mean values are in solid line, with error bars showing standard deviation; between 52 and 660 cells were tracked. (G) Tetracycline-inducible expression of mGreenLantern-p21. The cell size distribution with and without Tet-induction for 3 days was measured with coulter counter. (H) WB analysis of mGreenLantern-p21 with and without Tet-induction for 3 days, followed by 100 nM RSL3 for 24 h. (I) Senescence-associated β-galactosidase staining of mGreenLantern-p21 cells. Data shown are mean ± SD, n = 3. ANOVA with Tukey’s test. See also Figure S3 .

Article Snippet: The RPE1 mGreenLantern-p21 cells were established by cloning mGreenLantern in frame with p21 coding sequence into AAVS1-Tet plasmid (Addgene plasmid #158663, a kind gift from Masato Kanemaki).

Techniques: Control, Imaging, Flow Cytometry, Fluorescence, Standard Deviation, Expressing, Staining