Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 functions as a cytoplasmic ceRNA to sequester miR-562 and de-repress COL4A1. (A) Subcellular distribution of circSMAD4 in TC-hMDMs and patient-derived TAMs assessed by nuclear/cytoplasmic fractionation. (B) Representative immunofluorescence/ISH images showing circSMAD4 signals in macrophages (CD163) with nuclear counterstaining (DAPI). Scale bar, 50 μm. (C) Venn diagram of predicted circSMAD4-interacting miRNAs from circInteractome and circBank, yielding a shortlist including miR-562. (D) miR-562 levels following circSMAD4 knockdown in TC-hMDMs. (E–G) pri-miR-562, pre-miR-562, and miR-562 promoter reporter activity after circSMAD4 overexpression. (H) AGO2-RIP enrichment of circSMAD4 and miR-562 relative to IgG in TC-hMDMs. (I) AGO2 immunoblotting after circSMAD4 sense/antisense RNA pull-down in TC-hMDMs. (J) Predicted pairing between miR-562 and circSMAD4 (WT) and the corresponding mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (K) Dual-luciferase assays for circSMAD4-WT/MUT reporters in the presence of miR-562 mimics or inhibitor. (L) Intersection of miRNA target predictions (miRTarBase, miRmap, TargetScan, and miRDB) identifying candidate miR-562 targets. (M) COL4A1 mRNA levels after miR-562 mimics or inhibitor in TC-hMDMs. (N) Predicted miR-562 binding site within the COL4A1 3′UTR (WT) and mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (O) Dual-luciferase assays for COL4A1 3′UTR WT/MUT reporters with miR-562 mimics or inhibitor. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Derivative Assay, Fractionation, Immunofluorescence, Knockdown, Activity Assay, Over Expression, Western Blot, Mutagenesis, Sequencing, Luciferase, Binding Assay

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 facilitates IGF2BP2-dependent stabilization of m6A-marked transcripts. (A) Venn diagram intersecting ENCORI-predicted IGF2BP2 targets with DEGs from shIGF2BP2 versus shNC and shcircSMAD4 versus shNC mRNA-seq, identifying shared candidates. (B) MeRIP–qPCR showing m6A enrichment on COL4A1, SPI1, and ACTA2 candidate regions (CRDs) in shNC and shIGF2BP2 cells. (C) IGF2BP2-RIP–qPCR showing IGF2BP2 binding to COL4A1, SPI1, and ACTA2 CRDs in shNC + Vector, shcircSMAD4 + Vector, shNC + IGF2BP2, and shcircSMAD4 + IGF2BP2 groups. (D) Biotin-circSMAD4 pull-down followed by qPCR showing enrichment of COL4A1, SPI1, and ACTA2 CRDs in Vector + shNC, circSMAD4 + shNC, Vector + shIGF2BP2, and circSMAD4 + shIGF2BP2 groups. (E–G) Schematics of m6A-site mutations introduced into COL4A1, SPI1, and ACTA2 reporters. (H–J) Dual-luciferase assays for CRD reporters (WT and m6A-mutant) in Vector, circSMAD4, and IGF2BP2 groups. (K–M) MeRIP–qPCR for WT and m6A-mutant CRD reporters in Vector, circSMAD4, and IGF2BP2 groups. (N–P) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 knockdown with Vector or IGF2BP2 overexpression. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). (Q–S) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 overexpression with shNC or shIGF2BP2. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Binding Assay, Plasmid Preparation, Luciferase, Mutagenesis, Knockdown, Over Expression

Journal: Life Metabolism

Article Title: Gestational progesterone restores menstrual cycle in PCOS patients via enhancing ovary estrogen production

doi: 10.1093/lifemeta/loag004

Figure Lengend Snippet: Progesterone decreases FSHR levels through its transcription factor GATA2. (a) Expression levels of GATA2 in the granulosa cells of control, PCOS-like model mice, and progesterone-treated mice ( n = 6). (b) Expression levels of GATA2 in the KGN cell line after treatment with varying concentrations of progesterone ( n = 3). (c and d) The mRNA and protein levels of FSHR in control conditions and following GATA2 overexpression ( n = 3). (e) Expression levels of GATA2 and FSHR after treatment with progesterone and disturbance of GATA2 ( n = 6). (f) Schematic diagram of the dual-luciferase assays, along with the results of these assays using the promoters of FSHR ( n = 6).

Article Snippet: A dual-luciferase assay kit (Vazyme, DL101) was used to analyze transient expression.

Techniques: Expressing, Control, Over Expression, Luciferase