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: Genes & Diseases

Article Title: ADAR1p110 promotes hepatocellular carcinoma metastasis via the miR-451a/TUBA1A axis

doi: 10.1016/j.gendis.2025.101770

Figure Lengend Snippet: ADAR1p110 regulates the expression of TUBA1A by inhibiting the expression of miR-451a. (A) Changes of RNA editing ratio after ADAR1p110 overexpression or knockdown. (B) Schematic diagram of TUBA1A editing. (C) Statistics of microRNA editing events in WT and ADAR1p110-overexpressing HCC cells. (D) miRNAs with editing counts greater than 5 and editing ratio greater than 5% in ADAR1p110-overexpressing or ADAR1p110 knockdown cells. (E) miRNAs were down-regulated after ADAR1p110 overexpression and up-regulated after ADAR1 knockdown. (F) The mRNA expression of TUBA1A was negatively correlated with that of miR-451a analysis based on the TCGA-LIHC dataset ( n = 370). (G) The expression level of miR-451a in tumor and non-tumor tissues from the TCGA-LIHC dataset (Tumor n = 369, Non-tumor n = 49). (H) Kaplan–Meier overall survival curves of TCGA-LIHC patients with low or high expressed miR-451a (Low miR-451a n = 248, High miR-451a n = 113). (I) The expression of miR-451a in the indicated cells was verified by qRT‒PCR ( n = 3). (J) The expression of TUBA1A in the indicated cells was verified by qRT‒PCR ( n = 3). (K) The relative expression of miR-451a and TUBA1A in anti-AGO2 antibody precipitated RNA ( n = 3). (L) Schematic diagram of miR-451a binding with the WT and mutated 3′-UTR of TUBA1A. (M) Luciferase activities of TUBA1A-WT or TUBA1A-MUT were determined in the presence of the NC mimic or the miR-451a mimic ( n = 3). The data are presented as the mean ± SD. P values were computed using the unpaired Student's t -test (G, I, J, K, M), one-way ANOVA test (I), Pearson's correlation test (F), and log-rank tests (H). ns: not significant. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: A luciferase assay kit (Yeasen, China) was used to perform the luciferase reporter assay following the manufacturer's protocol.

Techniques: Expressing, Over Expression, Knockdown, Binding Assay, Luciferase

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Schematic illustration of in vivo tumor immunotherapy enhanced by mRNA/HNPs through intravenous injection. H18 lipid, DOPE, cholesterol, DMG-PEG 2000 and mRNA were mixed to form mRNA/H 18 NPs with the special multilamellar concentric nanostructures. Following intravenous administration, mRNA/H 18 NPs demonstrated preferential adsorption of complement C3 proteins to form a characteristic protein corona, resulting in specific mRNA transfection in the spleen, especially in splenic dendritic cells. When encapsulating tumor antigen-encoding mRNA, the mRNA/H 18 NPs achieved precise transfection of the antigen mRNA in splenic dendritic cells. This targeted delivery stimulated dendritic cell maturation and subsequent antigen presentation, initiating robust T cell priming. The activated antigen-specific cytotoxic T lymphocytes then infiltrated into tumor tissues, ultimately inducing tumor cell elimination.

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Injection, Adsorption, Transfection, Immunopeptidomics

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Preparation and Characterization of Optimal mRNA/H 18 NPs. (A) Schematic illustration of mRNA/H 18 NPs preparation. (B) The size distribution and (C) zeta potential of optimized mRNA/H 18 NPs. (D) The apparent p K a of mRNA/H 18 NPs. (E) Cryo-EM image of optimized mRNA/H 18 NPs. Scale bar = 50 nm. (F) Representative image and percentage of bioluminescence in major organs of mice following intravenous injection of mLuc/H 18 NPs. (G)-(I) Stability test for mRNA/H 18 NPs. (G) Size and PDI of mRNA/H 18 NPs when stored at 4 °C for different days (0, 3, 5, 7). (H) Left: Total bioluminescence flux in the spleen of mice 6 h after intravenous injection of mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). Right: Representative bioluminescence images of major organs of mice 6 h after intravenous injection of different mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). (I) Size and PDI of mRNA/H 18 NPs when diluted with PBS by different times. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: Zeta Potential Analyzer, Cryo-EM Sample Prep, Injection

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo splenic DC-specific transfection of mRNA/H 18 NPs and in vitro protein corona analysis of mRNA/H 18 NPs. (A) EGFP protein expression in splenic cell subsets of C57BL/6J mice 24 h post intravenous injection of different formulations. (B) The top 5 most abundant plasma proteins adsorbed on mRNA/H 18 NPs (C3: Complement C3; Ighm: Immunoglobulin heavy constant mu; Hbat1: Alpha-globin; Itih4: Inter alpha-trypsin inhibitor, heavy chain 4; Cnn2: Calponin). (C) Heatmap plot of major proteins in the protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. PBS group was used as a negative control. (D) Quantification of major adsorbed protein categories of different formulations. (E) Complement C3 abundance in protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. (F) Bioluminescence images of major organs and (G) Quantification of total bioluminescence flux in the spleen from C57BL/6J mice 6 h after intravenous injection of mLuc/H 18 NPs (mLuc dose of 0.25 mg kg −1 ). Mice were pre-treated with cobra venom factor (CVF) or PBS. (H) Fluorescence quantification of Cy5 mRNA delivered by uncoated or complement C3-coated Cy5-mRNA/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. (I) Bioluminescence intensity of luciferase protein translated from mRNA delivered by uncoated or complement C3-coated mLuc/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Transfection, In Vitro, Expressing, Injection, Clinical Proteomics, Negative Control, Combined Bisulfite Restriction Analysis Assay, Fluorescence, Incubation, Blocking Assay, Luciferase