Journal: Smart Medicine

Article Title: LINC00973/DTX3L Axis Promotes Non‐Small Cell Lung Cancer Progression and Serves as a Therapeutic Target

doi: 10.1002/smmd.70029

Figure Lengend Snippet: LINC00973 is upregulated in NSCLC and related to poor prognosis. (A) Heatmap showing the expression profiles of differentially expressed lncRNAs based on RNA‐sequencing data from paired NSCLC tumor tissues and adjacent normal tissues. (B) Volcano plot illustrating the distribution of differentially expressed lncRNAs. (C) qRT‐PCR analysis of LINC00973 expression in NSCLC cell lines (A549, H1299, and PC9) and the normal HBE cell line. (D) qRT‐PCR analysis of LINC00973 expression in paired tumor and adjacent normal tissues from 61 NSCLC patients. (E) Differential expression of LINC00973 in tumor and normal tissues of NSCLC patients based on TCGA data. (F) Kaplan–Meier survival analysis showing the association between LINC00973 expression levels and overall survival in NSCLC patients. Statistical significance was assessed using the log‐rank test. Data are presented as mean ± SD. * p < 0.05; *** p < 0.001.

Article Snippet: Human NSCLC cell lines A549, obtained from Co‐bioer Biosciences (Nanjing, China) and NCI‐H1299, sourced from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Quantitative Proteomics

Journal: Smart Medicine

Article Title: LINC00973/DTX3L Axis Promotes Non‐Small Cell Lung Cancer Progression and Serves as a Therapeutic Target

doi: 10.1002/smmd.70029

Figure Lengend Snippet: LINC00973 interacts with DTX3L protein and regulates the AKT pathway in NSCLC cells. (A) Schematic representation of the TRAP experiment. (B and C) Silver staining (B) and Western blot analysis (C) confirm GST expression in TRAP‐processed samples. (D) qRT‐PCR analysis of LINC00973 enrichment in TRAP‐pulled‐down products. (E) TRAP followed by Western blot analysis validating the interaction between LINC00973 and the DTX3L protein. (F) RIP assays showing the association of LINC00973 with DTX3L and PARP9 proteins. (G and H) Subcellular localization and co‐localization of LINC00973 and DTX3L in NSCLC cells assessed by RNA‐FISH combined with immunofluorescence (G) and subcellular fractionation analysis (H). (I and J) Heatmap (I) and volcano plot (J) displaying differentially expressed genes between control and LINC00973‐depleted A549 cells. (K) Pathway enrichment analysis of differentially expressed genes following LINC00973 knockdown. (L) Representative downregulated genes identified by RNA‐sequencing in A549 cells after LINC00973 depletion. (M and N) qRT‐PCR validation of selected differentially expressed genes in NSCLC cells following LINC00973 modulation and DTX3L‐related analyses. (O and P) qRT‐PCR analysis of differentially expressed genes in NSCLC cells after DTX3L silencing. (Q) Western blot analysis of AKT pathway‐related proteins in NSCLC cells with LINC00973 knockdown or overexpression ( n = 3, ** p < 0.01; *** p < 0.001).

Article Snippet: Human NSCLC cell lines A549, obtained from Co‐bioer Biosciences (Nanjing, China) and NCI‐H1299, sourced from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China).

Techniques: Silver Staining, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Fractionation, Control, Knockdown, RNA Sequencing, Biomarker Discovery, Over Expression

Journal: Smart Medicine

Article Title: LINC00973/DTX3L Axis Promotes Non‐Small Cell Lung Cancer Progression and Serves as a Therapeutic Target

doi: 10.1002/smmd.70029

Figure Lengend Snippet: Engineered exosomes for targeted delivery of LINC00973 siRNA suppress NSCLC progression. (A and B) When examining exosomes derived from HEK293T cells, TEM (A) and NTA (B) were employed for comprehensive characterization. (C) Western blot analysis of exosomal marker proteins. (D) qRT‐PCR analysis of LINC00973 expression in NSCLC cells treated with exosome‐delivered siRNA. (E) Western blot analysis of DTX3L expression and AKT pathway activation in exosome‐treated NSCLC cells. (F and G) Uptake of DiI‐labeled exosomes by NSCLC cells examined by confocal microscopy (F) and flow cytometry (G). (H) qRT‐PCR analysis of LINC00973 expression in A549 cells treated with RGD‐293T‐EX‐si‐LINC00973. (I) The in vivo distribution of engineered exosomes in mice. (J) In vivo imaging of organs and tumors in mice 24–72 h after tail vein injection of engineered exosomes. (K) Observations on tumor images across various experimental groups. (L) qRT‐PCR analysis of LINC00973 expression in tumor tissues from different experimental groups. (M) Western blot analysis of DTX3L and p ‐AKT protein levels in tumor tissues. (N) Representative images of TUNEL‐positive cells in tumor tissues from each experimental group. (O) IHC staining of indicated proteins in tumor tissues from different groups. Data are shown as means ± SD (** p < 0.01; *** p < 0.001; ns, not significant).

Article Snippet: Human NSCLC cell lines A549, obtained from Co‐bioer Biosciences (Nanjing, China) and NCI‐H1299, sourced from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China).

Techniques: Derivative Assay, Western Blot, Marker, Quantitative RT-PCR, Expressing, Activation Assay, Labeling, Confocal Microscopy, Flow Cytometry, In Vivo, In Vivo Imaging, Injection, TUNEL Assay, Immunohistochemistry

Journal: Smart Medicine

Article Title: LINC00973/DTX3L Axis Promotes Non‐Small Cell Lung Cancer Progression and Serves as a Therapeutic Target

doi: 10.1002/smmd.70029

Figure Lengend Snippet: In vivo anti‐LINC00973 siRNAs within self‐assembled exosomes suppress NSCLC progression. (A) Schematic illustration of the in vivo assembly and delivery of anti‐LINC00973 siRNA‐loaded exosomes. (B) The relative expression level of LINC00973 after co‐incubation of exosomes with A549 cells. (C) Absolute expression level of anti‐LINC00973 siRNA in exosomes. (D) Absolute quantitative analysis of anti‐LINC00973 siRNA levels in serum exosomes from mice. (E) Following a single intravenous dose (5 mg/kg) of anti‐LINC00973 construct, the distribution kinetics of its siRNA was analyzed across major mouse organs. (F) Schematic overview of the exosome‐based antitumor therapeutic strategy. (G) Images of tumors from various experimental groups of mouse models, as indicated. (H) qRT‐PCR analysis of LINC00973 expression levels in tumor tissues from different experimental groups. (I) Protein levels of DTX3L and p ‐AKT in tumor tissues were assessed with Western blot analysis. (J) Representative images of TUNEL staining of tumors in each group. (K) IHC staining showing the expression of indicated proteins in tumor tissues across different experimental groups. Data are shown as means ± SD (** p < 0.01; *** p < 0.001).

Article Snippet: Human NSCLC cell lines A549, obtained from Co‐bioer Biosciences (Nanjing, China) and NCI‐H1299, sourced from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China).

Techniques: In Vivo, Expressing, Incubation, Construct, Quantitative RT-PCR, Western Blot, TUNEL Assay, Staining, Immunohistochemistry