Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: CLCN3 was upregulated in human LUAD and facilitated tumor proliferation and migration. (a, b) Through IHC and IF analysis, the expression of CLCN3 was examined in a tissue microarray of 30 paraffin-embedded LUAD tissues and adjacent normal tissues (ANT) ( n = 30). (c, d) In human LUAD cell lines as well as in human bronchial epithelial cell lines, the basic protein expression of CLCN3 was measured ( n = 3). (e) The basic RNA level of CLCN3 was detected in human LUAD cell lines and a human bronchial epithelial cell line ( n = 3). (f) The protein expression of CLCN3 was inhibited after CLCN3 knockdown in H1299 and A549 cells. (g) RNA-seq was constructed after CLCN3 knockdown in H1299 cells. Locomotion and growth were significantly enriched as illustrated by GO analysis. (h) Knockdown of CLCN3 suppressed the clonogenicity of H1299 and A549 cells ( n = 3). (i) CLCN3 knockdown suppressed the invasion of H1299 and A549 cells ( n = 3). * P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Migration, Expressing, Microarray, RNA Sequencing Assay, Construct

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK was identified and validated as a CLCN3 promoter-binding transcription factor. (a) To identify putative transcription factors that bind to the CLCN3 promoter, nuclear protein extracts were subjected to incubation with a biotin-labeled DNA probe that included the promoter region. Following the SDS-PAGE separation and silver staining, a protein band with differential expression was identified after which it was excised and subjected to mass spectrometry (MS) analysis. (b) By employing either a synthesized probe or a nonspecific probe (NSP) in the nuclear protein/DNA complex, western blot analysis detected binding between HNRNPK and the CLCN3 promoter. (c) ChIP assays were performed and an HNRNPK antibody was used to immunoprecipitate the fragmented DNA in H1299 cells. The HNRNPK protein band was substantially enriched in the IP group in contrast to the IgG control. (d) The ChIP-seq data showed that the recruitment of HNRNPK was mostly enriched in the upstream and downstream TSS of the CLCN3 promoter region (NM_001829, chr4: 169620033-169620797, region -538/+226 bp). (e) The CLCN3 RNA level was inhibited following HNRNPK knockdown ( n = 3). (f) HNRNPK knockdown disrupted the promoter activities of the pGL4.10-CLCN3-538 reporter plasmid (region -538/+226 bp) in H1299 cells, and the binding motifs 'GCGAGG/CTAATG' from ChIP-seq data were enriched in this region ( n = 3). (g) GO analysis revealed that cell proliferation and migration were the biological functions of HNRNPK. (h) KEGG pathway analysis illustrated that cell growth and death, cell motility, transcription, translation, and other signaling pathways were mainly enriched after HNRNPK knockdown. (i) The luciferase activity was considerably reduced in HNRNPK knockdown cells transfected with a pGL4.10-CLCN3-538 plasmid (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Binding Assay, Incubation, Labeling, SDS Page, Silver Staining, Expressing, Mass Spectrometry, Synthesized, Western Blot, ChIP-sequencing, Plasmid Preparation, Migration, Luciferase, Activity Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK/CLCN3 axis facilitated LUAD progression through interaction between tumor cells and CAFs. (a) Primary human CAFs and paired NFs were extracted from fresh LUAD samples. The expression of CAF markers was increased in CAFs compared to NFs, which was in accord with the typical characteristic of CAFs. (b) The levels of CLCN3 were detected in the culture supernatants of LUAD cells, and the data indicated that decreased extracellular CLCN3 secretion could be induced by HNRNPK knockdown. (c-e) The supernatants (HNRNPK knockdown and control) of LUAD cells were incubated with CAFs for 24 h. We confirmed that the PI3K-AKT signaling pathway was significantly enriched in CAFs. (f) The levels of p-AKT, α-SMA, FAP, and COL1A1 were decreased when CAFs were stimulated with the supernatants of HNRNPK-knockdown cells. After CLCN3 knockdown in CAFs, the levels of p-AKT, α-SMA, FAP, and COL1A1 were also effectively inhibited. (g) Due to the activation inhibition, the CAFs co-cultured with HNRNPK-knockdown LUAD cells were then considered as inhibited CAFs. The analysis by ELISA revealed that the TGF-β1 production of inhibited CAFs was significantly decreased ( n = 7). (h) The supernatants of inhibited CAFs attenuated the expression of HNRNPK protein in the H1299 nucleus, and the attenuation effect was reversed after TGF-β1 treatment. (i) The supernatants of inhibited CAFs attenuated the fluorescence intensity of HNRNPK in the H1299 nucleus, which was also reversed after the addition of exogenous TGF-β1. (j, k) The supernatants of inhibited CAFs markedly attenuated the clonogenicity and invasion of LUAD cells, and this phenomenon was further reversed after the addition of TGF-β1 ( n = 3). *P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Expressing, Incubation, Activation Assay, Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK regulated the expression and function of CLCN3 in vivo . (a) In mouse xenograft models, stable HNRNPK-knockdown cells (H1299) and rescue model cells were administered to the nude mice via subcutaneous injection into their left flank. The representative figure of tumors formed in each group is shown ( n = 7). (b, c) The tumor weight and tumor growth curve were suppressed when HNRNPK was knocked down, and this inhibition was reversed by CLCN3 overexpression ( n = 7). (d) In tumor xenografts, the expression of CLCN3 was decreased following HNRNPK knockdown, however, this reduction was reversed by CLCN3 overexpression ( n = 7). (e) Nude mice received an injection with stable HNRNPK-knockdown cells (H1299) and rescue model cells into their tail veins. HNRNPK knockdown effectively decreased the average radiance of lung metastatic lesions, and the reduction was rescued by CLCN3 overexpression ( n = 7). (f) In paraffin sections of nude mouse lung metastases, we found that HNRNPK knockdown caused a reduction of lung metastasis area, which was rescued by CLCN3 overexpression ( n = 7). * P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Expressing, In Vivo, Injection, Inhibition, Over Expression

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: A schematic representation of the relationship between CLCN3 and HNRNPK in LUAD progression. Firstly, CLCN3 was upregulated in human LUAD and facilitated tumor proliferation and migration. Secondly, HNRNPK was validated as a CLCN3 promoter-binding transcription factor, and the binding motif 'GCGAGG' and binding site '-538/-248 bp' were identified. Finally, the expression and function of CLCN3 were regulated by HNRNPK in vitro and in vivo , and the HNRNPK-CLCN3 axis facilitated LUAD progression in a feedback way through CAF-tumor interaction in the TME.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Migration, Binding Assay, Expressing, In Vitro, In Vivo

Journal: Oncology Letters

Article Title: CPSF3 is a promising prognostic biomarker and predicts recurrence of non-small cell lung cancer

doi: 10.3892/ol.2019.10659

Figure Lengend Snippet: Correlation of CPSF3 mRNA expression, and its DNA methylation and DNA copy number variants in TCGA-LUAD and in Cancer Cell Line Encyclopedia-LUAD cell lines. Correlation analysis revealed that (A) CPSF3 mRNA expression was not significantly correlated with DNA methylation at CpG site cg12057242, while (B) it was negatively correlated with its DNA methylation at CpG site cg25739938 in TCGA-LUAD. (C) Correlation analysis indicated that CPSF3 mRNA expression had no association with its DNA methylation in 53 LUAD cell lines. (D) Correlation analysis indicated that CPSF3 mRNA expression was positively correlated with its DNA copy number variants in TCGA-LUAD. (E) CPSF3 was positively correlated with its DNA copy number variants in LUAD cell lines. TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; CPSF3, cleavage polyadenylation specific factor 3.

Article Snippet: LUAD cell line data (based on The Global Bioresource Center; http://www.atcc.org ) were downloaded to examine the association between CPSF3 expression and its DNA methylation.

Techniques: Expressing, DNA Methylation Assay

Journal: Frontiers in Immunology

Article Title: Targeting FCRLA to induce necrosis in lung adenocarcinoma: a novel strategy for prognosis and therapy via MPT-Driven pathways

doi: 10.3389/fimmu.2025.1596179

Figure Lengend Snippet: Candidate genes were selected based on DEG and MPTDNRG feature modules. (A) Univariate Cox regression analysis was performed for the 82 candidate genes. With HR ≠ 1 and a P < 0.05 as the criteria, 25 genes were screened, and the results are shown in a forest plot. (B) Least absolute shrinkage and selection operator regression analysis was performed on 19 genes screened using the univariate Cox + PH assumption test. (C) Expression of three prognostic genes in lung adenocarcinoma (LUAD) and control samples.

Article Snippet: Human LUAD cell lines A549 and NCI-H1975 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Selection, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Targeting FCRLA to induce necrosis in lung adenocarcinoma: a novel strategy for prognosis and therapy via MPT-Driven pathways

doi: 10.3389/fimmu.2025.1596179

Figure Lengend Snippet: FCRLA knockdown inhibits the proliferation ability of LUAD cells. (A) The relative expression levels of FCRLA , CD79A , and RASGRP2 mRNA in BEAS, A549, and NCI-H1975 cells were detected using RT-qPCR. (B) Changes in the relative expression levels of FCRLA mRNA 48 h after transfection with siRNA-1, 2, 3 interference sequences in A549 and NCI-H1975 cells were detected using RT-qPCR. (C) Relative FCRLA protein expression levels in A549 and NCI-H1975 cells 48 h after transfection with siRNA-1, 2, 3 interference sequences were detected using western blotting. (D) Changes in the proliferation ability of A549 and NCI-H1975 cells after different treatments were detected by EDU staining and CCK8 experiments (200×, scale bar: 100 μm). Compared between the two groups, *P < 0.05, ***P < 0.001, ****P < 0.0001, ns represents no statistical significance between the two groups.

Article Snippet: Human LUAD cell lines A549 and NCI-H1975 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: Targeting FCRLA to induce necrosis in lung adenocarcinoma: a novel strategy for prognosis and therapy via MPT-Driven pathways

doi: 10.3389/fimmu.2025.1596179

Figure Lengend Snippet: FCRLA knockdown induces necrosis in LUAD cells. (A) Changes in cell necrosis after treatment with each group of LUAD cells were detected using flow cytometry with FITC/PI double staining. (B) Changes in cell necrosis after treatment of each group in LUAD cells were detected using PI/Hoechst33342 cell viability staining (100×, Scale bar: 50 μm). Compared between the two groups, ****P < 0.0001, ns represents no statistical significance between the two groups.

Article Snippet: Human LUAD cell lines A549 and NCI-H1975 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Flow Cytometry, Double Staining, Staining

Journal: Frontiers in Immunology

Article Title: Targeting FCRLA to induce necrosis in lung adenocarcinoma: a novel strategy for prognosis and therapy via MPT-Driven pathways

doi: 10.3389/fimmu.2025.1596179

Figure Lengend Snippet: FCRLA knockdown leads to mitochondrial damage in lung cancer cells. (A) The changes in mitochondrial structure and morphology after treatment of each group in LUAD cells were observed using electron microscopy (10000×, scale bar: 1 μm). (B) The changes in mitochondrial membrane potential in A549 and NCI-H1975 lung cancer cells were detected using the JC-1 fluorescent probe (200×, Scale bar: 50 μm). (C) After FCRLA knockdown in A549 and NCI-H1975 cells, the cells were treated with cyclosporin A for 24 h, and the relative expression level changes of CypD protein were detected using western blotting. Compared between the two groups, **P < 0.001, ***P < 0.001, ****P < 0.0001.

Article Snippet: Human LUAD cell lines A549 and NCI-H1975 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Electron Microscopy, Membrane, Expressing, Western Blot

Journal: Scientific Reports

Article Title: TRIM29 promotes epithelial–mesenchymal transition, angiogenesis, and stromal remodeling in lung adenocarcinoma: integrated validation at histologic, transcriptomic, and protein levels

doi: 10.1038/s41598-026-45469-2

Figure Lengend Snippet: TRIM29 function in the EMT pathway and TRIM29–drug sensitivity in DepMap LUAD. TRIM29 knockdown versus control in NCI-H441 and NCI-H650 cells ( A ). Wound healing assay according to TRIM29 knockdown in NCI-H441 and NCI-H650 cells ( B ). Heatmap of compound–cell line responses in the PRISM dataset ( C ). Bar chart showing the mechanism-of-action frequency among the top 50 compounds ( D ). EMT, epithelial–mesenchymal transition; DepMap, Cancer Dependency Map; PRISM, Profiling Relative Inhibition Simultaneously in Mixtures; MOA, mechanism of action

Article Snippet: Human LUAD lines (NCI-H441 and NCI-H650; Korean Cell Line Bank) were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO2.

Techniques: Knockdown, Control, Wound Healing Assay, Inhibition