Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib enhances the cytotoxicity of PBMCs in EGFR-mutated NSCLC cells, but not in NSCLC cells with wild-type EGFR. Cell lysis measured by Calcein staining in four NSCLC lines: (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC-9. NSCLC cells were cultured with PBMCs at different E/T ratios. * P<0.05 vs. control. E/T, effector/target; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Lysis, Staining, Cell Culture, Control

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Expression of B7H5 following treatment with Gefitinib in NSCLC cells. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 expression after treatment with gefitinib in NSCLC cells (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC-9. (E) Immunofluorescence determined B7H5 expression (green colour) following treatment with or without gefitinib in NSCLC cells (magnification, ×200). ** P<0.01 vs. control. NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Control

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Changes in B7H5 expression in mutant and wild-type NSCLC cell lines after EGFR siRNA interference. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 and EGFR expression following transfection with an EGFR siRNA in NSCLC cells. (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC9. (E) Immunofluorescence analysis B7H5 (green colour) and EGFR (red colour) expression following transfection with EGFR siRNA in NSCLC cells (magnification, ×200). ** P<0.01, *** P<0.001 vs. negative control. siRNA, small interfering RNA; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Mutagenesis, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Immunofluorescence, Negative Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib treatment increases B7H5 expression in wild-type NSCLC cell lines expressing EGFR mutants. The levels of EGFR and B7H5 as determined by western blot analysis in (A) NCI-H1299 and (B) NCI-H358 cells. (C) Immunofluorescence analysis of B7H5 expression (green colour) after transfection with the EGFR mutation plasmid or the EGFR mutation plasmid combined with gefitinib in wild-type EGFR NSCLC cells (magnification, ×200). ** P<0.01, *** P<0.001. NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, Mutagenesis, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: B7H5 affects the killing effect of gefitinib combined with PBMCs in EGFR-mutated NSCLC cells. (A and B) Cell Counting Kit-8 assay and cytotoxicity test models of mixed cell culture to determine the cell viability and killing effect of PBMCs after treatment with or without B7H5 siRNA in (A) NCI-H358 and (B) NCI-H1299 cells. (C) Expression of B7H5 in NCI-H358 and NCI-H1299 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200; B7H5 expression: Green colour). (D and E) The mixed cell culture toxicity test model was used to test the efficacy of gefitinib against EGFR-mutated NSCLC cells prior to and after B7H5 siRNA interference in (D) HCC827 and (E) PC-9 cells. (F) Expression of B7H5 in HCC827 and PC-9 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200). (G) Killing effect of gefitinib combined with PBMCs with and without transfection with B7H5 siRNA on EGFR-mutated NSCLC cells in HCC827 and PC-9 cells. * P<0.05 vs. Negative control (A-E); * P<0.05 vs. control (G). siRNA, small interfering RNA; E/T, effector/target; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Cell Counting, Cell Culture, Expressing, Transfection, Immunofluorescence, Negative Control, Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib affects PBMC-mediated cytolysis in cells transfected with EGFR mutants and wild-type EGFR. (A) The killing effect of gefitinib combined with PBMCs was observed with and without transfection of the EGFR mutation plasmid. (B and C) Western blot analysis detected EGFR levels and the killing effect of PBMCs after transfection with or without the EGFR mutation plasmid in wild-type EGFR NSCLC (B) NCI-H358 and (C) NCI-H1299 cells. (D) Immunofluorescence analysis of EGFR expression (red colour) after transfection with or without the EGFR mutation plasmid in wild-type EGFR NSCLC cells (magnification, ×200). * P<0.05 vs. control. siRNA, small interfering RNA; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer; E/T, effector/target.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Interference with CD28 expression markedly decreases the killing activity against EGFR-mutant NSCLC cells. (A) CD28 expression was determined by western blot analysis and flow cytometry. (B) Mixed cell culture toxicity assay to determine the cytotoxic activity after transfection with or without CD28H siRNA; (C) combined efficacy of PBMCs and gefitinib, (D) combined efficacy of PBMCs and gefitinib with and without CD28H siRNA interference in EGFR-mutant NSCLC cells HCC827. (E) Mixed cell culture toxicity assay to determine the cytotoxic activity after transfection with or without CD28H siRNA; (F) combined efficacy of PBMCs and gefitinib, (G) combined efficacy of PBMCs and gefitinib with and without CD28H siRNA interference in EGFR-mutant NSCLC cells PC-9. * P<0.05 vs. Negative control. siRNA, small interfering RNA; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer; E/T, effector/target; NC, negative control.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Activity Assay, Mutagenesis, Western Blot, Flow Cytometry, Cell Culture, Transfection, Negative Control, Small Interfering RNA

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) was highly expressed in non‐small cell lung cancer (NSCLC) tissues and cells. (a) USP22 expression in NSCLC tissues was evaluated by immunohistochemistry (IHC) assay. (b, c) USP22 mRNA and protein levels in NSCLC tissues and adjacent normal tissues were measured by qRT‐PCR and western blot assays. (d, e) The mRNA and protein levels of USP22 in 16HBE, PC9, and HCC827 cells were determined by qRT‐PCR assay and western blot assay, respectively. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Deficiency of ubiquitin‐specific peptidase 22 (USP22) repressed non‐small cell lung cancer (NSCLC) cell migration and aggravated apoptosis and ferroptosis. (a) The protein level of USP22 in sh‐NC or sh‐USP22‐transfected PC9 and HCC827 cells was measured by western blot. (b) The migration of PC9 and HCC827 cells with sh‐NC or sh‐USP22 transfection was evaluated by transwell assay. (c) The apoptosis of PC9 and HCC827 cells transfected with sh‐NC or sh‐USP22 was analyzed by flow cytometry analysis. (d–g) The levels of reactive oxygen species (ROS), MDA, Fe 2+ , and GSH in sh‐NC or sh‐USP22 transfected PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Migration, Transfection, Western Blot, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ferroptosis promoted the effect of gefitinib on non‐small cell lung cancer (NSCLC) cells. (a) The migration ability of PC9 and HCC827 cells treated with DMSO or 2.5‐μM gefitinib was assessed by transwell assay. (b) The apoptosis of DMSO or 2.5‐μM gefitinib‐treated PC9 and HCC827 cells was analyzed by flow cytometry analysis. (c, d) After PC9 and HCC827 cells were treated with DMSO, gefitinib, Fer‐1, or gefitinib+Fer‐1, the oxidized C11‐BODIPY fluorescence intensity was examined by C11‐BODIPY staining. (e–g) After PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, Fer‐1, or gefitinib+Fer‐1, the levels of MDA, Fe 2+ , and glutathione (GSH) were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) overexpression promoted gefitinib resistance and repressed ferroptosis in gefitinib‐treated non‐small cell lung cancer (NSCLC) cells. (a) USP22 protein level in gefitinib or dimethyl sulfoxide (DMSO)‐treated PC9 and HCC827 cells was measured by western blot. (b) The effect of USP22 on gefitinib resistance was assessed by CCK‐8 assay. (c–i) PC9 and HCC827 cells were treated with DMSO+pcDNA, gefitinib+pcDNA, or gefitinib+USP22. (c, d) The migration and apoptosis of PC9 and HCC827 cells were evaluated by transwell assay and flow cytometry analysis, respectively. (e, f) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (g–i) The levels of MDA, Fe 2+ , and glutathione (GSH )in PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Over Expression, Western Blot, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) stabilized murine double minute 2 (MDM2) and modulated MDM2 expression. (a) Ubibrowser software predicted the relation between USP22 and MDM2. (b) Pull‐down assay was conducted to analyze the level of MDM2 ubiquitination. (c) Co‐IP assay was performed to explore the interaction between USP22 and MDM2. (d) The expression of MDM2 in non‐small cell lung cancer (NSCLC) tissues and normal tissues was estimated through IHC assay. (e, f) The mRNA and protein levels of MDM2 in NSCLC tissues and normal tissues were measured by qRT‐PCR and western blot, respectively. (g, h) The mRNA and protein levels of MDM2 in 16HBE, PC9, and HCC827 cells were examined by qRT‐PCR and western blot, respectively. (i) After PC9 and HCC827 cells were treated with DMSO, gefitinib, gefitinib+USP22, or gefitinib+USP22 + sh‐MDM2, MDM2 protein level was measured by western blot. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Expressing, Software, Pull Down Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Murine double minute 2 (MDM2) knockdown reversed the effects of Ubiquitin‐specific peptidase 22 (USP22) overexpression on gefitinib sensitivity and ferroptosis in gefitinib‐treated NSCLC cells. (a) IC50 of gefitinib in PC9 and HCC827 cells transfected with pcDNA, USP22, or USP22 + sh‐MDM2 was estimated through CCK‐8 assay. (b–h) PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, gefitinib+USP22, gefitinib+USP22 + sh‐NC, or gefitinib+USP22 + sh‐MDM2. (b, c) The migration and apoptosis of PC9 and HCC827 cells were explored by transwell assay and flow cytometry analysis. (d, e) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (f–h) The levels of MDA, Fe 2+ , and glutathione (GSH) in PC9 and HCC827 cells were determined with commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Knockdown, Over Expression, Transfection, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) enhanced gefitinib resistance of non‐small cell lung cancer (NSCLC) cells in vivo. (a) The protein levels of USP22 and murine double minute 2 (MDM2) in PBS, gefitinib, and gefitinib+USP22 treated PC9 cells were measured by western blot. (b) Xenograft tumor volume was monitored every 5 days. (c) Xenograft tumor weight was examined after 25 days. (d) The expression of USP22 and MDM2 in xenograft tumors was examined through immunohistochemistry (IHC) assay. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: In Vivo, Western Blot, Expressing, Immunohistochemistry

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que suppressed the proliferation potential of HCC827 cells. For MTT assays, cells were incubated with Que of 25 μM (Que L), 50 μM (Que M), and 100 μM (Que H) for 96 hours. Every 24 hours, cells were subjected to the testing. For colony formation assays, cells were cultured in medium containing 100 μM Que for 2 weeks. ( A ) Detection results of MTT assays. ( B ) Detection results of colony formation assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Incubation, Cell Culture, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que inhibited the invasion and migration potentials, and the signaling transduction of Src/Fn14/NF-κB pathway in HCC827 cells. For scratch assays, cells were incubated with Que of 100 μM (Que H) for 48 hours. For Transwell assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. For western blotting assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. ( A ) Detection results of scratch assays. ( B ) Detection results of Transwell assays. ( C ) Detection results of western blotting assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Migration, Transduction, Incubation, Western Blot, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Overexpression of Src blocked the anti-NSCLC function of Que in HCC827 cells. Cells transfected with negative control vector (NC) or Src expression vector (Src) were treated with Que of 100 μM (Que H) and subjected to MTT assay for 96 hours (cells were collected every 24 hours), and colony formation was assay at 2 weeks, Transwell assay at 24 hours, and scratch assay at 48 hours. ( A ) Detection results of Src level. ( B ) Detection results of MTT assays. ( C ) Detection results of colony formation assay. ( D ) Detection results of Transwell assay. ( E ) Detection results of scratch assay. # P <0.05 versus Que H group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, Expressing, MTT Assay, Transwell Assay, Wound Healing Assay, Colony Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Que inhibited the growth and metastasis potential of NSCLC in vivo in by inhibiting Src signaling. Mice were injected with different HCC827 cells and administrated with Que of 100 mg/kg body weight for 3 weeks. ( A ) Detection results of tumor volume. ( B ) Detection results of hematoxylin and eosin detection of tumor tissue. ( C ) Detection results of western blotting detection of E-cadherin and N-cadherin. * P <0.05 versus Que H+Src group. Scale bar, 100 μm.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: In Vivo, Injection, Western Blot

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: RNF125 inhibits the proliferation and colony formation of LUAD cells. (A) RT-qPCR shows RNF125 expression in six LC cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437). RT-qPCR and western blotting demonstrate plasmid-mediated RNF125 (B) knockdown or (C) overexpression in NCI-H1395 and HCC-827 cells. MTT assays show the effect of RNF125 (D) knockdown or (E) overexpression on the proliferation of NCI-H1395 and HCC-827 cells. Colony formation analysis shows the effect of RNF125 knockdown or overexpression on colony formation of (F) NCI-H1395 and (G) HCC-827 cells. Data are presented as mean ± SD. **P<0.01 vs. shNC or vector. LUAD, lung adenocarcinoma; RT-qPCR, reverse transcription-quantitative PCR; RNF125, ring finger protein 125; sh, short hairpin RNA; NC, negative control; OE, overexpression; OD, optical density.

Article Snippet: LUAD cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437), NK-92 and Jurkat T cells were purchased from iCell Bioscience, Inc. NCI-H1975 (cat. no. iCell-h156), NCI-H2228 (cat. no. iCell-h351), NCI-H1395 (cat. no. iCell-h154), HCC-827 (cat. no. iCell-h068), NCI-H1437 (cat. no. iCell-h284) and Jurkat T (cat. no. iCell-h117) cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd.).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: Knockdown of RNF125 enhances immune evasion in LUAD. (A) The PPI network of RNF125 interactors was constructed using the GeneMANIA database. (B) Gene Ontology Biological Process and Reactome pathway enrichment analysis of RNF125 interactors was performed using the DAVID database. (C) Co-IP assays show the interactions between RNF125 and PD-L1 in NCI-H1395 and HCC-827 cells. (D) Western blotting determines that knockdown of RNF125 reduces PD-L1 protein expression levels in NCI-H1395 and HCC-827 cells. (E) Correlation analysis between RNF125 expression and immune cell infiltration in LUAD was performed using the TIMER2.0 database. (F) Co-culture of T cells and RNF125-silenced cancer cells decreased IL-2 secretion from T cells. **P<0.01 vs. shNC. (G) Co-culture of NK cells and RNF125-silenced cancer cells attenuated NK cell-mediated lysis of NCI-H1395 and HCC-827 cells. **P<0.01 vs. sh1-RNF125; †† P<0.01 vs. sh2-RNF125. Data are presented as mean ± SD. PPI, protein-protein interaction; RNF125, ring finger protein 125; PD-L1, programmed cell death 1 ligand 1; LUAD, lung adenocarcinoma; sh, short hairpin RNA; NC, negative control; NK, natural killer; TPM, transcripts per million.

Article Snippet: LUAD cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437), NK-92 and Jurkat T cells were purchased from iCell Bioscience, Inc. NCI-H1975 (cat. no. iCell-h156), NCI-H2228 (cat. no. iCell-h351), NCI-H1395 (cat. no. iCell-h154), HCC-827 (cat. no. iCell-h068), NCI-H1437 (cat. no. iCell-h284) and Jurkat T (cat. no. iCell-h117) cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd.).

Techniques: Knockdown, Construct, Co-Immunoprecipitation Assay, Western Blot, Expressing, Co-Culture Assay, Lysis, shRNA, Negative Control

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: MBNL1 is an upstream modulator of RNF125 in LUAD. (A) Potential RNF125-binding RBPs were predicted using two RBP databases (RBPDB and RBPmap) and overlapped with RBPs involved in lung cancer progression. (B) Correlation analysis between RNF125 and the indicated RBPs in LUAD was performed using the TIMER2.0 portal. RT-qPCR and western blotting show MBNL1 and RNF125 expression in NCI-H1395 cells 48 h after transfection of (C) MBNL1-specific siRNAs or (D) plasmids containing MBNL1 coding sequences (MBNL1 OE). **P<0.01 vs. siNC or empty vector. (E) RIP-PCR and agarose gel electrophoresis showed that RNF125 transcripts were detected in the MBNL1 antibody RIP products from NCI-H1395 cells. After treatment with actinomycin D, RT-qPCR analysis showed the % of remaining RNF125 mRNA relative to 0 h at the indicated time points in (F) MBNL1-silenced (**P<0.01 vs. si1-MBNL1; † P<0.05, †† P<0.01 vs. si2-MBNL1) or (G) MBNL1-overexpressing NCI-H1395 cells (**P<0.01 vs. empty vector). (H) MTT assays determine the viability of NCI-H1395 cells 48 h after co-transfection of MBNL1 OE and shRNF125 plasmids. (I) Representative images (right) of Transwell assays in the presence of Matrigel and the mean number of invaded cells (left) are shown. Scale bar, 100 µm. **P<0.01. Data are presented as mean ± SD. MBNL1, muscleblind-like 1; RNF125, ring finger protein 125; LUAD, lung adenocarcinoma; RBP, receptor binding protein; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; OE, overexpression; NC, negative control; sh, short hairpin RNA; TPM, transcripts per million.

Article Snippet: LUAD cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437), NK-92 and Jurkat T cells were purchased from iCell Bioscience, Inc. NCI-H1975 (cat. no. iCell-h156), NCI-H2228 (cat. no. iCell-h351), NCI-H1395 (cat. no. iCell-h154), HCC-827 (cat. no. iCell-h068), NCI-H1437 (cat. no. iCell-h284) and Jurkat T (cat. no. iCell-h117) cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd.).

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Cotransfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Over Expression, Negative Control, shRNA

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: Schematic diagram of the function and molecular regulatory mechanism of RNF125 in LC. RNF125 is downregulated in lung cancer, and its low expression is associated with advanced-stage disease. RNF125 inhibits the LUAD cell growth and invasiveness and enhances the chemosensitivity of LUAD cells to cisplatin. RNF125 acts as an E3 ubiquitin ligase of PD-L1. Knockdown of RNF125 suppresses PD-L1 degradation, thereby impairing Tcell activation and antitumor cytokine secretion. Mechanistically, the RBP MBNL1 serves as an upstream regulator of RNF125 by stabilizing RNF125 mRNA. The figure contains elements from Servier Medical Art ( https://smart.servier.com/ ) under a Creative Commons 3.0 license. MBNL1, muscleblind-like 1; RNF125, ring finger protein 125; PD-L1, programmed cell death 1 ligand 1; LUAD, lung adenocarcinoma.

Article Snippet: LUAD cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437), NK-92 and Jurkat T cells were purchased from iCell Bioscience, Inc. NCI-H1975 (cat. no. iCell-h156), NCI-H2228 (cat. no. iCell-h351), NCI-H1395 (cat. no. iCell-h154), HCC-827 (cat. no. iCell-h068), NCI-H1437 (cat. no. iCell-h284) and Jurkat T (cat. no. iCell-h117) cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd.).

Techniques: Expressing, Ubiquitin Proteomics, Knockdown, Activation Assay