Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The relative miR-221 and AEG-1 mRNA levels in HCC cell lines. The miR-221 ( A ) and AEG-1 ( B ) relative mRNA levels analyzed in HCC cell line panels, the relative miR-221 expression in miR-221 mimic- ( C ), miR-221 inhibitor- ( D ), and AEG-1 siRNA- ( E ) transfected HCC cells. The relative mRNA levels of AEG-1 in miR-221 mimic/inhibitor- ( F ) and AEG-1 siRNA-transfected groups in HCC cells ( G ). The RNU6 and GAPDH were used as internal controls. Error bars presented as mean ± s.d and p -values represented as *** p < 0.001, **** p < 0.0001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1 and miR-221 regulate angiogenesis and cell cycle regulatory mRNA expressions in the HCC cell lines. The regulatory mRNAs expressions which regulate angiogenesis (LSF and MMP9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HepG2, Huh7, and Hep3B cells by using qRT- PCR. The GAPDH was used as an internal control. Error bars presented as mean ± s.d and p -values represented as * p < 0.05, ** p < 0.01, and *** p < 0.001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1/miR-221 regulates the apoptosis and autophagy regulatory mRNAs in the HCC cell lines. The apoptosis and autophagy regulatory mRNAs (OPN, Bcl-2, PTEN, LC3A, PI3K, and Akt) expressions were analyzed in miR-221 mimic-, miR-221 inhibitor, and AEG-1 siRNA-transfected HCC cells by using qRT-PCR, and GAPDH used as an internal control. Error bars presented as mean ± s.d. and p -value represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Silencing of AEG-1 and miR-221 inhibits HCC cell migration and proliferation in vitro. The effect of the miR-221/AEG-1 measured on HCC cell migration and cell proliferation in the miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their control-transfected HepG2, Huh7, and Hep3B cells by invasion ( A ), migration ( B ), and MTT assay ( C ) in vitro. Images analyzed using Image J (NIH) (scale bar, 100 µm). Error bars presented as mean ± s.d and p -value represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Migration, In Vitro, Control, Transfection, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effects of AEG-1/miR-221 on HCC cell cycle regulation and apoptosis. The effects of AEG-1/miR-221 on apoptosis ( A ) and cell cycle ( B ) analyzed in the miR-221 mimic-, and miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HCC cells in vitro by flow cytometry analysis. Error bars presented as the mean ± s.d. and p -values represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, In Vitro, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effect of AEG-1/miR-221 was analyzed on cell cycle and angiogenesis regulatory proteins in HCC cell lines. The relative AEG-1 protein expression in THLE-2, HepG2, Huh7, and Hep3B cells ( A ) and miR-221 mimic-, miR-221 inhibitor-, and AEG-1 siRNA-transfected HCC cells ( B ). The regulatory protein expressions which regulate angiogenesis (LSF and MMP 9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their controls-transfected HepG2 ( C ), Huh 7 ( D ), and Hep3B ( E ) cells by western blot. p -Values presented as * p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001 and error bars presented as the mean ± s.d. ns—(non-significant) compared to the controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Ectopic expression of AEG-1/miR-221 dysregulates apoptosis and autophagy regulatory protein levels in HCC cell lines. The relative expressions of apoptosis (OPN and Bcl-2), autophagy (LC3A/B), PTEN, and PI3K/Akt proteins were analyzed by western blotting in the mock control, miR-221 mimic, miR-221 inhibitor, AEG-1 siRNA, and corresponding controls transfected HepG2 ( A ), Huh7 ( B ), and Hep3B ( C ) cells using β-actin as an internal control. Error bars are represented as mean ± s.d. and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the control group. ns: non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Control, Transfection

Journal: Macromolecular bioscience

Article Title: Polyvinylpyrrolidone-Encapsulated Total Flavonoids Nanoparticles for Enhancing Antioxidant and Anticancer Activities.

doi: 10.1002/mabi.202500106

Figure Lengend Snippet: FIGURE 4 (a) Acute antioxidant stress model induced by H2O2 treatment created with BioRender. com. Both the normal and cancer cells were pretreated with TFs and TFNPs of different concentrations for 1 h, and then exposed to 500 µm H2O2 for 5 h. Flow cytometric analysis of fluorescence intensity of DCFH-DA fluorescence intensity to measure the intracellular ROS levels of (b) normal cells (HL-7702 and BEAS-2B) and (c) cancer cells (HepG2, Hep3B, A549, and H1975) treated with 100 µg/mL TFs/TFNPs in the presence of H2O2. CCK-8 assay results of (d) HL-7702 cells, (e) HepG2 cells, (f) Hep3B cells, (g) BEAS-2B cells, (h) A549 cells, and (i) H1975 cells survival under H2O2 induction at TFs and TFNPs concentrations of both 50 and 100 µg/mL. Data were obtained from one representative experiment out of three independent experiments. Statistical analysis was performed using two-way ANOVA. All data were presented as the mean ± Standard Error of the Mean, SEM, *p < 0.05, **p < 0.01.

Article Snippet: Human hepatoma cell lines HepG2 (ATCC HB-8065) and Hep3B (ATCC HB-8064), human lung cancer cell lines A549 (ATCC CCL-185) and H1975 (ATCC CRL-5908), human liver cell line HL-7702 (ATCC CRL-11928), as well as human lung epithelial cell line BEAS-2B (ATCC CRL-9609), were obtained from the American Type Culture Collection (ATCC).

Techniques: Fluorescence, CCK-8 Assay

Journal: Macromolecular bioscience

Article Title: Polyvinylpyrrolidone-Encapsulated Total Flavonoids Nanoparticles for Enhancing Antioxidant and Anticancer Activities.

doi: 10.1002/mabi.202500106

Figure Lengend Snippet: FIGURE 5 (a) Experimental flow chart of long-term antitumor effect of TFs/TFNPs. CCK-8 viability analysis for long-term anticancer effects of TFs and TFNPs on (b) HepG2 cell, (c) Hep3B cell, (d) A549 cell, and (e) H1975 cells. (f) Experimental flow chart of TFs/TFNPs combined with long-term oxidative stress stimulation on tumor and healthy cells. Survival rate measured by cell viability assay for (g) HepG2 cell, (h) Hep3B cell, (i) A549 cell, and (j) H1975 cells in a long-term oxidative stress model. Data were obtained from one representative experiment out of five independent experiments. One-way or two-way ANOVA was used for statistical analysis. All data were presented as the mean ± SEM *p < 0.05, **p < 0.01.

Article Snippet: Human hepatoma cell lines HepG2 (ATCC HB-8065) and Hep3B (ATCC HB-8064), human lung cancer cell lines A549 (ATCC CCL-185) and H1975 (ATCC CRL-5908), human liver cell line HL-7702 (ATCC CRL-11928), as well as human lung epithelial cell line BEAS-2B (ATCC CRL-9609), were obtained from the American Type Culture Collection (ATCC).

Techniques: CCK-8 Assay, Viability Assay

Journal: Advanced Science

Article Title: ISG15 Enhances the Activity of γ‐Glutamate Cysteine Ligase to Suppress Apoptosis in High Fat Diet‐Promoted Hepatocellular Carcinoma

doi: 10.1002/advs.202416401

Figure Lengend Snippet: PA transcriptionally upregulates ISG15 through HMGA1. A) mRNA levels of Irf3 in murine HCC tissues. B,C) Western blots (B) and the relative integrated density values (C) of Hmga1, Irf3 and phosphorylated Irf3 in murine HCC tissues. D) Potential transcription factor (TF) candidates for Isg15 under HFD challenge screened through JASPAR, TFDB and hTFtarget databases. Ten candidates were found in RNA‐seq data to detect differently expressed genes in HFD versus NCD samples, and Hmga1 was identified. E) mRNA levels of Hmga1 in murine HCC tissues. F) Western blots of endogenous ISG15 in Hep3B and HepG2 cells at 24 h after transfection of the plasmid expressing Flag‐HMGA1. G) Western blots of ISG15 (top) and HMGA1 (bottom) in Hep3B cells under PA (100 µM) or IFN‐γ treatments for 24 h. H) mRNA levels of ISG15 and HMGA1 in Hep3B and HepG2 cells under PA (100 µM) or IFN‐γ treatment for 24 h. Compared with the control group, the ISG15 levels were significantly elevated both by PA and IFN‐γ treatments; whereas the HMGA1 level was only increased in the PA‐treated group. I) mRNA levels of HMGA1 in HMGA1 ‐KO Hep3B cells. J) Western blots of HMGA1 in HMGA1 ‐KO Hep3B cells. K) Western blots of ISG15 in HMGA1 ‐WT and HMGA1 ‐KO cells treated with 100 µM of PA for 24 h. L) mRNA levels of ISG15 in HMGA1 ‐WT and HMGA1 ‐KO cells treated with 100 µM of PA for 24 h. M) Overexpression of HMGA1 (plasmid transfection gradient: 0, 0.1, 0.2, 0.4, 0.8 µg) enhanced the luciferase activity driven by the ISG15 promoter (2 kb upstream of ISG15 ) in Hep3B cells. N) ChIP analysis of HMGA1 occupancy on the different segments on ISG15 promoter in Hep3B cells with or without 100 µM of PA treatment. O) Deletion of indicated ISG15 promoter fragments attenuated the luciferase activity induced by HMGA1 in Hep3B cells. Compared with the WT group, in ISG15 promoter constructs with these two sites deleted, HIGMA1‐induced promoter activity was significantly attenuated. Data shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human HCC cell lines Hep3B (China Center for Type Culture Collection, CCTCC, Wuhan, China) and HepG2 (Procell Biotech, Wuhan, China), and HEK293T (Procell Biotech) were cultured in DMEM media (Hyclone, Logan, UT) containing FBS.

Techniques: Western Blot, RNA Sequencing, Transfection, Plasmid Preparation, Expressing, Control, Over Expression, Luciferase, Activity Assay, Construct

Journal: Advanced Science

Article Title: ISG15 Enhances the Activity of γ‐Glutamate Cysteine Ligase to Suppress Apoptosis in High Fat Diet‐Promoted Hepatocellular Carcinoma

doi: 10.1002/advs.202416401

Figure Lengend Snippet: ISG15 abrogates fat stress induced cytotoxicity in HCC cells. A) Viability of ISG15 ‐WT/KO Hep3B and HepG2 cells in PA‐free condition determined by MTT analysis. B) Viability of ISG15 ‐WT/KO and ISG15‐rescued Hep3B and HepG2 cells treated with 300 µM of PA. C) Colony formation of ISG15 ‐WT/KO and ISG15‐rescued Hep3B and HepG2 cells under 300 µM of PA treatment for 72 h (left), and quantification results (right). D) MDA contents of ISG15 ‐WT/KO and ISG15‐rescued Hep3B and HepG2 cells with or without 300 µM of PA treatment. E) Western blots and the quantitative ratio of the cleaved to full‐length of Caspase‐9/Caspase‐8 in ISG15 ‐WT/KO Hep3B and HepG2 cells under 300 µM of PA treatment for 48 h. F) Western blots and the quantitative ratio of the cleaved to full‐length of Caspase‐8 in ISG15 ‐KO and ISG15‐rescued Hep3B and HepG2 cells under 300 µM of PA treatment for 48 h. G) Apoptosis of ISG15 ‐WT/KO and ISG15‐rescued Hep3B and HepG2 cells under 300 µM of PA treatment for 48 h determined by FACS analysis. Data shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human HCC cell lines Hep3B (China Center for Type Culture Collection, CCTCC, Wuhan, China) and HepG2 (Procell Biotech, Wuhan, China), and HEK293T (Procell Biotech) were cultured in DMEM media (Hyclone, Logan, UT) containing FBS.

Techniques: Western Blot

Journal: Advanced Science

Article Title: ISG15 Enhances the Activity of γ‐Glutamate Cysteine Ligase to Suppress Apoptosis in High Fat Diet‐Promoted Hepatocellular Carcinoma

doi: 10.1002/advs.202416401

Figure Lengend Snippet: ISG15 inhibits apoptosis of HCC cells by increasing glutathione and reducing ROS. A) ROS levels of ISG15 ‐WT/KO Hep3B and HepG2 cells under 300 µM of PA treatments for 24 h determined by FACS analysis. B) Representative DCFH‐DA staining (Green fluorescence) images for ROS of ISG15 ‐WT/KO and ISG15‐rescued Hep3B cells treated with PA for 24 h. Cell nuclei were stained with Hoechst (blue fluorescence). C) Quantification results of ROS in Hep3B (left) and HepG2 (right) cells. D) Viability by MTT analysis of ISG15 ‐WT/KO Hep3B (left) and HepG2 (right) cells treated with PA (300 µM), or pre‐treated with glutathione (10 mM) or NAC (10 mM) for 12 h before PA treatment. (E) Total glutathione levels in ISG15 ‐WT/KO and ISG15‐rescued Hep3B (left) and HepG2 (right) cells treated with 300 µM of PA for 24 h. F) γ‐glutamate cysteine ligase activities in ISG15 ‐WT/KO and ISG15‐rescued Hep3B (left) and HepG2 (right) cells under 300 µM of PA treatment for 24 h. G) Total glutathione levels in two ISG15 overexpressing UBA7 ‐KO Hep3B cell lines (KO 1# and KO 2#) under PA treatments for 24 h. H) γ‐glutamate cysteine ligase activities in two ISG15 overexpressing UBA7 ‐KO Hep3B cell lines (KO 1# and KO 2#) under PA treatments for 24 h. I) Total glutathione levels in Hep3B and HepG2 cells overexpressing ISG15 mutants under 300 µM of PA treatments for 24 h. Compared with the control group, overexpressing ISG15 mutants increased total cellular glutathione levels with or without PA treatments. J) Co‐immunoprecipitation of GCLM and ISG15. HEK293T cells were co‐transfected with Flag‐GCLM and/or Myc‐ISG15‐GG/AA as indicated, anti‐Flag immunoprecipitates (top) and total lysates (bottom) were subjected to immunoblot with anti‐Flag antibody to reveal foreign GCLM, and anti‐Myc antibody to reveal foreign ISG15. K) Co‐immunoprecipitation of endogenous Gclm and Isg15 in mouse HCC tissues. Anti‐Gclm immunoprecipitate and total lysate of mouse tissues were subjected to immunoblot with anti‐Gclm and anti‐Isg15 antibodies. Data shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human HCC cell lines Hep3B (China Center for Type Culture Collection, CCTCC, Wuhan, China) and HepG2 (Procell Biotech, Wuhan, China), and HEK293T (Procell Biotech) were cultured in DMEM media (Hyclone, Logan, UT) containing FBS.

Techniques: Staining, Fluorescence, Control, Immunoprecipitation, Transfection, Western Blot

Journal: Advanced Science

Article Title: ISG15 Enhances the Activity of γ‐Glutamate Cysteine Ligase to Suppress Apoptosis in High Fat Diet‐Promoted Hepatocellular Carcinoma

doi: 10.1002/advs.202416401

Figure Lengend Snippet: ISG15 upregulates glutathione production by promoting the GCLC‐GCLM interaction. A) Co‐immunoprecipitation of GCLM/GCLC and ISG15. ISG15 ‐WT/KO Hep3B (left) and HepG2 (right) cells were treated with PA as indicated, anti‐GCLC immune‐precipitates (top) and total lysates (bottom) were subjected to immunoblot with GCLC, GCLM and ISG15 antibodies. 100 µM of PA was added 24 h before cell lysis. B) ISG15 ‐KO Hep3B (left) and HepG2 (right) cells were co‐transfected with HA‐ISG15‐GG/AA as indicated. Anti‐GCLC immunoprecipitates (top) and total lysates (bottom) were subjected to immunoblot with GCLC, GCLM, and ISG15 antibodies. C) Representative structures of ISG15 (green) and GCLM (blue), and residues on the interaction interface are as labeled; and the primary sequences of ISG15‐Δ85‐93 and ISG15‐84AAA. D) Co‐immunoprecipitation of GCLM and indicated ISG15 constructs. HEK293T cells were co‐transfected with Flag‐GCLM and HA‐ISG15 mutants as indicated. Anti‐Flag immunoprecipitates (top) and total lysates (bottom) were subjected to immunoblot with anti‐Flag and anti‐HA antibodies. E,F) Co‐immunoprecipitation of GCLC/GCLM and indicated ISG15 constructs in ISG15 ‐KO (E) and dual knockout of ISG15 and UBA7 Hep3B cells (F). Cells were co‐transfected with HA‐ISG15 mutants as indicated. Anti‐GCLC immunoprecipitates (top) and total lysates (bottom) were subjected to immunoblot with anti‐GCLM and anti‐GCLC antibodies. G) γ‐glutamate cysteine ligase activities in ISG15 ‐KO Hep3B (left) and HepG2 (right) cells expressing HA‐ISG15 mutants with or without 300 µM of PA treatment. H) Total glutathione levels in ISG15 ‐KO Hep3B (left) and HepG2 (right) cells expressing HA‐ISG15 mutants with or without 300 µM of PA treatment. I) Viability of ISG15 ‐WT/KO Hep3B (left) and HepG2 (right) cells overexpressing HA‐ISG15 mutants with 300 µM of PA treatment for 48 h determined by MTT assay. Data shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human HCC cell lines Hep3B (China Center for Type Culture Collection, CCTCC, Wuhan, China) and HepG2 (Procell Biotech, Wuhan, China), and HEK293T (Procell Biotech) were cultured in DMEM media (Hyclone, Logan, UT) containing FBS.

Techniques: Immunoprecipitation, Western Blot, Lysis, Transfection, Labeling, Construct, Knock-Out, Expressing, MTT Assay