Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 1. The lncGCLC expression in MCLR-exposed cells and population samples. (A) Changes of lncGCLC expression in WRL68 cells after exposure to 0 or 10 µg/L of MCLR for 0, 10, 15, and 25 passages. * p < 0.05 compared with passage-matched control cells. (B) Comparison of lncGCLC expression in WRL68, HepG2, and SMMC7721 cells. ** p < 0.01 compared with WRL68 cells. (C) Com- parison of lncGCLC expression in tumor tissue and adjacent normal tissue from HCC patients with MC exposure (n = 30), ** p < 0.01. (D) LncGCLC expression in HCC patients with high MC exposure (serum MCs ≥0.14 µg/L, n = 17) was lower than that in those with low MC exposure (serum MCs < 0.14 µg/L, n = 13), ** p < 0.01. Data are presented as the mean ± SD of three independent experiments.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Comparison

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 2. Knockdown of lncGCLC promoted the proliferation of MCLR-treated WRL68 cells. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) The expression of lncGCLC was detected using qRT-PCR. (B) Cell proliferation activity was detected by CCK-8. (C) Cell apoptosis was analyzed using flow cytometry. (D) Apoptosis rate in each group. (E) The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow cytometry. (F) Cell cycle distribution quantification. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Transfection, Expressing, Quantitative RT-PCR, Activity Assay, CCK-8 Assay, Cytometry, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 3. Knockdown of lncGCLC promoted the migration and invasion of MCLR-treated WRL68 cells. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) Representative images of a cell migration assay (100×), scale bar = 200 µm. (B) Quantification of cell migration. (C) Representative images of a cell invasion assay (Original magnification ×100, scale bar = 200 µm). (D) Quantification of cell invasion. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Migration, Plasmid Preparation, Transfection, Cell Migration Assay, Invasion Assay, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 4. Knockdown of lncGCLC promoted the growth of MCLR-induced malignantly transformed WRL68 cells in nude mice. Vector- or sh-lncGCLC-transfected cells were treated with or without 10 µg/L of MCLR for 25 passages. (A) Representative images of a soft agar assay (Original magnifica- tion ×200, scale bar = 100 µm). (B) Quantification of colony formation in soft agar. (C) Tumorigenicity test of BALB/c nude mice was performed. Solid tumors were removed after the sacrifice at 22 days. (D) Representative image showing subcutaneous tumor size. (E) The weights of solid tumors. The values given are mean ± SD (n = 3 for both male and female nude mice per group). (F) Tumor volume was monitored every 3 days after injection of WRL68 cells. (G) Pathological changes of the tumor tissue in nude mice in each group (HE stains, original magnification ×400, scale bar = 50 µm). ND—not detected. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Transformation Assay, Plasmid Preparation, Transfection, Soft Agar Assay, Injection, Control

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 5. Verification of the interrelationships among lncGCLC, miR-122-5p, and GCLC. (A) The mRNA expression level of neighboring genes which are located nearly 1.5 Mb downstream of lncGCLC in lncGCLC knockdown cells. * p < 0.05 and ** p < 0.01 compared with vector control cells. (B) The expression level of lncGCLC in the nuclear and cytoplasmic fractions of WRL68 cells. (C) The correlation between the lncGCLC expression and the GCLC expression in HCC tissues (n = 30). Correlation coefficient (r) and P value were calculated by Pearson correlation analysis. (D) Comparison of GCLC expression in tumor tissue and adjacent normal tissue from HCC patients with MC exposure (n = 30), ** p < 0.01. (E) GCLC expression in HCC patients with high MC exposure (serum MCs ≥0.14 µg/L, n =17) was lower than in those with low MC exposure (serum MCs < 0.14 µg/L, n = 13), ** p < 0.01. (F) After exposure to 10 µg/L of MCLR for 0, 10, 15, and 25 passages, expression of lncGCLC, miR-122-5p and GCLC mRNA was detected in WRL68 cells. * p < 0.05 and ** p < 0.01 compared with passage-matched control cells. (G,H) Expression of GCLC protein in P0, P10, P15 and P25 MCLR-induced malignantly transformed WRL68 cells. ** p < 0.01 compared with passage-matched control cells. (I) Changes of miR-122-5p in both vector- and sh-lncGCLC-transfected WRL68 cells treated with 0 or 10 µg/L of MCLR for 25 passages. (J–L) Changes of GCLC mRNA (J) and protein (K,L) expression in both vector- and sh-lncGCLC-transfected WRL68 cells treated with or without 10 µg/L of MCLR for 25 passages. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Knockdown, Plasmid Preparation, Control, Comparison, Transformation Assay, Transfection

Journal: Toxics

Article Title: Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

doi: 10.3390/toxics11020162

Figure Lengend Snippet: Figure 6. Knockdown of lncGCLC reduced GSH levels and induced oxidative DNA damage in MCLR-treated WRL68 cells. Alterations in GCL activity (A), GSH (B), and 8-OHdG content (C) in vector- or sh-lncGCLC-transfected cells exposed to 0 or 10 µg/L of MCLR for 25 passages. Data are presented as the means ± SD of three independent experiments in each group. * p < 0.05 compared with the control group; # p < 0.05 compared with the sh-NC group; † p < 0.05 compared with the sh-NC + MCLR group.

Article Snippet: The human normal liver cell lines WRL68 and human hepatoma cell lines (HepG2 and SMMC7721) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Activity Assay, Plasmid Preparation, Transfection, Control