Journal: Experimental & Molecular Medicine

Article Title: T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

doi: 10.1038/emm.2017.166

Figure Lengend Snippet: TCIRG1 knockdown has anti-tumorigenic effects on hepatocellular carcinoma (HCC) cells. ( a ) qRT-PCR analysis of TCIRG1 in 14 hepatic cell lines, including two normal (THLE3 and MIHA) and 12 hepatoma cell lines (Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization (mean±s.d.; n =3, ** P <0.01; *** P <0.001). ( b ) Endogenous protein expression of TCIRG1 in hepatic cell lines was analyzed by western blotting. GAPDH was used as a loading control, and the numbers under the blot indicate the relative expression level of each protein. ( c ) Clonogenic assays were performed in SNU475 and Huh7 cell lines. Upper panel: representative images of colonies. Lower panel: the data from three randomly selected images shown in the form of a graph (mean±s.d.; n =3, * P <0.05, ** P <0.01). ( d ) SNU475 and Huh7 cell lines were transfected with negative-control siRNA or TCIRG1-specific siRNA. Cell number was determined using trypan blue-based cell counting (mean±s.d., n =3, ** P <0.01). ( e ) SNU475 and Huh7 cell lines were transfected with negative-control siRNA or TCIRG1-specific siRNA. Cell growth was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. ( f ) Bromodeoxyuridine (BrdU) incorporation assays were carried out using SNU475 and Huh7 cell lines (mean±s.d.; n =3, * P <0.05, ** P <0.01). ( g ) Fluorescence-activated cell sorting (FACS) analysis was conducted after negative-control siRNA or TCIRG1-specific siRNA transfection. When TCIRG1 was knocked down, G1/S arrest was observed in SNU475 and Huh7 cell lines. The percentage indicates the distribution of cells in the different phases of cell cycle (mean±s.d., n =3, * P <0.05). ( h ) Negative-control siRNA (NC) or TCIRG1-specific siRNA (siTCIRG1) were transfected into cells. FACS analysis was performed using PI and Annexin V staining. The bar graphs indicate the percentage of annexin V-positive cells (mean±s.d., n =3, * P <0.05). ( i ) Following transfection, cells were treated with 3-MA (5 m M ), and FACS analysis was performed. The bar graph indicates the percentage of Annexin V negative and PI-positive cells (mean±s.d., n =3, *** P <0.001).

Article Snippet: Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475 HCC cell lines were acquired from the Korean Cell Line Bank (KCLB, Seoul, South Korea).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Control, Transfection, Negative Control, Cell Counting, BrdU Incorporation Assay, Fluorescence, FACS, Staining

Journal: Experimental & Molecular Medicine

Article Title: T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

doi: 10.1038/emm.2017.166

Figure Lengend Snippet: TCIRG1 knockdown attenuates the metastatic potential of hepatocellular carcinoma (HCC) cells. ( a , b ) TCIRG1 knockdown inhibited migration ( a ) and invasion ( b ) of SNU475 and Huh7 cell lines in vitro . The number of migrated and invaded cells was determined. Three randomly selected fields were captured, and the results were graphically presented (mean±s.d., n =3, ** P <0.01, *** P <0.001). Representative images are shown. ( c ) Wound healing assay. The bar graphs show the ratios of the recovered area (mean±s.d., n =3, *** P <0.001). ( d , e ) Effect of TCIRG1 knockdown on cell migration and invasion in vitro . TCIRG1 knockdown inhibited migration ( d ) and invasion ( e ) of ras -transformed NIH-3T3 mouse fibroblasts. The number of migrated and invaded cells was determined. Three randomly selected fields were captured, and the results were presented in the form of a graph (mean±s.d., n =3, ** P <0.01). Representative images. ( f ) Western blot analysis of epithelial–mesenchymal transition (EMT) markers. The protein level of TCIRG1, E-cadherin, N-cadherin, Fibronectin, Vimentin, Snail and Slug was detected with their specific antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. The numbers under the blot indicate the relative expression level of each protein. ( g ) Analysis of the NCBI GEO database. GEO data sets with the accession numbers GSE39791 and GSE77314 were used for expression analysis of TCIRG1 and CDH1 . Left panel, CDH1 expression in GSE39791 and GSE77314. Right panel, negative correlation of TCIRG1 and CDH1 expression in GSE39791 (Pearson’s correlation coefficient, r =−0.21, ** P <0.01) and in GSE77314 (Pearson’s correlation coefficient, r =−0.36, *** P <0.001).

Article Snippet: Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475 HCC cell lines were acquired from the Korean Cell Line Bank (KCLB, Seoul, South Korea).

Techniques: Knockdown, Migration, In Vitro, Wound Healing Assay, Transformation Assay, Western Blot, Control, Expressing

Journal: British Journal of Cancer

Article Title: Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance

doi: 10.1038/s41416-020-01240-6

Figure Lengend Snippet: a The diagram illustrates how miR424 was identified from the miRNA sequencing data and predicted by Venn screening from miRNA database websites. b Sequence alignment of the human miR424 seed sequence with the 3’-UTR of CBX4. The mutated sequence in the matched binding sites for the gene that was used to create the firefly luciferase reporter constructs is shown at the bottom of the gene set. A luciferase reporter assay demonstrated that miR424 inhibited the transcription of the wild-type but not the mutant 3’-UTRs of CBX4. c The expression of endogenous CBX4 was inhibited in miR424-overexpressed Huh7-SR and PLC-SR cells. d In contrast, CBX4 levels were increased in Huh7 and PLC cells with miR424-TUD. All data were compared with the respective controls, and the mRNA level was detected by qRT-PCR. CBX4 mRNA expression was normalised to that of GAPDH mRNA; and three independent experiments were conducted. e Relative miR424 expression in HCC tissues and matched adjacent normal tissues as assessed by qRT-PCR. f , g Relative expression data of miR424 in HCC cases were further analysed. The negative relationship between miR424 expression and liver cirrhosis ( f ) and size ( g ). h , i Kaplan–Meier curves of disease-free survival (DFS) ( h ) and overall survival (OS) ( i ). Survival of the high and low miR424 expression groups assessed using log-rank (Mantel–Cox) test in HCC, which were divided according to a cut-off of 2.5, the median value of CBX4 mRNA expression relative to GAPDH mRNA. j From the TCGA database, tumour purity was highly negatively correlated with miR424 expression. k Linear regression and correlation between the miR424 and CBX4 mRNA levels in 341 HCC tissues from the TCGA database. l The negative linear regression and correlation analysis for the relation of the mRNA levels of CBX4 and those of miR424 in 106 HCC patients by qRT-PCR.

Article Snippet: The human HCC cell lines PLC (catalogue number: CRL-8024TM) was obtained from the Global Bioresource Center of American Type Culture Collection (ATCC) and Huh7 (catalogue number: JCRB0403) was purchased from Health Science Research Resources Bank (Osaka, Japan).

Techniques: Sequencing, Binding Assay, Luciferase, Construct, Reporter Assay, Mutagenesis, Expressing, Quantitative RT-PCR