HEPN1, a novel gene that is frequently down-regulated in hepatocellular carcinoma, suppresses cell growth and induces apoptosis in HepG2 cells Mei Chung.

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HEPN1, a novel gene that is frequently down-regulated in hepatocellular carcinoma, suppresses cell growth and induces apoptosis in HepG2 cells Mei Chung Moh, Lay Hoon Lee, Xiaodong Yang, Shali Shen Journal of Hepatology Volume 39, Issue 4, Pages 580-586 (October 2003) DOI: 10.1016/S0168-8278(03)00359-3

Fig. 1 Expression of HEPN1 in 23 HCC patients. (A) One-step RT-PCR products were analysed by gel electrophoresis. (B) The expression level of HEPN1 in each sample was determined by Real-time RT-PCR. Results were normalised and converted into percentage against the expression level of GAPDH. The average level of expression (mean±SD, n=23) is represented on the right, *P<0.0001. N, non-tumorous liver tissue; T, HCC liver tissue; and β-actin, internal control. Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)

Fig. 2 Expression of HEPN1 in four HCC cell lines. One-step RT-PCR was used to amplify HEPN1 mRNA and GAPDH (an internal control). The expression of HEPN1 in four HCC cell lines, HepG2, Hep3B, Huh7 and SK-Hep1, was compared to that in the non-tumorous liver tissue from patient number one (N1). Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)

Fig. 3 Characteristics of HEPN1. (A) Sequences of HEPN1 cDNA and the predicted amino acid (GenBank Accession No. AY275431). The full-length cDNA of HEPN1 is approximately 1.4 kb long. The ORF, start codon, stop codon, and the Kozak sequence are highlighted. The predicted peptide has 88 amino acids with molecular weight about 10 kDa. (B) Localisation of HEPN1 in human genome. HEPN1 is mapped to chromosome 11q24.2. (C) Subcellular localisation of HEPN1 in HepG2 cells. HepG2 cells transfected with HEPN1-V5 construct. Anti-V5 immunostaining visualised that HEPN1 is predominantly distributed in the cytoplasm. Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)

Fig. 4 Growth inhibition of HepG2 cells by HEPN1. (A) Transient transfection for 48 h. Light microscopic photographs demonstrate the density (100×) and morphology (400×) of HepG2 cells transfected with HEPN1-pcDNA (right) in contrast to the control, cells transfected with pcDNA3.1 vector alone (left). Cell viability (mean±SD, n=12) was detected by MTT assay and computed into percentage against the control; *P=0.001. Real-time RT-PCR results show the expression level of HEPN1 in the transfected HepG2 cells. GAPDH was used as control. The RT-PCR products were analysed by gel electrophoresis to verify the specificity. (B) Colony formation. HepG2 cells transfected with the plasmid HEPN1-EGFP (right) and pEGFP-N2 vector (left) were cultured in the presence of 800 μg/ml of G418 for 2 weeks before confocal microscopy. The density and morphology of cells in a colony were visualised by green fluorescence. The number of colonies presented at the bottom of each picture was counted manually. Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)

Fig. 5 Induction of apoptosis in HepG2 cells by HEPN1. HepG2 cells were transfected with either pEGFP-N2 vector alone (labeled as EGFP) or HEPN1-EGFP construct. Between 8 and 24 h after transfection, cells were analysed by fluorescent microscopy at every 2 h. Green fluorescence visualised cell number, cell morphology and distribution of HEPN1. Microphotographs are presented at each time point and indicated as 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, respectively. An entire microscopic view at 24 h is compared to that at 12 h (inset), showing the reduction of cell number when HEPN1 was re-expressed. Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)

Fig. 6 Results of Annexin V assay. The fluorescent rings represent positive binding of Annexin V with PS in the cell membrane, an early indication of apoptosis. (A) HepG2 cells transfected with HEPN1-pcDNA construct; and (B) HepG2 cells transfected with the empty vector pcDNA3.1. Journal of Hepatology 2003 39, 580-586DOI: (10.1016/S0168-8278(03)00359-3)