Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma

Figure 1 Effect of long interspersed nuclear element ORF-1 protein on epirubicin, cisplatin and paclitaxel cytotoxicity. HepG2 cells were treated with the indicated amounts of epirubicin (A, B), cisplatin (C, D) or paclitaxel (E, F): 0 nmol/L, 0.5 nmol/L, 1 nmol/L, 2 nmol/L, 5 nmol/L, 10 nmol/L, 20 nmol/L, 50 nmol/L, 60 nmol/L. A: HepG2 cells were transfected with the fludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p expression vector, or the empty vector; B: HepG2 cells were transfected with the control siRNA vector or the LINE-1 ORF-1p siRNA vector; C: HepG2 cells were transfected with the FLAG-LINE-1 ORF-1p expression vector, or the empty vector; D: HepG2 cells were transfected with the control siRNA vector or the LINE-1 ORF-1p siRNA vector; E: HepG2 cells were transfected with the FLAG-LINE-1 ORF-1p expression vector, or the empty vector; F: HepG2 cells were transfected with the control siRNA vector or the LINE-1 ORF-1p siRNA vector. The assays were performed three times with similar results. ^aP < 0.05 vs control. 1: Control; 2: Epirubicin; 3: Cisplatin; 4: Paclitaxel. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2 Long interspersed nuclear element ORF-1 protein promotes the proliferation of hepatocellular carcinoma cells. A: HepG2 cells were stably transfected with the fludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p or the empty vector; B: HepG2 cells were stably transfected with the control siRNA or LINE-1 ORF-1p siRNA; C: BEL-7402 cells were stably transfected with the FLAG-LINE-1 ORF-1p or the empty vector; D: BEL-7402 cells were stably transfected with the control siRNA or LINE-1 ORF-1p siRNA; E: SMMC-7721 cells were stably transfected with the FLAG-LINE-1 ORF-1p or the empty vector; F: SMMC-7721 cells were stably transfected with the control siRNA or LINE-1 ORF-1p siRNA. The relative cell numbers (A_{490 nm}) were determined by MTT assays, relative cell numbers (A, B) with mean ± SD (n = 3). ^aP < 0.05 vs FLAG; ^cP < 0.05 vs control siRNA. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 3 Long interspersed nuclear element ORF-1 protein promotes apoptosis and anchor-independent growth of HepG2 cells. HepG2 cells were transfected with the empty vector or f ludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p, or with the control siRNA or LINE-1 ORF-1p siRNA. A, B: The rates of apoptosis are shown in the figures (A). The assays were performed three times with similar results (B); C, D: Colony numbers shown in the photographs (C), or mean ± SD (n = 3) of triplicate independent experiments (D). ^aP < 0.05 vs FLAG; ^cP < 0.05 vs control siRNA; ^eP < 0.05 vs FLAG-LINE-1 ORF-1p. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 4 Long interspersed nuclear element ORF-1 protein regulates the transcription of Luciferase reporters. HepG2 cells were transfected with the fludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p or FLAG empty vector, LINE-1 ORF-1p siRNA or control siRNA. HepG2 cells were co-transfected with Luc-p21 (A), Luc-SBE (B), Luc-Sp1 (C), or Luc-ARE (D). The assays were performed three times with similar results. ^aP < 0.05 vs FLAG; ^cP < 0.05 vs control siRNA. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5 Long interspersed nuclear element ORF-1 protein modulates the expression of genes related to cell proliferation and apoptosis regulation. A: HepG2 cells were transfected with the fludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p vector, or the empty vector. The assays were performed three times with similar results. ^aP < 0.05 vs FLAG; B: HepG2 cells were transfected with control siRNA or LINE-1 ORF-1p siRNA. The bar graph shows the relative protein expression level of each protein compared with GAPDH. The assays were performed three times with similar results. ^aP < 0.05 vs FLAG; ^cP < 0.05 vs control siRNA. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 6 Long interspersed nuclear element ORF-1 protein modulates the expression of genes related to drug resistance. A: HepG2 cells were transfected with the fludarabine + high-dose cytarabine + G-CSF human long interspersed nuclear element-1 (FLAG-LINE-1) ORF-1p vector, or the empty vector; B: HepG2 cells were transfected with control siRNA or LINE-1 ORF-1p siRNA. The bar graph shows the relative protein expression level of each protein compared with GAPDH. The assays were performed three times with similar results. ^aP < 0.05 vs FLAG; ^cP < 0.05 vs control siRNA. MDR: Multidrug resistance; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7 Long interspersed nuclear element ORF-1 protein expressed in clinical specimens and HepG2 cells. Representative immunohistochemical staining (A) and immunoblots (B) of human long interspersed nuclear element-1 (LINE-1) ORF-1p proteins in HepG2 cells, which were stably transfected with empty vector or LINE-1 expression vector, and in human cancerous liver tissues (C1-C10). Original magnification: × 40. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.