Inhibitory effects of LOXL2 knockdown on cellular functions of liver cancer stem cells

Abstract

Background: Lysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.

Methods: CD133⁺HepG2 and CD133⁺Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knockdown in CD133⁺HepG2 and CD133⁺Hep3B cells. Protein and mRNA levels were quantified by Western blotting, immunofluorescence and reverse transcription-PCR (RT-PCR).

Results: Knockdown of LOXL2 decreased spheroid formation, migration and invasion (P<0.05), also induced apoptosis (P<0.05) and cell cycle arrest (P<0.05) in CD133⁺HepG2 and CD133⁺Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (P<0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3B) and autophagy gene ATG5 in CD133⁺HepG2 and CD133⁺Hep3B cells (P<0.01).

Conclusions: The results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

Keywords: Hepatocellular carcinoma (HCC); apoptosis; liver cancer stem cells (LCSCs); lysyl oxidase-like 2 (LOXL2).

Figure 1

Cell sorting and the CD133 positivity after cell culture of CD133-positive subsets in HepG2 and Hep3B hepatoma cell lines. (A) The CD133-positive subsets in HepG2 and Hep3B cell lines were sorted by flow cytometry, the proportion of the subsets were 19.8%, 72.4%, respectively. About 60% of CD133⁺ cells presenting strongly positive were sorted. The sorted cells were cultured with DMEM/F12, EGF, bFGF, and B27. (B) The CD133 positivity of CD133⁺HepG2 and CD133⁺Hep3B cell lines which detected by flow cytometry were 74%, 90.5%, respectively. EGF, recombinant epidermal growth factor; bFGF, basic fibroblast growth factor.

Figure 2

Knockdown of LOXL2 impairs the spheroid formation of LCSCs. CD133⁺ (A) HepG2 and (B) Hep3B cells for sphere-forming assay were transfected with shRNA LOXL2-LV or NC-LV to observe cell survival upon LOXL2 knockdown. After culturing for 10–15 days, the number of tumor spheroids (>50 µm) was counted. LOXL2 knockdown impaired the cell sphere-forming, especially the CD133⁺Hep3B cells presenting with growth retardation. CD133⁺Hep3B cells in the following study were shifted to transiently transfection with LOXL2-siRNA or NC-siRNA. (C) Western blotting and (D) PCR were performed to determine transfection efficiency of the two ways. Relative intensity values for the proteins were obtained using Image J software. The data were analyzed upon three independent experiments and shown as mean ± SD. **, P<0.01; ***, P<0.001; ****, P<0.0001. NC-LV, lentiviral vector against negative control; LOXL2-LV, lentiviral vector against LOXL2; LOXL2, lysyl oxidase-like 2; LOXL2-siRNA, siRNAs against LOXL2; NC-siRNA, siRNAs against negative control; LCSCs, liver cancer stem cells; SD, standard deviation.

Figure 3

Knockdown of LOXL2 inhibits LCSCs migration and invasion. The transwell (A) migration and (B) invasion assays of CD133⁺HepG2 cells were performed upon LOXL2 knockdown by transfected with shRNA lentiviral vector (LOXL2-LV/NC-LV). After culturing for 24 hours in the transwells, cells were fixed and stained by 0.1% crystal violet. The average number of migrating and invading cells are showed in graphs. The transwell (C) migration and (D) invasion assays of CD133⁺Hep3B cells were performed upon LOXL2 knockdown by transfected with siRNAs (LOXL2-siRNA/NC-siRNA). Scale bar: 50 µm. The data were analyzed upon three independent experiments and shown as mean ± SD. *, P<0.05; **, P<0.01. NC-LV, lentiviral vector against negative control; LOXL2-LV, lentiviral vector against LOXL2; LOXL2, lysyl oxidase-like 2; LOXL2-siRNA, siRNAs against LOXL2; NC-siRNA, siRNAs against negative control; LCSCs, liver cancer stem cells; SD, standard deviation.

Figure 4

Knockdown of LOXL2 induces apoptosis and cell cycle arrest in LCSCs. CD133⁺HepG2 and CD133⁺Hep3B cells for flow cytometry analysis were transfected with shRNA lentiviral vector (LOXL2-LV/NC-LV) or siRNAs (LOXL2-siRNA/NC-siRNA), respectively, to knockdown LOXL2. Cells for apoptosis were gated by Annexin V-FITC and PI staining. Knockdown of LOXL2 induced the apoptosis of CD133⁺ (A) HepG2 and (B) Hep3B cells. LOXL2 knockdown also induced cell cycle arrested in CD133⁺ (C) HepG2 and (D) Hep3B cells. Data represented as % of total cells. The data were analyzed upon three independent experiments and presented as mean ± SD. *, P<0.05; **, P<0.01. NC-LV, lentiviral vector against negative control; LOXL2-LV, lentiviral vector against LOXL2; LOXL2, lysyl oxidase-like 2; LOXL2-siRNA, siRNAs against LOXL2; NC-siRNA, siRNAs against negative control; LCSCs, liver cancer stem cells; SD, standard deviation.

Figure 5

Knockdown of LOXL2 downregulates BIRC3 and MDM2 in LCSCs. Western blotting and PCR of CD133⁺ (A,B) HepG2 and (C,D) Hep3B cells were performed upon LOXL2 knockdown to determine the expression of BIRC3 and MDM2 by transfected with shRNA lentiviral vector (LOXL2-LV/NC-LV) or siRNAs (LOXL2-siRNA/NC-siRNA) respectively. Relative intensity values for the proteins were obtained using Image J software. (E,F) Immunofluorescence staining was performed for BIRC3 and MDM2 in CD133⁺HepG2 cells upon LOXL2 knockdown. Scale bar: 200 µM. Mean fluorescence intensity values were obtained using Image J software. The data were analyzed upon three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. BIRC3, baculoviral inhibitor of apoptosis protein repeat-containing 3; MDM2, murine double minute 2; NC-LV, lentiviral vector against negative control; LOXL2-LV, lentiviral vector against LOXL2; LOXL2, lysyl oxidase-like 2; LOXL2-siRNA, siRNAs against LOXL2; NC-siRNA, siRNAs against negative control; LCSCs, liver cancer stem cells.

Figure 6

Knockdown of LOXL2 inhibits the expression of autophagy marker LC3B and autophagy gene ATG5 in LCSCs. Western blotting and PCR of CD133⁺ (A,B) HepG2 and (C,D) Hep3B cells were performed upon LOXL2 knockdown to determine the expression of LC3B and ATG5 by transfected with shRNA lentiviral vector (LOXL2-LV/NC-LV) or siRNAs (LOXL2-siRNA/NC-siRNA) respectively. Relative intensity values for the proteins were obtained using Image J software. (E,F) Immunofluorescence staining was performed for LC3B and ATG5 in CD133⁺HepG2 cells upon LOXL2 knockdown. Scale bars 200 µM. Mean fluorescence intensity values were obtained using Image J software. The data were analyzed upon three independent experiments. *, P<0.05; **, P<0.01. LC3B, microtubule-associated protein 1 light chain 3 B; NC-LV, lentiviral vector against negative control; LOXL2-LV, lentiviral vector against LOXL2; LOXL2, lysyl oxidase-like 2; LOXL2-siRNA, siRNAs against LOXL2; NC-siRNA, siRNAs against negative control; LCSCs, liver cancer stem cells.