Effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2

Abstract

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepato-cellular carcinoma cell line HepG2 were elucidated. The human ANXA10 gene was subcloned into the lentiviral vector, PGC-FU, to generate the lentiviral expression vector, PGC-FU-ANXA10. The corrected ANXA10 was confirmed by endoenzyme digestion, and sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells, and lentiviral particles were produced. The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting. HepG2 cells were infected, and divided into PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group. The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively. Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively. The recombinant PGC-FU-ANXA10 vector was successfully constructed, the ANXA10 protein was detected by using Western blotting, and virus titer was 2×10⁸ TU/mL. The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%. At 72 h after infection, the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05); the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%, significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05), but there was no significant difference between PGC-Fu group and HepG2 cell group; the apoptosis rate in PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group was (51.92±1.41)%, (19.00±1.12)% and (3.59±0.89)% respectively. The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05). The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells. The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.

Keywords

ANXA10 / overexpression / hepatocellular carcinoma / lentiviral vector / cell proliferation / cell apoptosis

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