Transfection Of Apoptin Gene Into Human Liver Cancer Cells Induces Their Apoptosis

Objective This study was designed to investigate effect of the expression of apoptin gene on human liver cancer cells.Methods An apoptin gene was linked to the C terminal of enhanced green fluorescent protein gene to generate pEGFP-VP3 without reading frame shifted. The structure of vector was identified by endonuclease analyzing and base sequencing. In order to obtain transient expression of EGFP and EGFP-VP3 in human liver cancer cells, two plasmids, pEGFP-C2 and pEGFP-VP3, were respectively transfected into HepG2, a human liver cancer cell line, by a non-lipidsome lipid transfection method. Non-transfected HepG2 was used as a blank control. The location and quantity of EGFP and EGFP-VP3 in HepG2, cellular growth and apoptosis were observed under fluorescent microscope. Apoptosis in HepG2 was assayed by Annexin V-PE dying.Results EGFP was fairly well-distributed in both plasm and nucleus of HepG2. No change was found in cellular morphology. Proliferation and mitosis of transfected cells was shown. Apoptosis in transfected cells did not increase by contrast with natural apoptosis in non-transfected ones. In comparison with EGFP, EGFP-VP3 mainly assembled in nucleus as fluorescent granules. It was found that EGFP-VP3 granules in the nucleus became coarse for a period of six days. The cellular morphology became small and round, and finally pieces. Apoptosis increased in HepG2 transfected with pEGFP-VP3. There was significant difference between two transfected groups (p<0.01). In 48 hours after the transfection, apoptosis occurred in 50% of HepG2 transfected with pEGFP-VP3. In a few cells EGFP-VP3 did not concentrate in the nucleus. And no apoptosis in the cells was shown by Annexin V-PE dying.Conclusion (1) The apoptin gene links to EGFP gene to generate pEGFP-VP3, which can express EGFP-VP3 after transfected HepG2, (2) under fluorescent microscope, the location, expressive amount and changes can be shown by meansof EGFP-VP3. (3) Apoptin mainly locates in the nucleus of HepG2 and concentrates during apoptosis of HepG2, and finally becomes pieces. (4) Transfection of apoptin gene into HepG2 induces apoptosis and it is necessary for apoptosis that apoptin locates in the nucleus of HepG2. (5) There is a threshold of apoptin to induce HepG2 apoptosis.