Effect and mechanism of an autophagy gene beclin 1 on cervical cancer HeLa cells

Abstract

To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3.1(+)-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1(+) group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. (1) The mRNA expression of beclin 1 and caspase-9: pcDNA3.1(+)-beclin 1 group were 994.72 ± 468.76 and 12.88 ± 2.71, pSUPER-beclin 1 group were 0.18 ± 0.63 and 0.11 ± 0.08, pcDNA3.1(+) group were 0.57 ± 0.12 and 4.28 ± 3.25, pSUPER group were 0.67 ± 0.29 and 2.77 ± 1.27, and HeLa group were 0.74 ± 0.25 and 3.67 ± 3.78, respectively. The eukaryotic expression vector pcDNA3.1(+)-beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells (P < 0.05), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9(P < 0.05). (2) The cell proliferations: pcDNA3.1(+)-beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells (P < 0.05). (3) The rate of apoptosis: pcDNA3.1(+)-beclin 1 group was (28.2 ± 2.3)%, pcDNA3.1(+) group was (14.6 ± 4.6)%, pSUPER-beclin 1 group was (5.7 ± 2.0)%, pSUPER group wa (16.2 ± 3.1)%, and HeLa group was (11.2 ± 3.0)%. The pcDNA3.1(+)-beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate (P < 0.05). (4) The activity of autophagy: more autophagy cells were identified in pcDNA3.1(+)-beclin 1 group; the rate of autophagy of five group were (10.3 ± 1.5)% in pcDNA3.1(+)-beclin 1 group, (3.6 ± 0.8)% in pcDNA3.1(+) group, (1.2 ± 0.3)% in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1.1)% in HeLa group, there was statistical significances between test groups and control groups (P < 0.05). (5) Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1(+)-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups (P < 0.05). The tumor size was smaller from 21st day after injection in pcDNA3.1(+)-beclin 1 group than in control groups (P < 0.05). From 28th day after injection, the tumor weigh was (0.52 ± 0.08) g in pSUPER-beclin 1 group, apparently more than HeLa group (0.37 ± 0.12) g and pSUPER group (0.34 ± 0.24) g (P < 0.05). While in pcDNA3.1(+)-beclin 1 group the tumor weighed (0.18 ± 0.12)g, which was lower than HeLa group and pcDNA3.1(+) group(0.34 ± 0.18) g (P < 0.05). Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.