Beclin 1-mediated macroautophagy involves regulation of caspase-9 expression in cervical cancer HeLa cells.
OBJECTIVE
To investigate the role of Beclin 1 in HeLa cells and to obtain further insight into the relationship between autophagy and apoptosis.


METHODS
Beclin 1 silencing was achieved using RNA interference. The expression of gene was measured using quantitative real time RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. The ultrastructural analysis was under the electron microscope.


RESULTS
In pSUPER-Bec transfectants (Beclin 1 gene partially silenced) the expression of mRNA and protein of Beclin 1 were significantly suppressed in comparison to pSUPER-non (scramble RNA control) or untreated cells in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in pSUPER-Bec transfectants compared with pSUPER-non transfectants. Meanwhile pcDNA3.1-Bec transfectants (Beclin 1 gene overexpressed) showed reduction of cell proliferation but augmentation of cell programmed death compared with vector vehicle. The autophagy-promoting activity of beclin 1 in HeLa cells is associated with inhibition of HeLa cellular proliferation, in vivo tumorigenesis in nude mice. The expression pattern of caspase-9 was extraordinarily similar to that of Beclin 1in siRNA against Beclin 1 transfectants and constructive expression of Beclin 1transfectants.


CONCLUSIONS
siRNA against Beclin 1 transfectants promoted the cell proliferation but overexpression of Beclin 1 promoted the autophagy cell death, and in the process of autophagy triggered by Beclin 1 expression followed accordingly the regulation of the expression of caspase-9. We conjecture that the autophagy gene Beclin 1 may be the critical molecular switch that plays an important role in fine tuning the autophagy and apoptosis through caspase-9, and defection of autophagy or apoptosis may be an important mechanism in tumorigenesis.
Wang
Zan-hong
ZH
Department of Obstetrics and Gynecoligy, West China Second Hospital, Sichuan University, Chendu, Sichuan, 610041, China.
Xu
Li
L
Duan
Zhen-ling
ZL
Zeng
Li-qin
LQ
Yan
Nai-hong
NH
Peng
Zhi-lan
ZL
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D001343:Autophagy; D000071186:Beclin-1; D053453:Caspase 9 / Q000235:genetics*; D002453:Cell Cycle; D005260:Female; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014162:Transfection; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms / Q000235:genetics*
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D053453:Caspase 9