[Construction of shRNA expression vectors for autophagy gene beclin 1 and their downregulation effect on caspase-9].
The shRNA expression vectors were constructed and transfected via lipofectamine into HeLa cells. Real time-ploymerase chain reaction (RT-PCR) and Western blot were used for detecting the expression of mRNA and protein of Beclin1 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis and cell cycle of HeLa, and proliferation was analyzed by MTT assay. The expression of caspase-9 in transfection cells was also detected by RT-PCR and Western blot. The constructed vectors significantly inhibited the expressin of mRNA and the protein of Beclin1 in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in these cells. After transfection of the constructed vectors into HeLa cells, the expression of caspase-9 was effectively inhibited. All of these indicate that autophagy and apoptosis are two types of programmed cell death, that autophagy gene Beclin 1 plays an important role in these two types, and that defect of autophagy and apoptosis may be important in tumor genesis.
Wang
Zanhong
Z
Department of Obstetrics and Gynecology, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Peng
Zhilan
Z
Duan
Zhenling
Z
Yan
Naihong
N
D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000235:genetics*; D000071186:Beclin-1; D053453:Caspase 9 / Q000378:metabolism*; D015536:Down-Regulation; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053453:Caspase 9