Short-interfering RNA-mediated silencing of proliferating cell nuclear antigen inhibit proliferation and induce apoptosis in HeLa cells.
Proliferating cell nuclear antigen (PCNA) is an important protein for DNA polymerase delta in the nucleus, and shown to have a fundamental role in cellular proliferation. It is overexpressed to support cell growth in cervical carcinoma. To study its role in stress response, we design and use short hairpin RNA (shRNA) to inhibit PCNA expression in HeLa cells and validate its effect on cell proliferation. In this study, three PCNA-shRNA expression vectors are constructed and introduced into HeLa cells, and the cell cycle is analyzed by flow cytometry. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay), and caspase cleavage is studied also. Expression of PCNA is assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it is found that expression of PCNA decreased in shRNA-transfected cells, downregulation of PCNA inhibit cell growth and induce apoptosis in HeLa cells. PCNA downregulation also increase cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in HeLa cells effectively and, therefore, could be used as a new potential anticancer tool for therapy of human cervical carcinoma.
Hao
H
H
Department of Pathogenic Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Xin
T
T
Nancai
Y
Y
Yanxia
W
W
Qian
L
L
Wei
M
M
Yandong
Y
Y
Hanju
H
H
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D049109:Cell Proliferation; D020552:Comet Assay; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference / Q000187:drug effects*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering