Inhibition of acid-induced apoptosis by targeting ASIC1a mRNA with short hairpin RNA.
OBJECTIVE
To study the role of acid-sensing ion channel (ASIC) 1a in the cell death and apoptosis induced by extracellular acid in C6 glioma cells.


METHODS
The stable ASIC1a-silenced C6 cell line, built with RNA interference technology, were confirmed by RT-PCR and Western blot analysis. The cell viability following acid exposure was analyzed with lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptotic cells dyed with Annexin-V and propidium iodide were measured with a flow cytometer, while the changes of cell cycle were also assayed.


RESULTS
The downregulation of ASIC1a proteins by stable transfection of short hairpin RNA decreased the cell death percentage and increased cell viability following acid exposure with LDH and the MTT assay. The rate of apoptosis was lower in the ASIC1a-silenced cell line than that in the wild-type C6 cell line. The percentage of sub-G0 cells was lower in the ASIC1a-silenced C6 cells than that in the wild-type cells.


CONCLUSIONS
Extracellular acid induced cell death and apoptosis via ASIC1a mechanisms in the C6 glioma cells.
Weng
Xie-Chuan
XC
Beijing Institute of Pharmacology and Toxicology, the Academy of Military Medical Sciences, Beijing 100850, China. wengxc2002@yahoo.com.cn.
Zheng
Jian-Quan
JQ
Jin
Qing-E
QE
Ma
Xiao-Yun
XY
D062565:Acid Sensing Ion Channels; D000143:Acids / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D005910:Glioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006863:Hydrogen-Ion Concentration; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D062565:Acid Sensing Ion Channels; D000143:Acids; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015222:Sodium Channels