RNA interference targeting of sphingomyelin phosphodiesterase 1 protects human granulosa cells from apoptosis.
OBJECTIVE
Sphingomyelin phosphodiesterase 1 (SMPD1) plays an essential role in initiating the female germ cell death signal. To evaluate whether RNA interference has potential as a new approach in germ cell protection, we tested the effect of SMPD1 knockdown on human granulosa cells in vitro.


METHODS
We designed and synthesized three small interference RNA (siRNA) sequences targeted on SMPD1 and transfected them into human luteinizing granulosa cells (hGC) in vitro. Forty-eight hours after transfecting with siRNAs, hGC were treated with mitomycin C (MMC) to induce apoptosis. mRNA was detected with quantitative RT-PCR and protein was detected with Western blot. Methyl thiazolyl tetrazolium (MTT) assay was used to measure cell survival rate and detection of apoptotic rate of cells with Annexin V-PI staining by flow cytometer (FCM). Study groups were compared with liposome (lipofectamine 2000), MMC control and negative control siRNA.


RESULTS
After treatment with siRNA targeted to SMPD1, significant SMPD1 suppression occurred. After knockdown expression of SMPD1, the survival rate of hGC increased from 32.3% to 40.3%, and the apoptosis rate decreased from 68.3% to 44%.


CONCLUSIONS
siRNA targeted on SMPD1 can protect hGC cells from apoptosis. These results reveal SMPD1 as a significant and effective target site for RNAi in the protection of human germ cells, which may have a direct bearing on future therapeutic research.
Gao
Jun
J
Reproductive Center, The First Affiliated Hospital, Guangzhou, China.
Zhang
Ren-Li
RL
Zhou
Can-Quan
CQ
Ma
Yun
Y
Zhuang
Guang-Lun
GL
D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002478:Cells, Cultured; D015536:Down-Regulation; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D034741:RNA, Small Interfering; D016685:Mitomycin; C543066:SMPD1 protein, human; D013108:Sphingomyelin Phosphodiesterase