[Effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells].
OBJECTIVE
To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs).


METHODS
Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs (Par-4-siRNA-1 and -2) targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups: non-transfected hBMSCs (normal control group), blank Pae-4 plasmid transfected hBMSCs (Par4 control group), Par4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups: non-transfected normal hBMSCs, glutamate (an apoptosis inducer) + non-transfected hBMSC group, glutamine + Par-4-siRNA-1 hBMSC group, and glutamate + Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression.


RESULTS
The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SiENA-2 hBMSCs, and Par-4 control hBMSCs were 0.12 +/- 0.03, 0.33 +/- 0.09, and 0.97 +/- 0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate + Par-4-siRNA-1 hBMSCs was (37.2 +/- 6.3)%, significantly lower than that of the glutamate + non-transfected hBMSC group [(58.9 +/- 8. 9)%, F = 58.26, P < 0.01). The Smac protein expression level of the glutamate + non-transfected hBMSC group was significantly higher than that of the normal control group (P < 0.01); however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected hBMSC group (P < 0.01). The caspase-3 activity of the glutamate + Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected BMSC group (P < 0.01).


CONCLUSIONS
Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.
Lu
Chao
C
Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. luchao8009@163.com
Chen
Ji-Qing
JQ
Zhou
Guo-Ping
GP
Wu
Sheng-Hua
SH
Guan
Ya-Fei
YF
Yuan
Chuan-Shun
CS
Huang
Song-Ming
SM
Guo
Xi-Rong
XR
Chen
Rong-Hua
RH
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055815:Young Adult
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C101296:prostate apoptosis response-4 protein; D053148:Caspase 3