[Effects of small interfering RNA (siRNA) against Par-4 gene on the apoptosis of human bone marrow mesenchymal stem cells].
OBJECTIVE
The prostate apoptosis response factor-4 (Par-4) gene was originally identified by differential screening for genes that are up-regulated when prostate cells are induced to undergo apoptosis. Par-4 was found to possess potent apoptotic activity in various cellular systems in response to numerous stimuli. The aim of this study was to explore the effects of small interfering RNA (siRNA) against Par-4 gene on the apoptosis of human bone marrow mesenchymal stem cells (hBMSCs) exposed to glutamate.


METHODS
Primary culture of hBMSCs was carried out and siRNAs targeted Par-4 gene (Par-4-SiRNA) were chemically synthesized. Eukaryocytic expression vector was built and were transfected into hBMSCs with liposome. After selecting with G418, the stable cell clones were treated with glutamate. The expression of Par-4 mRNA was determined by real-time PCR. The apoptosis of hBMSCs was quantified by flow cytometry. Western blotting was used to detect the protein levels of phosphorylated Akt1 (Thr308). Relative Caspase-3 activity was determined by colorimetric assay.


RESULTS
The Par-4-SiRNA-1 and Par-4-siRNA-2 could markedly down-regulate the mRNA levels of Par-4 gene in hBMSCs. With the transfections of Par-4-SiRNA-1 and Par-4-SiRNA-2, the levels of Par-4 mRNA were respectively decreased by 88% and 67%. Both Par-4-SiRNA-1 and Par-4-SiRNA-2 inhibited significantly the apoptosis of hBMSCs induced by glutamate, in which the percentages of apoptotic cells were respectively decreased to 38.80% +/- 3.97% (P < 0.01) and 45.49% +/- 4.32% (P < 0.01) from 60.30% +/- 6.82%. Western blot assays demonstrated that, glutamate down-regulated the expression of phosphorylated Akt1 proteins in hBMSCs (89.07 +/- 6.42 and 28.30 +/- 5.65, respectively, P < 0.01). However, Par-4-SiRNA-1 and Par-4-SiRNA-2 could markedly recover the down-regulation of Akt1 proteins induced by glutamate (63.56 +/- 6.75 and 45.59 +/- 4.88, respectively, P < 0.01). And the relative Caspase-3 activity which was enhanced by the treatment with glutamate (0.1428 +/- 0.0495 and 0.8616 +/- 0.1051, P < 0.01), was suppressed by Par-4-SiRNA-1 and Par-4-SiRNA-2 (0.8616 +/- 0.1051 and 0.6581 +/- 0.0555, respectively, P < 0.01).


CONCLUSIONS
SiRNA against Par-4 gene could inhibit the apoptosis of hBMSCs induced by glutamate, and its inhibitory effects may be mediated by the up-regulation of phosphorylated Akt1 and the suppression of the relative Caspase-3 activity.
Lu
Chao
C
Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
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 / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering
D051017:Apoptosis Regulatory Proteins; D034741:RNA, Small Interfering; C101296:prostate apoptosis response-4 protein; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; D053148:Caspase 3