[Effects of short hair RNA on the expression of SMN1 gene in human mesenchymal stem cells].
OBJECTIVE
To construct a plasmid expressing short hairpin RNA (shRNA) targeting SMN1 gene in mammalian cells and to investigate the effect of shRNA on the expression of SMN1 gene in human mesenchymal stem cells (hMSCs), thus providing experimental basis for the establishment of SMA cell model.


METHODS
Five shRNAs targeting SMN1 gene was designed according to the SMN1 cDNA sequence provided by GenBank, and cloned to the downstream of U6.1 promoter in the pRNAT-U6.1/Neo vector, thus forming different recombinant plasmids: pshRNA-SMN1, pshRNA-SMN2, pshRNA-SMN3, pshRNA-SMN4, and pshRNA-SMN5 expressing specific shRNAs targeting SMN1 gene. The plasmid psh-RNA-0 was used as controls. The recombinant plasmid pshRNA-SMN1 was transfected into human mesenchymal stem cells (hMSCs) by Lipofectomine (TM) 2000, and its effects on the mRNA expression and protein expression of fl-SMN were observed by RT-PCR and Western blotting.


RESULTS
The successful construction of recombinant plasmid pshRNA-SMN was identified by enzyme cutting and DNA sequencing. After transfection, the expression levels of fl-SMN mRNA and protein in hMSCs of the pshRNA-SMN1-1 group and pshRNA-SMN1-4 group were significantly lower than that of the control group (both P < 0.05) with an interfering efficiency of 64.05% and 61.04% in mRNA level, and 52.97% and 61.57% in protein level respectively. However, the expression of Delta7-SMN mRNA was not effected.


CONCLUSIONS
The constructed recombinant plasmid called pshRNA-SMN1 inhibits the expression of fl-SMN mRNA and protein in hMSCs efficiently.
Yang
Xiao-su
XS
Department of Neurology, Xiangya Hospital, Zhongnan University, Changsha 410008, China.
Hu
Yi-min
YM
Xiao
Bo
B
Yang
Qi-dong
QD
Xie
Ying
Y
D015153:Blotting, Western; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein