[Recombinant adenovirus expressing siRNA is generated to inhibit the expression of RARbeta in rat mesenchymal stem cells treated by all-trans retinoic acid].
To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-beta (RARbeta) gene, and to detect its effect on RARbeta expression and neuronal differentiation of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSCs). First, we designed four pairs of siRNA sequence for rat RARbeta gene and annealed complementary oligonucleotides in vitro, then cloned double-stranded DNA in pSES-HUS vector containing U6/H1 double-promoter and recombinated with the backbone vector to construct pAd-siRARbeta plasmid. We infected MSCs by using adenovirus Ad-siRARbeta which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARbeta. We used combination of ATRA and MNM to induce MSCs into neural-like cells, then performed Real-time PCR and immunoflourencence to detect neuronal specific markers of induced neural cells. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in adenovirus vector. We could observe more than 60% RFP-positive MSCs at 24 h after adenovirus infection. The expression of RARbeta was significantly increased to 16.5 +/- 2.34 fold in ATRA treated MSCs (P < 0.05) and located in nucleus. Three of four pairs siRNA could effectively inhibit the expression of RARbeta with inhibition efficacy of (66.26 +/- 9.12)%, (48.70 +/- 5.78)%, (64.09 +/- 0.53)% (P < 0.05), especially siRNA-pool group with inhibition efficacy of (78.09 +/- 4.24)% (P < 0.01). Combination of ATRA and MNM induced MSCs into neural-like cells which expressed neuronal specific markers, Nestin, NSE, MAP-2, and Tau. Immunoflourencence result showed that about 50-88 present of cells were positive for Nestin, NSE, Tjul, however, adenovirus medicated expression of siRARbeta could effectively inhibit the expression level of neural specific proteins and the ratio of positive stained cells (P < 0.05). Therefore, we successfully constructed the recombinant adenovirus vector containing siRNA for rat RARP gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARbeta in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.
Key Laboratory of Developmental Diseases in Childhood of Ministry of Education, Children's Nutritional Research Center, the Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
Yang
Y
Bi
Min
M
Gong
Yun
Y
He
Yun
Y
Zhang
Jie
J
Chen
Tingyu
T
Li
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D015536:Down-Regulation; D057809:HEK293 Cells; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*
D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082925:retinoic acid receptor beta; D014212:Tretinoin