Objective       To construct the adenovirus vector containing small interfering RNA (siRNA) targeted against human inositol-requir

ing enzyme 1α (IRE1α) gene

and determine its effect on the proliferation and apoptosis of mouse C2C12 myoblasts C2C12 under endoplasmic reticulum (ER) stress.       Methods       The siRNA sequence targeting IRE1α gene was synthesized and cloned into the shuttle plasmid pSES-HUS to generate the vector pSES-HUS-IRE1α siRNA

which was later homogenously recombined with the adenovirus backbone plasmid pAdEasy-l in

E. coli

BJ5183. Then the recombinant adenovirus was transfected into the 293 packing cells by lipofectamine-mediated transfection to amplify the recombinant adenovirus Ad-IRE1α siRNA. The C2C12 cells were infected with this adenovirus

and the expression of IRE1α at mRNA and protein levels were detected by RT-PCR and Western blotting respectively. The effects of the recombinant adenovirus on the proliferation and apoptosis of C2C12 after the treatment of ER stress inducer Tm were detected by flow cytometry and MTT assay. The normal control

and the cells treated by the Tm

Tm+Ad-RFP served as control. The expression levels of cleaved Caspase-3 and chop were detected by Western blotting.       Results       The recombinant adenovirus Ad-IRE1α siRNA with high titer of 4.3×10

11

PFU/mL was successfully obtained. Both the IRE1α mRNA and protein levels were significantly decreased in the C2C12 cells after 48 h of infection with Ad-IRE1α siRNA. Flow cytometry showed that in the stress condition the C2C12 cells infected by Ad-IRE1α siRNA had more cells at S phase than the cells treated by Tm and Tm+Ad-RFP (increased by 12.62% and 14.80% respectively

P

<

0.05)

and the apoptotic rate of the Ad-IRE1α siRNA group was decreased by 16.64% and 16.26% (

P

<

0.05) when compared with the 2 control groups. MTT assay and Western blotting results of cleaved Caspase-3 and chop were in accordance with those results shown by FCM.       Conclusion       The recombinant adenovirus Ad-IRE1α siRNA significantly decreases the expression of IRE1α at mRNA and protein levels in C2C12 cells. Infecti

ng C2C12 cells with Ad-IRE1α siRNA promotes the proliferation and suppresses the apoptosis in the cells under the stress condition.