Inhibition of the expression of Survivin in human osteosarcoma cell line MG63 by specific antisense RNA / 中国矫形外科杂志

ABSTRACT

[Objective]To investtgate the specific inhibition of Survivin gene expression by antisense RNA in human osteosarcoma cell line MG63.[Method]Total RNA was extracted from osteosarcoma cell line MG63 cells using Trizol reagent.Coding sequence of Survivin was amplified from MG63 mRNA by RT-PCR and then cloned into inducible eukaryotic vector pMDNA3.After the reducible smmvm antisense vector was construted,MG63 cells were transfected with control pMDNA3 vector or pMDNA3-anti-Survivin and selected by G418.Both of the control and Survivin antisense transfectants were treated with ZnSO_4.These cells cultured on cover slips were observed through immunohisto-chemistry staining,HE staining and electrn microscopy.At the same time,the above cells were cultured and the numbers were counted every other day,thus growth curve was drawn.Apoptosis was analyzed by Annexin V stammg.[Result]cDNA coding Survivin about 420 bp was generated by reverse transcription-PCR Survivin PCR product was cloned reversal into pMDNA3 vector.The MG63 stable tansfectants with either control vector pMDNA3,or pMDNA3-anti-Survivin were established.ZnSO_4 induction of Survivin antistense RNA suppressed the expression of endogenous Survivin mRNA.The cell growth curve showed MG63/pMDNA3-anti-survivm with ZnSO_4 induction proliferated slowly than other kinds of cells(P