Down-regulation Of STAT3 Expression Using Vector-based SiRNA Suppresses Growth Of Human Lung

Objective: To develop an RNAi approach that specificallytargets the STAT3 gene sequence by short interfering RNA (siRNA), andto assess the inhibitory effect of pSUPER–siRNA–STAT3 on thegrowth of A549 cells. Methods: A siRNA targeting STAT3 genesequence were constructed based on pSUPER vector, the sequence weredigested with the restriction endonucleases BglII and HindIII, then it wascloned into pSUPER which contain RNA polymerase Ⅲ to constructpSUPER–siRNA–STAT3. The eukaryotic expression plasmid werecotransfected into A549 cells with either the RNAi plasmid pSUPER–siRNA–STAT3 or control plasmid to assess the inhibitory effect ofRNAi on STAT3 gene expression. At 24h,48h,72h post transfection, thelevel of STAT3 mRNA was detected by RT-PCR and cells viability byfluorescence microscope. Results: The siRNA eukaryotic expressionvector against STAT3 mRNA has been successfully conctructed, whichwas transfected into A549 cells. The IeveI of STAT3 mRNA in A549cells was inhibited by the specific siRNAs.The decrease of STAT3mRNA expression began to appear 24 hours after transfection.And themost apparent interfering efficiency was 85.32%and 75%,48h aftertransfection,which was markedly higher than that in the cells transfectedwith the controI siRNAs.Both siRNAs from different Ioci had interferingeffect on STAT3 mRNA expression,Compared with those controlcells ,the apoptotic cells were significantly higher in siRNA transfectedcells by fluorescence microscope. Conclusion: siRNA transcriptingplasmids pSUPER are successfully constructed.pSUPER–siRNA–STAT3 could significantly inhibit STAT3 expression,suppress the growthof A549 cells. The RNA interfering technique targeted on STAT3 mayProvide a new method in the gene therapy of lung cancer.