Construction of stable expression of siRNA-STAT3 gene in Hep-2 cell line

OBJECTIVE To construct Hep-2 cell line with stable transfection of siRNA-STAT3 gene, and to explore the relationship between STAT3 expressionand the pathogenesis of laryngeal cancer. METHODS Specific STAT3 oligonucleotides were designed and synthesized. These oligonucleotides were annealed form the double strands DNA fragments. Then the fragments were cloned into pGPU6/GFP/Neo vector. The recombinant PGPU6/GFP/Neo-siRNA-STAT3 plasmid was confirmed by enzyme digestion and sequence analysis at the same time. Positive clones were selected out by G418 and p-STAT3 expression was identified by Western-blot analysis. The growth curve of the Hep-2 cells was drawn based on MTT assay and the growth ability of single Hep-2 cell was measured according to the plate colony formation assay respectively. RESULTS PGPU6/GFP/NEO-siRNA-STAT3 expression vector was successfully constructed. Western-blot analysis identified that p-STAT3 expression declined obviously in siRNA- STAT3 group compared with that in negative control and blank group. The growth curve explained that the Hep-2 cells of siRNA-STAT3 group began to show obvious inhibitory effect until they were inoculated in the culture plate for 3 days(P =0.001).There were pronounced inhibitory effect after 5 days(P=0.000). The results also showed time-effect relationship of the inhibition. Furthermore, the cell colony formation rate of siRNA- STAT3 group was less than the negative control and blank group(P =0.000). CONCLUSION We successfully selected out Hep-2 cell line expressed siRNA-STAT3 gene stably and efficiently in this study. Down regulation of STAT3 correlated with the inhibition of growth of Hep-2 cells.