OBJECTIVE:　The　aim　of　this　study　was　to　construct　a　eukaryotic　expression　plasmid　with　a　short　hairpin　RNA　(shRNA)　targeting　Livin　in　order　to　obtain　a　stably　transfected　Hep-2　cell　line　with　a　reduced　expression　of　Livin.　METHODS:　The　shRNA　targeting　Livin　mRNA　was　designed,　and　a　shRNA　plasmid　and　a　negative　control　plasmid　were　constructed.　After　amplification　in　E.　coli,　restriction　endonuclease　digestion　and　sequence　confirmation,　the　plasmids　were　transfected　into　Hep-2　cells　using　Lipofectamine　2000.　The　stably　transfected　cell　line　was　screened　using　G418,　and　inhibition　of　Livin　mRNA　and　protein　levels　were　detected　using　real-time　PCR　and　western　blotting,　respectively.　RESULTS:　pGenesil-Livin-shRNA　eukaryotic　expression　plasmid　was　successfully　constructed　and　identified　by　sequencing.　Green　fluorescent　protein　(GFP)　expression　was　observed　in　Hep-2　cells　transfected　with　shRNA　plasmids　by　fluorescence　microscopy.　The　expression　levels　of　Livin　mRNA　and　protein　decreased　significantly　in　Hep-2　cells　transfected　with　the　shRNA　recombinant　plasmid.　The　mRNA　level　was　reduced　by　47.17　%,　and　the　protein　level　was　reduced　by　34.25　%.　CONCLUSION:　The　shRNAeukaryotic　expression　plasmid　targeting　Livin　was　successfully　constructed,　which　could　significantly　inhibit　the　expression　of　Livin　in　laryngeal　cancer　Hep-2　cells.　This　provides　a　basis　for　future　research　on　the　function　of　Livin　in　Hep-2　cells,　and　gene　therapy　for　laryngeal　cancer　(Fig.　5,　Ref.　10).　Text　in　PDF　www.elis.sk.