Construction Of SiRNA Expression Vectors Targeting Stathmin And Its Influence On Biological Behaviour In LM8 Cells

Partâ… Construction and Effect Identification of siRNA ExpressionVectors Targeting Stathmin GeneBackground and purpose:The RNA interference and RNA-mediatedposttranscriptional gene silencing have opened an unanticipated new window onthe regulation of gene expression and a highly effective tool for knockingdown gene expression in many organismsand cells.In addition,RNA interferenceand RNA silencing may conceivably be exploited for human gene therapeutics ofcarcinomas and possibly bring greater clinical impact than other methods.Toconstruct small interfering RNA(siRNA)expressing vector targeting mousestathmin gene and observe the interfering effect in LM8 cell line after thevector transfection.Methods:The specific siRNA sequence was designed according to the stathminsequence of mouse in GenBank.The designed oligos targeting stathmin were cloned into the pGenesil-1 vector which was lineated after BamHâ… and Hindâ…¢digestion.The recombinant vectors were confirmed by enzyme digestionanalysis and DNA sequencing.The recombinant vectors of pGenesil-1-stathmin1 and 2 and pGenesil-1-HK were transfected by liposome-mediated transfectioninto the LM8 osteosarcoma cell line.The stable transfected cells were selectedby G418.There are 5 groups including C1(LM8 control),C2(LM8+ liposome),C3(LM8+pGenesil-1-HK),C4(LM8+pGenesil-1-stathminl)and C5(LM8+pGenesil-1-stathmin2).The expression of stathmin at the levels of mRNA andits protein were detected by real-time quantitative PCR and Westernblotting,respectively.Results:The siRNA expression vectors of pGenesil-1-stathminl andpGenesil-1-stathmin2 were successfully constructed and confirmed by theenzyme digestion analysis and the DNA sequencing.Stathmin mRNAand its proteinafter transfaction were obviously down-regulated.Conclusion:pGenesil-1-stathmin expression vectors targeting stathmin weresuccessfully constructed.The RNAi inhibitory effect on the LM8 cellstransfected by pGenesil-1-stathminl and pGenesil-1-stathmin2 expressionvectors was confirmed,which laid a basis for their application in the treatmentof osteosarcoma.Partâ…¡siRNA Expression Vectors Targeting Stathmin GeneInfluence on Biological Behaviour in LM8 CellsBackground and purpose:Stathmin is expressed at high levels in many kindsof human cancers.Inhibition of stathmin expression in malignant cells results in reduced cellular proliferation and high apoptosis.This study is to observesmall interfering RNA(stathmin siRNA)expressing vector to influence onbiological behaviour in LM8 cell line after the vector transfection.Methods:The designed siRNA expression vector targeting stathmin gene hadbeen constructed in the first part.The effective recombinant vectors ofpGenesil-1-stathmin and the controlled pGenesil-1-HK were transfected byliposome-mediated transfection into the LM8 osteosarcoma cell line.Thecellular growth rates were assayed by Trypan blue staining.The cellular growthinhibiting rates in vitro were assayed by MTT colorimetry.The cellular growthactivities were assayed by vitro clonogenic assays.The morphologicalassessment of apoptosis was studied by Hoechst staining and HE staining.Thecell cycle was analyzed by FCM.C3H mice-transplanted tumors show the formationrate and growth speed of tumours of the three kinds of cells.Results:After the siRNA expression vectors of pGenesil-1-stathmin wassuccessfully transfected,the cellular growth inhibiting rates increasedobviously.The cellular growth activities decreased obviously in soft agarculture.Hoechst and HE staining showed their classic apoptosis characteristicwith higher apoptosis rate.Their percentage of GJM phase of cells wassignificantly higher than those in the negative control group transfected withpGenesil-1-HK and the nontransfected group.The formation rate and growthspeed of C3H mice-transplanted tumor in pGenesil-1-stathmin transfected miceslowed down significantly.Conclusions:The RNAi inhibitory effect on the LM8 cells transfected bypGenesil-1-stathmin expression vectors effectively influenced on itsbiological behaviour and inhibited its growth which laid a basis for itsapplication in the gene therapy of osteosarcoma. Partâ…¢Synergistically Enhancive Effect of Stathmin Genesilencing on Sensitivity of LM8 Cells to DocetaxelBackground and purpose:Stathmin is an important factor that promotesdepolymerization of the microtubules that make up the mitotic spindle andregulate cellular proliferation.Docetaxel is an effective chemotherapeuticdrug that results in more sever mitotic abnormalities through stabilizationof the microtubules of the mitotic spindle and increases apoptosis of cells.This study is to investigate the enhancive effect of small interference RNAtargeting stathmin gene on sensitivity of LM8 cells to Docetaxel.Methods:siRNA eukaryotic express vector targeting stathmin gene wassuccessfully transfected into LM8 osteosarcoma cells.The stable transfectedLM8 osteosarcoma cells have been gained.The controlled LM8 cells andtransfected LM8 cells were induced by Docetaxel and Platinol,respectively.The cellular growth rates were assayed by Trypan blue staining.The cellulargrowth activities were assayed by vitro clonogenic assays.The 50% inhibitingconcentration(I C₅₀)of Docetaxel and Platinol were measured by MTT colorimetry.The cell cycle and apoptosis rate which were influenced by Docetaxel wereanalyzed by FCM.Results:Docetaxel and Platinol with different concentration inhibited twogroups of cellular growth,but it is more obvious for Docetaxel to inhibitgrowth of transfected LM8 cells;Clonogenicity of two groups of cells withDocetaxel and Platinol were reduced,but it is drastical for Docetaxel toreduce clonogenicity of transfected LM8 cells in soft agar culture.The 50%inhibiting concentration(IC₅₀)of Docetaxel and Platinol on transfected LM8cells were lower than IC₅₀on controlled LM8 cells,but reduction of IC₅₀ofDocetaxel was more obvious.Their percentage of G₂/M phase and the rate of apoptosis of transfected cells which were induced by Docetaxel were higher.Conclusion:Stathmin gene silencing could improve sensitivity of LM8 cellsto Docetaxel and synergistically induced cellular apoptosis.This novelcombination may provide an attractive therapeutic strategy.