The Experimental Research On Silencing Of HSP70Gene By RNA Interference In Laryngeal Carcinoma

Object: Explore the effects of RNA interference (RNAi) to HSP70on the biologicalactivity of human Laryngeal carcinoma cell line Hep-2.Methods: The expressions of HSP70and HSF1proteins were detected by theimmunohistochemical SP method in6specimens with leukoplakia of laryngeal(leukoplakiaof laryngeal group)，6specimens with laryngeal papilloma (laryngeal papilloma group) and7specimens of vocal cord polyp (control group). To detect the expression of HSP70inHep-2cells by immunofluorescence stain; The HSP70gene siRNA eukaryotic expressionvector was constructed, The pHSP70-shRNA vector was transfected into Hep-2cells line bycationic liposome method. Using flow cytometry to detect the distribution of Hep-2cellcycle, RT-PCR，immunofluorescence and other techniques study of siRNA silencing ofHSP70expression on the downstream genes CyclinD1, C-myc, Gadd45and P21expressioninfluence.Results:1.Immunohistochemical results show, the positive expression of HSP70wasbrown granules which located in cytoplasm and nucleus. The expression of HSP70invocal cord polyp was low. It mainly located in the nuclear. The expression of HSP70inlaryngeal papilloma and vocal leukoplakia were strong. H-score method for semiquantitative results show： The expression of HSP70in laryngeal papilloma group（4.25±0.53）increased significantly than in vocal cord polyp group（3.63±0.58），Theexpression of HSP70in vocal leukoplakia group（5.43±0.40）were the strongest，thedifferences were significantly（P<0.01）. The positive expression of HSF1mostly localized incytoplasm，a small mount of it located in the nucleus. The trend of HSF1expression issimilarto the HSP70. Linear regression analysis of HSP70protein and HSF1expressions invocal cord polyp,laryngeal papilloma and leukoplakia of laryngeal showed positive linearcorrelation (r=0.867, P <0.01).2. Immunohistochemical results show， HSP70showed high expression in Hep-2cellsand was significantly higher in nasopharyngeal epithelial immortalized cell line NP-69.3. Flow cytometric detection results show，transfection of recombinant plasmid,the proliferation of Hep-2cell is inhibited and the apoptosis is increased, the percentage of G1phase increased, while the percentage of G2phase decreased obviously.4. Immunohistochemcal results show，the expression of HSP70in experimental group(pHSP70-shRNA plasmid) than the control group (pRNAT-U6.1/Neo control plasmids)was decreased, accompanied by the expression of downstream genes C-myc, CyclinD1,Gadd45decreased.5.Realtime PCR results show，the expression of HSP70and CyclinD1mRNA intransfection group was significantly lower than that in the control group, while theexpression of P21mRNA was significantly higher than that of the control group.Theexpression of CyclinD1mRNA in transfection group was down-regulated and the expressionof P21mRNA was upregulated,which may associate the gene silencing of HSP70.Conclusion:1.There is over expression of HSP70in epithelial laryngeal cancer tissue，which may contribute to the genesis of human laryngeal squamous cell carcinoma.2.Recombinant plasmid pHSP70-shRNA down-regulation of HSP70protein inhibits theproliferation of human laryngeal carcinoma Hep-2cells and induces the apoptosis of Hep-2cells. The knockdown of HSP70in turn caused the changes in gene expression related withcell proliferation and apoptosis，which may promote laryngeal carcinoma cells apoptosis.