The Effect Of HSP90Î' Gene Silencing By SIRNA On The Growth Inhibition And Chemosensitivity Of JURKAT Cells

Objective To investigate the effect of Hsp90β(Heat shock protein 90 beta) gene silencing by small interfering RNA (siRNA) on the growth inhibition and chemosensitivity of Jurkat cells.Methods 1.The cell line overexpressing Hsp90βgene was distinguished by means of Real-Time PCR from the cells of Jurkat, Raji, K562 and HL-60. 2.According to Hsp90βgene cDNA sequence Hsp90B1(NM₀03299.1), three specific interference sequences and a random controlled sequence were inserted into the pSOS-HUS, respectively. The recombinant eukaryotic expression plasmids pSOS-Hsp90βi1, pSOS-Hsp90βi2, pSOS-Hsp90βi3 and pSOS-Hsp90βicontrol were constructed and transfected into Jurkat cells by Lipofectamine ? LTX with PLUS ? Reagent. The gene silencing efficiency of recombinant plasmid pSOS-Hsp90βi was monitored by real-time PCR and effective Hsp90β-specific RNAi sequences were screened as well. Western blotting was used to detect the protein expression of Hsp90β. 3.The changes in Hsp90βsiRNA, the cell growth and apoptotic rate were determined by MTT assays and flow cytometry. 4. The gradient concentrations of Vincristine, Adriamycin and Etopodide were applied respectively in the cell culture medium of Jurkat before and after the Hsp90βsiRNA transfection. The cell sensitivity to chemotherapy drugs was measure by MTT.Results 1. Real-Time PCR results revealed that Jurkat cell had a highest expression level of Hsp90βof four leukemia cell lines (Raji, K562, HL-60), the expression difference was statistically significant (P<0.05); 2. Recombinant plasmid pSOS-Hsp90βi1, 2, 3, eukaryotic vector targeting Hsp90βwas successfully constructed. The expression of Hsp90βin the cells transfected with pSOS-Hsp90βi2 were inhibited significantly at both mRNA and protein levels (P<0.05). 3. MTT assays and flow cytometry showed that Hsp90βsiRNA inhibited the proliferation of Jurkat cells and induced apoptosis of leukemia cells. 4. pSOS-Hsp90βi2 transfection can increase the sensibility of Jurkat cells to anticancer drugs (for example, VCR, ADM and VP16). The IC50 of drugs were lower than that of two control groups (p<0.05).Conclusion Hsp90βis expressed higher in Jurkat cells than that in other leukemia cell lines(Raji, K562, HL-60); The successfully constructed eukaryotic expression vector pSOS-Hsp90βsiRNA. Highly interference pSOS-Hsp90βi2 was screen; The chemically synthesized specific siRNA targeting Hsp90βcould effectively reduce the expression of Hsp90βgene, inhibit growth and increase the sensitivity of Jurkat cells to VCR, ADM and VP16. In conclusion, it laid a good foundation for further prepare a condition for targeted gene therapy in leukemia.