靶向Hsp90β的siRNA对Jurkat细胞生长的抑制作用

Inhibitory effect of small interfering RNA targeting heat shock protein 90β on growth of Jurkat cells

目的探讨RNA干扰技术沉默热休克蛋白90β(heat shock protein90beta,Hsp90β)基因表达对Jurkat细胞生长的抑制作用。方法根据Hsp90β基因全长cDNA序列Hsp90B1(NM_003299.1),设计并构建针对Hsp90β基因的siRNA真核表达载体,应用LipofectamineTMLTX转染入Jurkat细胞后,Real-time PCR检测Hsp90βmRNA水平的变化,筛选出沉默效果最好的Hsp90βsiRNA干扰片段;Western印迹法检测Hsp90β蛋白表达水平;MTT法和流式细胞仪检测干扰前后对Jurkat细胞生长抑制作用。结果成功构建Hsp90β基因特异性的真核表达载体pSOS-Hsp90βi,转染Jurkat细胞后发现pSOS-Hsp90βi2的沉默效果最明显,Jurkat细胞Hsp90βmRNA及蛋白表达均明显降低(P<0.05)。Hsp90β基因表达沉默后,Jurkat细胞增殖明显受抑(P<0.01),实验组与对照组的细胞凋亡率分别为:(2.69±0.34)%、(3.63±0.38)%、(19.56±0.62)%,细胞凋亡率比空白对照组及转染pSOS-Hsp90βi control组明显增加,差异具有显著意义(P<0.05)。结论靶向Hsp90β的siRNA下调Hsp90β表达对人白血病Jurkat细胞有明显的生长抑制作用。

Objective To study the inhibitory effect of small interfering RNA （siRNA） targeting heat shock protein 90 beta （Hsp90β） gene on growth of Jurkat cells. Methods Three specific interference sequences and a random controlled sequence were inserted into the pSOS-HUS according to the cDNA sequence Hsp90B1（NM_003299.1）of Hsp90β gene. Recombinant eukaryotic expression plasmids,including pSOS-Hsp90βi1,pSOS-Hsp90βi2,pSOS-Hsp90βi3 and pSOS-Hsp90βi control,were constructed and transfected into Jurkat cells by LipofectamineTM LTX. Gene silencing efficiency of recombinant plasmid pSOS-Hsp90βi was monitored by real-time PCR and effective Hsp90β-specific RNAi sequences were screened. Hsp90β expression level was measured by Western blotting. Inhibitory effect of Hsp90β gene on growth of Jurkat cells was detected by MTT assay and flow cytometry,respectively,before and after interfered by siRNA. Results The recombinant plasmid pSOS-Hsp90βi1,2,3 eukaryotic vector targeting Hsp90β was successfully constructed. The expression of Hsp90β in Jurkat cells transfected with pSOS-Hsp90βi2 was significantly inhibited at both mRNA and protein levels （P0.05）. MTT assay and flow cytometry showed that Hsp90βsiRNA inhibited the proliferation of Jurkat cell （P0.01） 19.56% of Jurkat cells apoptosis were also observed （P0.05）. Conclusion Chemically synthesized specific siRNA targeting Hsp90β can effectively reduce the expression of Hsp90β gene and growth of leukemia Jurkat cells.