Stress response changes of NIH-3T3 cells with HSP90alpha expression inhibition by RNA interference / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish a heat shock protein 90alpha (HSP90alpha) expression-inhibited cell line and study the effect of lowered HSP90alpha level on cell stress response.</p><p><b>METHODS</b>The recombinant plasimid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90alpha was subcloned, purified and identified by DNA sequence analysis before introduced into mouse fibroblast cell line NIH-3T3 by electroporation. After G418 selection, the positive clones were identified by immunofluorescence and Western blotting. NIH-3T3 cells were subjected to hyperthermia at 44 degrees C for 40 min to simulate oxidative stress, and flow cytometry was performed to analyze the effect of low-level HSP90 on DNA damage under stress condition.</p><p><b>RESULTS</b>Immunofluorescence and Westen blotting showed lowered HSP90 levels in the transfected cells. Compared with the control cells, cells subjected to hyperthermia displayed intensified DNA damage.</p><p><b>CONCLUSION</b>Low-level HSP90alpha causes the cells to be more vulnerable to oxidative stress condition, and HSP90 content can be associated with cell protection against such condition.</p>