Effect of HSP90α RNA interference on biological behaviour of liver cancer cell line HepG2

Objective To establish a cell line stably inhibiting the heat shock protein 90α(HSP90α)expression by siRNA interference and to explore the effect of HSP90α siRNA on proliferation and cell cycle of liver cancer cell line HepG2. Methods The recombinant plasmid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90α was subcloned,purified and identified by DNA sequence analysis.Then the recombinant plasmid pSilencerHSP90 was introduced into HepG2 cell by electroporation.After G418 selection,positive clones were isolated and cultured to form a cell line stably inhibiting HSP90α expression(siRNA interference group).The non-transfected HepG2 cells were chosen as control group.The positive clones were identified by quantitative RT-PCR and Western blot.CCK-8 assay and flow cytometry(FCM) were applied to determine the cell growth and cell cycle,respectively. Results The sequence of specific siRNA was correct by sequence analysis.The levels of HSP90α mRNA and protein expression were reduced in siRNA interference group.The CCK-8 assay results showed that the cell growth in siRNA interference group was significantly inhibited after 48 h compared with control group.The FCM results showed that there was an obvious G2/M phase arrest in siRNA interference group. Conclusion The cell lines stably low expressing HSP90α is successfully established.The decreased expression of HSP90αgene can inhibit the cell proliferation,and cause the cell cycle arrest.Successful cloning of the recombinant plasmid may help to find a new gene therapy for hepatoma.