AR Mediates The Mechanism By Which Hsp70-1 Regulates C-Fos In Prostate Cancer Cells

Prostate cancer is one of the most common cancers in men and the second leading cause of cancer related deaths in the North America.In China it is estimated that the incidence will be the third in male urogenital system malignant tumor in the near future.According to incomplete statistics,there are 66 million people worldwide are diagnosed with prostate cancer each year and about 25 million people die from this disease every year.Heat shock protein(Hsp)70-1 is overexpressed in many types tumor and correlated with tumor malignancy,progression,poor prognosis and metastasis.So,the study of Hsp70-1 is more and more important.Hsp70-1 is highly expressed in prostate cancer,which plays a crucial role in prostate cancer for resistance to conventional therapies.A large number of researches on Hsp70-1 are still in the role of molecular chaperones,but its downstream gene is rarely reported.It has been reported c-Fos is often overexpressed in tumor cells and its up-regulated phenomenon occurs simultaneously with Hsp70 in a variety of cell stress and physiological changes.However,how Hsp70-1 functions and regulates c-Fos remains unclear.Our previous experiments found that Hsp70-1 could promote the expression of c-Fos.Therefore,utilizing multiple approaches,we are making an attempt to investigate the molecular mechanism of the regulation of Hsp70-1 on c-Fos through AR-indepedent pathway as well as AR-depedent pathway.It is well known that Hsp70-1 regulates AR,so we need to further investigate the effect of AR on Hsp70-1 regulating c-Fos,which might be helpful to understand more about Hsp70-1,served as a potent target during the treatment of prostate cancer.To solve the above problems,the experiment content is divided into the following sections:First,the assays of Transfection,RT-PCR,Real-time PCR,Western blot were performed to explore the expression of Hsp70-1 and c-Fos in normal cell and cancer cells of prostate.Then,in the early screening of stably transfected cell line LNCaP-Hsp70-1(LnD3),we found that the expression of c-Fos could be increased by Hsp70-1.In contrast to by treating with inhibitor or siRNA of Hsp70-1,the effects will be reduced or even disappeared.However,after transfection of eukaryotic expression vector of Hsp70-1 in vitro,the methods of Transfection,RT-PCR,Real-time PCR,Western blot were performed to explore the expression and regulation of Hsp70-1 on c-Fos and the target genes of c-Fos in prostate cancer cells.The result showed that overexpression of Hsp70-1 in AR-indepedent as well as AR-depedent cells increased the expressions of c-Fos and the target genes of c-Fos,both in mRNA and protein levels.Second,In AR independent cell lines,to examine the mechanisms of Hsp70-1 regulating c-Fos in the absence of AR,we constructed a plasmid of c-Fos-promotor,using Luciferas,Nuclear run on and Q-PCR means to explore the effects of Hsp70-1 on c-Fos transcriptional level.It was found that overexpression of Hsp 70-1 in PC-3 cell lines,transcriptional activity of c-Fos had been increased,and the difference was significant,while suppressing the expression of Hsp70-1,the transcriptional activity also decreased.These results indicated that Hsp70-lmediated c-Fos at the transcriptional level.Third,In AR dependent cell lines,in view of the function of Hsp70-1 on AR,we speculated that the regulation of Hsp70-1 on c-Fos may also be associated with AR.In order to study the impact of AR on c-Fos.We used stably AR-transfected LNCaP-AR(LNA1A)and AR-transient transfected PC-3-AR cells,and the methods of RT-PCR,Real-time PCR,Western blot and siRNA.It has been observed that the expression of c-Fos was found to be up-regulated in LNA1A but almost not in LnD3(LNCaP,overexpressing Hsp70-1)cells when knocked out AR.These indicate that Hsp70-1 regulates c-Fos by AR in LNCaP cells.So,AR plays an important role in the regulation of Hsp70-1 on c-Fos.It also prompts us that the regulation of Hsp70-1 on c-Fos may have AR-indepedent pathway and/or AR-depedent pathway.To further analyze the role of AR in regulating c-Fos,AR-transient transfected PC-3-AR cells down-regulated in PC-3-AR.Hence,we suggested thatRecent investigation indicated that HSF2 could bind to c-Fos promoter region.So,we tested the expressions of HSFs in LNA1A and PC-3-AR cell lines,suggesting that the expression levels of HSF2 and c-Fos were consistent in both cells.Then,we co-transfected the AR and HSF2 promoter/shRNA-HSF2 and further confirmed AR regulated c-Fos by HSF2.In general,we provided evidence that Hsp70-1 mediated c-Fos at transcriptional level in the absence of AR.On the other hand,AR influences c-Fos through HSF2 in the presence of AR.