Study The ERβGene Function Domain Inhibiting Proliferation In Human Prostate Cancer Cell

Morbidity of prostate cancer(PCa) has been the first place of the male malignant tumour in the USA.The fatality of it has been the second only of lung cancer. The morbidity of PCa has been increasing year by year in our country,and prostate cancer(PCa) has been the third place in male urinary system tumor. So prostate cancer are becoming one of the serious diseases gradually threatened our countries gerontal men. Evidence indicates the onset risk of prostate cancer relates to high rstrogen(E), and Diethylstilbestrol has direct cytotoxic effects both on hormone dependent and independent prostate cancer cells,this promote estrogen recepter(ER) plays an importtant role in the development of prostate cancer. Evidence indicates compare with normal prostate tissue both hormone dependent and independent prostate cancer the transcription of estrogen receptorβ(ERβ)is inhitbited. ERβis expressed in normal prostatic tissue but no expressed in over 75% of the prostate cancers, yet absenced obviously in the invasion prostate cancer tissues. It's found in clinical the expression of ER-? had been decreased with the increased of the Gleason grade. So, the expressed absence of ERβwas intimate correlated with the genesis and development of prostate cancer, and might be a potential cancer inhibitor. The research of ER?-/- mouse found that the prostate glandular epithelium appeared visible hyperplasy. Our laboratory has found out that ERβcould inhibit the prostate cancer cell PC-3 and PC-3M proliferation and induce apoptosis .The mechanism of apoptosis induced by ERβwas related with the suppress of cyclinD1 and down regulate of the Bcl-XL , AKt and Survivin and the expression regulate of P21 and Caspase3 , Caspase9.Objective: To find out the target site of ERβwhich inhibit the prostate cancer cell PC-3M proliferation and induce apoptosis , provide new idea and experiment evidence for the PCa therapy.Methods: 1. The construction and identification of recombinant plasmid: To take ERβdomains gene using PCR;then clone them into eukaryotic expression plasmid pEGFP-C1 using gene recombination technique; identified the results by endonuclease digested and DNA auto sequencing.2 . To transfect the recombinant plasmids into the prostate cancer cell PC-3M and then observe the green fluorescent in these cells. It was provided that the successful tronsfected that the strong expression of GFP in these cells .3. MTT chromatometry used to observe proliferation ability of PC-3M cell which transfected the recombinant plasmids.4. To detect the gene expression related to apoptosis and proliferation by RT-PCR.5. To detect the gene expression related to apoptosis and proliferation by western blotResults: 1. RT-PCR showed that the expression of Caspase-3,P53 and P21 was increased .In contrast , the expression of C-myc and CyclinD1 was inhibited.Compare with hERβcomplete gene, the hinge domain (D) and the ligand binding domain(LBD,E/F) has more significally effect on these genes.2. MTT experiment showed that the hinge domain (D) and the ligand binding domain(LBD,E/F) of ERβcould inhibit PC-3M cell growth obviously,this effect depends on time and dose..3. Western blot showed that the protein level of CyclingD1 expression was decreased.In contrase, the expression of actived Caspase3 and Caspase-9 increased. The hinge domain (D) ligand and the binding domain(LBD,E/F) of ERβhas more significally effect than the compelet gene.Conclusion: 1. The combinant plasmids of ERβ13 function domains gene were constructed successfully.2. Creat high expression plasmids above-mentioned cyto-model was created by transfecting them into PC-3M cell.3. Growth inhibiting essay proved thath ERβ-D,DF,EF,and F could inhibit the proliferation of PC-3M cell ,this effect depends on time and dose.4. The mechanism of hERβ-DF,hERβ-EF,hERβ-F inhibit the proliferation of PC-3M cell is related with the suppress of CyclinkD1, upregulation P53 and P21. In addtion,they could induce apoptosis by increasing C-myc,Caspase-3 and Caspase-9.5. The hinge domain (D) and ligand binding domain(E/F) of ERβmay play an important role of ERβwhich induce cell apoptosis and inhinbit cell proliferation,and the effect of them is better than complete gene of ERβ.