Effects Of Omi/HtrA2 Gene Overexpression And Small Interference RNA Targeting PED/PEA-15 Gene On Biological Behavior Of Prostate Cancer

1. Expression and significance of PED/PEA-15 in prostate cancer Objective To study the expression of PED/PEA-15 in prostate cancer, and interpret the role of it in the occurrence and development of prostate cancer. Methods The expression of PED/PEA-15 in prostate cancer cell (PC-3) and normal prostate tissue were respectively assayed by means of RT-PCR and immuneohistochemistry techniques. Results PED/PEA-15 high express in PC-3 cells, 73.17% prostate cancer tissue express PED/PEA-15, by contrast, the normal prostate tissue showed no or weak expression of it. Conclusions Anti-apoptotic factor PED/PEA-15 can inhibit apoptosis, and its expression might be involved in prostate cancer development.2. The effect of PED/PEA-15 on prostate cancer cells (PC-3) apoptosis Objective To study the effect of PED/PEA-15 on human prostate carcinoma cells (PC- 3) apoptosis through constructing its eukaryotic expressing vectors. Methods PED/PEA-15 gene was amplified by means of RT-PCR technique and was cloned into pEG FP-N1 vect- or. The constructed vector was transiently transfected into PC-3 cells under the induction of liposome. Using flow cytometry technique to determine cancer cells apoptosis. Results Enzyme digestion analysis and DNA sequencing confirmed that the PED/PEA-15 eukaryo- tic expression vector was constructed successfully. High expression of PED/PEA-15 can inhibit PC-3 cells apoptosis and degrade KillerTRAIL's proapoptotic effect. Conclusions PED/PEA-15 can inhibit PC-3 cells'apoptosis, and its expression might be involved in pr- ostate cancer's development.3. Effect of small interference RNA targeting PED/PEA-15 on the apoptosis of prostate cancer cell (PC-3) Objective To approach the effect of PED/PEA-15 on prostate cancer cell (PC-3) apopto- sis by means of constructing PED/PEA-15 specific siRNA vector. Methods Using the s- oftware of Invivogen company, computer aided designing PED/PEA-15 specific siRNA s- equence. The synthesized siRNA sequence was cloned into psiRNA-hH1neo vector. The constructed psiRNA-PED/PEA-15 was transiently transfected into PC-3 cells and the inh- ibited effect of psiRNA-PED/PEA-15 on expression of PED/PEA-15 was detected using RT-PCR and Western blot, the effect of psiRNA-PED/PEA-15 on cancer cells'apoptosis was determined by flowcytomtry and MTT techniques. Results Enzyme digestion analy- sis and DNA sequencing confirmed that the PED/PEA-15 specific siRNA expression vect- or was successfully constructed. The apoptosis of PC-3 cells and the expression of PED/P- EA-15 were both down-regulated after the transfection of psiRNA-PED/PEA-15. Conclu- sions PED/PEA-15 can inhibit the apoptosis of PC-3 cells, and that its expression might be involved in prostate cancer development.4. Effect of PED/PEA-15 and XIAP on prostate cancer cell (PC-3) apoptosis Objective To study the effect of antiapoptosis factors PED/PEA-15 and XIAP on prosta- te cancer cells (PC-3) apoptosis. Methods Expression of XIAP and PED/PEA-15 in pro- state cancer cells (PC-3) were respectively assayed using RT-PCR technique. PED/PEA-15 and XIAP specific siRNA vectors were designed and constructed and then were transientl- y cotransfected into PC-3 cells under induction of liposome. Effects of siRNA sequences on PED/PEA-15 and XIAP transcription were assayed by RT-PCR technique, effects of XIAP and PED/PEA-15 on cancer cell apoptosis was determined by flow cytometry and microscope observation. Results PED/PEA-15 and XIAP were both highly expressed in PC-3 cells. Enzyme digestion analysis and DNA sequencing confirmed that PED/PEA-15 and XIAP specific siRNA expression vectors were constructed successfully. The designed siRNA sequences of PED/PEA-15 and XIAP could specificly inhibit theirs transcription. The cotransfected with PED/PEA-15 and XIAP specific siRNA vectors PC-3 cells were more sensitive to doxorubicin and its apoptosis rate also increased significantly. Conclus- ions PED/PEA-15 and XIAP might be involved in the development of prostate cancer. 5. Immunohistochemical analysis of Omi/HtrA2 expression in prostate cancer and benign prostatic hyperplasiaTo study the expression and significance of the serine protease Omi/HtrA2 in p- rostate cancer and benign prostatic hyperplasia. The expression of Omi/HtrA2 was assayed by means of immunohistochemical technique in prostate cancer, benign prostatic hyperpla- sia and normal prostate tissue specimens. Omi/HtrA2 mRNA levels of in vivo prostate can- cer and benign prostatic hyperplasia were assayed by semi-quantitative RT-PCR technique. Omi/HtrA2 expression was positive in 30 of 42 prostate cancer specimens, and the positive rate was lower in well differentiated group than in poorly and moderately differentiated gr- oups (P < 0. 05). By contrast, the cells in benign prostatic hyperplasia and normal prostate groups showed no or weak expression of Omi/HtrA2. The Omi/HtrA2 mRNA level of pro- state cancer is much higher than benign prostatic hyperplasia. Prostate cancer cells in vivo may need Omi/HtrA2 expression for apoptosis, and Omi/HtrA2 might be involved in pros- tate cancer development.6. Experimental study on Omi/HtrA2 promoting prostate cancer cell (PC-3M) apopt- osisObjective To study the expression of Omi/HtrA2 in human prostate cancer cell line (PC- 3M) and to approach the effect of Omi/HtrA2 on PC-3M apoptosis by means of construct- ing the Omi/HtrA2 specific siRNA expressing vector. Methods The expression of Omi/- HtrA2 in human prostate cancer cell line (PC-3M) was detected by means of RT-PCR and immunohistochemical techniques. According to the software of Invivogen company, com- puter aided design Omi/HtrA2 specific siRNA sequence and then synthesized and cloned into the expression vector psiRNA-hH1neo. The constructed psiRNA-Omi/HtrA2 was tra- nsiently transfected into human prostate cancer cell line (PC-3M) and the inhibited effect of psiRNA-Omi/HtrA2 on expression of Omi/HtrA2 in PC-3M cells was detected using RT-PCR and Western Blot; the apoptosis of cancer cells was determined by flow cytome- try and MTT techniques. Results The expression level of Omi/HtrA2 was up-regulated in PC-3M cells. Enzyme digestion analysis and DNA sequencing confirmed that the Omi/- HtrA2 specific siRNA expression vector was constructed successfully. The apoptosis of PC-3M and the expression of Omi/HtrA2 were down-regulated in prostate cancer cells af- ter the transfection of psiRNA-Omi/HtrA2. Conclusions prostate cancer cells may need Omi/HtrA2 expression for apoptosis, and Omi/HtrA2 might be involved in prostate cancer development.7. Omi/HtrA2 promotes prostate cancer cell death by degrading the anti-apoptotic protein PED/PEA-15BACKGROUND & OBJECTIVE: Abnormal apoptosis is one of the key factors for the development of neoplasms. Omi/HtrA2 is a novel gene involved in regulation of apoptosis. PED/PEA-15 is a ubiquitously expressed cytosolic protein exerting a broad anti-apoptotic action. This study was designed to explore the effect of Omi/HtrA2 on PED/PEA-15 expr- ession and prostate cancer cells (PC-3) apoptosis. METHODS: Omi/HtrA2 expression and specific siRNA vectors were respectively constructed and were transiently transfected into PC-3 cells under induction of liposome. Effect of Omi/HtrA2 on PED/PEA-15 expression and PC-3 cells apoptosis was assayed by Western blot and ELISA techniques. Caspase-8 activity was assayed by caspase-8 colorimetric assay kit which can be inhibited by PED/P- EA-15. Using RT-PCR and Western blot techniques assayed the effect of Omi/HtrA2 spec- ific siRNA sequence on its transcription and translation. Effect of Omi/HtrA2 inhibited by siRNA on PC-3 cells apoptosis was determined by flowcytometry technique. RESULTS: Enzyme digestion analysis and DNA sequencing confirmed that the Omi/HtrA2 expression and specific siRNA vectors were successfully constructed. High express PED/PEA-15 can inhibit caspse-8 activity and hinder cancer cells apoptosis. The PC-3 cells transfected with Omi/HtrA2 specific siRNA vectors were less sensitive to Cisplatin. CONCLUSIONS: Omi/HtrA2 can inhibit PED/PEA-15 expression and might play an important role in the development of prostate cancer.8. Effect of high expressed Omi/HtrA2 on PED/PEA-15 knockout prostate cancer cell apoptosis.Objective To study the mechanism of Omi/HtrA2 promoting prostate cancer cells apopt- osis and find out the new method of prostate cancer gene treatment. Methods Omi/Htr- A2 eukaryotic expression and PED/PEA-15 specific siRNA vectors were respectively con- structed. Under the induction of liposome, PED/PEA-15 specific siRNA vector was transf- ected into prostate cancer cell line (PC-3). After being selected by G418, the subclone cell line was obtained. Cellular gene silence of PED/PEA-15 was identified by RT-PCR and Western blot techniques. Omi/HtrA2 eukaryotic expression vector was transfected into su- bclone cells to high express it under induction of liposome. Effect of Omi/HtrA2 on subcl- one cells apoptosis was assayed by flow cytometry and Western blot technique. Results Enzyme digestion analysis and DNA sequencing confirmed that Omi/HtrA2 eukaryotic ex- pression and PED/PEA-15 specific siRNA vectors were successfully constructed. RT-PCR and Western blot revealed that there was no PED/PEA-15 expression in subclone cells, by the contrast, high level mRNA and protein of PED/PEA-15 was detected in PC-3 cells (P < 0. 01). High express Omi/HtrA2 in subclone cells can increase its apoptosis. Conclusions PED/PEA-15 can affect Omi/HtrA2 proapoptotic function. Omi/HtrA2 might be involved in prostate cancer development.