The Influence Of Knocking Down HIF-1α Gene With SiRNA On Biological Behavior, Chemosensitivity Of Docetaxe1 And Radiosensitivity Of PC3 Cell

Prostate cancer is most common malignant tumours in urinary system. Radical prostatectomy, hormonal therapy, radiotherapy and chemotherapy are considered as major therapeutic approaches for prostate cancer. Patients with recurrence or metastasis after radical prostatectomy and patients who lost opportunity of operation usually choose hormonal therapy that is effective in the initial stage. But after 14 30 months from hormonal therapy given, prostate cancer would progressed into androgen-independent and hormone-refractory prostate cancer, hormonal therapy would become unefficient, how to treat it is a difficult problem and hot topic. The RNA interference （RNAi） is used to knock down expression of gene targeted with small RNA （including small interference RNA, siRNA）. However, the siRNA derived from mammals has not been found in vivo so far. If investigators transfer the chemosynthesis siRNA targeting some gene into tumour cells by liposomes or other assistant transfection methods, mRNA with homologous sequences would be degradation and the expression of gene targeted would be knocked down efficiently. RNAi has provided firenew research path for prostate cancer for the past few years.Chemosensitivity, radiosensitivity and other therapy sensitivity of malignant tumours including prostate cancer is relation to oxygen content of tissue. Biological pathways are regulated by hypoxia inducible factor-la （HIF-la） including hypoxia adaptation, angiogenesis, glucose transport, glycolysis, proliferation, apoptosis, et al. Our preceding research and others reports have confirmed that the mRNA expression and protein expression of HIF-la in prostate cancer is high, it also involves in malignant biological behaviour of prostate cancer. The thread of this topic:First of all, we should design several chemical synthesis siRNAs targeting HIF-la, and transfer them into human prostate cancer cell line PC3 by liposomes to observe their effect and screen out the most effective siRNA. Then we should identify its the influence on biological behaviour of PC3 cells in vitro. Finally, on the basis of the effective siRNA we can obtained, we use the RNAi combination with chemotherapy and radiotherapy to study the influence of the HIF-la-siRNA on chemosensitivity and radiosentivity in vitro. To explore the effective methods of treatment for prostate cance is our goals in this study.This study has four parts.Partâ… Screening of effective siRNA targeting HIF-1αgeneObjective:To design and produce siRNA targeting HIF-la gene and select out the most effective siRNA for subsequent research.Methods:Seven chemical synthesis Oligos of siRNA targeting human HIF-la mRNA （NM₀01530） and two control siRNAs were designed.40nM was choosed as the final concentration for RNAi. The Real-Time-PCR was used to detect the expression of HIF-1α-mRNA in PC3 cells at 24,48, and 72 hours after transfection. The western-blotting was used to measure the expression of HIF-1αprotein at 48 hour post transfection.Results:At 24 hours after transfection, HIF-1α-siRNA1 and HIF-1α-siRNA5 had displayed results expected of interference and inhibition ratio were all over 70%on the level of mRNA. The chronergy test of RNAi indicated HIF-1α-siRNA1 and HIF-la-siRNA5 had the highest inhibition ratio on mRNA level at 24h-48h after transfection. HIF-1α-siRNA1 also showed the best inhibition result on the protein level of HIF-1αat 48 hours after transfection and its inhibition ratio was over 70%. Conclusions:HIF-1α-siRNA1 （target cDNA site location:792-812） had best inhibition result among seven chemical synthesis siRNAs. The inhibition ratios on mRNA and protein level were all over 70%. Accordingly, it was chosen for subsequent research. The peak of inhibition can be reach at 24 48 hours after transfection.Partâ…¡Influence of knocking down HIF-la gene with siRNA on the biological behavior of human prostate cancer PC3 cellObjective:To explore the influence of knocking down HIF-1αgene with siRNA on the the biological behavior of human prostate cancer PC3 cell, including cell proliferation, migration force, invasiveness, apoptosis.Methods:PC3 cell was divided into three groups:PC3 group, PC3+NC siRNA group and PC3+HIF-1α--siRNAl group. Each group was transfected with HIF-1α-siRNA1 which was confirmed in the preceding study, by using cationic liposomes Lipofectamine 2000. CCK-8 test was used to measure the proliferation of PC3 cell; Scarification test was used to observe migration ability; Transwell test was used to observe invasiveness; Annexin V-FITC/PI test on Flow cytometer was used to measure cell apoptosis.Results:（1） Survival rate of HIF-1α-siRNA1 group is lower significantly than two control groups at 24h,48h,72h and 96h after transfection （P<0.01）, while there was no difference between control groups （P>0.05）. The interference effect reached its peak at 48h⁷2h after transfection. （2） Scarification test demonstrated that cell migrate of HIF-1-α-siRNA1 group was more slowly than two control groups （P<0.01）. The difference between NC siRNA group and PC3 group was not obvious （P>0.05）. （3） Transwell test showed that the cell number of HIF-1α-siRNA1 group that went through the Matrigel gel were significantly less than two control groups （P<0.01）, There is no difference between PC3 group and NC siRNA group. （4） Cell apoptosis ratios of PC3+ HIF-1αsiRNAl group, PC3 group and PC3+NC siRNA group at 48h after transfection was 11.2%,2.4%and 2.5%respectively. The apoptosis rate of PC3+HIF-1αsiRNA1 group was higher than two control groups （P<0.01）.Conclusions:（1） Knocking down of HIF-1αgene inhibit effectively the proliferation in the human prostate cancer PC3 cell; （2） Knocking down of HIF-la gene improve the ability of migration and invasion in the human prostate cancer PC3 cell; （3） Knocking down of HIF-la gene may have reinforcement of apoptotis, in some sense, in human prostate cancer PC3 cells.Partâ…¢Influence of knocking down HIF-1αgene on the chemosensitivity of Docetaxel in PC3 cell lineObjective:To evaluate the influence of knocking down of HIF-la gene with siRNA on the chemosensitivity of Docetaxel in PC3 cell line.Methods:PC3 cell was divided into three groups as previous study, in addition, each chemotherapy group was followed by one group without chemotherapy. Docetaxel was given as final concentration of 0.25 u M at 24h after transfection. CCK-8 test was used to measure the proliferation of PC3 cell. Flow cytometer was used to measure cell apoptosis, cycle distribution and expression of Multidrug resistance gene/P glucose protein （MDR/P-gp）. Results:（1） Inhibition ratio of PC3+HIF-1α-siRNA1+DTX group is higher than PC3+DTX group and PC3+NC siRNA+DTX group （P<0.05, at 48h）, while there was no difference between two control groups （P>0.05）. Cell apoptosis rate of PC3+ HIF4a-siRNA1+DTX group, PC3+DTX group and PC3+NC siRNA+DTX group was 21.3%,15.6%and 14.8%respectively at 48h after docetaxel given. The apoptosis rate of PC3+HIF-1α-siRNA1+DTX group was the highest （P<0.01）. Flow cytometry analysis showed there was less G0/G1 phase arrest in PC3+HIF-1α-siRNA1 group than two control groups without chemotherapy. There is no difference of expression of MDR/P-gp among the groups before and after chemotherapy.Conclusions:HIF-la-siRNA1 can enhance chemosensitivity of docetaxel in PC3 cell in vitro by knocking down of HIF-la gene expression. The possible mechanisms involved in chemosensitization of HIF-1α-siRNAl include increasing apoptosis rate and releasing cell cycle arrest of G0/G1 but no downregulating expression of MDR/P-gp.Part IVInfluence of knocking down HIF-1αgene with siRNA on the radiosensitivity of PC3 cell linesObjective:To evaluate the influence of knocking down HIF-1αgene with siRNA on the radiosensitivity of PC3 cell line, and to probe the mechanisms may be involved.Methods:PC3 cell was divided into three groups as previous study. radiosensitization of each group was measured by clongenic assay through different doses of 6MV X-ray. We obtained the values of D0, Dq, SF2, N and the delineated dose-survival curve by computer using single-target multi-hit model. CCK-8 Determination was used to evaluate proliferation of PC3 cell after single dose irradiation of 6Gy. With single dose irradiation of 6Gy, apoptotic rate and cycle distribution distribution at 72 hours after irradiation, cycle distribution before irradiation, were analyzed by flow cytometry. Results:（1） Analysis of clongenic assay showed radiosensitizing effect in PC3+HIF-1α-siRNA1 group. The values of D0, SF2, Dq and N in PC3+HIF-1αsiRNA1 group were lower than the others Sensitizing Enhancement Ratio was 1.24. （2） The outcomes of CCK-8 tests showed that the proliferation of PC3 cell transfected with HIF-la-siRNA1 was rather slow than two control groups after radiotherapy with single dose irradiation of 6Gy （P<0.01）, but there was no difference between two groups （P>0.05）. （3） Apoptotic rate was more higher in PC3+HIF-1αsiRNA1 group compared with two control groups at 72 hours after radiotherapy （P<0.01）. （4） Flow cytometry analysis showed there was less G0/G1 phase arrest in HIF-1α-siRNA1 group before irradiation than two control groups.Conclusion:（1） HIF-1α-siRNA1 can enhance radiosensitivity of PC3 cell in vitro by inhibition HIF-1αexpression. （2） The possible mechanisms involved in radiosensitization of HIF-1α-siRNAl include enhancement of oxidative stress injury and weakening the ability of reconstruction after radiotherapy, increasing apoptosis rate and releasing cell cycle arrest of G0/G1.