| Objective: To explore PLCε,Notch1/Hes1,and AR protein of androgen independent prostate cancer,androgen independent prostate cancer and BPH organization.Methods: 12 cases of androgen independent prostate cancer organization,35 cases of androgen dependent prostate cancer,20 cases of BPH were collected.PLCε,Notch1/Hes,AR protein of all organizations were detected through immunohistochemistry,and semi-quantitative analysed.PLCε,Notch1,Hes1,and AR protein expression of different organizations was compared with each other.The correlation of each protein expression with the clinical feature,and the correlation of Notch1,Hes1,or AR with PLC ε were analysed.Results: Immunohistochemistry showed: compared with BPH tissue,the PLCε,Notch1,Hes1 protein of prostate cancer tissue were significantly higher than that of BPH,and PLCε,Notch1,Hes1 in androgen independent prostate cancer organization protein were slightly higher than that of the prostate cancer tissue,with statistical significance.AR was significantly increased in androgen independent prostate cancer organization,compared with the BPH or prostate cancer tissues expression.In the prostate cancer tissue,the expression of PLCε,Notch1,hes1 and AR in patients of older than 60 years were higher than those of the 60 years and lower age,the difference was statistically significant;The PLCε,Notch1,hes1 and AR expression in patients with T2-T4 stage were higher than those in Ta-T1 patients,the PLCε,Notch1,hes1 and AR in Gleason 7 or over 7 were higher than those in the lower Gleason.In androgen independent prostate tissues and androgen dependent prostate cancer tissue,Notch1,hes1,AR and PLCε were positively correlated.Conclusion: The expression of PLCε,Notch1/hes1 and AR in androgen independent prostate cancer is significantly higher than that of androgen dependent prostate cancer or BPH.Objective: To explore the influence of PLC ε gene knocked out to biological behavior of PC-3 cells.Methods: The PLCε gene was knocked out through CRISPR/Cas.Two groups were designed,PC-3 cell lines group,PLC ε knocked out PC-3 cell lines group.Cells were respectively cultured at the conditions 37℃ and 5% CO2,24,48 or 72 hours.The PLC ε m RNA and the protein were detected.Cells’ growth rate,apoptosis,cycle,repair ability and invasion ability were detected.Results: The PLCε m RNA and the PLCε protein of PLCε knocked out PC-3 cell lines had not been detected confirmed the success of the PLCε knockoued.Compared with the PC-3 group,the PLCε gene knocked out inhibits PC-3 cells’ proliferation.The apoptosis of the PLCε gene knocked out group PC-3 was higher than that of the PC-3 group,at each time point,the differences between the two groups were statistically significant.The proportion of S stage cells of PLC ε knocked out PC-3 cell was higher than that of the PC-3 group.The reparing rate or invasion of the PLC ε knocked out PC-3 group was lower than that of the PC-3 group at 24,48,or 72 hours.The differences were statistically significant.Conclusion: After PLC ε knockouting,PC-3 cells’ proliferation is restrained,and the apoptosis is increased,cell cycle was blocked at S phase,and the repare or invasive ability was restricted.Objective: To explore whether metformin influence PC-3 cells’ biological behavior.Methods: Cell lines were cultured at the environment of 37℃ and 5% CO2 in RPMI-1640 respectively.Two groups of cells,with metformin,0,10,20,or 40 m M concentrations of metformin were cultured 24 h,48h or 72 h.Cells’ growth rate,cells’ apoptosis a,cycle,cells’ repare ability and invasion ability were detected.Results: Along with the increasing concentration of metformin,the PC-3 groups cells’ proliferation inhibition was metformin dose dependent.The PLCε knocked out cells’ proliferation inhibition has no obvious changes.At 20 mM concentration condition,PC-3 groups’ cells proliferation gradually reduced with the time,but the PLC ε knocked out groups cells proliferation had no obvious changes with the time.The difference of the two groups was statistical significant.At 20 m M of metformin,all cell lines were cultured 48 h,The apoptosis of PLCε knocked out PC-3 groups cells was higher than that of PC-3 cells.The proportion of S phase PC-3 groups was decreased by metformin.There are less cells of PC-3 groups that were blocked at the cell cycle at S phase than that of the PLCε knocked out group,comparison between the two groups was statistical difference(P<0.05).There was no obvious change of PLC ε knocked out group when cultured with metformin.PC-3 group’s repair rate or invasion ability was reduced but was higher than that of the PLCε knocked out PC-3 group.There was statistical difference between the two groups.Conclusion: Metformin can restrain the proliferation and repare and attack ability,and can block the cell cycle at S phase of PC-3.Objective: To explore whether metformin down-regulated Notch1/HES1 and AR by inhibiting PLCε.Methods: The cell lines culturing was as same as the part three.PLC ε,Notch1,Hes1,AR,PCNA,Cyclin D1,and c-fos m RNA was detected through RT-PCR,and PLCε,Notch1,Hes1,AR,PCNA,Cyclin D1,and c-fos protein was detected through Western Blot.Results: With increasing concentration of metformin,PLCε,Notch1,Hes1,AR,PCNA,Cyclin D1,c-fos m RNA of the PC-3 group had gradually reduced.PLC ε m RNA was not detected in the PLC ε knocked out group,and along with the increasing concentration of metformin,Notch1,Hes1,AR,PCNA,Cyclin D1,c-fos m RNA expression has no obvious change,compared between the two groups,there were statistical difference.And with 20 m M concentration condition,PC-3 groups’ PLC ε,Notch1,Hes1,AR,PCNA,Cyclin D1,c-fos m RNA expression had gradually reduced with the time increasing.Western blot results show: The protein expression is consistent as the expression of the molecules.Conclusion: Metformin down-regurates Notch1/hes1 and AR by inhibiting PLC ε.Objective: To explore whether metformin enhances the chemotherpeutic sensitivity of PC-3 cells to docetaxel.Methods: Cell culture: PC-3 cell lines was divided into the control group,the metformin group(20mm),the docetaxel group(50ng/ml),the metformin combined docetaxel group(metformin 20 m M,docetaxel 50 ng/ml),all groups were cultured 24 h,48 and 72 hours.The PLCε,Notch1,Hes1,AR protein expression of docetaxel group were detected.Cells’ growth,cells apoptosis rate,cycle,cells repair ability and invasion ability were detected.Results: In the docetaxel group,the Western blot results showed that the change of PC-3 PLCε,Notch1,Hes1 protein expression is not obvious,but AR protein expression was reduced with the time.The control group’s cells has the highest proliferation,cells’ proliferation of metformin group was lower than those of the control group,cells’ proliferation of docetaxel group was lower than that of the metformin group,and cells’ proliferation of metformin combined docetaxel group was lower than that of docetaxel group.The metformin group,the docetaxel group,or metformin combined docetaxel group was compared to control group respectively,the all differences were statistical significant.The apoptosis rate of drug treatment groups increased with,apoptosis rate of metformin group is higher than that of control group,the apoptosis rate of docetaxel group was higher than that of metformin group,and the apoptosis rate of metformin combined docetaxel was higher than that of metformin combined docetaxel group.The all differences were statistical significant.All repair rates or invasion activity of drug treatment groups were lower than that of the control group at any time point;The repair rates or invasion activity of metformin group was lower than that of the control group;The repair rates or invasion activity of docetaxel was lower than that of metformin group;The repair rates or invasion activity of metformin combined docetaxel group was lower than that of docetaxel group.Conclusion: Metformin enhances the chemotherapeutic sensitivity of PC-3 cells to docetaxel. |
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