Study Of The Effects And Mechanisms Of Hypoxia And Stretch On Proliferation Of Prostate Stromal Cells

ObjectiveIn this study,prostate stromal cells（WPMY-1）were cultured in both hypoxic and normoxic environment.Besides,mechanical stretch device was used to apply stimulation to WPMY-1 cells.The first aim is to study the effects of hypoxia on cell proliferation and to expound HIF-1αsignals and relevant genes’expression under hypoxia environment.The second aim is to explore the effects of mechanical stretch on cell proliferation and oxidative stress.Through the above studies,a theoretical basis could be provided for the prevention and treatment of benign prostate hyperplasia.Methods1.The WPMY-1 cells were cultured and sub-cultured.And cells of 3⁵ generations were divided into either normoxia group or hypoxia group.The oxygen concentrations in the two incubators were 21%O₂ and 3%O₂,respectively.When the total time reached 12 hours,24 hours and 48 hours,cell proliferation was detected by CCK-8 and the expressions of HIF-1α,PCNA,Bcl-2,Bax proteins were detected by Western Blot.2.The hypoxic intervention time was 48h,24h,and 12h in turn and the cells were collected at the same time by reverse order method.The apoptosis of the cells was detected by Annexin V/PI,and the oxidative stress level in the cells was detected by the DCFH-DA probes.RT-qPCR and Western Blot were used to detect the expressions of HIF-1α,AKT,ERK mRNA and proteins,respectively.3.In Phase II,cells were divided into four groups:control,hypoxic,stretch,hypoxic plus stretch.The stretch scheme was 10%deformation,0.5 Hz frequency lasting for 4 hours,followed by biochemical tests of WPMY-1 cells according to the above methods.Results1.The proliferation of WPMY-1 cells increased significantly both in a hypoxic and normoxic environment with prolonged culture time,48h cell proliferation was significantly higher compared to 12h and 24h in each group（both P<0.01）.When compared between groups,only 48h had a significant difference（P<0.01）.Hypoxic intervention had no significant effect on apoptosis at different time（P>0.05）;24h hypoxic intervention significantly increased levels of intracellular oxidative stress（compared with normoxia group and 12h hypoxia,both P<0.01）,and 48h hypoxic intervention significantly decreased levels of intracellular oxidative stress（compared with 24h hypoxia,P<0.01）.2.The expression of HIF-1αprotein increased significantly after 24 hours and 48hours of hypoxic culture compared with 12 hours（P<0.05,P<0.01,respectively）;There was no significant difference in expression of PCNA protein at any time points in hypoxic culture（P>0.05）;The expression of Bcl-2 protein increased significantly in hypoxic culture for 24 hours compared to 12 hours（P=0.05）;The expression of Bax protein increased significantly after 24 hours and 48 hours of hypoxic culture compared with 12 hours（both P<0.05）.3.HIF-1αmRNA significantly increased in hypoxic intervention at 48h compared with the normoxia group（P<0.05）;AKT mRNA significantly decreased in hypoxic intervention at 48h compared with the normoxia group（P<0.05）;There was no significant difference in ERK mRNA in hypoxia group and normoxia group.The AKT protein is significantly lower in the 24h hypoxic intervention group and an increase in48h hypoxic intervention compared with 24h（P<0.05）.There was a significant decrease in ERK protein in hypoxia for 12h,24h,and 48h compared to the normoxia group（P<0.05,P<0.01,P<0.01 respectively）.4.After hypoxia and stretch intervention,the cell proliferation in the hypoxia group was still higher than that in the normoxia group（P<0.01）,the cell proliferation in the stretch group was significantly lower than that in the normoxia group and the hypoxia group（P<0.01）,the cell proliferation in the hypoxic-binding stretch group was significantly higher than that in the normoxia group and the stretch group（P<0.01）.The apoptosis rate in each group was not statistically significant（P>0.05）.The oxidative stress in the hypoxia group was significantly lower than that in the normoxia group（P<0.05）.After hypoxia combined with stretch intervention,the level of oxidative stress decreased significantly（compared with other groups,all with P<0.01）.Conclusion1.Hypoxia may promote the proliferation of WPMY-1 cells through increased oxidative stress levels and the activation of HIF-1α,as well as expressions of downstream genes Bcl-2 and Bax.2.Hypoxia may cause elevated oxidative stress in prostate stromal cells and affect AKT and ERK genes expression.3.Mechanical stretch could inhibit cell viability and oxidative stress levels.