EP₁ Receptor Regulates The Growth Mechanism Of Prostaglandin E₂ Induced Osteosarcoma Growth

Osteosarcoma（OS）is a common primary and malignant tumor in the skeletal system of children and adolescents.With the development of techniques,such as chemotherapy,targeted therapy,surgical treatment,immunotherapy,traditional Chinese medicine treatment,and limb reconstruction,the survival rate of OS patients treated with limb function maintenance therapy has increased significantly,but the proliferation,migration,and new tumor blood vessel formation mechanisms of the tumor cells in OS patients are relatively complicated,and the tumor development progress still involves complicated network signal transduction pathways.Therefore,the pathogenesis of OS still needs in-depth research.In recent years,the role of inflammatory mediators and related cytokines in the development and formation of OS tumors has received increasing attention from researchers.Cyclooxygenase（COX）is an important inflammation mediator.During the conversion of arachidonic acid（AA）to prostaglandin H2（PGH2）,COX is the rate-limiting enzyme.PGE₂ is the main metabolite produced by AA catalyzed by COX-2.As an inflammatory mediator,PGE₂ is widely distributed in the body.In the process of tumor development,leucotriene and PGE₂ regulates the proliferation,differentiation and apoptosis of tumor cells in multiple signal transduction pathways in autocrine and paracrine manners.Through specific binding with EP receptors,PGE₂ exerts its physiological effects.EP receptors can be divided into four sub-types:EP₁,EP₂,EP₃,and EP₄.The EP₁ receptor belongs to the family of G protein-coupled receptors,and the EP1receptor can mediate Ca2⁺-regulated signal transduction.The EP₁ receptor is associated with various cancers,such as liver cancer,skin cancer,colon cancer,and breast cancer.In human Hep G2 and Hep3B liver cancer cells,the expression level of EP₁ receptor is the most abundant among the four EP receptors.The specific antagonist of EP₁ receptor ONO-8713 inhibits the proliferation activity of human Hep G2 and Hep3B liver cancer cells in a dose-dependent manner,and plays a role in inducing the apoptosis of human Hep G2 and Hep3B liver cancer cells.In tissues of patients with breast cancers,the EP₁receptor participates in the process of vascular endothelial growth factor（VEGF）regulation.In addition,the EP₁ receptor also plays a great role in the formation of neovascularization in tumor tissues,as well as the differentiation and proliferation of tumor cells.However,it is still not clear whether PGE₂ is involved in the malignant proliferation of tumor cells in the development progress of osteosarcoma,and certain experimental evidence is still needed to determine whether targeting the EP₁ receptor can develop an effective,highly selective,and low-toxic anti-osteosarcoma treatment method in clinical practice.In this subject,a nude mouse model bearing osteosarcoma was established,the human osteosarcoma cells were taken as the research object,and techniques such as flow cytometry,enzyme-linked immunoreaction,TUNEL,immunohistochemistry,immunoblotting,and laser confocal were adopted to observe the role of the EP₁receptor in the growth of tumor cells,hoping to provide a certain experimental basis for the development of an effective,highly selective,and low-toxic anti-tumor treatment method in clinical practice.Objective:A nude mouse model bearing osteosarcoma was established,the human osteosarcoma cells were taken as the research object,and techniques such as flow cytometry,enzyme-linked immunoreaction,TUNEL,immunohistochemistry,immunoblotting,and laser confocal were adopted to explore the role of the EP₁receptor in the growth of tumor cells.Methods:（1）Specific primers for EP₁ m RNA were designed,and the expression levels of EP₁m RNA in MG63,OS732,143B,U-2OS cells,and human fetal osteoblasts（h Fob1.19）were evaluated with PT-PCR.（2）Western Blot was used to evaluate the expression levels of EP₁in MG63,OS732,143B,U-2OS,and human fetal osteoblasts（h Fob1.19）.（3）20,000 MG63 cells were seeded per well in a 24-well culture plate.The treatment group was treated with PGE₂（5μM）and 17-PT-PGE₂（5μM）,and the control group was treated with DMSO.The effect of the EP₁receptor agonist on MG63 cells was examined with the PKC activity assay kit.（4）10,000 MG63 cells were seeded per well in a 96-well culture plate.The treatment group was treated with PGE₂（5μM）and 17-PT-PGE₂（5μM）,and the control group were treated with DMSO.The effect of the EP₁receptor agonist on the proliferation of MG63 cells was detected by using the MTT assay after 48 hours.（5）20,000 MG63 cells were seeded per well in a 24-well culture plate.The treatment group was treated with 17-PT-PGE₂（5μM）and 17-PT-PGE₂（5μM）+SC51809（2μM）.After 48 hours of the treatment,the PKC activity in MG63 cells was detected by using the PKC activity assay kit.（6）10,000 MG63 cells were seeded per well in a 96-well culture plate.The treatment group was treated with 17-PT-PGE₂（5μM）and 17-PT-PGE₂（5μM）+SC51809（2μM）.Subsequently,the proliferation of MG63 cells was detected by using the MTT assay after 48 hours.（7）20,000 MG63 cells were seeded per well in a 24 well culture plate.The treatment group was treated with PGE₂（5μM）and 17-PT-PGE₂（5μM）,and the control group was treated with DMSO.The apoptosis was detected with the TUNEL fluorescence staining method 48 hours later.（8）20,000 MG63 cells were seeded per well in a 24 well culture plate.The treatment group was treated with 17-PT-PGE₂（5μM）and 17-PT-PGE₂（5μM）+SC51809（2μM）.The apoptosis was detected with the TUNEL fluorescence staining method 48 hours later.（9）The expression levels of EP₁ in MG63+PGE₂（5μM）,si RNA control+PGE₂（5μM）,and EP₁-si RNA+PGE₂（5μM）were detected with Western Blot.（10）10,000 MG63 cells were seeded per well in a 96-well culture plate.The treatment group was treated with MG63+PGE₂（5μM）,si RNA control+PGE₂（5μM）,and EP1-si RNA+PGE₂（5μM）.After 48 hours,the proliferation of the MG63 cells was detected with the MTT assay.（11）20,000 MG63 cells were seeded per well in a 24 well culture plate.The treatment group was treated with MG63+PGE₂（5μM）,si RNA control+PGE₂（5μM）,and EP₁-si RNA+PGE₂（5μM）.The apoptosis was detected by using the TUNEL fluorescence staining method 48 hours later.（12）Western Blot was used to detect the expression of apoptosis-related proteins in si RNA-control MG63 cells and EP₁-si RNA MG63 cells.（13）In male BALB/c nude mice,5 x 10⁷ MG63 cells were inoculated into the axilla of nude mice.After the inoculation,a ridge formed in the inoculation site.When the tumor diameter of nude mice reached 3-5 mm,the nude mice were randomly divided into a medication group（SC51890）and a control group.The mice in the medication group were administered 2 mg/kg daily for 21 days.The tumor was measured using a vernier caliper every three days during the administration,and the tumor volume was calculated according to the following method:tumor volume=0.44×long diameter×short diameter ².（14）The activity of SC51809 in vivo in MG63 tumor-bearing nude mice was detected by using the PKC activity detection kit.（15）The TUNEL fluorescence staining method was used to detect the apoptosis of tumor tissues in MG63 tumor-bearing nude mice with SC51809.（16）Western Blot was used to detect the effect of SC51809 on tumor apoptosis related proteins in MG63 tumor-bearing nude mice.Results:（1）The expression levels of EP₁ m RNA in MG63,OS732,143B,and U-2OS cells were significantly increased compared with that in the human fetal osteoblasts（h Fob1.19）（P<0.01）.（2）The expression level of EP₁in human osteosarcoma cells MG63 was higher than those in the human osteosarcoma cells OS732,143B,and U-2OS（P<0.01）.（3）The activity of PKC of the 17-PT-PGE₂ group and the PGE₂ group was higher than that of the control group.17-PT-PGE₂and PGE₂ enhanced the activity of PKC in MG63 cells（P<0.01）.（4）The proliferation levels of the 17-PT-PGE₂ group and the PGE₂ group were higher than that of the control group.17-PT-PGE₂ and PGE₂ enhanced the proliferation activity in MG63 cells（P<0.01）.（5）The PKC activity of the 17-PT-PGE₂group was higher than that of the17-PT-PGE₂+SC51809 group（P<0.01）,and SC51809 reduced the PKC activity of MG63 cells induced by 17-PT-PGE₂.（6）The proliferation level of the 17-PT-PGE₂ group was higher than that of the17-PT-PGE₂+SC51809 group（P<0.01）,and SC51809 reduced the proliferation of MG63 cells induced by 17-PT-PGE_2.（7）The number of apoptotic cells observed in the 17-PT-PGE₂ group and the PGE₂group was lower than that in the control group,and the apoptosis rate was calculated.The apoptosis rate observed in the 17-PT-PGE₂ group and the PGE₂ group was lower than that in the control group.17-PT-PGE₂and PGE₂ enhanced the anti-apoptotic activity in MG63 cells（P<0.01）.（8）The apoptosis level of the 17-PT-PGE₂ group was lower than that of the17-PT-PGE₂+SC51809 group（P<0.01）,and SC51809 reduced the anti-apoptosis in MG63 cells induced by 17-PT-PGE₂.（9）The expression level of EP₁ in human osteosarcoma cells EP₁-si RNA+PGE₂ was lower than those in MG63+PGE₂（5μM）and si RNA-control+PGE₂（5μM）cells.（10）The proliferation levels of the MG63+PGE₂（5μM）group and the si RNA control+PGE₂（5μM）group were higher than that of the EP₁-si RNA+PGE₂（5μM）group（P<0.01）,suggesting that silencing EP₁ receptor can reduce the proliferation of the MG63 cells.（11）The apoptosis level of the EP₁-si RNA+PGE₂ group was higher than those of the MG63+PGE₂（5μM）group and the si RNA control+PGE₂（5μM）group,（P<0.01）,suggesting that silencing EP₁receptor can induce the apoptosis of the MG63 cells.（12）The expression levels of Bax,cytoplasmic cyt c,cleaved caspase-3,cleaved caspase-8,and cleaved caspase-9 in human osteosarcoma cells EP₁-si RNA were significantly higher than those in human osteosarcoma MG63 cells and si RNA-control cells.Compared with the expression levels of Bcl-2 and mitochondrial cyt c in Human osteosarcoma cells EP₁-si RNA,the expression levels in MG63 cells and si RNA-control cells were significantly lower（P<0.01）.（13）SC51809 has an inhibitory effect on the tumor volume and the mean tumor weight in MG63 tumor-bearing nude mice.Compared with the control group,there is a difference on the tumor volume and tumor weight in MG63 tumor-bearing nude mice in the SC51809 group（P<0.01）.In-vivo results suggest that the EP₁ inhibitor SC51809 has an inhibitory effect on the tumor growth in MG63 tumor-bearing nude mice.（14）The activity level of PKC in the SC51809 group was lower than that in the control group（P<0.01）.SC51809 reduced the PKC activity of MG63 tumor-bearing nude mice.（15）The apoptosis rate observed in the SC51809 group was higher than that in the control group.SC51809 induced the apoptosis of MG63 cells in vivo（P<0.01）.（16）The expression levels of Bax,cytoplasmic cyt c,cleaved caspase-3,cleaved caspase-8,and cleaved caspase-9 in the SC51809 group were significantly higher than those in the control group,and the expression levels of Bcl-2 and mitochondrial cyt c in the SC51809 group were significantly lower than those in the control group（P<0.01）.Conclusions:（1）The expression level of EP₁ in MG63 cells is high,the EP₁receptor agonist and PGE₂can enhance the expression level of PKC and the activity of MG63 cells.The EP₁receptor inhibitors can inhibit the PKC expression and the proliferation of MG63 cells.（2）The EP₁ receptor agonist and PGE₂can inhibit the apoptosis of MG63 cells,and the EP₁ receptor inhibitor can inhibit the apoptosis of MG63 cells induced by 17-PT-PGE_2.Blocking EP₁ receptor can inhibit the proliferation of 17-PT-PGE₂-induced MG63 cells and promote the apoptosis of 17-PT-PGE₂-induced tumor cells.Silencing EP₁ can inhibit the proliferation of PGE₂-induced MG63 cells and promote the apoptosis of PGE₂-induced tumor cells,suggesting that EP₁ is involved in the proliferation and apoptosis of PGE₂ and17-PT-PGE₂induced MG63 cells.（3）SC51809 can inhibit the tumor growth in tumor-bearing nude mice,reduce the activity of PKC in tumor tissues,induce tumor apoptosis,and mediate the regulation of apoptosis-related proteins.