| Glioma is the most common intracranial malignant tumor,accounting for more than 50% of the primary tumor in brain.Glioma has the characteristics of high incidence,high malignancy,high recurrence rate,poor prognosis and high fatality rate.The etiology and pathogenesis of glioma are complicated,and it is not yet fully understood.With the development of molecular biology,cell biology,tumor immunology and genetics,people have a better understanding of the occurrence and development mechanism of glioma.The development of glioma is a complicated and multistep process.Prostaglandin E2(PGE2)is an important cell growth and regulatory factor that participates in almost all cellular metabolic activities and mediates a variety of physiological and pathological functions.PGE2 has now identified four receptor subtypes of EP1,EP2,EP3 and EP4.EP4 is the most widely studied EP receptor subtype,and its role in tumor is clear.There are two important signaling pathways downstream of EP4,namely c-AMP and PI3 K signaling pathways.The role of PGE2 in tumor development has been widely confirmed.For example,PGE2 is abnormal in many types of tumors,including hepatocellular carcinoma,gastric cancer,lung cancer,colorectal cancer,oral squamous cell carcinoma,breast cancer,etc,and there was a correlation between tumor size,stage,metastasis,prognosis and recurrence.Snail is an inhibitory transcription factor found in recent years.The study shows that Snail binds to the promoter region of E-cadherin gene and inhibits the transcription of E-cadherin gene,and participates in the tumor progression.c-myc is a multifunctional early response gene with a wide range of biological effects,regulating cell proliferation,differentiation,and apoptosis.Studies have shown that c-myc is abnormal expression in a variety of malignant tumors,affecting the proliferation and metastasis of tumors,and participating in the occurrence and progression of tumors.What is the expression of PGE2 and its receptor in human glioma and what are the specific regulatory mechanisms? No related studies have been reported.Based on the above,in this study,the expression of PGE2 and its receptors and the relationship with the clinicopathology of glioma were observed in human glioma tissues,which preliminarily verify the role of PGE2 and its receptors in the occurrence and progression of glioma.On this basis,a series of in vitro cell experiments were conducted.At the same time,EP receptor selective agonist,EP receptor selective antagonists and signaling pathway inhibitors were used to investigate the molecular mechanism of PGE2 in regulating human glioma.Our study could provide a certain idea and experimental basis for the molecular mechanism research of the development of glioma.Objective: To investigate the expression and meaning of PGE2 and its receptors in human glioma and investigate the control effect and mechanism of PGE2 and its receptor on human glioma,which might provide certain ideas and experimental basis for the molecular mechanism research of glioma progress.Methods: A total of 60 cases of primary brain glioma patients admitted to the first affiliated hospital of Anhui Medical University from January 2014 to December 2016 were collected.At the same time,20 cases of cerebral contusion tissue were collected in the same period as the control.Then the following experiments were conducted: 1)The expression level of PGE2 in glioma tissues were detected by ELISA assay.2)The expression level of PGE2 in serum of glioma patients were detected by ELISA assay.3)The expressions of PTGES2?CD34?VEGF?EP1?EP2?EP3 and EP4 in glioma tissues were detected by immunohistochemistry.4)The expression of EP4 in glioma tissues were detected by immunofluorescence.5)The expressions of EP3?EP4?Snail and c-myc were detected by western blot.Finally,all of the above results were analyzed with appropriate statistical methods.The human glioma cell line U87 was cultured by conventional methods.In investigating the effect of PGE2 on proliferation of glioma cells,groups were divided into: Control group(same volume solvent instead of PGE2),PGE2 1 ?M group,PGE2 5 ?M group and PGE2 10 ?M group.In investigating effect of PGE2 on Invasion and metastasis of glioma cells,groups were divided into: Control group(U87+same volume solvent instead of PGE2),PGE2 group(U87+ PGE2 5 ?M).In investigating effect of L-902,688 z on biological behavior of glioma cells,groups were divided into: Control group(U87+same volume solvent instead of PGE2),PGE2 group(U87+ PGE2 5 ?M),L-902,688 z group(U87+L-902,688 z 5 ?M).In investigating effect of CJ-42794 on biological behavior and signal molecular expression of glioma cells,groups were divided into: Control group(U87+same volume solvent instead of PGE2),PGE2 group(U87+ PGE2 5 ?M),CJ-42794 group(U87+ PGE2 5 ?M+CJ-42794 5 ?M).Three to six compound holes were set in each set,and cells were cultured for 24 to 72 hours in cell incubator in 37?.Then the following experiments were conducted: 1)The effect of PGE2 on proliferation of glioma cells were detected by MTT assay.2)The effect of PGE2?L-902,688 z and CJ-42794 on invasion and metastasis of glioma cells was measured by cell adhesion experiment.3)The protein expressions of EP4,Bcl-2,Bcl-xl,Bax,caspase-3,caspase-9,cytochrome C,c AMP,PI3 K,c-myc and Snail were detected by western blot assay.4)The effects of L-902,688 z and CJ-42794 on proliferation of glioma cells were measured by CCK8 assay.5)The apoptosis of glioma cells was measured by flow cytometry assay.Finally,all of the above results were analyzed with appropriate statistical methods.Results: 1)The tissue and serum expression level of PGE2 in glioma patients was significantly increased compared with control group(P<0.01).2)The tissue and serum levels of PGE2 in glioma patients were associated with tumor size and degree of differentiation(P<0.05).3)Immunohistochemical results showed that the expression level of PTGES2 was significantly increased in glioma tissues than in normal brain tissues(P<0.05),and is associated with pathology classification of glioma.4)Immunohistochemical results showed that the expression of VEGF was detected in 44 of 60 cases of glioma tissue samples,accounting for 73.3%.VEGF expression was positively correlated with PTGES2(P<0.001).5)The MVD of PTGES2 positive expression tissues was higher than the MVD of PTGES2 negative expression tissues(P<0.01).6)Results showed that the expressions of both EP1 and EP2 in normal brain tissues and glioma tissues were not obvious,and there was no statistically significant difference between the two groups with EP1 and EP2 expression(P>0.05).7)Immunohistochemical results showed that,the expression of EP3 in glioma tissues was significantly increased compared with the control group.In detail,in the 60 cases of glioma tissue samples,54 cases of EP3 were positive,accounting for 90% compared with three of the 20 normal brain tissue samples were positive,accounting for 15%(P<0.01).Staining intensity(+,++,+++)in glioma tissues was 6,22,26,respectively,in normal brain tissues was 3,0,0,respectively.8)Immunohistochemical results showed that the expression of EP4 in glioma tissues was significantly increased compared with control group.In detail,the positive expression rate of EP4 in glioma tissues was 96.7%(58/60)compared with 15%(3/20)in normal brain tissues(P<0.01).Staining intensity(+,++,+++)in glioma tissues was 8,21,29,respectively,in normal brain tissues was 3,0,0,respectively.9)Western blot results showed that the expression of Snail in glioma tissues is significantly increased compared in normal brain tissues,and is associated with the WHO classification(P<0.01).10)Western blot results showed that the expression of c-myc in glioma tissues is significantly increased compared in normal brain tissues,and is associated with the WHO classification(P<0.01).11)MTT results showed that the proliferation of glioma cells was significantly increased by PGE2 compared with control group(P<0.01).12)The cell adhesion experiment results showed that the migration and adhesion of glioma cells was significantly increased by PGE2 compared with control group(P<0.01).13)Western blot results showed that the expression of Bcl-2 and Bcl-x was significantly increased,the expression of Bax,caspase-3,caspase-9 and cytochrome C was significantly decreased,compared with control group(P<0.01).14)Western blot results showed that the membrane level of EP4 was significantly increased in PGE2 group compared with control group(P<0.01),the protein level of Snail and c-myc was significantly increased in PGE2 group compared with control group(P<0.01).15)The proliferation,migration and adhesion of glioma cells were obviously increased in PGE2 group compared with in control group(P<0.01),and L-902,688 z has the similar effect of PGE2.The expression of Bcl-2 was increased,caspase-3 and cytochrome C expressions were decreased in PGE2 group compared with control group,and L-902,688 z has the similar effect of PGE2.16)The proliferation,migration and adhesion of glioma cells were obviously increased in PGE2 group compared with in control group(P<0.01),while the proliferation,migration and adhesion of glioma cells were obviously inhibited in CJ-42794 group compared with PGE2 group(P<0.01).The expression of Bcl-2 was obviously increased in PGE2 group compared with control group,while was inhibited in CJ-42794 group compared with PGE2 group(P<0.01).In addition,the expression of EP4(-membrane),CJ-42794,c AMP,PI3 K,Snail and c-myc was significantly decreased by CJ-42794(P<0.01).17)In investigating the related signaling pathways of PGE2 in regulating glioma,results showed that the proliferation was increased,the apoptosis was decreased by PGE2,and dbc AMP has the similar effects.There was no obvious effect of wortmannin on proliferation of glioma cells,but has an effect on apoptosis of glioma cells.So the role of EP4-PI3 K pathway in PGE2 mediated glioma remains to be further studied.Conclusions:(1)PGE2 is abnormal expression in human glioma,and the abnormal expression of PGE2 in glioma was related to the clinicopathology of glioma including tumor size,degree of differentiation and pathological grading.(2)PGE2 plays an important role in the regulation of glioma through EP4 receptor,and participates in the occurrence and progression of glioma.(3)PGE2 may participate in the regulation of glioma through PGE2/EP4/c-AMP /c-myc or PGE2/EP4/PI3K/Snail signaling pathway. |
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