Expression,Function And Mechanism Of PDPN In Glioma

Background: Gliomas are the most common primary malignant brain tumors and the most difficult to treat.Conventional radiotherapy and chemotherapy are often ineffective.Therefore,it is urgent to find new treatment methods to improve the prognosis of glioma patients.Molecular targeted therapy is a promising treatment method,but it highly depends on accurate molecular marker detection and precise molecular typing.So far,although many molecular markers have been found in gliomas,due to the high heterogeneity of gliomas,it is still necessary to continuously search for new molecular markers for molecular typing,prognosis prediction,or developing new targets.Podoplanin(PDPN)is a transmembrane mucin-type glycoprotein that is specifically expressed on the surface of glomerular podocytes and is involved in maintaining the normal morphology of podocyte foot processes.It plays an important physiological role in maintaining the integrity of the glomerular basement membrane and ensuring normal glomerular permeability.Recently,it was found that PDPN is also abnormally expressed in various malignant tumors and is associated with lymph node metastasis and poor prognosis in malignant tumors,making it a potential tumor molecular marker.However,the expression characteristics of PDPN in glioma tissue,its correlation with clinicopathological parameters,and its relationship with patient survival time have not been validated in tissue specimens;its molecular mechanism,biological characteristics,and function in the development of gliomas have not been fully understood.Based on the above background,this study aims to explore the following issues: first,the expression characteristics of PDPN in gliomas and its correlation with clinical prognosis;second,the prognostic prediction value of PDPN in traditional histopathological typing and the 2021 fifth edition of the World Health Organization Classification of Tumors of the Central Nervous System(WHO CNS5);third,exploring the biological function of PDPN in the development of gliomas and the possible signaling pathways involved.Methods: In this study,227 specimens of gliomas were collected from surgical treatment at the Second Affiliated Hospital of Anhui Medical University and the Cancer Hospital of the Chinese Academy of Medical Sciences from 2009 to 2016,of which 22 cases had matched non-tumor brain tissue.Immunohistochemical staining was used to detect the expression of PDPN,IDH,ATRX and p53 in tissue samples.Sanger sequencing was used to detect the TERT promoter mutation in tissue samples.The correlation between PDPN and clinical pathological parameters such as IDH,TERT,ATRX,p53,gender,age,Karnofsky performance status(KPS score)and WHO grading was analyzed by chi-square test.According to the WHO CNS5 classification criteria,227 cases of gliomas were divided into four groups: IDH-mutant astrocytoma,IDH wild-type glioblastoma,oligodendroglioma and IDH wild-type astrocytoma.Then,the prognostic predictive value of PDPN in traditional histology classification and WHO CNS5 molecular typing was analyzed by Kaplan-Meier method.Finally,Cox regression univariate and multivariate analysis were used to explore the main factors affecting the prognosis of patients with glioma.In the functional experiment,we constructed U87 MG and U118 MG cell lines with transient knockout of PDPN,detected the effect of PDPN on the proliferation of glioma cells by CCK-8 technique,and detected the effect of PDPN on the migration and invasion of glioma cells by Transwell migration and invasion in vitro.In the in vivo experiments,we constructed a stably knocked down PDPN U87 MG cell line,and used the subcutaneous tumor formation experiment of nude mice to detect the effect of PDPN on the subcutaneous tumor formation ability of nude mice.In mechanism studies,we first predicted the biological function of PDPN and possible signal transduction pathways using GO analysis and KEGG analysis.Then,by knocking down PDPN,the changes of downstream molecules in U87 MG and U118 MG cells were detected by Western blot technique.Finally,Akt/mTOR pathway agonist SC79 combined with PDPN knockdown was used to observe cell viability,migration and invasion ability of U87 MG and U118 MG cells,so as to explore the possible signal pathway involved in PDPN.Results: The positive rate of PDPN expression in 227 cases of glioma tissues was44.9%(28.1% of WHO grade 2,32.4% of WHO grade 3,55.1% of WHO grade 4),and no positive expression was detected in 22 paired non-tumor brain tissues,the positive rate of PDPN expression was statistically different between glioma and non-tumor brain tissue(p < 0.001).The high expression of PDPN was positively correlated with age of onset(p < 0.001)and WHO grade(p = 0.001),but not significantly correlated with clinicopathological parameters such as gender(p = 0.802)and KPS score(p =0.754).In addition,the high expression of PDPN was positively correlated with IDH wild-type status and TERT promoter mutation status(p < 0.001),negatively correlated with 1p/19 q combined deletion and ATRX deletion status(p < 0.01),and had no significant correlation with p53(p > 0.05).After further typing according to the WHO CNS5 classification criteria,it was found that the positive rate of PDPN expression in oligodendroglioma was significantly lower than that in astrocytoma(11.8% vs 48.0%;p < 0.004).PDPN was positive in 11.9% of IDH mutant astrocytoma,11.8% in oligodendrogliomas,43.2% in IDH wild-type astrocytoma,and 71.3% in IDH wildtype glioblastomas.And,there were significant differences in PDPN expression levels between different molecular subtypes(p < 0.001).In IDH wild-type diffuse astrocytoma(WHO grade 2-4),the positive rate of PDPN expression gradually increased with the increase of WHO grade(37.9% for grade 2,53.3% for grade 3,71.3% for grade 4;p = 0.003).However,there was no significant correlation between PDPN expression and WHO grade in IDH mutant astrocytoma(WHO 2-4),oligodendroglioma(WHO 2-3)and IDH wild-type astrocytoma(WHO2-3).In addition,among WHO grade 4 gliomas,the positive rate of PDPN in IDH wild-type glioblastomas was significantly higher than that in IDH mutant astrocytoma(71.3% vs16.7%;p < 0.001).The results of Kaplan-Meier survival analysis showed that the high expression of PDPN was positively correlated with the poor prognosis of patients in both low-grade glioma group and glioblastoma group(p = 0.004,p = 0.001).After subdividing according to the WHO CNS5 classification,it was found that in IDH mutant astrocytoma,IDH wild-type glioblastoma and IDH wild-type astrocytoma,the overall survival of patients with high expression of PDPN was significantly shorter than that of patients with low PDPN expression patients(p = 0.01,p = 0.012 and p =0.017).In addition,among patients with low-grade glioma and glioblastoma who received postoperative radiotherapy and chemotherapy,the median survival time of patients with high expression of PDPN was also significantly shortened(p < 0.001,p =0.001).Univariate Cox regression analysis showed that age,KPS score,WHO grade,postoperative radiotherapy and chemotherapy,IDH,TERT promoter mutation,p53 and PDPN were all important factors affecting the prognosis of patients.Multivariate Cox regression analysis showed that WHO grade,postoperative radiotherapy and chemotherapy,and high expression of PDPN were independent predictors of poor prognosis in glioma patients.In vitro and in vivo experiments showed that knockdown of PDPN could inhibit the proliferation,migration and invasion of glioma cells and inhibit the subcutaneous tumorigenesis of nude mice.Western blot results showed that knockdown of PDPN could down-regulate the expression of p-Akt and p-mTOR,but did not affect the expression of Akt and mTOR.Using the Akt/mTOR pathway agonist SC79 can partially reverse the effect of PDPN knockdown on the malignant phenotype of glioma cells,suggesting that PDPN is involved in the Akt/mTOR signal transduction pathway and plays a regulatory role by affecting the phosphorylation status of Akt and mTOR.Conclusion: The expression level of PDPN in glioma tissue is significantly higher than that in non-tumor brain tissue.PDPN is positively correlated with IDH wild-type status and TERT promoter mutation status,negatively correlated with 1p/19 q codeletion and ATRX deletion status,and has no significant correlation with p53.The expression level of PDPN in astrocytes is significantly higher than that in oligodendrocytes.In IDH wild-type diffuse astrocytoma,the expression level of PDPN is positively correlated with the pathological grade.High expression of PDPN is associated with adverse prognosis in patients and is an independent risk factor,which can be used as a predictive marker for adverse prognosis in glioma.Knockdown of PDPN can inhibit the proliferation,migration,and invasion of glioma cells and inhibit glioma growth in vivo.PDPN participates in the Akt/mTOR signaling pathway and exerts a carcinogenic effect by regulating the phosphorylation status of Akt and mTOR.PDPN is expected to be a potential target for targeted therapy in glioma.