Effect And Mechanism Of OLFML2A Gene Regulation Of Glioma Proliferation And Apoptosis

Background and objective: Glioma is the most common and lethal primary tumor of the central nervous system,with a high malignancy degree and an extremely poor prognosis.Malignant proliferation and apoptosis resistance is one of the most important mechanisms of glioma genesis and development.It is crucial to reveal the regulatory mechanisms of tumor cell proliferation and apoptosis for predicting malignant degree of tumor,reflecting biological characteristics of tumor and guiding clinical treatment and prognosis evaluation.Glioma is a highly heterogeneous tumor.Further study of the molecular mechanism of glioma is helpful to reveal the heterogeneity of tumor cells,explain the mechanism of malignant proliferation and apoptosis of tumor cells,which is significant to the molecular classification,prognosis evaluation and targeted therapy of glioma.Our research group have previously analyzed the TCGA(The Cancer Genome Atlas)database and found that the expression level of Olfactomedin-like 2A(OLFML2A)was elevated in glioma,and its expression level was correlated with the pathological grade of glioma and the prognosis of patients.However,the molecular function of OLFML2 A and its underlying mechanism of action in glioma remains unclear.In this study,the effects of OLFML2 A on proliferation and apoptosis of glioma were investigated by conducting experiments of cell lines,animal model and human tissue specimens,to provide a new genetic target for the diagnosis,molecular classification and treatment of glioma.Methods: The expression level of OLFML2 A in glioma and its relationship with tumor pathological grade was determined by immunohistochemistry(IHC).Lentivirusmediated RNA interference(RNAi)was used to knock down the expression of OLFML2 A in glioma cells,and the effects of OLFML2 A gene on proliferation and apoptosis were further investigated by Celigo,MTT assay and flow cytometry.Gene chip microarray analysis and ingenuity pathway(IPA)analysis were used to explore the potential regulatory mechanism of OLFML2 A gene on glioma,and the relevant mechanisms were further verified at the molecular level by qRT-PCR and Western blot.In order to investigate the effect of OLFML2 A on glioma proliferation in vivo,subcutaneous tumor-forming animal models in nude mice and brain orthotopic transplantation animal models in rats were established,and underlying regulatory mechanisms were verified at the animal tumor tissue.Results: Immunohistochemistry staining showed that OLFML2 A was elevated in glioma compared with normal brain tissue.Meanwhile,the expression level of OLFML2 A was significantly higher in high-grade glioma(HGG)than in low-grade glioma(LGG).The results of Celigo,MTT assay and and flow cytometry analysis indicated that OLFML2 A downregulation could inhibit the proliferation and promote the apoptosis of glioma via using U251 and U87 MG cell lines.Gene chip microarray analysis showed that the expression levels of 1911 genes were altered following OLFML2 A knockdown,of which 658 genes were up-regulated and 1253 genes were down-regulated.Then,functional analysis was performed via IPA,and the differentially expressed genes(DEGs)were observed to be enriched in cancer,cell cycle,cell death and cell survival,cellular growth and proliferation,suggesting that OLFML2 A was significantly involved in proliferation and cell cycle regulation processes.Canonical pathway analysis using IPA was conducted and found that the Wnt/ β-catenin signaling pathway is a potential downstream regulatory pathway for OLFML2 A.Molecular interaction network using Ingenuity pathway analysis have shown that amyloid precursor protein(APP)is a potential regulator of OLFML2 A regulating the Wnt/ β-catenin signaling pathway.Western blot and qRT-PCR analysis further confirmed that OLFML2 A gene knockdown could up-regulate APP expression and inhibit Wnt/ β-catenin signaling pathway at the cellular and molecular level.The results of nude mouse tumor model and rat C6 brain orthotopic transplantation model showed that downregulation of OLFML2 A gene can inhibit the growth of glioma cells in vivo,and further confirmed that OLFML2 A gene knockdown can up-regulate the expression of APP and inhibit the Wnt/β-catenin signaling pathway by Immunohistochemistry staining in animal tumor tissue.Conclusions: OLFML2 A is elevated in glioma tissues and the expression level of OLFML2 A is positively correlated with tumor grade.Downregulation of OLFML2 A could inhibit proliferation and promote the apoptosis of glioma cells,and increase the expression of APP.Mechanistically,knockdown of OLFML2 A regulates glioma proliferation and apoptosis via surpression of Wnt/β-catenin signaling pathway,and APP is a potential intermediate molecule that mediates this regulatory process.The effect and mechanism of OLFML2 A regulation of glioma proliferation and apoptosis provides a new target for glioma molecular targeted therapy.