The Effect Of OLFML2A In Autophagy And TMZ Chemosensitivity Of GBM Cells

Background and objective:Glioma is one of the most common fatal primary tumors in the central nervous system.It is very troublesome to treat and patients have a poor prognosis.In recent decades,there has been no breakthrough in the treatment of glioma.Analyzing of the TCGA database,we found that the expression level of OLFML2 A is significantly related to the pathological grade of glioma and the prognosis of patients.There had been no report on the role of OLFML2 A in glioma before.This study intends to explore the functional role of OLFML2 A in gliomas through bioinformatics analysis and cell-level experiments.Methods:The first part of the study was to download the glioma expression profile chip data from the TCGA database to evaluate the relationship between the expression of OLFML2 A and the clinical characteristics of glioma,and to analyze the significance of the expression level of the target gene at different levels in different clinical data.Statistics correlation between gene expression level and clinical data was analyzed as well.The second part of the study was to use the U251 cell line as the research object,uses lentiviral transfection to knock down the expression level of OLFML2 A,use CCK-8 to test the effect of the OLFML2 A expression level on cell proliferation,and detect the effect on apoptosis by flow cytometry;The effects of OLFML2 A on autophagy of U251 cells were observed by staining with pyridine orange(AO).Western blot was used to detect the expression of apoptosis markers Bax,Bcl-2,Caspase-3 and the changes of autophagy-related proteins LC3 B,P62,AMPK,ULK1.In the third part of the study,U251 cells were used as the research object,and the CCK-8 experiment was used to detect the effect of OLFML2 A on the degree of inhibition of U251 cells by Temozolomide(TMZ).Single-cell gel electrophoresis experiment(comet experiment)was used to detect the degree of DNA damage of U251 cells after OLFML2 A gene knockdown by TMZ.Results:1.The expression level of OLFML2 A in glioma tissue was significantly higher than that in normal brain tissue,and the expression level was positively correlated with the degree of malignancy;Moreover the expression level of OLFML2 A was positively correlated with age and pathological stage,and the expression level of OLFML2 A was significant for the survival time of glioma patients,patients with higher expression levels of OLFML2 A had shorter average survival time and a worsen prognosis.2.Knockdown of OLFML2 A gene can inhibit the proliferation of U251 cells,and inhibit the expression of P62,increase the expression of LC3 B II,AMPK,ULK1,and induce autophagy.Moreover,knockdown of OLFML2 A gene can increase the expression of Bax and Caspase-3,inhibit the expression of Bcl-2,and induce cell apoptosis.3.CCK-8 results showed that compared with the control group,TMZ had a more obvious inhibitory effect on U251 cells in the knockdown group.Comet experiments showed that TMZ caused more severe DNA damage in knockdown group cells.Conclusions:The expression level of OLFML2 A is directly proportional to the malignant degree of glioma,and the higher the expression level,the worsen the prognosis of the patient.Down-regulating the level of OLFML2 A gene can inhibit the proliferation of glioma U251 cells,promote their apoptosis,and induce autophagy.Down-regulating the level of OLFML2 A gene can enhance the toxic effect of TMZ on glioma cells.