Identification Of ALDH1A3 Role On Glioma Progression

Objectives:Glioma is the most comman and lethal brain tumor in the world.Glioma places a huge financial burden on the community, the families of patients and health care systems. Malignant glioma remains a major therapeutic challenge owing to several factors, such as high invasion of the normal brain, resistance to chemotherapy and radiotherapy, and intratumoral cellular heterogeneity. At present, the exact pathogenesis of glioma is not clear. Since the implementation of cancer genome mapping and advances in molecular biotechnology,a large number of genetic information of malignant tumors was revealed. The study on gliomas has advanced from the histopathological level to the key driving genes and molecular targets,including the genetic mutation, signaling pathway transduction,microenvironment change and so on. However the complexity of glioma has been underestimated by genomic research. Glioma undergo aerobic glycolysis rather than oxidative metabolism, even in the presence of oxygen. This metabolic modification provides a survival advantage and facilitates synthesis of biological macromolecules substance which required for cancer cell growth.It was unclear that aldehyde dehydrogenase 1 A3(ALDH1A3) expression pattern and influence on progression in malignant glioma.The discovery of vital biomarkers against glioma would increased our understanding on the molecular mechanism and development of therapeutic target.The specific differentially expressed genes and the characteristic gene changes following knockdown ALDH1A3 were investigated.Methods:Not only have the methylation status of ALDH1A3 CpG island been identified by pyrophosphate sequencing, the protein expression level of ALDH1A3 also have been measured with paraffin section immunohistochemitry in 115 glioma and 12 normal brain tissues. ALDH1A3 promoter methylation status and total surviving curve is analyzed by Kapplan-Meier. Real-time fluorescence quantitative PCR assay was performed to detect the expression of ALDH1A3 in glioma cell lines. Subsequently,we established the model of ALDH1A3-shRNA U87 cell line by stably transfected lentiviral vector and established brain transplantation model in nude mices. The changes of biological function following down-regulation of ALDH1A3 were detected by mitochondrial membrane potential ,intracellular ATP level,glucose level,apoptosis level ,cell cycle, cell proliferation and key enzyme involved in glycosis and invasive ability in vitro .For further study the role of ALDH1A3 on glioma,high-throughput transcriptome sequencing, bioinformatics analysis and validation were carried out.Results:Not only the hypermethylation status of ALDH1A3 promoter but also low protein expression of ALDH1A3 predicted a better prognosis. Our results showed that ALDH1A3 was frequently overexpressed in glioma tissues and cell lines. Knockdown of ALDH1A3 in glioma cell line U87 which expressing high levels of ALDH1A3 resulted in decreases in intracellular mitochondrial membrane potential and cellular ATP levels, inhibitions of cell proliferation, PKM2 , HK2 protein levels and invasion ability in vitro. Knockdown of ALDH1A3 increased apoptosis and caused cell cycle arrest at G2/M. Knockdown of ALDH1A3 also enhanced tumorigenicity in brain of nude mices.The 104 significantly common DEGs in samples have been detected which including 33 upregulated and 71 downregulated DEGs. The 104 DEGs were divided into 24 functional categories based on GO analysis. There are 8 significant signal pathways involed in various cellular fuction including Apoptosis, Cellular invasive, FoxO signaling pathway, Cell cycle, Glycolysis and Gluconeogenesis pathway, Metabolic pathways, Jak-STAT signaling pathway, Retinol metabolism.The protein interaction uncovered 4 DEGs including HSP90AB, Fox03, STAT6, PKM2.The four protein was validated by Western-Blot method.Conclusions:ALDH1A3 overexpression is a common feature of gliomas. The expression levels of the ALDH13 proteins increase with the malignancy degrees of gliomas, and correlate with the pathological grades of the tumors. Our results indicate ALDH1A3 promoter methylation correlation with the prognosis in primary glioma. ALDH1A3 promoter status and expression level was an independent predictor for DFS and OS of glioma patients. The biological behaviors including apoptosis, proliferation, cell cycle,intracellular mitochondrial membrane potential , cellular ATP levels ,cellular glucose ,glycosis enzyme PKM2 and HK2 and invasive ability have been regulated by ALDH1A3. These result suggested that ALDH1A3 have been involved in glycosis and energy metabolism.It could be used as candidate for glioma diagnosis and treatment.In this study, the DEGs of ALDH1A3-shRNA were thoroughly analyzed.The 104 significantly common DEGs in samples have been detected and focused on 8 signal pathways. The protein interaction analysis revealed 4 DEGs including HSP90AB1, Fox03, STAT6, PKM2, while further research is needed to explor mechnism in glioma progression.