The Roles Of ARPP-19,LC3B,P62 Expression In Glioma

Part One: The roles of ARPP-19 in gliomaObjectiveGlioma is the most common and aggressive type of human primary brain tumor with a poor outcome.The molecular mechanisms understanding glioma development and progression are still poorly understood.c AMP-regulated phosphoprotein 19(ARPP-19),belongs to the family of alpha-endosulfine(ENSA),and is identified in the mammalian brain as a substrate for protein kinase A(PKA).ARPP-19 plays an important regulatory role in the central nervous system,including the signal transduction of many neural factors,the expression regulation of the genes related to nervous system.ARPP-19 was an important regulator to modify the growth and physiological status of synapses.On the other hand,ARPP-19 also plays a role in the pathologic process of neuronal system dieases.For example,Kim et al revealed that decreased levels of ARPP-19 may contribute to the progression of Down syndrome and Alzheimer's disease.In addition,the studies have also demonstrated a novel role of ARPP-19 in human cancer development and progression.Recent studies have demonstrated a novel role of ARPP-19 in regulation of cell mitosis and cancer progression.Therefore,ARPP-19 can be considered a promoter of oncogenes.However,no study has been conducted to determine the role of ARPP-19 in human glioma cells,and analyzed the expression and clinical significance of ARPP-19 in human glioma.In this study,we investigate the role of ARPP-19 in human glioma cell line A172 using immunohistochemical staining and protein immunoblotting,and to further explore the role of ARPP-19 in the genesis and development of human glioma.A retrospective study was performed to examine the expression of ARPP-19 protein in 81 cases of human glioma tissue,and to explore the correlation ARPP-19,and to analyze the relationship between the pathological parameters and the survival rate of patients.Our aims are to clarify the clinical significance of the ARPP-19 in glioma patients.We hoped that ARPP-19 can be used as biological markers to analyze the survival rate of high-grade glioma,and to find a new treatment target and strategy for high-grade glioma.MethodsIn this study,the human glioma cell line(A172)was used in the cell experiment research in vitro,and to explore the role of ARPP-19 genes in A172 cell line.After the transfection of synthetic ARPP-19 si RNA,the expression of ARPP-19 was detected in A172 glioma cell line,and the proliferation,non-anchorage-dependent growth and malignant behavior of the transfected A172 cell line were examined by cell soft agar cloning assay.Trans-well chamber and cell scratch wound healing experiments were performed to examine the migration and invasion of A172 cells after the transfection with ARPP-19 si RNA.Western Blot was used to detect the expression of cyclin D1 and c-myc proteins in A172 cells transfected with ARPP-19 si RNA and control group cells(?-actin as the internal reference gene).At the same time,81 cases of human glioma clinical tissue samples was collected.The protein expression of ARPP-19 was detected by immunochemistry.The overall survival of these patients was recorded by telephone and outpatient follow-up.The correlation between the clinical pathological parameters and patient's disease-free survival(RFS)and overall survival(OS)were analyzed using Pearson chi-square,Kaplan Meier curve(Kaplan Meier curves).ResultsAfter the transfection with ARPP-19 si RNA in A172 cells,ARPP-19 m RNA expression was significantly decreased.The cell growth curve of A172 cells 5 days after the transfection with ARPP-19 si RNA was detected,and the results showed that the number of cell growth was significantly decreased.Consistent with the results of cell growth,the formation of soft agar clones in A172 cells after the transfection of ARPP-19 si RNA was also significantly reduced.In addition,the ability of metastasis and cell invasion have a significant degree of reduction in the A172 cell line after the si RNA transfection,which due to the reduced expression level of ARPP-19.Concomitantly,when the ARPP-19 expression level was knocked down in the A172 cells,the ability of scratched wound healing was greatly weakened.Moreover,we found that the expression level of cyclin D1 and c-myc decreased after knockdown of ARPP-19 in A172 cells.ConclusionOur results suggested that ARPP-19 had the effect of promoting the proliferation and metastasis of human glioma.These effects of ARPP-19 may be related to significantly up-regulate the expression of cyclin D1 and c-myc genes.The protein expression level of ARPP-19 was significantly correlated in malignant degree of glioma.High expression of ARPP-19 in gliomas was generally related to high-grade malignancy of the tumor,including high-grade tumors and poor prognosis.Part Two:The roles of LC3 B,p62 expression in gliomaObjectiveCell autophagy is a basic physiological metabolic mechanism to maintain cell stability and metabolic balance,and by which cells degrade their unwanted molecules and components.Simultaneously autophagy also play a important role in the development,progression and anti-cancer therapy resistance of many types of human cancers.As previously reported,the formation of autophagosomes is a key step in the process of autophagy.LC3 is associated with the membrane of autophagosomes,and involved in the autophagy.Therefore,LC3 is widely used as an autophagy marker in the study of autophagy.In the other way,with the progress of biotechnology,the detection of LC3 by immunohistochemistry,immunoblotting and other methods using specific antibodies was more common.Many studies revealed high LC3 expression associates with poor prognosis in diverse cancer.In the process of development of human malignancies,cell autophagy can promote tumor cells to adapt to the malignant environment,and thereby contributing to the malignant behavior of malignant tumors.Therefore,the high-degree malignancy tumor with poor prognosis is often accompanied by high LC3 expression.Many studies revealed high LC3 expression associates with poor prognosis in diverse cancer patients through autophagy.In malignant gliomas,a few studies documented the association of autophagy with cancer aggravation.But there was no one studying systematically about the relation between LC3 and clinical behaviors of glioma patients.LC3 consists of three isoforms: LC3 A,LC3B,and LC3 C.In immunohistochemical testing,LC3 A shows three patterns: diffuse cytoplasmic,cytoplasmic /juxtanuclear,and stone-like pattern,and which pattern represented the functional LC3 A involved in autophagy remains confusing.In all tissues,LC3 C is much lower than that of LC3 A and LC3 B.So LC3 B was selected to study the relation between LC3 and clinical behavior of glioma patients here,which has a high expression level and has a defined punctate pattern by immunohistochemical testing participating in autophagy.p62 plays a key role in cell autophagy,which is involved in the process of cell autophagy as an essential substrate for some of the enzymes.Therefore,p62 is commonly used to study the cell autophagy.In order to understand the correlation between P62 and LC3 B,we also detected the P62.In the study,we performed a retrospective study to examine the expression of LC3 B and p62 protein in 81 cases of human glioma tissue,and the correlation between LC3 B and p62 was analyzed in 81 cases.In addition,the correlation between the clinical pathologic parameters and the survival rate of patients was illustrated.Our aims is to clarify the clinical significance of the above proteins in glioma patients,and hoped that LC3 B and p62 can be used as a biological index to measure the survival rate of high-grade glioma,and to find a new treatment target and strategy for high-grade glioma.MethodsWe collected 81 cases of human glioma clinical tissue samples from Department of Pathology of the Second Affiliated Hospital of Anhui Medical University from 2009 to 2015.The expression of LC3 B and p62 protein was detected using immunohistochemical technique in human gliomas tissues from 81 patients.The correlation between the expression level of LC3 B and p62 protein and the clinical pathological parameters and patient's disease-free survival(RFS)and overall survival(OS)were analyzed using Pearson chi-square,Kaplan Meier curve(Kaplan Meier curves).ResultsIn this study,we detected the expression of two autophagic protein LC3 B and p62 in 81 glioma tissues by Immunohistochemistry analysis.LC3 B and p62 expressed high in high-grade glioma tissues,and expressed low in low-grade glioma tissues.High LC3 B and p62 protein level were also associated with advanced tumor stages,worse relapse-free survival(RFS)and overall survival(OS)in glioma patients,but not with patients' age,gender or KPS.Furthermore,we found a significant positive correlation between LC3 B and p62 expression in gliomas,suggesting that LC3 B and p62 may play a role in gliomas and contribute to tumorigenesis and progression of gliomas malignant deterioration.ConclusionOur results suggested that the expression of LC3 B and p62 protein was closely related to autophagy,which can promote the development of malignant glioma and the prognosis of patients with poor prognosis.Highly expressed cell autophagy-related proteins,LC3 B and p62,are predictive of high malignancy and poor prognosis of gliomas.Therefore,therapies based on autophagy,or drugs targeting to the LC3 B and p62 genes,are a potential can be used to treat the advanced glioblastoma.