The Expression And Correlation Of NDRG2, AKT2 Protein And Their Phosphoralated Forms In Gliomas

Background Human gliomas are the most common primary central nervous system neoplasm. The vast majority of these tumors developing from glial cells and the minority from neuron cells are collectively called gliomas. The patient's prognosis was still not satisfactory with poor effect of traditional treats,such as surgical operation, radiotherapy and chemotherapy. Gene therapy has been thought of as a promising method to treat gliomas nowadays for higher understanding that tumors are gene related diseases .NDRG2 (N-myc downstream regulated gene 2)is a novel gene found firstly by my teacher professor Deng Yanchun in 1999 and its expression level is decreased apparently in gliomas than in normal tissues. A series of researches have showed that the level of NDRG2 was lower in all kinds of tumors than in their counterpart normal tissues. Interfering some tumor cell lines with recombinant protein of NDRG2 or transfection of tumor cell lines with NDRG2 could inhibited proliferation and progression of these cells. So we regard NDRG2 as a candidate tumor suppressor gene,and are going to study its mechanisem of being downregulated and inhibiting tumors,wishing that it may be a tool to help curing tumors.Extensive expression of NDRG2 in all kinds of tissues implies its important meaning.NDRG2 may function by acting a role in some signal transduction path. PI3K-AKT signaling pathway is often activated in gliomas and encourages tumors'malignant progression.It has reported that NDRG2 is a substrate of AKT and is phosphorylated by AKT in rats'skeletal muscle cells when investigating insulin signaling pathway, so we design this experiment to investigate the expression level of NDRG2 and AKT2 protein and their phosphoralated forms in different pathology grades gliomas and to study the quantitive changes of these proteins through PI3K activity inhibition in both U87 and U251 cell lines in order to get more information available to more research of action mechanism of NDRG2.Methods1.Every 8 samples belonging toâ…¡,â…¢,â…£pathology grade gliomas were collected and total protein was extracted from them respectively.BCA method was applied to test concentration of total protein.At the same time,glioma cell lines U87 and U251 were cultured,and 10uM LY294002, a PI3K signaling pathway inhibitor, was used to inhibit the activity of PI3K for 1 hour.Total protein was extracted from two cell lines before and after the inhibition with the same ways.2. Western blot analysis was applied to detect the expression level of NDRG2 and AKT2 protein and their phosphoralated forms in all of tissue samples. At last,all data was analyzed statistically.3. The level of five kinds of proteins was still detected in U87 and U251 cell lines before and after the inhibition and analyzed.Result1.The level of NDRG2 protein decreased apparently in gliomas with the increasing of grades and the variance was significant statistically ( P<0.05 ) .The AKT2 protein is expressed in all samples ,while the AKT2 phosphorylated at Ser474 site (P-AKT2-SER474) is only expressed in 58% gliomas samples.The levels of the NDRG2 phosphorylated at Ser332 (P-NDRG2-SER332) and the NDRG2 phosphorylated at thr348 (P-NDRG2-THR348) were different in gliomas from every grade and the levels of two kinds of protein in gradeâ…¢were both the highest of the three grades.2. Both P-NDRG2-SER332 protein and P-NDRG2-THR348 protein highly expressed in the glioma samples in which P-AKT2-SER474 lowly or didn't express,while both proteins lowly or didn't express in the glioma samples in which P-AKT2-SER474 highly expressed.3. After phosphorylation inhibition of PI3K,the level of NDRG2 protein, P-NDRG2-SER332 protein and P-NDRG2-THR348 protein decreased in both U87 cell line and U251 cell line.AKT2 protein expressed in both cell lines whenever before or after PI3K activity inhibition,but P-AKT2-SER474 expressed only in U87 cell line,and its level decreased after inhibion of PI3K activity.Conclusion1.The expression level of NDRG2 in gliomas correlates with the pathology grades of gliomas,which supports NDRG2 may a tumor suppressor gene.2.In gliomas,the phosphorylation of NDRG2 doesn't depend on the phosphorylation of the AKT2,and the competitive relation between the phosphorylation of NDRG2 and that of AKT2 maybe exist.3.NDRG2 protein is a probable molecule in PI3K signaling pathway,but other molecules in downstream of PI3K must phosphorylate it except for AKT2.