Functional Studies Of NonO In Erythroid Differentiation Process

From the fertilized egg to multicellular organisms individual, it must undergo cell proliferation, migration, apoptosis and differentiation process, the process of cell proliferation associated with cell differentiation, it is increasingly recognized that cancer is not only the abnormal proliferation of cells but also the result of disorders of cell differentiation. It even suggested that differentiation of tumor itself is a disease. Compared with the structure of normal cells, the function and metabolism abnormalities of tumor cells stopped dividing and exceed the normal proliferative capacity. Study of cell differentiation and dedifferentiation (canceration) is an important topic of life sciences research field, because its mechanism is important for us to understand tumor occurrence and it can provide an important basis for prevention and treatment of cancer.Murine erythroleukemia (Murine erythroleukemia, MEL) cells remain in the early stages of differentiation. MEL resemble to the normal immature red blood cells. MEL contains a cell nucleus, do not express hemoglobin, and can be induced differentiate to erythroid cells which express hemoglobin in vitro by sodium butyrate (SB), dimethylsulfoxide (DMSO) and six methyl allyldiethylamide (HMBA). This process is similar to the normal differentiation of erythroid progenitor cells, therefore, it is the best model for study of cell differentiation. In this paper, SB-induced MEL differentiation model was used for subsequent tests. Mouse fetal liver is the major hematopoietic organs which is a good model for in vivo studies of erythroid differentiation.Our previous study have found that the differences expression of NonO protein in fetal liver differential proteomics. NonO protein has been reported closely relate with melanoma, papillary renal cell carcinoma, prostate cancer, and red blood cell cancers and other tumors. So the study of this protein is very significant for further study of cancer.In our experiments we used a variety of biological methods. First Wright’s stain and benzidine staining was used to test MEL cell differentiation induced by sodium butyrate. By using of Western blot and immunocytochemistry we can test the expression changes of NonO protein during the process of differentiation and NonO protein’s localization in MEL cells. Immunocytochemistry can also detect colocalization of NonO with regulatory factors associated with erythroid. Lentivirus packaging technology construct NonO stable expression cell lines of MEL for further testing proliferation activity and differentiation of MEL cells with high expression of NonO.By these experiments we found NonO protein is upregulated during MEL the process of cell differentiation induced by sodium butyrate, which indicates that NonO protein expression may be associated with increasing cell differentiation and reversal of malignant leukemia cells. Through immunocytochemistry we first analysis NonO protein and negative regulator of erythropoiesis PU.1are colocalize in MEL cells, which also shows NonO protein is related with differentiation of erythroid cells. In addition, we found that NonO protein cannot directly cause MEL differentiation only by high expression experiment, but can promote MEL differentiation induced by sodium butyrate, which indicate that besides drug of sodium butyrate there are maybe other factors involved in this process. High expression of NonO protein can inhibit cell proliferation activity, and change the red lines regulatory factor GATA1expression and c-Myc. All of these can indicate that NonO is an important regulatory factor of erythroid differentiation.