Establishment Of Expression Of C/EBPα Gene In Raji Cell Line And The Research On Its Biological Activity

Background and ObjectiveLeukemia is the common term for a diverse group of malignancies, defined by accumulation of abnormal hematopoietic progenitor cells, which fail to undergo terminal differentiation.The second-hit hypothesis proposes that most acute leukemias are the consequence of a collaboration of several types of mutations. ClassⅠmutations often affect genes of RTKs, which confer a proliferative and survival advantage to hematopoietic progenitors. ClassⅡmutations are usually chromosomal translocations involving hematopoietic transcription factors, with consecutive impairment of differentiation and apoptosis of cells. The CCATT/enhancer binding protein alpha, C/EBPa, is a key transcription factor involved in normal hematopoietic system and leukemia. C/EBPa act as an inhibitor of growth and apoptosis, and induce myeloid progenitors to differentiate. It's reported that C/EBPa was oncogene and tumor suppressor gene in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) respectively. And "Just the right" amount of C/EBPa is needed for maintenance of normal haematopoiesis. "Too much or too little" of C/EBPa can contribute to leukaemogenesis. Hematopoietic lineages are specified in a stepwise process of binary decisions, starting with multipotent progenitors which branch into a common lymphoid and a common myeloid progenitor that differentiate in turn into additional intermediate progenitors. Each lineage exhibits a distinct gene expression pattern that is laid down and maintained by a set of more than a dozen lineage restricted transcription factors that are part of a "transcription factor network" .It has long been assumed that differentiation is an irreversible process.While, it's reported that enforced expression of C/EBPa in B cells leaded to their rapid and efficient reprogramming into macrophages.And we also can't explain some phenomenons like relapsed ALL could transform into AML, CML at blast crisis(CML-BC) may be AML or ALL. The relationship between C/EBPa and transformation of ALL is unknown.In this paper, we detected C/EBPa gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in HL60, NB4, K562, Raji, MOLT-4,6T-CEM cells, and the entire C/EBPa coding sequence was screened in positive cells. By using retroviral vector pMSCV-IRES-GFP, the stable expression of the C/EBPa gene homo-ALL cell line (Raji cell) was established, and we can study the biological function of C/EBPa gene in Raji cell.Materials and Methods1. We withdrawed total RNA from the HL60, NB4, K562, Raji, MOLT-4 and 6T-CEM cells, and detected C/EBPa gene by RT-PCR. For the positive cells, we made TA clone to sequence the entire C/EBPa coding sequence.2. The pMSCV-C/EBPA-IRES-GFP (pMIG-C/EBPa) retroviral vector was transformed into DH5a. We confirmed the target gene and its sequence by double enzyme (BglⅡand XholⅠ) and direct sequencing.3. The pMIG and pMIG-C/EBPa retroviral vector were inducted into the packaging cell Phoenix 293 by LipofectamineTM2000 (Invitrogen). After transfection 48 hours and 72 hours, gently removed the supernatant and either filtered through a 45 M filter or centrifuged 5 min at 500g at 4℃to remove living cells. The supernatant was used to transfect Raji cell, and we sorted the Green Fluorescent Protein (GFP) positive cells by Fluorescence-Activated Cell Sorting (FACS) to establish the stable expression of C/EBPa gene Raji cell. By RT-PCR, we detected the expression of C/EBPa gene.4. To study the biological differences in C/EBPa positive-Raji cell, we used multiple methods such as cytomorphology, FACS, MTT (Multiply-Table Tournament).5. The statistical analysis was performed with the software SPSS13.0, and the significance was set at P value≤0.05.Results1. RT-PCR and direct sequencing results showed that HL60 and NB4 cells were positive for C/EBPa gene, other cells were negative. Comparison with the NCBI database (NM 007678.3), the coincidence rate of HL60 cell entire C/EBPa coding sequence was 99%.2. The C/EBPa gene was insected into pMIG vector by using the technology of digestion by double enzyme (BglⅡand XholⅠ) and direct sequencing.3. We used FACS to sort the GFP+ cells at 72h post-transfection of Raji cells by retroviral supernatant. After a few days cell culture, the GFP+ cells were taken account 93.8% and 79.6% in Raji+pMIG cells and Raji+pMIG-C/EBPa cells, respectively. By RT-PCR, we confirmed that C/EBPa gene was positive in Raji+pMIG-C/EBPa cells, and negative in Raji and Raji+pMIG cells.4. Cytomorphology showed that the nuclein of Raji+pMIG-C/EBPa cells was loose compared to the other two cells. All the three cells were PAS and POX negative.5. RT-QPCR results showed that Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPa cells were Pax5 and PU.1 positive (P value was 0.450 and 0.186), and negative for MPO, G-CSFR and GM-CSFR gene. 6. FACS results showed that CD19 positive rate was (97.76±1.48)%,(97.93±0.64)%,(96.98±1.80)% in Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPa cells separately(F=0.398,P=0.688), and their mean value was 7003.5±276.48,5803.5±159.10,4808.5±327.39. However,they were all negative for CD33 and CD14.7. MTT showed that the cell proliferation rate of Raji+pMIG-C/EBPa cells was significantly slower that that in Raji cells and Raji+pMIG cells (P< 0.001).8. Above all, we knew that C/EBPa gene may decrease proliferation of the cell. And this was maybe the reason for why we didn't find a cell-type tansformation we expected.We did FACS after transfection 72h, it was found that, CD19 positive rate of the cell was decreased, and CD 14 and CD33 were still nagetive. By sorting the CD 19 negative cells, we did RT-PCR. Apart fromβ-actin and C/EBPa gene were positive, the others were all nagetive including Pax5 and PU.1.ConclusionWe detected C/EBPa gene in several leukemia cells and the entire coding sequence of the positive cell HL60 was not found mutation. The stable expression C/EBPa gene Raji cell line was obtained and the gene may decrease proliferation of the cell.Meanwhile, CD 19 positive rate of the cell was decreased after 72h of transfection. More conculsions needed convinced by more experiments.