The Establishment Of Overexpression Of CEBPα Combined With PU.1 In Jurkat Cell Line And The Exploration Of Its Biology

Background and objectiveLeukemia is a group of highly heterogeneity disease, defined by accumulation of abnormal hematopoietic progenitor cells, which fail to undergo terminal differentiation and resist to die. It is widelyspread that the causes of leukemia followed by a series of destructive hits. The most popular second-hit hypothesis claims that most acute leukemia are the consequence of a collaboration of several types of mutations. Class I mutations often influence the function of RTKs, which contains a proliferative and survival advantage to normal hematopoietic progenitors. Class II mutations are usually chromosomal translocations or the formation of fusion protein, which involving hematopoietic transcription factors, with consecutive impairment of differentiation and apoptosis of cells. This shows that the transcription factors play a vital role in the maintenance of normal biological characteristics of hematopoietic stem cells. In clinical practice, we found some phenomena that some AL patients with both myeloid and lymphoid marker at diagnose. CML patients at blast crisis (CML-BC) may transform to ALL or AML. And it is still unclear about it and what the probable mechanism.Several studies proved that, CCAAT/enhancer binding protein alpha (C/EBPa) and PU.1 play a major role during the commitment of hematopoietic stem cells, towards granulocytic and monocytic differentiation, and their reciprocal ratio was found to be essential for lineage determination. The CCAAT/enhancer binding protein alpha, C/EBPa, is a key transcription factor involved in normal hematopoietic system and leukemia. CEBPa gene is a membership of leucine zipper, which is located in the long arm of the nineteenth chromosome, contains 3318bp. Several lines of evidence indicate that C/EBPa functions in terminally differentiated, growth-arrested cells and inhibitor of apoptosis. 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 the maintenance of normal hematopoiesis. "Too much or too little" of C/EBPa can lead to leukemia. Hematopoietic lineages are specified in a stepwise process of binary decisions, starting with multipotent progenitors which branch into a common lymphoid progenitor and a common myeloid progenitor that differentiate in turn into additional intermediate progenitors. Each lineage owns 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 with the burn of IPS (Induced pluripotent stem cell) technology, this theory has been greatly challenged. A series study of Thomas suggested that normal primary B/T lymphoma cell can be induced into functional macrophage by overexpression of CEBPa and PU.1. Profeesor Feng also indicated that the combination of the two factors, as well as PU.1 and C/EBPβ, induced the up-regulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells, which distantly related to blood cells. And these mac-1 positive cells, like functional macrophage cells can phagocyte of bacteria. Therefore, whether the expression level of these transcription factors can influence the type of acute leukemia is still unknown. If so, it will be a potential treatment for ALL patients.Our precious study implied that by enforced expression of CEBPa in B cell lymphoma cell line Raji, CD 19 (as a marker of B cell) gradually decreased, but mac-1 positive cells did not appear. Thomas paper also indicated that the malignance of most B lymphoma/leukemia cell lines can be greatly reduced, while most of them can not be induced into macrophage-like cells when by enforced expression of CEBPa. So we combined PU.1, assumed that the two factors can highly induce ALL cell line into functional macrophage cells, and provide a potential theory for induce-treatment in non-APL. We first established the overexpression lentivirus vectors (PWPXLD-PU.1+CEBPa-eGFP, PWPXLD-PU.1-eGFP, PWPXLD-CEBPa-eGFP. By using lentivirus vector, the stable expression of the C/EBPa, PU.1, CEBPa+PU.1 and only GFP positive T-ALL Jurkat cell lines were established, and we then study the biological function of these gene in Jurkat cell.Materials and Methods1. Designing the primers of these entire gene, then made TA clone to sequence the entire C/EBPa coding sequence. Cutting the T-target gene and backbone vector with Double enzymes (Pme1 and Spe1) and then connect the two parts by Solution I with right percentage.2. The lentivirus vectors were transformed into TOP 10. We confirmed the target gene and its sequence by double enzyme cutting (Pme1 and Spe1) and direct sequencing.3. These lentivirus expression vectors combined with helper vectors were transfected into the packaging cell 293 T using PEI transfection reagent. To collect virus surpernatant at 24 hours,48 hours and 72 hours after transfected, gently removed the supernatant and either filtered through a 45 M filter or centrifuged 2 hours at 28000 rpm at 4℃. Virus titer was detected.4. The supernatant virus was used to infect Jurkat cell, and then we sorted the enhancer Green Fluorescent Protein (eGFP) positive cells by Fluorescence-Activated Cell Sorting (FACS) to establish the stable expression of C/EBPa, PU.1, CEBPa+PU.1 gene cells. We detected the expression of C/EBPa、and PU.1 gene by RT-PCR. To make sure these genes were stable expressed in Jurkat cells.5. To study the biological of these target genes positive-Jurkat cells, we used multiple methods such as cytomorphology, FACS, CCK-8, qRT-PCR detect related genes.6. The statistical analysis was performed with the software SPSS for windows 19.0, and the significance was defined at P value<0.05.Results1. T vector with a target gene (PU.1, CEBPa) was cut by double enzymes, then validated by agarose gel electrophoresis. If the T-A clone was right that proved by agarose gel electrophoresis, direct sequencing by Life Company, comparing the DNA sequence with NCBI gene.2. The CEBPa, PU.1,PU.1+CEBPa gene was inserted into PWPXLD-eGFP vector by using the technology of digestion by double enzyme (Pmel and Spel). All these steps were proved enforced expression lentivirus vectors were successfully made.3. These lentivirus vectors combined with helper-plasmid PMD2G and PSPAX2 were transfected into the packaging cell 293T using PEI transfection reagent. Virus titers were detected, MOI as follows:GFP NC:1.87×10*9 TU/ml; PU.1:3.0×10*9 TU/ml; CEBPa:1.3×10*9TU/ml; CEBPa+PU.1:3.94×10*9 TU/ml.4. Jurkat cells were infected by virus. To detect the GFP positive cells at 48h post-infection of Jurkat cells by lentivirus supernatant. The efficiency of infect were: PWPXLD-GFP:96.8%; PWPXLD-PU.1:96%; PWPXLD-CEBPa:81.6%; PWPXLD-CEBPa+PU.1:75%; respectively. We confirmed related genes expressed in Jurkat cells by RT-PCR.5. Cytomorphology showed that the nuclein of Jurkat-PU.1 cells was loose compared to the other cells. Cells with only PU.1 become larger in volume and particle increased. Jurkat pluse CEBPa showed nuclein loose, but no particle in it. While other groups indicate no change in cytomorphology compared with GFP control group.6. We extracted RNA from virus infected cells at day 8.We used bone marrow cells as positive control and Jurkat cell as negative control. RT-PCR results showed that only Jurkat-PU.1 group showed myeloid related gene positive like mac-1, CD64, GM-CSFR and CD 16 gene, weakly express G-CSFR,M-CSFR. While T cell related gene Rag1, Rag2 and lck unchanged. E2A a transcription factor as lymphocytes showed greatly downregulated, T cell specific transcription factor GATA-3 and Notch-1 slightly decreased. Jurkat-CEBPa showed T cell related gene lck greatly decreased, while expression of rag1 and rag2 showed no difference when compared with Jurkat plus only GFP cells. But, myeloid line related gene mac-1, GM-CSFR and the transcription factors seem to be indifferent. However, Jurkat-PU.1+CEBPa, showed weakly expression macrophage cell related gene like mac-1, CD64, but negative for CD 16, M-CSFR, G-CSFR. And lck significantly reduced, but rag1, rag2, GATA-3 and Notch-1 gently decreased. T cell related genes and transcription factor showed no change.7. FACS results showed that half of Jurkat-PU.1 were upregulated of mac-1, while slightly downregulate of CD3. CD3 negative cells were about 70.4% and 43.3% for highly CEBPa positive Jurkat and PU.1+CEBPa-Jurkat, respectively. Both of them were mac-1 negative. While GFP control cells were showed no response. We found that only PU.1 positive cells showed significantly increase in size and granularity when analysed the size and granularity in these genes transfected cells at day 4. Moreover, we compared the size and granularity of different level fluorescence intensity PU.1-Jurkat cells, higher fluorescence intensity means bigger size and more granularity.7. CCK-8 showed that the cell proliferation rate of Jurkat+C/EBPa cells was significantly slower that in Jurkat-PU.l, Jurkat-CEBPa, Jurkat-PU.1+CEBPa, and Jurkat-GFP. And with the higher expression of CEBPa, the more cells commit to apoptosis.While,PU.l promote growth.8. Above all, We established the stable expression genes Jurkat cells with CEBPa, PU.1, CEBPa+PU.1. And found that T-ALL cell line Jurkat cells can been reprogramed into mac-1 positive cells and CD3 negative cells by overexpression of PU.1 and CEBPa. Jurkat-PU.1+CEBPa cell showed CD3 partly downregulated, a few cells with mac-1 positive. This was maybe because of low expression of these genes when put them in one vector in Jurkat cells.