The Role Of PADI4 In Atra-induced Differentiation Of HL-60 Cells And Its Regulation Of Signaling Pathways

Background Leukemia is a type of malignant clonal hematopoietic stem cell disease and it is one of the cancers that leads to the death due to the enhancement of self-renewal of leukemia cells, out of control of proliferation, differentiation disorders and blocked apoptosis which stagnates at the different phases of cell development. Epidemiological survey demonstrate that the annual incidence of leukemia is about 3-4 / 105, which accounts for approximately 5% of the total incidence of cancers. The number of leukemia will increase more than 40000 cases in China each year, including more than 20,000 cases of children. Bone marrow transplantation(Bone marrow transplantation, BMT) is the most effective treatment for curing acute leukemia, however it is restricted by expensive cost, high risk and insufficient of the HLA-identical donors. Moreover, there are more complications after transplantation and high mortality at early stage, as a result, it can be difficult to generalize and use in domestic. Currently, leukemia belongs to the malignant clone diseases of blood system which can be hardly to be cured thoroughly. Nonetheless, combination chemotherapy still be the main treatment of acute leukemia. Therefore, it is more urgent to research the treatment of leukemia. Recent researches had discovered that all-trans-retinoic acid(All-Trans-Retinoic Acid, ATRA) which also known as Vitamin A acid, other names such as retinoic acid, vitamin A and formic acid. And its chemical name is(13E) and the formula is C20H2802, which is mainlyresponsible for the derivative of vitamin a acid. It is also the middle metabolizing production of vitamin A maintaining the growth and development. In addition, it can induce the VSMC differentiation and inhibit the VSMC proliferation. Retinoic acid receptor(retinoic acid receptor, RAR) is an important nuclear receptors, including three sub-types(α, β, γ). The RARa existing in the VSMC can combine with ATRA to induce the differentiation of the VSMC. Krüppel-like factor 4(krüppel-like factor, KLF4) is a nuclear transcription factor which contains the zinc finger structure, which plays an important role in VSMC proliferation, differentiation, migration and apoptosis process. Therefore, ATRA has a strong function of induction of cell differentiation and immune regulation and it must be the preferred clinical medicine to treate the malignant diseases, such as acute promyelocytic leukemia(APL) and myelodysplasia syndrom, etc. However, the specific mechanism is not definite. Peptidyl arginine deiminase-PADI4(Peptidylarginine deiminase 4) contains 2238 base pairs, encoding 663 amino acids, molecular weight of 67 KDa, which is widely distributed in animal tissues. The enzyme of encoding has the ability to convert catalyze arginine residues to citrulline residues and the process is known as citrullinated. The process requires the assistance of Ca2+, citrulline which is converted to protein residues conformational be also changed that leading to the change of the activity of the enzyme. Studies have shown that in different solid tumors the expression of PADI4 is high. The expression of HL-60 is different between the various types of leukemia cell lines. PADI4 rarely expresses in hematopoietic cells, but the expression can be detected when the HL-60 converts to granulocytes and macrophages by the induction of ATRA. Thus we consider that the differentiation and apoptosis of leukemia cell has the relationship with the PADI4. However, under the status of physiological, the function and mechanisms of differentiation process of PADI4 can be not clear. It has been reported that ATRA can induce cell leukemia HL-60 to myeloid differentiation. Some scholars found that during the treatment of leukemia, MAPK, PI3 K / Akt and NF-κB signaling pathway involved in the regulation of gene expression, cell proliferation and apoptosis process. Thereinto, ERK1/2 is the family member of MAPK and the molecular weight is 44 k Da and 42 k Da.Phosphorylation of P44/42 has the function of mediated transcriptional activationand it also takes part in the proliferation and differentiation of cells. PI3K/Akt pathway which widely exists into the cells is mainly involved in cell growth, proliferation, differentiation regulation. Therefore, this study intends to preliminary discuss the transformation rule of the differentiation process of leukemia cell according to the differentiation model of inducing HL-60 cell of ATRA. Furthermore, this study will further explore whether the signal pathway of MAPK,P12K/AKt and NF-κB can fulfill their impact during PAD14 and ATRA induce the cell differentiation progress of HL60 in depth, which can reveal the molecular mechanism of the differentiation of leukemia cell affected by PADI4. As a result, it can be able to provide the important theoretical evidence for treatment of leukemia, which makes a significant contribution to treat leukemia.Objective This paper explores the all-trans retinoic acid-induced promyelocytic myeloid differentiation process to change the expression and function of the mechanism PADI4 leukemia cells HL-60 cells based on the above research background. According to the extra body experiment we discuss whether the PADI4 is related to the differentiation of leukemia and HL-60 cell. It is more important to observe the change of expression of PADI4 when the HL-60 cell convert to granulocyte differentiation by the induction of ATRA and regulate the impact of signal pathway of MAPK,PI3K/AKt and NF-κB after the expression of PADI4. The result is to verify the relationship between PADI4 and leukemia, which provides a new therapeutic target for the clinical treatment of leukemia.Methods The impact of cell cycle, differentiation, apoptosis of HL-60 affected by ATRA.ATRA induced HL-60 cells for 24 h, 48 h, 72 h, 96 h. Using Wright-Gimesa stained to observe the transformation of cell morphology; Using flow cytometry to detect the change of expression of differentiation antigen CD11 b on the cell surface; Using the analysis of RT-PCR to analyze the expression tendency of PADI4 in gene level, and using the method of western blot to detect the change of expression of PAKI4 in protein level; Detecting the transformation of CD11 b by flow cytometry after the interference of the technology of Si RNA and the inhibitorof PADI4, Cl-amidine. Using western blot to detect the change of PADI4 P44/42, P65, P105 and P55 in protein level. Each experiment was repeated for 3 times, the experimental results based on mean ± standard deviation, mean analysis using t-test, P<0.05 had statistical significance. Try to use the software of spss17.0 to statistical analysis.Results ATRA can be able to accelerate the cell differentiation and apoptosis of leukemia cell. Compared with the control group the dye of wright-gimesa shows that the cell nucleus be shrank, nucleolus is disappeared and cytoplasm becomes more, the proportion of nucleoplasm decreases apparently, nuclear dented and leaf, rod-shaped nucleus, lobocytes phenomenon increases significantly by means of induction of ATRA of HL-60 cell. In addition, the expression of cell surface molecules CD11 b be increased from 2.1% to 48.9%(P<0.05); With the prolongation of ATRA induction, PADI4 has a increasement tendency in gene and protein level; After disturbing or inhibiting the expression of PADI4, the protein level of phosphoryltion P44/42 decreases. Besides, the change of protein level, including P65, P105, P55, is not obvious.Conclusions In the process of ATRA which induces the HL-60 cell to directional granulocyte differentiation, PADI4 promotes the HL-60 cell to granulocyte differentiation. Moreover, PADI4 can be more likely to promote the phosphorylation of P44/42 in the signal pathway of MAPK to play a role.