Through Negative Regulation Of AKT Pathway Rig-1 Involved In Atra And Infα Induced U937 Cell Proliferation Inhibition, Differentiation, Apoptosis And Cell Cycle Arrest

The normal regulation of hematopoiesis depends on the balance among various factors, regulatory factors are too many or negative regulatory factors are too few will cause out of control of hematopoiesis. If the hematopoietic cells or their progeny cells are malignant hyperplasia, it becomes the malignancy of blood system. Mid-70s, mainly using chemotherapeutic drugs of inhibiting the proliferation of malignant cells to treat the leukemia, In addition to malignant proliferation, leukemic cells have the biologic characteristics of blocked differentiation, apoptosis disorders and metastasis. In APL (acute promyelocytic leukemia) patients, unnatural accumulation of promyelocytic cells in bone marrow show the characteristics of granule cells significantly blocked. Since 1986, Chinese scholars firstly using ATRA (all-trans retinoic acid) to treat APL, ATRA has become an antitumor drug and as a material of differentiation therapy has demonstrated very good results in the treatment of APL. anti-proliferation activity of ATRA mainly comes from the growth inhibition and induction of differentiation, moreover, in addition to inhibit the proliferation, ATRA also promotes apoptosis in different cell lines.Rig-I (Retinoic acid-inducible gene-I) is a RNA helicase with DExD / H box, composed with the 925 amino acid residues, N terminal contains 2 CARD domain (caspase recruitment domains), C terminal has an RNA helicase domain. Rig-I plays an important role in innate immunity, it can identify the virus RNA within the cell, and induce the production of IFNs (typeⅠinterferon through the activation of IRF3 and NF-kB, now has become a hot topic in immunology. However Rig-I was firstly isolated from NB4 of APL cell line which is induced differentiation with ATRA in the absence of virus infection, and is closely related with the ATRA-induced granulocytic differentiation pathways. Compared with the research of anti-virus in innate immunity. So far, it is still not clear of the structure and function, signal transduction of Rig-I in hematopoietic cell. Especially, It has not report about the function and signaling pathway of Rig-I in U937 (human acute monocytic leukemia cell line; AML-M5) belonging to AML (acute myeloid leukemia) with NB4 (APL cell line; AML-M3) under the action of ATRA and IFN (interferon). In this study, first of all, on the basis of preparation of the mouse and human Rig-I antibody, establishing the induced expression of human Rig-I and regulatory Rig-I RNAi U937 cell lines separately, studied the effect of cell proliferation, differentiation, apoptosis and cell cycle on U937 under the action of ATRA and IFN, in the process of above, Rig-I function and signaling pathways have been studied.The main results were as follows:1. The mRig-I (full-length gene of Rig-I in mouse) and hRig-I-N (850 bp sequence of human Rig-I N terminal) were built into the prokaryotic expression vector, expressed in E. coli, fusion proteins of mRig-I and hRig-I-N were purified and separately prepared for the polyclonal antibodies of good specificity, the prepared antibodies can identify the expression of respective antigens from prokaryotic and eukaryotic expression system and Combined with mouse spleen cells and immunofluorescence technique of U937 cells indicate that polyclonal antibodies of mRig-I and hRig-I-N have a good immune specificity.2. By PCR, hRig-I gene was constructed into the pTRE2-hyg response plasmid of the Tet-Off inducible expression system, after transformed into 293 (Tet-Off) cells and verified the expression of Rig-I, the constructed plasmid was electroporated into the U937 (Tet-Off) cells, cells were selected and isolated, after withdrawal of Tetracycline, clones which can be well induced expression of Rig-I were picked by Western and combined with immunofluorescence verified Rig-I expression, the U937 can be induced expression of Rig-I was obtained by using Tet-Off inducible expression system.3. Two designed shRNA miR-30 partial sequences of human Rig-I were amplify to complete sequences of miR-30 structure and constructed into the p201 lentiviral vector, after indicated have obvious RNAi effect in 293T, the two complete shRNA sequences were built into response plasmid of pTRE2-GFP-hyg and electroporated into the U937 (Tet-Off) cells, these cells were selected and isolated, clones were picked and verified the RNAi regulation by using the expression of GFP report gene and Western blot technique, the regulatable Rig-I RNAi stable U937 was established based on the Tet-Off system.4. ATRA, IFNα, IFNγalone inhibited the U937 cell proliferation, in combination, in particular, ATRA and IFNαcombination significantly inhibited the cell proliferation, cell cycle analysis also showed the same result, most blocked in the G0/G1 period; ATRA alone has obvious effect on cell differentiation, ATRA and IFNαcombination have the synergy effect on differentiation and apoptosis. Therefore, ATRA and IFNαcombination show a synergistic effect on the proliferation inhibition, differentiation, apoptosis and cell cycle arrest of U937. 5. The Rig-I expression of U937 induced by ATRA or IFNγalone is not obvious, but the induction of the Rig-I is significantly by IFNαalone, especially it was show a synergistic effect on the induced expression of Rig-I by ATRA and IFNαcombination. Compared with the upregulated expression of Rig-I in U937 combination ATRA with IFNα, under the same conditions, Rig-I silent U937 cells showed the opposite results, showing significant proliferation, differentiation and apoptosis reduction, cycle arrest partially relieved. Both positive and negative Research showed that upregulated expression of Rig-I involved in proliferation inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells induced by ATRA and IFNαcombination.6. Research on the cell signaling pathway of U937 showed that Rig-I expression induced by the combination of ATRA and IFNαcan inhibit the phosphorylation and activation of not only AKT but also P70 and 4EBP1 which is the downstream of AKT-mTOR pathway, also inhibit the phosphorylation of transcription factor FOXO3α, another AKT downstream target protein, to promote P27 cycle arrest protein expression, Under the same conditions the silence of Rig-I can promote activation of AKT and its downstream pathway, The AKT and AKT signaling pathway play a decisive role on proliferation, differentiation, apoptosis and cell cycle of tumor cell, therefore, through negative regulation of AKT pathway Rig-I involved in ATRA and IFNαinduced cell proliferation inhibition, differentiation, apoptosis cell cycle arrest of U937.