The Study Of Notch Signaling’s Function In Tumor: Regulation Tumor Cells EMT And Macrophage Polarization

Tumor is seriously harmful to human health. And metastasis and invasion propertiesof tumor cells make it difficult to completely heal tumor with surgical excisation.Therefore effective therapeutic strategies were particularly significant. Previous treatmentdrugs for cancer were often targeted to certain oncogenes or tumor suppressor genes toinhibit proliferation and promote apoptosis of tumor cells.This therapeutic strategy hasmade a clinical effect, however the genesis and development of tumor is not only theproblem of tumor cells themselves. Inflammatory microenvironment of tumor plays animportant role in tumor formation, in which a large number of stromal cells, endothelialcells and immune cells are performing important regulatory functions. How to improve theinflammatory microenvironment, active innate immune system and stimulate the killer Tcell immune response will become the new strategy for cancer therapy and makebreakthroughs.Notch signaling pathway is one of the main signals for development of organisma multicellularis. Notch signaling ligands bind to the surface receptors of adjacent cell,resulting in the release of NICD into the nuclear, which will interact with RBP-J and otherco-active transcription complex components to promote expression of downstream gene.Through lateral inhibition, lateral induction and selfrenew maintenance, Notch plays animportant role in cell proliferation, differentiation and apoptosis. Abnormal expression ofNotch often induced various diseases, and even caused cancer in normal tissues. In avariety of tumor tissues, the activation degree of Notch is associated with tumor cellproliferation, apoptosis, invasion and metastasis. Therefore drugs targeted to Notch havealso become one of effective method in clinical treatment and research.FHL1C is an inhibitor of Notch signaling, which contains two LIM domainsmediated protein-protein interaction in N-terminal and a RBP-J specific binding site inC-terminal. FHL1C could interact with RBP-J and rectuit PcG protein family to inhibittranscription of Notch downstream genes. However FHL1C protein does not contain anuclear localization signal. It is not clear whether there are other chaperones interactedwith it, which mediates FHL1C to locate into the nuclear and perform functions underphysiological and pathological conditions.In addition, Notch signal could regulate in T/B lymphocytes differentiation,maturation and differentiation of dendritic cells and different functional activation of bonemarrow-derived macrophages. Depending on the different stimulus to macrophage, itcould trigger different immune response and play different roles—anti-tumor andtumor-promoting functions—in tumor immunity. Therefore, the function of macrophage intumor development gradually becomes popular in oncology research and will be atherapeutic method in the future. The previous studies in our laboratory found that, Notchsignal can promote macrophages M1type polarization and inhibit tumor formation andplay an anti-tumor function, but the molecular mechanisms of macrophage polarization isremains unclear.This topic preliminarily explored the regulation mechanisms of Notch signalingpathway in tumor genesis and development from the molecular level and cellular level.The main results were as follows: ⅠNotch regulate EMT of tumor cell1) With co-immunoprecipitation and mammalian two-hybrid assay, we identifiedinteraction between FHL1C with tight junction proteins ZO-1. PSG truncated fragment ofZO-1altered cellular localization after overexpression FHL1C in cells. These indicate thatFHL1C lead ZO-1to translocate into the nuclear mediated by interacting;2) It has been demostrated that FHL1C could promote the EMT of MCF-7cells andenhance its migration ability through qRT-PCR and wound healing. Further studiesshowed that the promoting function of FHL1C in tumor cells EMT is Notch signalindependent;ⅡNotch regulate polarization of macrophage1) Notch signaling could promote the expression of miR-125a/miR-99b cluster inmacrophage. Overexpression miR-125a of macrophage enhanced the ability of killingtumor cells and resistance to pathogen invasion. overexpression of miR-99b can alsoactivate macrophages inflammatory response. miR-125a could decrease the expression ofits target gene FIH1, thus enhancing Hif-1α activity and promote iNOS synthesis; on theother hand, miR-125a inhibited IRF4translation thereby inhibiting the expression of MR.These results indicate Notch signaling pathway could promote macrophage M1polarization and inhibit M2polarization through regulating miR-125a/miR-99bexpression;2) After overexpression of miR-125a or miR-99b, the number of macrophages inbone marrow increased significantly. And miR-125a/miR-99b expression levels graduallyincreased during differentiation from BM cells to monocytes and macrophages. Theseresults indicate that miR-125a/miR-99b could promote macrophage differentiation;3) The expression of Pri-miR-125a, miR-99b and the host gene Spaca6were downregulation after miR-125a overexpression, suggesting that miR-125a could promote theexpression of itself.In summary, our results showed that FHL1C could promote EMT and migration oftumor cells through a Notch independent way; Notch signal could promote macrophagesM1polarization by up-regulation of miR-125a/miR-99b. The above reveals an important regulatory role of Notch signaling and its related molecule in tumor genesis anddevelopment, providing a theoretical basis for molecular and cellular therapy of tumors,which has practical clinical value.