Study On The Function And Regulation Mechanism Of GFI1 In The Development Of Lung Cancer

Background:GFI1,as a zinc finger protein,is widely expressed in many hematopoietic,lymphatic and myeloid progenitor cells as well as mature lymphoid and myeloid cells,acting as a transcriptional inhibitory factor.C-terminal zinc finger domain mediates its interaction with DNA,while the N-terminal SNAIL / GFI-1(SNAG)domain helps to exercise its inhibitory effect.GFI1 plays an important role in the early development of lymphoid hematopoietic system,which expresses in hematopoietic stem cells and can regulate the self-renewal of hematopoietic stem cells.GFI1 can help the cells from the original matrix during the transformation of endothelial cells into hematopoietic cells.GFI1 is associated with a variety of hematological disorders,including neutropenia and leukemia.GFI1 plays an important role in the development of the peripheral nervous system in the late stage of embryo,mainly regulating the development of inner ear hair cells,retinal nerve cells and pulmonary neuroendocrine cells.Recently,GFI1 and MYC have been found to play an important role in the development and progression of medulloblastoma.We first discovered that GFI1 is widely expressed in lung cancer cells.And we further explored its specific functions and mechanisms in the development and progression of lung cancer.Aim:To clarify the expression characteristics of GFI1 in lung cancer patients and lung cancer cell lines.To elucidate the function of GFI1 in ectopic expression in lung cancer cell lines and its specific mechanism for its function.To investigate the epigenetic mechanism of GFI1 expression in lung cancer cells.To determine whether it may become a marker of early diagnosis and the target of treatment in lung cancer.Methods: 1.The expression of GFI1 in different lung cancer cell lines was detected by PCR and western blot.The expression of GFI1 in different types of lung cancer tissues was detected by immunohistochemistry.To explore the specific pattern of GFI1 expression in different types of lung cancer cell lines and pathogens,and to clarify the relationship between GFI1 and lung cancer progression.2.The human lung cancer cell lines overexpressing GFI1 were injected into BALB / C nude mice by tail vein injection.The survival curves of mice were observed and the function of GFI1 in tumor formation was studied.3.Microarray was used to detect the changes of gene expression in A549 GFI1 compared with the control group,and verified by real-time quantitative PCR.4.GFI1 is forcibly expressed in a low expression of GFI1 in lung cancer cell lines,and was knocked down in the cell line with high expression rate.A series of experiments on cell behavior were carried out,including woundhealing test,proliferation experiment,invasion experiment,cell death ELISA,in vitro tumorigenesis experiments.To clear the expression of GFI1 role in which specific aspects of tumor process.5.Explore the downstream pathway by western and the recovery experiments after the downstream pathway is inhibited.6.Luciferase assay and ChIP was used to clear the promoter region of GFI1.3C and DNA methylation was used to study the specific epigenetic mechanism of GFI1 expression.Result:GFI1 is associated with distant metastatic lymph node metastasis and poor prognosis.Hematopoietic system-specific transcription factor GFI1 expressed ectopicly in lung cancer cell lines and patient tissue.And the higher the expression of GFI1 in patients with higher lymph node metastasis.Survival time of patients with high GFI1 expression was significantly shorter.BALB/C nude mice were injected into A549 overexpressing GFI1 through tail vein injection,the survival time of the mice in the experimental group was shorter.This suggests that GFI1 promotes tumor formation in animals.GFI1 helps cells escape anoikis.The morphology of A549 cells overexpressing GFI1 significantly changed,tended to turn round.The adhesion between cells and cells,and between cells and the substrate significantly reduced.While the expression of GFI1 in H1155 was knocked down,the cell adhesion was significantly improved.After overexpressed GFI1 in HUVEC,HBEC,A549 and H460,the viability of cells in the low-adsorbed culture dish was significantly enhanced.The expression of GFI1 was knocked down in the small cell lung cancer cell line H209,then the apoptotic level of the cells increased.In the subsequent study,overexpression of GFI1 could promote the increase phosphorylation level of Erk.u0216,phosphorylation inhibitor of of Erk,could make the ability of cell resistance to anoikis to occur a certain recovery.These datas suggest that GFI1 can help cells improve their viability in the absence of a matrix by activating Erk phosphorylation.Interactions of GFI1 gene structure lead to increased expression of GFI1 in small cell lung cancer cell lines.The promoter region of GFI1 was mapped by using ChIP to study histone modification and verified by Luciferase reporter gene technique.The promoter of the GFI1 promoter was found near the third transcription start site.The difference of high chromatin spatial conformation of GFI1 in A549 and H209 was detected by 3C technique.The GFI1 promoter region in H209 forms a distinct loop structure,whereas A549 does not have this structure.The Luciferase assay showed that there was a silencer in the 1.5 kb upstream of the third promoter in GFI1.And loop structure of GFI1 chromatin in H209 can shield the role of upstream silencers.DNA methylation showed that methylation level was significantly higher in A549 and H460 cell lines with low expression of GFI1,while H1155 and H209 with high expression of GFI1 were very low.DNA methylation maybe an important mechanism for regulating the expression of GFI1.Conclusion:GFI1 was activated in lung cancer cells,and was closely related to lymph node metastasis.In the GFI1 function study,we found that GFI1 by activating Erk phosphorylation to help cells escape anoikis,and ultimately the formation of distant metastasis.The interaction of GFI1 chromatin shield the role of upstream silencers.DNA methylation maybe an important mechanism for regulating the expression of GFI1.Our study broadens the awareness of the development of lung cancer,and provides a theoretical basis for further clinical research and treatment.