Interleukin 7 Inhibit Autophagy Via P53 Regulated AMPK/mTOR Signaling Pathway In Non-small Cell Lung Cancer

Objective: Lung cancer is a leading cause which threatening the life of human beings,for the reason that lacking early diagnosis and effective therapeutic methods,the death rate of lung cancer still increasing.Interleukin 7(IL-7)is found to be essential in the development of B cell and T cell,besides,it play dual role in cancers.In non-small cell lung cancer(NSCLC),IL-7 facilitate lymphangiogenesis,promote proliferation,and anti-apoptosis.Whereas,IL-7 play anti-tumorigenesis role in other cancer.Autophagy regulate metastasis of cell,and have interaction with apoptosis and epithelial mesenchymal transition.In addition,autophagy affecting the reaction of tumor for chemotherapeutic drug.Thus,our article is aiming at exploring the mechanism of IL-7regulated autophagy in NSCLC.Methods: 1.The Western Blot was used for detection of protein expression level.A549 cell line was incubated in appropriate circumstance according to instruction.Cells were grouped into negative control,IL-7 24ng/ml for 8 hours and IL-7 24ng/ml for 12 hours.Nuclear and cytoplasmic protein were extracted for p53 protein detection and total protein were extracted for phosphorylated protein detection.Then cell were treated with anti-IL-7R antibody 100 ng/ml and IL-7 24 ng/ml 12 hour for protein detection.Moreover,p53 inhibitor PFT-α and AMPK inhibitor Compound C were used for cell incubation to observe the molecular reaction in signaling pathway.2.Quantitative Real-Time PCR were used for detection of p53 mRNA level after treated with IL-7,PFT-α,then using SPSS software for statistical analysis.3.Transmission electron microscopy was used for observation of autophagosome according to the groups designed above.Results: 1.In normal A549 cell line,p53 protein mostly located in nucleus,only small part of it located in cytoplasm.After treated with IL-7 for 12 hours,part of nuclear p53 translocated into cytoplasm.Besides,qRT-PCR results showed that an increasing level of p53 mRNA after treated with IL-7 for 12 hour.2.IL-7 caused decrease of p-AMPK protein and increase of p-mTOR protein.This effect could be blocked by A-IL-7R.3.After incubated with Compound C,a decrease of p-AMPK was observed andin company with an increase of p-mTOR.However,there is no any obvious change observed neither in nuclear nor in cytoplasmic p53 protein.4.P53 inhibitor PFT-α was used in our experiment,western blot results show that nuclear and cytoplasmic p53 decreased,qRT-PCR results showed that p53 mRNA level increased which may due to negative feedback.Detection for total protein show that PFT-α caused p-AMPK escalation and p-mTOR inhibition.5.TEM results show that IL-7 regulated autophagosome inhibition,and A-IL-7R could block this effect,PFT-α causing autophagosome activation,PFTα+IL-7 group also observed autophagoyosome activation,Copound C causing autophagosome inhibition,Compound C+IL-7 observed autophagosome suppression,the group was compared with negative control.Comclusions: 1.IL-7 regulating p53 protein translocate from nuclear to cytoplasm,and using A-IL-7R could block this effect.2.IL-7 activating AMPK/mTOR signaling pathway.Besides,we demonstrate that AMPK is an upstream factor of m TOR,p53 is the upstream factor which regulate AMPK and m TOR.3.IL-7 inhibit autophagy of A549 cells,A-IL-7R could block the effect of IL-7.Besides,PFT-α induce autophagy,Compound C inhibit autophagy indicating that IL-7 regulating p53 translocation from nuclear to cytoplasm and activating AMPK/m TOR to inhibit autophagy.