AIM2 Promotes The Growth And Proliferation Of Non-small Cell Lung Cancer By Inhibiting Mitochondrial Fusion And Its Mechanism

Backgrounds and Objectives:In the past few decades,lung cancer has been one of the most common malignant tumors in the world,and its morbidity and mortality have shown a rapid growth trend in China.Lung cancer is divided into Small Cell Lung Cancer(SCLC)and Non-Small Cell Lung Cancer(NSCLC).Among them,NSCLC originates from bronchial mucosa epithelium,accounting for about 85%of lung cancer patients.The incidence of lung cancer is concealed and malignant,most patients with lung cancer are in advanced stage.The overall 5-year survival rate of lung cancer is still less than 20%,therefore,it is necessary to continue to study and explore the molecular targets and mechanisms of action related to the development of lung cancer.Mitochondria is an organelle with a highly dynamic structure.The dynamic changes of mitochondrial fusion and fission finely regulate the calcium homeostasis,ATP and ROS production.The dynamic process of mitochondrial fusion and fission is mainly regulated by some large GTPases proteins,such as OPA1 and MFN1/2 involved in the regulation of mitochondrial inner and outer membrane fusion,and DRP1 and its receptor proteins Fisl,MFF,MiD49/51 involved in the regulation of mitochondrial fission.In recent years,more and more evidences indicate that mitochondrial structure and dysfunction are closely related to the occurrence and development of lung cancer.Targeted regulation of mitochondrial dynamics can change mitochondrial metabolism and its intracellular distribution,thus achieving the goal of tumor cell therapy.AIM2 is a member of the HIN-200 protein family induced by interferon and is an important molecule of the innate immune system,causing the maturation and secretion of the inflammatory factors IL-1? and IL-18.In addition,it can also induce cell pyroptosis.In addition,AIM2 is also involved in the regulation of the occurrence and development of a variety of tumors,and shows the dual role of tumor suppression and cancer promotion.The immune function of AIM2 has been well known,but its mechanism of action in tumors remains unclear.In this study,we aimed to explore the role of AIM2 expression in regulating mitochondrial dynamics and provide a new idea for lung cancer treatment.Methods and Results:1.AIM2 promotes the proliferation of NSCLC cells in vitro and in vivo through an inflammation-independent pathway.(1)According to Oncomine and TCGA database analysis,AIM2 is highly expressed in tissue samples of NSCLC patients;detection of AIM2 in NSCLC cells is higher expressein NSCLC cells at the cellular level by q-PCR and Western blotting(WB);Kaplan-Meier analysis showed a negative correlation between AIM2 expression and survival in lung cancer patients(2)In vitro knockdown or overexpression of AIM2 inhibits or promotes proliferation of NSCLC cells by cell counting,colony formation,and soft agar assay;in vivo by mouse tumor volume,tumor weight,immunohistochemistry(IHC)Ki-67 positive rate indicates that AIM2 knockdown inhibits tumorigenesis in mouse.(3)After the knockdown of AIM2 in NSCLC cells,by q-PCR,protein extraction form supernatant,the WB of mouse tumor tissues,cell count and WB with and caspase-1 inhibitor,show that IL-1? and caspase-1 did not participate in the growth of NSCLC cells;knockdown of AIM2 did not change the NF-KB-related protein.2.AIM2 regulates mitochondrial dynamics through MFN2 to promote the growth and proliferation of NSCLC cells.(1)k-NN software prediction,cell component separation and immunofluorescence indicate that AIM2 is localized to the mitochondrial outer membrane.(2)Knockdown or overexpression of AIM2 in pDsRed2-Mito stably transfected NSCLC cells,and observed under laser confocal microscopy,mitochondria are hyperfused or fragmented.After knocking down AIM2,WB detected mitochondrial dynamics-related protein,MFN2 was up-regulated.After overexpression of AIM2,MFN2 was down-regulated.And MFN2 was also significantly up-regulated in mouse tissues WB and IHC.(3)Knockdown or overexpression of MFN2 and DRP1 in NSCLC cells,analysis of mitochondrial morphology by laser confocal observation,cell count and colony formation,demonstrated that mitochondrial dynamics can regulate the proliferation of NSCLC cells.3.AIM2 regulates mitochondrial dynamics through MFN2,regulates ROS production,and then inhibits MAPK/ERK signaling pathway,leading to growth and proliferation of NSCLC cells.(1)Protein kinase assay results showed that phosphorylation of extracellular regulated protein kinase(ERK)was down-regulated after AIM2 knockdown;WB results in cells and mouse tumor tissues were also down-regulated by p-ERK1/2.(2)Knockdown or overexpression of MFN2 and DRP1 in NSCLC cells,WB detected p-ERK1/2 changes,and the results showed that mitochondrial dynamic can regulate MAPK/ERK..(3)Knockdown or overexpression of MFN2 and up or down of ROS levels.NAC or H2O2 treatment of AIM2 knockdown NSCLC cells,p-ERX1/2 down-regulated or up-regulated,MFN2 did not change.It indicates mitochondrial dynamics and regulates ROS production,thereby inhibiting MAPK/ERK signaling pathway.Conclusion and Significance:In this study,we explored the role of AIM2 in regulating mitochondrial dynamics and promoting the development of NSCLC from molecular,cellular,mouse models and tissue samples of tumor patients:AIM2 regulates mitochondrial dynamics through MFN2,regulates ROS production,and then inhibits MAPK/ERK signaling pathway,leads to the growth and proliferation of NSCLC cells.This project will help reveal the new functions and new roles of AIM2 in tumorigenesis and development,and elucidate the molecular mechanism of mitochondrial dynamic dysregulation in NSCLC cells,and provide a new idea for lung cancer treatment.