Effects Of Hypoxia-induced Exosomal MiR-31-5p On Invasion And Metastasis Of Lung Adenocarcinoma And Its Mechanism

Primary Lung cancer(Lung cancer)is one of leading malignant tumors threatening human health and life.Lung adenocarcinoma(LUAD)is the most common subtype of lung cancer.Due to the insidious early symptoms of lung cancer,most patients develop advanced disease when diagnosed,and tumor metastasis is one of the most important reasons.Exosomes secreted by various types of cells are small vesicles with membrane lipid bilayer structures,which carry a variety of bioactive factors.In recent years,The role of exosomes secreted by tumor cells in regulating intercellular communication has aroused heated debate among researchers.Hypoxia,as a key regulator of tumor invasion,affects the release of exosomes and their contents from tumor cells.micro RNAs(miRNAs)are the main bioactive molecules encapsulated in exosomes.It has been reported that exosomal miRNAs derived from tumor cells are transfer to recipient cells to regulate their gene expression to affect the physiological or pathological state of cells,and then change the aggressiveness of tumor.However,The functional role of exosomes secreted under hypoxia condition on LUAD remains unclear.This study aims to explore the role of hypoxic LUAD-derived exosomes(HExo)miRNAs in invasion and metastasis of LUAD and its related mechanisms,so as to provide a new theoretical basis for the progression of LUAD and a new and reliable diagnostic marker for clinical liquid biopsy of LUAD.Objectives 1.To investigate the effects of HExo on migration and invasion of LUAD cells.2.To investigate the mechanism of exosomal miR-31-5p regulating invasion and metastasis of LUAD.3.To investigate the expression and clinical significance of plasma derived exosomal miR-31-5p in patients with LUAD.4.To investigate the role of long non-coding RNA GCC2-AS1 in LUAD and its prognostic significance.Methods1.Exosomes were extracted from LUAD cell culture medium under hypoxic and normoxic condition by centrifugation method;Transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and Western Blot(WB)were used to characterize exosomes.2.To observe whether HExo can be transmitted to LUAD cells through uptake assay.The effects of HExo on the migration,invasion and EMT of LUAD cells were detected by scratch,Transwell invasion assay and WB,respectively.3.Illumina high-throughput sequencing technology was used to detect miRNA expression profiles in exosomes from hypoxic and normoxic LUAD cells,and to screen and verify differential miRNAs.4.RNA-digestion assay and GW4869 treatment were used to observe whether miR-31-5p was encapsulated in HExo.5.To investigate the promotion of invasion,metastasis and EMT of exosomal miR-31-5p on LUAD by in vitro functional experiment and in vivo lung metastasis model.6.The target genes of miR-31-5p were predicted by miRNA database query and literature survey,and double verification was performed by double luciferase assay and WB.7.To investigate whether the exosome miR-31-5p affects the progression of LUAD through target genes through rescue experiment of RNA interference and miR-31-5p inhibitor co-treatment;8.To detect the signaling pathways related to the regulation of exosomal miR-31-5pmediated the progression of LUAD.9.The expression levels of plasma derived exosomal miR-31-5p in patients with lung adenocarcinoma and healthy people were detected.The correlation between exosomal miR-31-5p and clinicopathological features of lung adenocarcinoma patients was analyzed.10.Receiver operating curve(ROC)was used to observe the potential clinical diagnostic value of plasma derived exosomal miR-31-5p in LUAD patients.11.The expression of GCC2-AS1 in LUAD was detected and analyzed.The biological effects of GCC2-AS1 on LUAD was observed.The prognostic significance of GCC2-AS1 in LUAD patients was analyzed.Results 1.Hypoxia can induce LUAD cells to secrete more exosomes.2.HExo can be taken up by LUAD cells and promotes the migration,invasion and EMT of LUAD cells.3.The expression of miR-31-5p in HExo was most significantly increased in LUAD cell lines and could be transmitted to other cells.4.The exosomal miR-31-5p promoted the migration and invasion of LUAD cells in vitro;In nude mouse lung metastasis model,it was found that the exosomal miR-31-5p promoted lung metastasis and EMT,and shortened the survival time of nude mice.5.Both the dual luciferase assay and WB results confirmed that the exosomal miR-31-5p could directly regulate the expression of SATB2.Rescue experiments demonstrated that the ability of exosomal miR-31-5p to regulate the migration and invasion of lung adenocarcinoma cells depended on SATB2.6.Exosomal miR-31-5p can activate MEK/ERK signaling pathway.7.The expression of exosomal miR-31-5p was significantly up-regulated in plasma of LUAD patients and metastatic disease,and it closely correlated with the N stage and TNM stage.ROC analysis of plasma derived exosomal miR-31-5p showed a high ability to distinguish patients with LUAD from healthy individuals.8.The long non-coding RNA GCC2-AS1 was significantly up-regulated in LUAD,and promoted the proliferation and invasion of LUAD cells;High expression of GCC2-AS1 is associated with poor prognosis in LUAD patients and can be identified as an independent prognostic marker.Conclusions 1.Hypoxic-derived exosomes can promote invasion,metastasis and EMT of LUAD both in vivo and in vitro.2.The exosome miR-31-5p targets the migration and invasion of LUAD mediated by SATB2 gene and activates ERK/MEK signaling pathway.3.Plasma derived exosomal miR-31-5p can be used as a biomarker for peripheral circulation diagnosis of LUAD.4.High expression of GCC2-AS1 promotes proliferation and invasion of LUAD,which can be used as an independent risk factor for worse prognosis in LUAD patients.