Study On The Role And Mechanism Of Exosomal MiR-10527-5p In Lymphatic Metastasis Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma(EC)is ranked as the seventh most frequently malignancy and the sixth leading cause of cancer-related deaths globally.Esophageal squamous cell carcinoma(ESCC),as the predominant subtype,comprises approximately 85%of esophageal cancer cases.Despite the combination of screening and surgical resection have effectively improved the prognosis of early-stage ESCC,the treatment of advanced-stage ESCC still disappointing.As the predominant metastatic manner,lymph node metastasis(LNM)is one of the most leading causes for the poor prognosis of ESCC patients.Therefore,identification of a precise molecular which could discriminate the LNM status and elucidate the underlying mechanisms of ESCC metastasis are essential for improving clinical outcomes of ESCC patients.Currently,imaging examination such as Computed Tomography(CT),Endoscopic Ultrasonography(EUS),are mainly methods for clinicians to predict the LNM status of ESCC patients preoperatively.However,due to the low specificity and sensitivity,some cases could not be detected,which result in false staging and inadequate therapy.With the rapid development of tumor biomarkers,such as carcinoembryonic antigen(CEA),had been proved to play an important role in the diagnosis of ESCC patients,but they cannot be used to judge the LNM status.Exosomes are nano-sized lipid bilayer extracellular vesicles ranging in size from 30 to 150 nm.It can be produced by multiple types of cells and serve as mediators in intercellular communications through transmitting information cargos,including proteins,lipids,RNAs.The contents of exosomes vary with different physiological and pathological states,which can reflect the state and difference of the origin cell,so it is an ideal carrier for tumor noninvasive examination.microRNAs(miRNAs)are a class of short endogenous non-coding RNAs of 1724 nt that promote the cleavage of the target mRNA or inhibit its translation through complementary base pairing at the 3’-untranslated region(UTR).Compared with circulating miRNAs,miRNAs in plasma exosomes are protected by the double lipid membrane of exosomes,which can avoid degradation by RNA enzymes and thus maintain their own stability.Although several studies have shown that exosomal miRNAs could be used for the early diagnosis and prognosis of ESCC patients,whether exosomal miRNAs could be used to determine the status of lymph node metastasis in ESCC patients preoperatively still not clear,which need further exploration.During the process of tumor metastasis,tumor derived exosomal miRNAs could be taken up by other tumor cells and regulate the migration,invasion,epithelial-mesenchymal transformation(EMT)of recipient cells by binding to their target genes.It could also be internalized by other cells in the tumor microenvironment,such as immune cells,vascular endothelial cells,and lymph endothelial cells to regulate immune response,angiogenesis and lymphangiogenesis.Lymphangiogenesis refers to formation of new lymphatic vessels.During the sequential processes of lymphatic metastasis,lymphatic vessels in the tumor periphery served as a route for tumor cells spread to regional lymph nodes.It is of great significance to explore the mechanism of ESCC lymphangiogenesis.Recently,emerging evidences had been demonstrated that exosomes served as key mediators of lymphangiogensis in various cancers,nevertheless,the roles and regulatory mechanisms of exosomal miRNAs in ESCC lymphangiogensis remain largely unclearly,which warrant further investigation.Objectives1.To identify the critical plasma exosomal miRNAs involved in lymphatic metastasis of ESCC and evaluate their efficacies in predicting the LNM status of ESCC patients preoperatively.2.To explore the functional roles and mechanisms of exosomal miR-10527-5p on lymphatic metastasis and lymphangiogensis of ESCC.Methods1.Analysis of the expression of plasma exosomal miRNA in ESCC patients and its clinical application1.1 Firstly,small RNA sequencing,GEO database,qRT-PCR were used to identify the critical plasma exosomal miRNAs associated with lymphatic metastasis of ESCC.Receiver operating characteristic(ROC)curve was carried out to evaluate the diagnostic potential of differential exosomal miRNAs in predicting the LNM status of ESCC patients.1.2 Analysis of the relationship between exosomal miR-10527-5p and LNM of ESCC:Chi-square test was performed to investigate the association between exosomal miR-105275p and clinicopathological parameters of ESCC patients.2.Analysis of the functional roles and mechanisms of exosomal miR-10527-5p on lymphatic metastasis and lymphangiogensis of ESCC.2.1 Analysis of the roles and downstream signaling pathways of exosomal miR-10527-5p on ESCC and HLECs cells:Eca109 and KYSE150 cells were transfected with miR-10527-5p overexpression lentiviruses or miR-10527-5p inhibitor.Wound-healing,transwell and Western blot were used to evaluate the functions of miR-10527-5p on migration,invasion,EMT progression and Wnt/β-catenin signaling pathway of ESCC cells.Exosomes derived from Eca109 cells were co-incubated with KYSE150 cells.PKH67 exosome labeling assay and qRT-PCR were applied to assess whether exosomal miR-10527-5p could be internalized by KYSE150 cells.Then,wound-healing,transwell and Western blot assays were carried out to verify whether exosomal miR-10527-5p mediated migration,invasion,EMT and Wnt/βcatenin signaling pathway.Exosomes derived from Eca109 cells were co-cultured with human lymphatic endothelial cells(HLECs).Then,tube formation and transwell assays were used to assess the functions of exosomal miR-10527-5p on tube formation and migration abilities of HLECs.Popliteal lymph node metastasis model was used to evaluated the regulatory function of exosomal miR-10527-5p on ESCC lymphangiogenesis in vivo.2.2 Analysis of the target gene of miR-10527-5p:Bioinformatics tools,qRT-PCR and Western blot were used to identify the candidate target genes of miR-10527-5p.Meanwhile,fluorescence in situ hybridization was used to detect the co-localization of miR-10527-5p and Rab10.After treatment with actinomycin D,qRT-PCR was used to evaluate the effect of miR10527-5p on the stability of Rab10 mRNA.Dual-luciferase reporter assay was used to detect the targeted inhibition of miR-10527-5p on Rab10 mRNA 3’-UTR.Rescue experiments were performed to detect whether Rab10 overexpression could reverse the inhibitory effects of exosomal miR-10527-5p on KYSE150 cells and HLECs.IHC assay was used to detect the expression of Rab10 in the foot-pad tumor tissues and ESCC tumor tissues.Finally,KaplanMeier Plot was used to analyze the prognosis value of Rab10 in ESCC patients.Results1.Exosomal miR-10527-5p and miR-493-5p could be used for predicting the LNM status of ESCC1.1 miR-10527-5p,miR-493-5p were found significantly reduced in plasma exosomes of LNM+group ESCC patients than LNM’ group.ROC curve analyses showed that both of them had strong capability in predicting LNM status of ESCC patients preoperatively.1.2 Plasma exosomal miR-10527-5p level was significantly negatively correlated with tumor size,T stage,LNM status in ESCC tissues.2.Exosomal miR-10527-5p inhibits migration,invasion and lymphangiogenesis of ESCC via Wnt/β-catenin signaling pathway by directly targeting Rab10 mRNA2.1 miR-10527-5p could be transferred from Eca109 cells to KYSE150 cells or HLECs via exosomes,thus suppressed migration,invasion and EMT of KYSE150 cells,as well as repressed migratory and tube formation capabilities of HLECs through Wnt/β-catenin signaling pathway,thereby prohibited lymphatic metastasis of ESCC in vivo.2.2 Rab10 was a candidate target gene of miR-10527-5p.The inhibitory effects of exosomal miR-10527-5p on KYSE150 and HLECs cells were reversed when reintroduction of Rab10.Kaplan-Meier Plot analysis showed that high Rab10 levels resulted in a poor survival in ESCC patients.Conclusions1.miR-10527-5p and miR-493-5p were significantly reduced in plasma exosomes of ESCC patients with LNM,and both of them could be used for predicting the LNM status of ESCC preoperatively.2.Plasma exosomal miR-10527-5p level was significantly negatively correlated with tumor size,T stage,LNM status in ESCC.tissues.3.Exosomal miR-10527-5p could inhibit migration,invasion and lymphangiogenesis of ESCC via Wnt/β-catenin signaling pathway.4.Rab10 is a direct target gene of miR-10527-5p.Re-expression Rab10 also could reverse the inhibitory effects of exosomal miR-10527-5p on KYSE150 and HLECs cells.