MiR-144 Delivered By Nasopharyngeal Carcinoma-derived Extracellular Vesicles Stimulates Angiogenesis Through The FBXW7/HIF-1α/VEGF-A Axis

Research background and objective:Nasopharyngeal carcinoma(NPC)is a malignant tumor occurring in the parietal epithelial of the nasopharynx.It is the most common type of cancer in otorhinolaryngology,especially in Asia.Nasopharyngeal carcinoma is characterized by regional concentration,complex pathogenesis,insidious onset,high metastasis and high invasiveness,etc.The clinical treatment of nasopharyngeal carcinoma is mainly radiotherapy,which has many adverse reactions.The overall survival was poor,especially in patients with metastatic nasopharyngeal carcinoma and recurrent nasopharyngeal carcinoma.Therefore,it is of great significance to explore its pathogenesis and find new drugs and therapeutic targets.Pathological angiogenesis is a fundamental factor in tumor progression and metastasis.This project intends to conduct an in-depth research on the role and mechanism of miR-144 delivered by nasopharyngeal carcinoma-derived exosome in endothelial angiogenesis.The research methods:Part Ⅰ Expression of miR-144 in nasopharyngeal carcinoma and its correlation with angiogenesis1.qRT-PCR was used to detect miR-144 m RNA expression levels in nasopharyngeal carcinoma tissues,chronic nasopharyngitis tissues,nasopharyngeal carcinoma cell lines and normal nasopharyngeal cell line.2.In situ hybridization verified the expression of miR-144 in nasopharyngeal carcinoma tissues and chronic nasopharyngitis tissues.3.Immunohistochemistry was used to detect the positive rate of CD31 expression in nasopharyngeal carcinoma and chronic nasopharyngitis tissues;correlation between CD31 and miR-144 expression was performed by pearson correlation analysis.Part Ⅱ Expression of miR-144 in exosomes of nasopharyngeal carcinoma cell lines1.The extracellular vesicles(EVs)in NP69,C666-1 and SUNE1 cells were isolated by differential centrifugation and ultracentrifugation.2.The morphologies of EVs were observed under a TEM.The particle diameter and concentration were measured with NTA using the Nanosight NS3000 system.The characteristic surface marker proteins CD9,CD63,and TSG101 and the endoplasmic reticulum membrane protein,calnexin,of EVs were determined using immunoblotting.Surface charge of different cell EV particles was analyzed with the use of Zetasizer Nano series Nano-ZS.3.Ribonucase and Triton X-100 were used to treat EVs that derived from NP69,C666-1 and SUNE1,respectively.The expression levels of miR-144 m RNA in EVs treated by different methods were detected by Taq Man miRNA to evaluate the stability of EVs membrane.4.The expression levels of miR-144 m RNA in NP69,C666-1 and SUNE1 cellsderived EVs were detected by qRT-PCR.Part Ⅲ Effects of miR-144 delivered by nasopharyngeal carcinoma-derived exosomes on human umbilical vein endothelial cells(HUVECs)1.The isolated EVs were labeled using PKH67 and incubated with cultured HUVECs.The uptake of PKH67-labeled EVs by HUVECs at different time points was traced under a confocal microscope.2.qRT-PCR was used to detect the expression level of miR-144 m RNA in HUVECs co-cultured with EVs of different cells.3.After miR-144 was differentially transfected with C666-1 and SUNE1 cell lines,the EVs were isolated and incubated with cultured HUVECs.qRT-PCR was used to detect the expression level of miR-144 m RNA in HUVECs.Transwell chamber experiments were used to detect the migration and invasion of HUVECs.Wound healing was used to determined the migration of HUVECs.The in vivo Matrigel plug assay and the in vivo Matrigel plug assay were evaluated to determine angiogenesis.Part Ⅳ Mechanism of angiogenesis promoted by miR-144 delivered by nasopharyngeal carcinoma-derived exosomes1.The in-silico analysis predicted the presence of miR-144 binding sites in the 3’UTR of FBXW7 m RNA,which was further confirmed by luciferase report.2.qRT-PCR was used to detect FBXW7 m RNA expression levels in nasopharyngeal carcinoma tissues,chronic nasopharyngitis tissues,nasopharyngeal carcinoma cell lines and normal nasopharyngeal cell line.Western Blot and immunohistochemistry were used to detect FBXW7 protein levels and the positive rate of FBXW7 expression in nasopharyngeal carcinoma tissues and chronic nasopharyngitis tissues,respectively.The correlation between miR-144 and FBXW7 in tissues was performed by pearson correlation analysis.3.After miR-144 was differentially transfected with C666-1 and SUNE1 cell lines,the EVs were isolated and incubated with cultured HUVECs.The protein levels of FBXW7 and HIF-1α were detected by Western Blot,and the m RNA expression levels of FBXW7 and HIF-1α were detected by qRT-PCR.The concentration of VEGF-A in supernatant of HUVECs was determined by ELISA.4.C666-1 and SUNE1 cell lines which were overexpressed with FBXW7 or silenced with HIF-1α were isolated and incubated with cultured HUVECs.The expression levels of FBXW7 and HIF-1α m RNA in HUVECs were detected by qRTPCR.The protein level of HIF-1α was detected by Western Blot,and the concentration of VEGF-A in supernatant of HUVECs was determined by ELISA.5.After down-regulation of miR-144 in C666-1 and SUNE1 cell lines,EVs was isolated and incubated with cultured HUVECs which were silenced FBXW7.Western Blot was used to detect the protein levels of FBXW7,HIF-1α and VEGF-A in HUVECs.Transwell chamber experiments were used to detect the migration and invasion of HUVECs.The in vivo Matrigel plug assay and the in vivo Matrigel plug assay were evaluated to determine angiogenesis.The results of the study:1.miR-144 was highly expressed in NPC tissues and cells,with a positive correlation detected between its expression and that of CD31.2.miR-144 was highly enriched in exosomes released from NPC cells.3.miR-144 delivery from NPC cells into HUVECs via exosomes can enhance the migration,invasion and angiogenesis of HUVECs.4.miR-144 delivered by NPC-derived exosomes stimulates angiogenesis through the FBXW7/HIF-1α/VEGF-A axis.Conclusion:Specifically,miR-144 could be shuttled to HUVECs via EVs derived from NPC cells,which ultimately strengthens their malignant phenotypes,providing a new target for the development of EV-based biomarkers and treatment modalities against NPC.