The Role Of Exosome MiR-550a-3-5p In Promoting Lung Cancer Brain Metastasis By Targeting YAP1

Lung cancer is one of the most common cancers and the leading cause of cancer-related death,which has become an important global health issue.With the progress of surgical techniques,chemotherapy,radiotherapy,and neoadjuvant therapy,the 5-year survival rate of patients with early lung cancer has been significantly improved.However,the early diagnosis rate remains low.There are no obvious and specific symptoms in the early stage of lung cancer.Brain metastases are a common complication of advanced lung cancer and a major cause of death.It is important to identify reliable predictive biomarkers for early diagnosis and prognosis of lung cancer.Proliferation and migration of human microvascular endothelial cells(HBMECs)are important steps in brain metastasis.Therefore,effective regulation of HBMECs function in clinical settings is a promising treatment for lung cancer with lung cancer brain metastasis.However,the mechanisms related to the proliferation and migration of HBMECs during brain metastasis of lung cancer remain unknown.The study aims to explore the potential regulatory mechanisms and identify new potential targets for lung cancer brain metastasis.As an important medium of intercellular communication,exosomes exert a vital role in information transmission between tumor cells and tumor microenvironment.Studying the potential mechanism of exosome in tumor development may provide new ideas for effective early diagnosis and treatment of lung cancer metastasis.Previous studies have shown that tumor-derived exosomes promote lung cancer metastasis by promoting epithelial-mesenchymal transformation of tumor cells,inducing angiogenesis,establishment of premetastasis microenvironment,and immune escape.In addition,exosomal mi RNAs are reported to be involved in tumorigenesis and development.However,there are few reports about key mi RNAs associated with brain metastasis in lung cancer,and their potential mechanisms in lung cancer brain metastasis remain unclear.Objective:1.Exosomes are derived from plasma from lung cancer patients with brain metastasis and patients without brain metastasis,as well as exosomes from human low metastatic lung cancer cell line 95 C and human high metastatic lung cancer cell line 95 D,which lay the foundation for subsequent research.2.Screening differentially expressed mi RNAs in exosomes to find biomarkers for brain metastasis of lung cancer.3.To investigate the biological role of differentially expressed exosomal mi R-550a-3-5p in the proliferation,migration,apoptosis and cycle of HBMECs.4.To clarify the biological mechanism of exosomal mi R-550a-3-5p in HBMECs.Methods:1.Plasma of lung cancer patients with brain metastasis and those without brain metastasis was collected,and human low metastatic lung cancer cell line 95 C and human high metastatic lung cancer cell line 95 D were cultured.Then exosomes were isolated from plasma and cells.The morphological characteristics of exosomes were observed by transmission electron microscope.The particle size of exosomes was analyzed by nano-particle tracking.The expression levels of exosome specific protein markers were detected by western blot assay.2.Differentially expressed mi RNAs in exosomes from lung cancer patients with brain metastasis and those without brain metastasis were screened by mi RNA sequencing.Target genes downstream of mi RNAs were screened by Target Scan,mi Randa and PITA databases.Functional analysis of differentially expressed mi RNAs was performed.The differentially expressed mi RNAs were verified by RT-q PCR.3.PKH67-labeled exosomes could be taken up by HBMECs.Isolated exosomes,mi R-550a-3-5p mimic and inhibitor were used to treat HBMECs.The proliferation,migration,apoptosis and cycle of HBMECs were detected by CCK-8,Transwell and flow cytometry.4.Protein expression cleaved PARP,p RB,CDK6,YAP1,CTGF and CYR61 were detected by western blot assay.The relationship between mi R-550a-3-5p and YAP1 was verified by dual-luciferase reporter gene assay.Results:1.The exosomes from lung cancer patients with brain metastasis and those without brain metastasis,from human low metastatic lung cancer cell line 95 C and from human high metastatic lung cancer cell line 95 D were cup-shaped or nearly round with a diameter of about 100 nm.The particle size of extracted exosomes showed no difference,and the main peaks were about 94 nm,90 nm,95 nm and 99 nm,respectively.Western blot showed that the extracted exosomes were abundant in exosome specific protein markers CD9,CD63 and HSP70,and negative in Calnexin.2.Sequencing results showed that 10 mi RNAs were up-regulated and 12 mi RNAs down-regulated in exosomes from lung cancer patients with brain metastasis.In addition,there were 21759 target genes downstream of the differentially expressed mi RNAs.KEGG analysis showed that differentially expressed mi RNAs were significantly enriched in MAPK,chemokine,PPAR and Wnt signaling pathways.RT-q PCR confirmed the high expression of mi R-550a-3-5p in plasma derived exosomes of lung cancer patients with brain metastasis,while the low expression of mi R-144-3p and mi R-27a-3p.These results indicate that the consistency rate between RT-q PCR and sequencing was 60%,which implies a relatively high reliability of the sequencing results.3.The extracted exosomes could be taken up by HBMECs,and the fluorescence intensity increases with the increase of culture time.The exosomes from highly metastatic lung cancer suppressed HBMECs proliferation.mi R-550a-3-5p inhibited HBMECs proliferation and migration,promoted cell apoptosis,and regulated cell cycle.4.mi R-550a-3-5p had the same effect as exosomes derived from highly metastatic lung cancer cells,and increased cleaved PARP protein expression,and decreased p RB,CDK6,YAP1,CTGF,and CYR61 protein expression.mi R-550a-3-5p directly targeted YAP1.Conclusion:1.Plasma extracted from lung cancer patients and exosomes derived from lung cancer cells are in line with the characteristics of typical exosomes,which can be further studied.2.10 up-regulated and 12 down-regulated mi RNAs,as well as MAPK,chemokine,PPAR and Wnt signaling pathways,may be closely related to brain metastasis of lung cancer.3.Exosomal mi R-550a-3-5p from highly metastatic lung cancer inhibited HBMECs proliferation and migration,promoted cell apoptosis,regulated cell cycle,and participated in lung cancer brain metastasis.4.Exosomal mi R-550a-3-5p targeted YAP1 and modulated the expressions of PARP,p RB,CDK6,CTGF and CYR61.