| Research BackgroundGlobally,lung cancer,including small cell lung cancer and non-small cell lung cancer,ranks first in morbidity and mortality among all malignancies.However,due to the causes of lung cancer,the specific pathway of lung cancer development has not been fully defined,and most patients can not be completely cured due to late staging or extensive metastasis,and the 5-year survival rate of patients is maintained at about15%.To study and determine the detailed mechanism of the occurrence and development of lung cancer is of great importance for clarifying the prevention and treatment of lung cancer and for the targeted precise treatment of molecular targets of lung cancer.In recent years,with the deepening of molecular biology and pathology studies,more and more mechanisms related to exosomes,such as tumor angiogenesis,changes in vascular microenvironment,as well as vascular invasion and metastasis,have been discovered.Recent studies have suggested that miRNAs in exosomes play an important role in tumor genesis and development.Tumor cells can avoid the tracking of the host immune system by regulating the expression level of miRNAs,successfully colonize and proliferate in the host body,and eventually invade and metastasize to surrounding or distant tissues and organs.In previous studies,we observed significant differences in miRNA expression profiles in serum exosomes of lung cancer patients and healthy people,and comprehensive analysis showed that miRNA-141 was significantly upregulated in serum exosomes of lung cancer patients.Further bioinformatics analysis revealed that miRNA-141 targeted and negatively correlated with GAX,suggesting that miRNA-141 may directly transcriptional regulation of GAX.GAX is a homeobox gene,and its high expression inhibits angiogenesis.This study aimed to clarify the mechanism of lung cancer cell-derived exosome miRNA-141 in lung cancer angiogenesis.PurposeThis study aims to clarify the role of miRNA-141 in exosomes on lung cancer angiogenesis.To reveal the molecular mechanism of exosome-mediated miRNA-141regulating angiogenesis;To explore the feasibility of miRNA-141 in exosomes as markers of malignant progression and therapeutic targets of lung cancer,and to provide new ideas and new targets for early diagnosis and targeted therapy of lung cancer.MethodPart Ⅰ1.MiRNA-141 in lung cancer cell A549 was overexpressed,and the exosomes of the cells were extracted and identified and the miRNA-141 in the exosomes was detected.2.Immunofluorescence detection of lung cancer cell-derived exosome miRNA-141 could enter the cytoplasm of HUVECs.3.The co-culture system of lung cancer cell-derived exosomes and human umbilical vein endothelial cells(HUVECs)was established to detect the effects of exosome miRNA-141 on the proliferation,migration and tubular differentiation of human umbilical vein endothelial cells.Part Ⅱ1.Bioinformatics database predicted multiple binding sites in the 3’untranslated region of miRNA-141 transcript and GAX.2.GAX expression changes were detected after RNAi and overexpression of miRNA-141 in HUVECs.3.MS2-RIP test and dual luciferase report test verified the interaction and regulatory effect of miRNA-141 and GAX.4.Reverse regulation of GAX expression level was performed with RNAi and overexpressed plasmid respectively to observe whether the effects of miRNA-141expression level changes of HUVECs could be blocked.5.The effects of exosome miRNA-141 on subcutaneous tumor growth and angiogenesis of lung cancer cells were detected in animal experiments.Part Ⅲ1.Blood samples from 98 patients with lung cancer were collected to analyze the correlation between the expression level of miRNA-141 in serum exosomes and the clinical characteristics of patients with lung cancer.2.To evaluate the possibility of the expression of miRNA-141 in serum exosomes as a prognostic factor of patients.ResultPart ⅠMiRNA-141 was overexpressed in lung cancer cell A549,and the exosomes were extracted and co-cultured with vascular endothelial cells HUVECs.The exosomes were taken up by HUVECs and then distributed around the nucleus under confocal microscope.CCK-8 assay showed that exosome miRNA-141 could significantly promote endothelial cell proliferation.The scratch experiment results showed that the addition of exosome miRNA-141 could significantly promote the scratch healing ability of endothelial cells.Transwell migration assay showed that the number of transmembrane migration cells in the miRNA-141 overexpression group was(216.7±23.6),significantly higher than that in the control group(85.1±4.1),and the difference was statistically significant(P<0.01).Compared with the control group,the tube structures formed in the experimental group were faster and more complex under inverted phase contrast microscope.The number of tube structures in the experimental group was(76.0±5.0)/well,significantly higher than that in the control group(36.0±2.6)/well,and the difference was statistically significant(P<0.01).Part ⅡBioinformatics database was used to predict the presence of multiple binding sites between miRNA-141 transcript and GAX’s 3’UTR.In situ hybridization was used to detect the expression level of GAX in lung cancer tissue microarray.According to the previous obtained miRNA-141 expression in lung cancer microarray,it was found that there was a negative correlation between the two.After RNAi and overexpression of miRNA-141 in lung cancer cell lines,realtime PCR detection of changes in GAX expression levels confirmed the negative correlation.MS2-RIP test and double luciferase report test were used to prove that there was a direct interaction between miRNA-141 and GAX,and the miRNA-141 mutant was constructed to detect the loss of binding between miRNA-141 and GAX,proving its specific binding.A miRNA-141 overexpressed A549 cell line(A549-miRNA-141)was constructed,and then tumor experiments were made with the original A549 cell line subcutaneously in nude mice,respectively.The results showed that the tumor growth rate of A549-miRNA-141 was faster than that of the control group.After the experiment,the mice were sacrificed and the tumor volume was measured.The mean tumor volume of the experimental group(A549-miRNA-141)(578±52mm~3)was significantly higher than that of the control group(A549)(348±28mm~3),the difference was statistically significant(P<0.01).Tumours in the two groups were stripped and tumor angiogenesis was observed.It was found that the abundance of blood vessels in the tumor cells of the experimental group was significantly different from that of the control group,indicating that miRNA-141 promoted tumor angiogenesis.Part ⅢAnalysis of the correlation between clinical information and serum exosome miRNA-141 expression level in 98 patients with lung cancer showed that the expression level of serum exosome miRNA-141 was correlated with clinical stage and lymph node metastasis.In the 98 patients,the median expression of miRNA-141was 2.15.Among them,52 patients with higher than or equal to the median value were classified as the miRNA-141 high expression group,and 46 patients with lower than the median value were classified as the miRNA-141 low expression group.All patients were followed up for 24 months.Twenty-five patients in the group with high miRNA-141 expression died,and 10 patients in the group with low miRNA-141expression died.Kaplan-Meier method was used to analyze the survival curves of the high expression group and the low expression group.The survival curves of the high expression group were significantly worse than the low expression group,and the difference was statistically significant(P=0.0088).Conclusion1.MiRNA-141 in exosomes can promote the proliferation,migration and tubular-like structure formation of HUVECs.2.Exosomes mediate miRNA-141 to bind GAX with negative correlation,miRNA-141 promotes the growth of A549 in nude mice,and miRNA-141 promotes angiogenesis.3.The expression level of miRNA-141 in serum exosomes was correlated with clinical stage and lymph node metastasis.The survival of high expression group of serum exosome miRNA-141 was significantly worse than that of low expression group. |
