Mechanism Of Exosomal MiRNA Promoting Brain Metastasis Of Lung Cancer By Destroying Blood Brain Barrier

In recent years,the rapid development of diagnosis and treatment of lung cancer has significantly prolonged the survival of patients,more and more brain metastases have been found.Brain metastasis of lung cancer leads to a series of neurological related symptoms,which seriously affect the life quality of the patients.After local and systemic therapy,the median survival time of patients with brain metastasis is only 6-8 months.The main factor of poor curative effect is that the existence of blood brain barrier(BBB),making it difficult for therapeutic drugs to reach the brain.Therefore,analyzing the role of BBB in the occurrence and development of lung cancer brain metastasis can provide a new perspective for its treatment,which has an important scientific value and clinical significance.The metastasis of tumor cells from the primary site to the target organ is a complex dynamic process.Studies have found that the primary tumor can induce the formation of a microenvironment conducive to metastasis in specific organs and tissues,that is,the formation of pre-metastatic niche(PMN).The important factor of brain PMN formation lies in the destruction of BBB,and the tight junction between brain capillary endothelial cells is an important structural basis for determining cerebral vascular permeability and maintaining BBB integrity.Previous studies have shown that the destruction of BBB caused by the increase of cerebrovascular permeability is an early event in the formation of brain PMN.During the formation of brain metastases of lung cancer,the tight junction between cerebrovascular endothelial cells is opened,and fenestras,pinocytic vesicles and basement membrane thickening can be seen.However,the internal mechanism of these changes remains to be further explored.The key to the formation of PMN is that tumor cells can reshape the microenvironment of premetastatic target organs by secreting cytokines,extracellular vesicles,or exosomes,to facilitate the realization of metastasis.In recent years,exosomes derived from tumor cells have attracted more and more attention as intercellular communication materials,which play an important role in mediating the formation of PMN.Tumor cells derived exosomes play an important signal transduction role in the tumor microenvironment,and they can also be used as biomarkers for early diagnosis and prognosis evaluation of tumors.Previous studies have shown that exosomes derived from tumor cells participate in the development of brain metastasis,and they usually carry important signal molecules such as mi RNAs,which can regulate the formation of brain PMN.However,there are still relatively few studies on the role of lung cancer cells derived exosomal mi RNAs in lung cancer brain metastasis.Therefore,we co-cultured exosomes of A549 and brain metastatic lung cancer cell line PC14 / B with human brain microvascular endothelial cells(HBMECs)to detect the permeability and tight junction proteins expression between HBMECs.The differentially expressed mi RNAs in the exosomes of A549 and PC14 / B cell lines were detected by exosomal mi RNA sequencing technology.It was determined that the key exosomal mi RNA was mi R-4746-5p,and its target was PPM1 D protein.Then,the effect of exosomal mi R-4746-5p on HBMECs and the mechanism of PPM1 D in it were explored.Through this study,we clarified the molecular mechanism of lung cancer cell exosomal mi R-4746-5p destroying the tight junction between endothelial cells by regulating PPM1 D,which could provide a new theoretical basis for lung cancer brain metastasis and a new target for its treatment.Part one:Effect of lung cancer cell derived exosomes on human brain microvascular endothelial cellAbjectives:To investigate the role of exosomes derived from A549 and brain metastasis prone lung cancer cell line PC14 / B in promoting the formation of brain pre-metastatic niches in brain metastasis of lung cancer by affecting the tight junction between human brain microvascular endothelial cells and destroying the blood brain barrier.Methods:The cell supernatants of A549 and PC 14 / B cell lines were collected,and the exosomes were extracted by exosome extraction kit.The extracted exosomes were identified by transmission electron microscope,particle size determination and Western Blot.The uptake of exosomes by HBMECs was observed by exosome endocytosis experiment.The BBB model in vitro was established and the exosomes of A549 and PC 14 / B were added.The fluorescein sodium permeability test was counducted to observe the changes of BBB permeability.The exosomes of A549 and PC 14 / B were co-cultured with HBMECs respectively,and then the expression of tight junction proteins between HBMECs were detected by immunofluorescence and Western Blot experiments.Results:1.Under transmission electron microscope,the exosomes of A549 and PC 14 / B showed three-dimensional tea-tray nanoparticles with obvious membrane structure.2.The results of particle size detection showed that the average particle size of the extracted exosomes of A549 and PC 14 / B cells was about 100 nm.3.Western Blot experiment showed that the exosomes of A549 and PC 14 / B cells expressed positive protein markers CD9,TSG101 and CD63,while the negative protein marker calnexin was not expressed.4.In the exosome endocytosis experiment,green fluorescence aggregation could be seen near the nucleus of HBMECs,suggesting that the exosomes were taken up by HBMECs.5.The sodium fluorescein permeability test displayed that the BBB permeability after the exosomes of PC 14 / B cells treatment was significantly higher than that of the control group and exosomes of A549 cells treatment group.6.From the immunofluorescence and Western Blot experiments,we could see that the expression of tight junction proteins(ZO-1,occludin and claudin-5)in exosomes of PC 14 / B cells treatment group were significantly down regulated compared with the control group and exosomes of A549 cells treatment group.Conclusion:The exosomes of A549 and PC 14 / B cells extracted by exosome extraction kit were qualified and could be used in subsequent experiments.HBMECs could uptake the co-cultured exosomes of A549 and PC14 / B cells.The exosomes of PC 14 / B cells could significantly increase the permeability between HBMECs and significantly down regulate the expression of tight junction proteins(ZO-1,occludin,claudin-5)to promote the formation of PMN in brain metastasis of lung cancer.Part two:Mechanism of exosomal mi R-4746-5p promoting lung cancer brain metastasis by destroying blood-brain barrierObjectives:To investigate the role of lung cancer cell exosomal mi R-4746-5p in promoting the formation of brain pre-metastatic niche in lung cancer brain metastasis by destroying the tight junction between HBMECs.Methods:1.The exosomes of A549 and PC 14 / B cells were analyzed by mi RNA sequencing.The expression of mi R-4746-5p was significantly increased in exosomes of PC 14 / B cells.HBMECs were transfected with lentivirus to construct mi R-4746-5p mimics HBMECs with high expression of mi R-4746-5p.2.RT-q PCR was used to determine the expression of mi R-4746-5p in HBMECs and mi R-4746-5p mimics HBMECs.4.In vitro BBB models were established with HBMECs and mi R-4746-5p mimics HBMECs respectively.The changes of BBB permeability were observed by fluorescein sodium permeability experiment.5.The expression of tight junction proteins between HBMECs and miR-4746-5p mimics HBMECs were detected by immunofluorescence and Western Blot.6.Co-cultured the exosomes of A549 and PC 14 / B cells with HBMECs.The expression of PPM1 D protein and PI3 K / Akt / m TOR signaling pathway proteins in HBMECs were detected by Western Blot.7.The expression of PPM1 D protein and PI3 K / Akt / m TOR signaling pathway proteins in mi R-4746-5p mimics HBMECs were detected by Western Blot.Results:1.The results of mi RNA sequencing and RT-q PCR showed that mi R-4746-5p was significantly overexpressed in the exosomes of PC 14 / B cells.2.Under fluorescence microscope,HBMECs transfected with lentivirus showed green fluorescence.RT-q PCR results showed that mi R-4746-5p was significantly overexpressed in mi R-4746-5p mimics HBMECs compared with HBMECs.3.The permeability of mi R-4746-5p mimics HBMECs cells was significantly higher than that of HBMECs.4.From the immunofluorescence and Western blot experiments,we could see that the expression of tight junction proteins(ZO-1,occludin,claudin-5)between mi R-4746-5p mimics HBMECs was significantly down regulated compared with HBMECs.5.When exosomes of PC14 / B cells were co-cultured with HBMECs,the expression of PPM1 D protein was significantly up regulated and the expression of pAkt and p-m TOR were down regulated.6.Compared with HBMECs,the expression of PPM1 D in mi R-4746-5p mimics HBMECs was significantly up regulated,and the expression of p-Akt and p-m TOR were down regulated.Conclusion:Mi R-4746-5p was the most differentially expressed mi RNA in the exosomes of A549 and PC 14 / B cells.A stable HBMECs cell line with high expression of mi R-4746-5p(mi R-4746-5p mimics HBMECs)has been established.Compared with HBMECs,the intercellular permeability of mi R-4746-5p mimics HBMECs was significantly reduced,and the expression of tight junction proteins(ZO-1,occludin,claudin-5)were significantly down regulated.The expression of PPM1 D protein was up regulated and PI3 K / Akt / m TOR signaling pathway was inhibited in mi R-4746-5p mimics HBMECs and HBMECs co-cultured with the exosomes of PC 14 / B cells.Mi R-4746-5p could regulate PI3 K / Akt / m TOR signaling pathway by PPM1 D,down regulating the expression of tight junction proteins(ZO-1,occludin,claudin-5)between HBMECs,destroying the BBB to promote the formation of PMN and the brain metastasis of lung cancer.Part three: Exploration of exosomal mi RNA in plasma as biomarker for predicting brain metastasis of lung cancerObjectives:To analyze the difference of plasma exosomal mi RNA expression in advanced lung cancer patients with or without brain metastases,screening out the plasma exosomal mi RNA that could be used to predict brain metastasis of lung cancer.Methods:26 patients diagnosed as advanced lung cancer were enrolled,including 14 patients with brain metastases and 12 patients without brain metastases.The plasma exosomes of patients were extracted,and the total RNA of plasma exosomes was isolated.Then the extracted patient plasma exosomal RNAs was sequenced and identified,and their differential expression was analyzed.The target genes were predicted by mi Randa and RNAhybrid software.We then compared the target genes with NR,Swiss-Prot,GO,COG,KEGG,KOG and Pfam databases by blast software to obtain the annotation information of them.RT-q PCR was used to determine the expression of mi RNAs significantly highly expressed in plasma exosomes.Results:1.The results of mi RNA sequencing analysis of plasma exosomes showed that a total of 892 mi RNAs were obtained.Compared with the exosomal mi RNAs in plasma of advanced lung cancer patients without brain metastases,31 mi RNAs were significantly up regulated,and 23 mi RNAs were significantly down regulated in patients with brain metastases.2.The results of GO classification of differentially expressed mi RNAs target genes showed that they were related to extracellular matrix and involved in intercellular adhesion,receptor regulation,antioxidation and so on.3.The target genes were involved in protein processing of endoplasmic reticulum,ubiquitin mediated by single element,RNA transport,regulation of cell adhesion and endocytosis.4.The KEGG pathway enrichment analysis of differentially expressed mi RNAs target genes showed that they were involved in the regulation of MAPK and Ras signaling pathways.5.There were 8 mi RNAs were significantly up regulated in plasma exosomes of advanced lung cancer patients with brain metastases,as follows: mi R-4746-5p、mi R-224-5p、mi R-151a-3p、mi R-99b-5p、mi R-27a-3p、mi R-27b-3p、mi R-223-3p、mi R-125b-5p.Conclusion:Mi R-4746-5p、mi R-224-5p、mi R-151a-3p、mi R-99b-5p、mi R-27a-3p、mi R-27b-3p、mi R-223-3p、mi R-125b-5p were significantly up regulated in plasma exosomes of patients with brain metastases,which may be used as biomarkers to predict brain metastasis of lung cancer,but its clinical significance and value still need to be further verified by expanding the sample size.