The Mechanism Of Cerebrospinal Fluid Exosome MiR-204-3p Promoting Malignant Progression Of Glioma And The Development Of Related Liquid Biopsy Microfluidic Chip

Glioma is the most common malignant tumor of the central nervous system.Traditional therapies such as surgery,radiotherapy and chemotherapy are ineffective in treating glioma and have a high recurrence rate,and immunotherapy is not as effective in treating glioma.The therapeutic resistance of glioma is related to its unique tumor microenvironment.Studying the formation mechanism of glioma tumor microenvironment and finding therapeutic approaches to target glioma tumor microenvironment are expected to improve the prognosis of glioma patients.The tumor microenvironment of glioma is composed of tumor cells,non-tumor cells and extracellular matrix,which is characterized by low oxygen and low pH.The non-tumor cells in the glioma tumor microenvironment mainly include neuronal precursor cells,vascular cells,fibroblasts and a variety of immune cells.Glioma cells and non-tumor cells are able to interact with each other through various pathways such as cytokines and extracellular vesicles,which together contribute to the formation of the glioma tumor microenvironment and help tumor cells evade immune surveillance and maintain the malignant phenotype.Exosomes are a class of extracellular vesicles with a diameter of 30-150 nm that are capable of delivering biomolecules such as proteins and nucleic acids between cells and mediating intercellular information exchange.Non-coding RNAs delivered by exosomes play an important role in intercellular communication in the glioma tumor microenvironment,and miRNAs account for the highest proportion of these exosome non-coding RNAs.In our previous study,glioma cells were able to regulate the function of mesenchymal stem cells,tumor-associated macrophages,and promote tumor progression through exosomal miRNAs.It has been reported that cells do not randomly load miRNAs into exosomes,but rather enrich some of them into exosomes through a special sorting process,and this sorting mechanism relies on the binding of RNA-binding proteins to specific sequences on miRNAs.hnRNP family members belong to the RNA-binding proteins,and the hnRNP A2/B1,hnRNP Q of this family have been reported to be involved in the exosome sorting process of miRNAs.Small ubiquitin-like modifications are post-translational modification that is widely found in eukaryotic organisms and is involved in various physiological processes such as protein degradation,DNA repair,and cell proliferation.The small ubiquitin-like modification can promote hnRNP A2/B1 mediated miRNA sorting process,but the exact mechanism is not clear.In addition,the hypoxic environment can also promote the secretion of exosomes and change the composition of exosome contents,and the related mechanisms need to be further investigated.Searching for miRNAs sorted into glioma exosomes,researching sorting mechanisms and the role of exosomal miRNAs in glioma progression will help us to gain insight into the glioma tumor microenvironment and develop targeted therapeutic strategies.The first part of this paper focuses on finding miRNAs enriched in cerebrospinal fluid glioma exosomes,investigating the mechanisms associated with their sorting into exosomes and promoting malignant progression of gliomas.By analyzing the sequencing data of cerebrospinal fluid exosomes and tumor tissue samples from glioma patients,we found that miR-204-3p was sorted to glioma exosomes.In glioma cells,miR-204-3p was able to inhibit cell proliferation through CACNA1C/MAPK pathway.Further studies showed that hypoxia could promote miR-204-3p transcription through upregulating SOX9.Also hypoxia can increase the small ubiquitin-like modification of hnRNP A2/B1 in glioma by upregulating UBC9,promote the transfer of hnRNP A2/B1 to the cytoplasm,accelerate the exosome sorting of miR-204-3p,and thus relieve the inhibitory effect of miR-204-3p on the proliferation of glioma cells.Glioma exosomes can be taken up by vascular epithelial cells,and miR-204-3p in exosomes can promote vascular epithelial tube formation and migration ability through ATXN1/STAT3 pathway.The small ubiquitin-like modification inhibitor TAK-981 was able to inhibit the exosomal sorting of miR-204-3p by downregulating the small ubiquitin-like modification level of hnRNP A2/B1,and showed good inhibitory effects on tumor growth and angiogenesis in animal experiments.Liquid Biopsy is a non-invasive diagnostic method using body fluid samples,which has been rapidly developed in the field of early diagnosis of tumors in recent years.In the first part of this paper,we found that miR-204-3p is enriched in cerebrospinal fluid exosomes of glioma patients and has the potential to be used as a target for early diagnosis of glioma.However,traditional exosome isolation and detection methods are costly and time-consuming,and these drawbacks have limited the clinical application of exosome liquid biopsy.Microfluidics,a class of devices that use microfluidic channels to manipulate minute amounts of fluid,has been widely used in liquid biopsy in recent years because of its low cost,fast detection speed,and small size.Studies have reported the use of microfluidic chips for the separation and purification of exosomes,but these methods have low separation efficiency and rely on special equipment,while there have been no reports related to the separation and detection of exosomes in cerebrospinal fluid by microfluidic devices.In the second part of this paper,we developed a microfluidic technology-based,anodic aluminum oxide nanofilm biopsy chip for cerebrospinal fluid exosomes to address the above issues.The experimental data confirmed that this new chip can isolate exosomes from various body fluids with an efficiency of up to 82%,while the device is inexpensive,fast,and able to handle small volume samples without special equipment.We have also demonstrated that the cerebrospinal fluid exosome miR-204-3p can be used as a diagnostic target for glioma through liquid biopsy microfluidic chip.These results provide a new technical approach and detection target for exosome liquid biopsy of glioma.Part I Study on the mechanism of exosomal miR-204-3p promoting malignant progression of gliomaObjectiveTo search for miRNA molecules that are sorted into glioma exosomes,to investigate the mechanism by which miR-204-3p is enriched into exosomes and promotes malignant progression of glioma,and to search for targeted therapeutic drugs.Methods1.Cerebrospinal fluid and tumor tissue samples from glioma patients were collected and sequenced to find miRNAs with exosomal sorting and enrichment processes in glioma.2.In vivo,in vitro phenotyping experiments were performed to confirm the role of miR-204-3p in glioma cells.TargetScan and other databases are used in combination with in vitro experiments to search for miR-204-3p downstream target molecules and related pathways.3.The StarBase database was used to find RNA-binding proteins capable of binding to miR-204-3p and its ability to mediate miR-204-3p exosomes sorting was verified using siRNA knockdown.4.The hnRNP A2/B1 fragment plasmid was constructed and the domain mediating its binding to miR-204-3p was verified with immunoprecipitation experiments.5.Exploring the role of miR-204-3p in the glioma tumor microenvironment using normal/knockdown miR-204-3p exosomes cultured with vascular epithelial cells.6.Glioma cells were treated with hypoxic environment to study its effect on miR-204-3p synthesis and sorting process.7.Searching for drugs that can inhibit the sorting process of miR-204-3p into exosomes,and to evaluate its anti-tumor effects by animal experiments.Results1.miR-204-3p was upregulated in cerebrospinal fluid of glioma patients,while miR-204-3p content was significantly downregulated in glioma tissues.2.miR-204-3p inhibits the proliferation of glioma cells and prolongs the survival of in situ tumor-forming animals.3.miR-204-3p targets CACNA1C and downregulates the expression of p-AKT and p-ERK,key molecules of the downstream MAPK pathway.4.hnRNP A2/B1 can bind to miR-204-3p through RRM1 motif and mediate its sorting into exosomes,miR-204-3p binds hnRNP A2/B1 via exosome-sorting sequence.5.Hypoxia promotes ubiquitination modification of hnRNP A2/B1 to the cytoplasm,which in turn upregulates hnRNP A2/B1-mediated sorting of miR-204-3p into exosomes.6.Exosomes from glioma are able to be taken up by vascular epithelial cells,and miR-204-3p in exosomes promotes angiogenesis via the ATXN1\STAT3 pathway.7.Hypoxia can upregulate the expression of the transcription factor SOX9,which can control the transcription of miR-204-3p by binding to the promoter region.8.The ubiquitination-like inhibitor TAK-981 inhibits glioma growth and reduces angiogenesis within tumor tissue in an animal model.Conclusion1.miR-204-3p in glioma cells was enriched towards exosomes by sorting.2.miR-204-3p in glioma cells can inhibit the proliferation of glioma cells in vivo/in vitro through the CACNA1C\MAPK pathway.3.hnRNP A2/B1 mediates the exosome sorting process of miR-204-3p.4.Hypoxia promotes hnRNP A2/B1 localization to the cytoplasm through upregulation of SUMOylation.5.Hypoxia promotes transcription of miR-204-3p through upregulation of SOX9.6.miR-204-3p in glioma exosomes promotes angiogenesis in the glioma tumor microenvironment.7.The SUMOylation inhibitor TAK-981 can inhibit glioma growth by inhibiting the exosomal sorting process of miR-204-3p.Part Ⅱ Development of a liquid biopsy chip for cerebrospinal fluid exosome miR-204-3pObjectiveDevelopment of a microfluidic-based liquid biopsy chip for cerebrospinal fluid exosomes to validate the ability of the cerebrospinal fluid exosome miR-204-3p identified in Part I as a diagnostic marker for glioma,providing a new detection tool and detection target for the clinical application of cerebrospinal fluid exosome liquid biopsy.Methods1.Design of microfluidic devices capable of exosome separation and exosome marker identification using anodic aluminum oxide as nanofiltration membranes.2.The filter membrane material was characterized accordingly using electron microscopy.3.Use Comsol to simulate the filtration process of the microfluidic device and optimize the filtration conditions.4.The microfluidic device was used to separate and identify exosomes in biological samples and verify the effectiveness of the separation device.5.Using the microfluidic device to isolate cerebrospinal fluid exosomes from patients and validate the ability of cerebrospinal fluid exosome miR-204-3p as a diagnostic marker for glioma.Results1.Microfluidic devices based on anodic alumina can efficiently and rapidly isolate exosomes from various types of biological fluid samples.2.The microfluidic chip constructed from plasma-treated 90 nm pore size anodized alumina can separate exosomes while maintaining high separation efficiency(up to 82%);the separation efficiency and particle size distribution of exosomes are correlated with the filtration speed.3.The microfluidic exosome separation device can rapidly and efficiently separate exosomes from cerebrospinal fluid,serum and other samples,and the molecular characteristics of the separated exosomes remain the same as before separation.The miR-204-3p was significantly upregulated in the exosomes of cerebrospinal fluid from glioma patients isolated by microfluidic chip.Conclusion1.A microfluidic exosome separation device based on anodic aluminum oxide nanochannel membrane was successfully constructed.2.0.1 μl/min is the optimal filtration rate for microfluidic devices,and plasma-treated 90 nm alumina membrane is the best membrane material for microfluidic devices.3.The microfluidic exosome isolation device can be used to isolate exosomes from a variety of body fluids,including cerebrospinal fluid,and the characteristic molecules of exosomes are well preserved.miR-204-3p can be used as a target for cerebrospinal fluid exosome liquid biopsy in glioma.