| BackgroundMeningeal carcinomatosis（MC）refers to the metastasis of various malignant tumors to the dura mater,pia mater,cerebrospinal fluid,and subarachnoid space.Generally,there is no tumor mass in the brain and spinal cord.It is a special type of metastatic cancer of the central nervous system（CNS）,which belongs to the serious complications of the central nervous system in the late stage of cancer patients.About 10%一 30%of patients with solid tumors will have a metastasis of the central nervous system,of which 4%～15%are meningeal carcinomatosis.Meningeal carcinomatosis is highly malignant and has a poor prognosis.Its incidence has been increasing in recent years.Early diagnosis and treatment are the keys to improving the curative effect and reducing the mortality of meningeal carcinomatosis.MC has a delitescence onset,and there is no specificity in neurological symptoms and imaging manifestations.Its diagnosis is challenging.Although there is an in-depth understanding of the risk and clinical manifestations of meningeal carcinomatosis,the occurrence and diagnosis of MC are still underestimated in clinical practice,and there is no good quantitative index for evaluating the tumor load in soft meningeal space.In the research process of solving this problem,the concept of the "tumor microenvironment"（TME）has attracted the attention of many researchers.TME is composed of tumor cells,surrounding stromal cells,extracellular matrix,and signal molecules.TME provides the environment for tumor growth.Tumor cells can interact with the microenvironment and change the cell spectrum and various molecular levels in the microenvironment.For MC,cerebrospinal fluid（CSF）is the direct medium in contact with the tumor in the arachnoid space.The analysis of CSF can provide us with an opportunity to further study the TME of MC,which is helpful to the diagnosis,disease monitoring,and treatment response evaluation of MC.At present,the research on TME is mainly focused on the extracranial metastasis of solid tumors,but there is little research on the metastasis of CNS,and there is no relevant research on the MC microenvironment.Exosomes are an important non-cellular component of TME,and they are also a research hotspot in recent years.Exosomes are a kind of extracellular vesicles（EV）.They are a kind of extracellular membrane microcapsules secreted by almost all cells,with a diameter between 30-150 nm.Exosomes contain a variety of endogenous substances,such as proteins,lipids,and genetic substances,which exist in our bodies in large quantities under physiological and pathological conditions.Exosomes are important mediators of intracellular communication among tumor cells,immune cells,and stromal cells.They play a key role in many cellular processes,such as antigen presentation,signal transduction,and waste disposal.The content of exosomes is stable and can be transported to recipient cells to play its biological role.Various important cellular signal molecules,such as DNA,RNA,and protein,can be carried and transmitted through exosomes.Therefore,exosome-derived RNA signal spectrum and proteomics are usually used to explain the molecular regulation mechanism of disease and can be used as biomarkers to reflect the status of tissue disease.In the past decades,many studies have been devoted to revealing the mechanism of exosomes in regulating tumorigenesis and development and exploring new biomarkers and therapeutic targets.More and more evidence also shows that exosomes secreted by host cells or tumor cells are involved in tumor genesis,growth,invasion,and metastasis.However,the precise function of exosomes is still unknown to a great extent.Many studies focus on the function of different cell components of exosomes.These different cell components can promote tumor progression by promoting tumor cell growth,cell invasion,metastasis,angiogenesis,and even tumorigenesis,especially noncoding RNAs（ncRNAs）,which is a research hotspot in recent years.Exosomal ncRNAs,such as microRNAs（miRNAs）,long noncoding RNAs（lncRNAs）,and circRNAs（circRNAs）are promising new tumor biomarkers and therapeutic targets.So,are exosomal RNAs involved in the occurrence and development of meningeal carcinomatosis?Do exosomal RNAs have potential as new biomarkers for the diagnosis and prognosis of meningeal carcinomatosis?It’s not clear yet.ObjectiveThis study intends to use high-throughput sequencing technology to analyze the expression profiles of exosomal RNAs in cerebrospinal fluid（CSF）of patients with meningeal carcinomatosis（MC）and healthy controls,and explore whether there are differences in the expression profiles of exosomal RNAs in CSF of patients with MC and healthy volunteers.GO（Gene Ontology）functional annotation analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway enrichment analysis was made to further clarify the biological functions of these differentially expressed RNA molecules and the signal pathways involved of the related genes,and the interaction network diagrams were constructed to explore the molecular mechanism of exosomal RNAs involved in the occurrence and development of MC.Method1.Research object:A total of 35 patients with meningeal carcinomatosis（MC）who were admitted to the Second Hospital of Shandong University from May 2018 to June 2021 were collected.The diagnostic criteria were based on "the expert consensus on diagnosis of meningeal carcinomatosis in the infectious diseases and CSF cytology" of the Chinese Medical Association.18 patients with meningeal carcinomatosis metastatic from lung adenocarcinoma were selected to form an MC group（18 patients had a clear history of lung adenocarcinoma,had newly emerging neurological symptoms,and signs,and positive cerebrospinal fluid cytology by lumbar puncture）.18 healthy volunteers matched in age and sex were selected as the healthy control group.This study was approved by the ethics committee of the Second Hospital of Shandong University.All MC patients and healthy volunteers signed informed consent.2.Specimen collection:The cerebrospinal fluid（CSF）samples of the MC group and the healthy control group were collected through the lumbar puncture and frozen in-80℃refrigerators for standby.CSF samples（at least 5 ml for each CSF sample）from 3 patients with meningeal cancer metastatic from lung adenocarcinoma and 3 healthy volunteers were randomly selected for high-throughput whole transcriptome sequencing of cerebrospinal fluid exosomal RNA.3.Extraction and identification of CSF exosomes:MinuteTM Hi-Efficiency Exosome Isolation Reagent was used to extract the exosomes from CSF samples of MC group and healthy control group;The exosomes were identified by transmission electron microscope scanning,and the exosome markers CD63 and TSG101 were identified by Western blot.4.Exosomal RNA extraction:The total RNA of CSF exosomes from 3 MC patients and 3 healthy volunteers were extracted by the Trizol method for high-throughput whole transcriptome sequencing.5.RNA-seq library construction and RNA sequencing5.1 Transcriptome sequencing of CSF exosomal mRNA,lncRNA,and circRNA:Total RNA was used for removing the rRNAs using Ribo-Zero rRNA Removal Kits（Illumina,USA）following the manufacturer’s instructions.RNA libraries were constructed by using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit（Illumina,USA）according to the manufacturer’s instructions.Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system（Agilent Technologies,USA）.10 pM libraries were denatured as single-stranded DNA molecules,captured on Illumina flow cells,amplified in situ as clusters,and finally sequenced for 150 cycles on Illumina HiSeq Sequencer according to the manufacturer’s instructions.5.2 Sequencing of CSF exosomal miRNA:Total RNA of each sample was used to prepare the miRNA sequencing library,which included the following steps:1)3’-adaptor ligation;2)5’-adaptor ligation;3)cDNA synthesis;4)PCR amplification;5)Size selection of～150 bp PCR amplicons（corresponding to～22nt miRNAs）.The libraries were denatured as single-stranded DNA molecules,captured on Illumina flow cells.amplified in situ as clusters,and finally sequenced for 50 cycles on Illumina NovaSeq 4000 sequencer following the manufacturer’s instructions.6.Bioinformatics analysis and partial validation test6.1 Analysis of mRNA sequencing data and validation test:The high-quality reads were aligned to the human reference genome（UCSC HG19）with hisat2 software.Then,under the guidance of the Ensembl gft gene annotation file,the FPKM（fragments per kilobase of exon per million fragments mapped）value of gene-level mRNA was obtained by using the cuffdiff software as the mRNA expression profile,and the fold change and P-value between the two groups of samples were calculated to screen the differentially expressed（DE）mRNA,and the GO and KEGG pathways of the DE mRNA were analyzed.RT-qPCR was used to verify the relative expression levels of 6 target exosomal mRNAs in another 15 pairs of CSF samples of the MC group and healthy control group.6.2 Analysis of lncRNA sequencing data:The high-quality reads were aligned to the human reference genome（UCSC HG19）with hisat2 software.Then,under the guidance of Ensembl gft gene annotation file,the FPKM value of transcriptional level lncRNA was obtained by using coffediff software as the expression profile of lncRNA,and the fold change and P-value between the two groups of samples were calculated to screen the DE lncRNA.According to the proximity relationship,the target genes of lncRNA were predicted,and the GO and KEGG pathways of target genes were analyzed.The miRcode database was used to predict the DE lncRNA-miRNA interaction,and the prediction network diagram of lncRNAmiRNA of 4 up-regulated and 5 down-regulated target lncRNAs was constructed by using Cytoscape software.6.3 Analysis of miRNA sequencing data:After sequencing by Illumina sequencer,image analysis,and base recognition,the original reads after quality control are harvested.Use cutadapt software（v1.9.3）to de-splice the original reads,remove the low-quality reads,and retain the reads with length>=15 nt to obtain the de spliced reads（i.e.trimmed reads）.Then,the trimmed reads of all samples were combined and the new miRNA was predicted by miRDeep2 software（v2.0.0.5）.Use Novoalign software（v3.02.12）to compare the trimmed reads of each sample to the combined human pre-miRNAs database,with a maximum of 1 mismatch allowed.Count the number of tags on each mature miRNA as the original expression of the miRNA,and standardize it by TPM（tag counts per million aligned miRNAs）.Differentially expressed（DE）miRNAs between two samples were filtered through Fold change.DE miRNAs between two groups were filtered by Fold Change and P-value.The miRNA target gene prediction software based on miRanda and TargetScan was used to predict the target genes of the top 10 known DE miRNAs,and the GO function analysis and KEGG pathway analysis of the target genes were carried out.The target gene prediction network diagram of the top 5 DE exosomal miRNAs was constructed by using Cytoscape software.6.4 Analysis of circRNA sequencing data and validation test:The high-quality reads were aligned to the reference genome/transcriptome with STAR software and circRNAs were detected and identified with DCC software and annotated with circBase database.Then,edger software was used for data normalization and DE circRNA screening.Go and KEGG analysis of DE circRNA-derived genes were carried out.RT-qPCR was used to verify the relative expression levels of 5 target exosomal circRNAs in another 15 pairs of CSF samples of the MC group and healthy control group.The miRNA target gene prediction software based on miRanda and TargetScan was used to predict the DE circRNA-miRNA interaction,and the prediction network diagram of circRNA-miRNA of 5 target DE circRNAs was constructed by using Cytoscape software.Results1.Exosome identificationUsing MinuteTM Hi-Efficiency Exosome Isolation Reagent,we isolated exosomes from cerebrospinal fluid（CSF）of the MC group and healthy control group.Lipid bilayer vesicles with a diameter of about 60-100 nm can be observed by transmission electron microscope.Western blot analysis confirmed the existence of exosome marker proteins CD63 and TSG101 and confirmed that the cystic vesicles we isolated were exosomes.2.RNA high throughput sequencing and analysisAfter extracting the total exosomal RNA,the CSF exosomal RNA of 3 MC patients and 3 healthy controls were sequenced by High-throughput Sequencing and Bioinformatics Analysis.2.1 Differences analysis of exosomal mRNA expression profiles in CSF of MC patients2.1.1 By comparing the exosomal mRNA expression profile in CSF of MC patients with that of the healthy control group,a total of 20308 exosomal mRNA were identified.Compared with the healthy control group,there were 3845 differentially expressed（DE）exosomal mRNAs in CSF of MC patients,including 896 up-regulated exosomal mRNAs and 2949 down-regulated exosomal mRNAs.2.1.2 GO analysis of up-regulated exosomal mRNAs showed that the first three GO terms enriched in Biological Process（BP）were:cellular response to chemical stimulus,interspecies interaction between organisms,and symbiont process;The first three GO terms enriched in Cell Component（CC）were:extracellular exosome.extracellular vesicle,and extracellular organelle;The first three GO terms enriched in Molecular Function（MF）were:protein binding,enzyme binding,and cell adhesion molecule binding.GO analysis of downregulated exosomal mRNAs showed that the first three GO terms enriched in BP were:multicellular organismal process,nervous system process,and system process.The first three GO terms enriched in CC were:intrinsic component of membrane,integral component of membrane,and plasma membrane;The first three GO terms enriched in MF were:signaling receptor activity,molecular transducer activity,and transmembrane signaling receptor activity.2.1.3 KEGG analysis of up-regulated exosomal mRNAs showed that the top five pathways were HIF-1 signaling pathway,Staphylococcus aureus infection,Complement and coagulation cascades,TNF signaling pathway,Herpes simplex infection;KEGG analysis of up-regulated exosomal mRNAs showed that the top five pathways were HIF-1 signaling pathway,Staphylococcus aureus infection,Complement and coagulation cascades,TNF signaling pathway,Herpes simplex infection;KEGG analysis of down-regulated exosomal mRNAs showed that the top five pathways were Neuroactive ligand-receptor interaction,Olfactory transduction,Nicotine addiction,Hedgehog signaling pathway,Calcium signaling pathway,and Basal cell carcinoma.2.1.4 According to the results of KEGG analysis and KEGG database retrieval,the pathway relationship network analysis diagram of the top 10 pathways enriched by DE exosomal mRNAs was constructed,which showed that the core downstream pathway was Calcium signaling pathway,and the main cancer-related signaling pathways upstream were HIF-1 signaling pathway and cAMP signaling pathway.2.1.5 Fluorescence quantification RT-PCR was used to verify the relative expression levels of six target exosomal mRNAs in another 15 pairs of cerebrospinal fluid mixed samples of MC group and healthy control group.The results showed that compared with the healthy control group,the mRNA expression levels of PI3K,CASP3,and CASP8 in the cerebrospinal fluid exosomes of the MC group were significantly up-regulated（P<0.05）,while the mRNA expression levels of NETRIN1,AMPA and SHANK were significantly down-regulated（P<0.05）,which was consistent with high-throughput sequencing data.2.2 Differences analysis of exosomal lncRNA expression profiles in CSF of MC patients2.2.1 By comparing the exosomal lncRNA expression profile in CSF of MC patients with that of the healthy control group,a total of 37684 exosomal lncRNA were identified.Compared with the healthy control group,there were 1183 DE exosomal lncRNAs in CSF of MC patients.including 12 up-regulated exosomal lncRNAs and 1171 down-regulated exosomal lncRNAs.2.2.2 Go analysis of the target genes of up-regulated secreted lncRNA showed that the first three GO terms enriched in BP were positive regulation of intracellular estrogen receptor signaling pathway,regulation of cholesterol esterification,and negative regulation of protein autophosphorylation;The first three GO terms enriched in CC were MLL3/4 complex,chylomicron,and very-low-density lipoprotein particle;The first three GO terms enriched in MF were phospholipase inhibitor activity,lipase inhibitor activity,and phosphatidylcholine binding.Go analysis of the target genes of down-regulated secreted lncRNA showed that the first three GO terms enriched in BP were protein localization to the plasma membrane,protein localization to cell periphery,and cell surface receptor signaling pathway;The first three GO terms enriched in CC were actin cytoskeleton,neuronal cell body,and plasma membranebounded cell projection;The first three GO terms enriched in MF were enzyme binding,protein self-association,and collagen binding.2.2.3 KEGG analysis of adjacent target genes of down-regulated exosomal lncRNAs showed that the seven pathways with significant enrichment were Amyotrophic lateral sclerosis（ALS）,Amphetamine addiction,Adipocytokine signaling pathway,Dopaminergic synapse,Peroxisome,Hypertrophic cardiomyopathy（HCM）,and Nicotine addiction.2.2.4 Based on the miRcode database,the target miRNA prediction network diagram of 4 up-regulated and 5 down-regulated exosomal lncRNAs was constructed by using Cytoscape software.2.3 Differences analysis of exosomal miRNA expression profiles in CSF of MC patients2.3.1 By comparing the exosomal miRNA expression profile in CSF of MC patients with that of the healthy control group,a total of 431 exosomal miRNAs were identified,of which 161 were predicted new miRNAs.Compared with the healthy control group,there were 14 DE exosomal miRNAs in CSF of MC patients,including 6 up-regulated exosomal miRNAs and 8 down-regulated exosomal miRNAs.2.3.2 Go analysis of the target genes of up-regulated exosomal miRNA showed that the first three GO terms enriched in BP were localization,generation of neurons,and nervous system development:The first three GO terms enriched in CC were transporter complex,cell junction,and transmembrane transporter complex;The first three GO terms enriched in MF were protein binding,cAMP binding,and phospholipid scramblase activity.Go analysis of the target genes of down-regulated exosomal miRNA showed that the first three GO terms enriched in BP were positive regulation of cellular extravasation,positive regulation of cellcell adhesion,and T cell differentiation;The first three GO terms enriched in CC were integral component of plasma membrane,intrinsic component of plasma membrane,and cell surface;The first three GO terms enriched in MF were solutecation symporter activity,solute sodium symporter activity,and symporter activity.2.3.3 KEGG analysis of target genes of up-regulated exosomal miRNAs showed that the top 5 pathways with significant enrichment were Proteoglycans in cancer,Glutamatergic synapse,Dopaminergic synapse,Hippo signaling pathway,and cGMP-PKG signaling pathway;KEGG analysis of target genes of down-regulated exosomal miRNAs showed that the top 5 pathways with significant enrichment were Wnt signaling pathway,Oocyte meiosis,Progesterone-mediated oocyte maturation,Pathways in cancer,and HTLV-I infection.2.3.4 MiRNA target gene prediction software based on MiRanda and TargetScan was used to predict the target genes of the top 10 DE miRNAs,and Cytoscape software was used to construct the target gene prediction network diagram of the top 5 up-and down-regulated exosomal miRNAs.2.4 Differences analysis of exosomal circRNA expression profiles in CSF of MC patients2.4.1 By comparing the exosomal circRNA expression profile in CSF of MC patients with that of the healthy control group,a total of 12503 exosomal circRNAs were identified,of which 8940 were newly identified circRNAs.Compared with the healthy control group,there were 39 DE exosomal circRNAs in CSF of MC patients,including 34 up-regulated exosomal circRNAs and 5 down-regulated exosomal circRNAs.2.4.2 Go analysis of the up-regulated exosomal circRNAs-derived genes showed that the first three GO terms enriched in BP were lipid storage,regulation of lipid storage,and androgen receptor signaling pathway;The first three GO terms enriched in CC were nucleoplasm,nuclear lumen,and membrane-enclosed lumen;The first three GO terms enriched in MF were transcription cofactor activity,transcription factor activity:transcription factor binding,and transcription factor activity:protein binding.2.4.3 KEGG analysis of the up-regulated exosomal circRNAs-derived genes showed that the top 10 pathways with significant enrichment were Phagosome,Allograft rejection,Graftversus-host disease,Type I diabetes mellitus and herpes simplex infection,Autoimmune thyroid disease,Staphylococcus aureus infection,Viral myocarditis,Aminoacyl-tRNA biosynthesis,and Leishmaniasis.2.4.4 Fluorescence quantification RT-PCR was used to verify the relative expression levels of five target exosomal circRNAs in another 15 pairs of CSF mixed samples in the MC group and healthy control group.The results showed that compared with the healthy control group,the expressions of five target exosomal circRNAs（chr6:31238163-31323269-,hsacirc0001451,chr1:231928640-231954263+,hsacirc0001861,and hsacirc0001181）in the MC group were significantly up-regulated,which was consistent with high-throughput sequencing data.2.4.5 The miRNA target gene prediction software based on MiRanda and TargetScan database was used to predict the DE circRNA-miRNA interaction.The circRNA-miRNA interaction prediction network diagram of five targets DE circRNAs was constructed by using Cytoscape software.ConclusionThrough the study of high-throughput sequencing,it is found that the cerebrospinal fluid（CSF）exosomes of patients with meningeal carcinomatosis（MC）and those of the healthy control group have their own characteristic RNA expression profiles.The differentially expressed（DE）exosomal RNA molecules screened by bioinformatics analysis,including mRNA,lncRNA,miRNA,and circRNA,may play an important role in the occurrence and development of MC.By establishing the differential expression profiles of CSF exosomal mRNA,lncRNA,miRNA,and circRNA in patients with MC,making GO function annotation and KEGG pathway analysis,and constructing their interaction prediction network diagram,we provided a theoretical basis for the subsequent exosome genomics,proteology and molecular biology research of MC,as well as provide potential candidate molecular diagnostic MC markers. |
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