Characteristics Of Plasma Exosomal MicroRNA In Luminal A Breast Cancer Patients Treated With Neoadjuvant Chemotherapy With AT Regimen

BackgroundExosome is small microvesicle with a size about 30-100 nm released from the fusion process between the plasma membrane and the late endocytic compartment,multivesicular endosome(MVE).Exosomes are widely distributed in blood,urine,ascites,cerebrospinal fluid and may even exist in all body fluid in eukaryotes.Recent studies on exsoome and microRNA they carried on discover that they are closely reated with the development and prognosis in breast cancer patients.Further investigations even show immense potential for exosome miRNAs to function as prognostic and diagnostic biomarkers.Till now,numerious studies demonstrate that tumor initiation,drug resistance,invasiveness and metastasis in breast cancer are partly mediated by exosome miRNAs due to the important role they played in cell-to-cell communication.While most researches are peformed in cell lines,there may be a discrepancy between in viov and invitron miRNA expressions.What's more,as a novel aspect,major exosome studies are based on prior results in plasma or pathologic tissues,which may narrow the horizon in this hotspot.Thus,we plan to explore the role of plasma exosomes miRNA in breast cancer patients more via second generation sequencing technologies and try to figure out their roles in drug resistance and prognosis.Objectives1)To identify the existence of exosome in plasma of breast cancer patients;2)To explore different plasma exosome extracting methods and work out a practical flowchat for clinical use;3)To compare exosome miRNA expression profiles in breast cancer patients;to elucidate their roles in drug resistance and prognosis.Methods1)Plasma exosomes were harvested by ultracentrifugation and exsosome precipitation kit;TEM and western blot were subsequently allied to verify the existence of exosomes2)Total RNA was extracted by Trizol or miRNeasy kit and microRNA library was constructed for second generation sequencing;expression profiles were compared between luminal A breast3)The target genes and signal pathways of exosome miRNA were further explored using DIANA Tools?GO and KEGG database.Results1)The amount of plasma exosomes harvested by precipitation kit was highest compared to traditional ultracentrifugation;blood smaples with limited volumn could still produce satisfactory results even kept for a long time;2)Total 1446 types of RNA were sequenced and identified with miRBase 21;miRNA with highest expression as miR16 and miR21 were confirmed as a potential marker in serum or tissues from breast cancer patients;3)Twenty-two miRNAs with different expression levels were identified between patients in PR and SD groups,among them 9 were upregulated and 13 downregulated in SD groups.Based on Kaplan Meire plot database,miR454 were found to be survival-related.Also 6 miRNAs with down trend and 99 with up trend after neoadjuvant therapy were identified in 5 PR patients;4)GO analysis indicates that target genes of there miRNAs are involved in RNA synthesis,metabolism,transcription and cell development and morphogenesis.KEGG pathway analysis demonstrated the downstream signaling pathways include drug metabolism,GnRH and Hedgehog pathways;5)Sevreal important node genes in the whole regulation network for different neoadjuvant outcome like CALM2 and GRB2 were picked out based on String and Cytosapce software,as well as PTEN and KRAS for regulation network responsed to neoadjuvant chemotherapy.6)Genes were further filtered based on the correlation coefficient of mRNA and microRNA expression levels in TCGA.GO analysis presented a similar result as their major functions lied in macromolecular metabolism and cell development.Major nodes like PPP2C A and IGF 1 were identified;7)MiRNA and target gene expression levels were tested in TCGA database,expression levels of miR184,miR144 and KITLG still diverse in patients with different AJCC stages.Conclusions1)The amount of plasma exosomes harvested by precipitation kit was highest and could be applied to smaples with limited volumn and longtime storage;2)Total 27 miRNAs with different expression levels were identified between patients in PR and SD groups,among them 16 were upregulated and 11downregulated in SD groups.Based on Kaplan Meire plot database,2 of 27 were found to be survival-related.Also 6 miRNAs with down trend and 99 with up trend after neoadjuvant therapy were identified in 5 PR patients;3)Sevreal important node genes in the whole regulation network for different neoadjuvant outcome were selected out as CALM2 and CREB1.while for regulation network responsed to neoadjuvant chemotherapy,PTEN and KRAS may play the vital part;4)MiRNA and target gene expression levels in TCGA database were analyzed.They construct a network with similar biological function while PP2CA and IGF1 were important nodes.Expression levels of miR184,miR144 and KITLG still diverse in patients with different AJCC stages.