Bioinformatics Analysis Of Differentially Expressed LncRNAs In Exosomes Derived From Oral Squamous Cell Carcinoma

Background: Oral squamous cell carcinoma is the most prevalent form of malignant tumor,rapid progress,extensive infiltration,and poor prognosis are its main characteristics;gold standard for oral squamous cell cancer diagnosis is tissue biopsies,however,at the time of treatment,the tumor may have metastasized.Thus,non-invasive diagnosis biomarkers with high sensitivity and specificity for the early detection and diagnosis of oral squamous cell carcinoma are urgently needed.Exosomes are nanometer-sized particles secreted from many types of mammalian cells both normal and pathological conditions.Due to exosomes contain a variety of ingredients,which can regulate micro-environment of cell,so exosomes are recognized as a novel mechanism and an important vehicle of communication.LncRNA is a group of non-coding RNAs with more than 200 nucleotides.Although lncRNA had been considered to have no biological function,recent studies have regarded it as an emerging regulator to biological functions,and aberrant expression of lncRNA was closely associated with many cancers.Therefore,exosomal lncRNAs which serve as early non-invasion diagnostic markers became a hot direction for cancer research.Objectives: The aims of this study were to isolate and confirm the exosomes derived from the supernatant of human oral squamous cell carcinoma cell line?CAL-27?and human immortalized oral epithelial cell?HOEC?,then to investigate the lncRNAs expression profile through high throughput sequencing and to analyze the diagnostic potential based on bioinformatics analysis.Methods:?1?Exosomes were purified from the supernatant of CAL-27 and HOEC by differential ultracentrifugation;?2?Exosomes were confirmed with Western blotting,transmission electron microscopy?TEM?and particle size analysis;?3?LncRNA expression profiles were explored by High throughput sequencing technology;?4?Significantly differential expression exo-lncRNAs were screened,and then identified and quantified by real-time quantitative PCR?RT-qPCR?,respectively.Results:?1?Western blotting showed that exosome-enriched protein CD63,ALIX and TSG101 were positive;TEM showed that were either round or oval in 30 nm to 150 nm;Particle size analysis showed that the most of exosomes were concentrated in 100 nm.?2?High throughput sequencing revealed that 52 differentially expressed lncRNAs were identified with 23 lncRNAs up-regulated and 29 lncRNAs down-regulated;?3?The GO annotation showed that the target gene of 52 differentially expressed lncRNAs were involved in many biological processes such as cellular process,cell part and binding.?4?KEGG pathways analysis showed 259 pathways,of which,50 pathways with meet the threshold requirements;?5?Three lncRNAs?NR₀26892.1,NR₁26435.1 and NR₀36586.1?have significantly differential expression between CAL-27-exo and HOEC-exo.RT-qPCR showed that three marked different exo-lncRNAs expression in cell and exosome have the same trend with sequencing?P<0.001?.Conclusion:?1?Abundant exosomes were secreted by CAL-27 and HOEC,and exosomes can be effectively isolated by differential ultracentrifugation.?2?Exosomal lncRNAs were differentially expressed between CAL-27 and HOEC.?3?Based on bioinformatics analysis,it was speculated that three lncRNAs?NR₀26892.1,NR₁26435.1 and NR-036586?might have the potential for detecting early oral squamous cell carcinoma,and provide the foundation for the establishment of early non-invasive diagnostic methods.