| Objectives: Oral squamous cell carcinoma(OSCC)is the most frequent malignant tumour in the head and neck region.Extracellular Vesicles(EVs)are secreted by tumor cells and enter into circulation.Tumour derived EVs carry biologically active proteins and nucleic acids from the parent cells.miRNAs in EVs are promising to be biomarkers for diagnostic and prognosis.We aim to find whether miRNAs in salivary extracellular vesicles could be served as biomarkers in diagnosis and postoperative monitoring of OSCC.Materials and Methods: 17 patients diagnosed OSCC were consented and enrolled in this study,and 11 age matched healthy volunteers without lesion and inflammation in oral cavity as control were collected.EVs were isolated from salivary samples by using the gradual centrifugation and Total Exosome Isolation Kit.Transmission electron microscopy(TEM)and western blot were used to confirm isolated EVs.We measured the protein concentration of EVs with Bicinchoninic Acid assay(Beyotime biotechnology,China).Total RNA were extracted from salivary EVs by using Trizol agent,and measured by Nano Drop 1000 spectrometer.Differential relative expression of miRNA was detected by quantitative reverse transcriptase polymerase chain reaction(q PCR).The values of miRNA relative expression were normalized by cel-miR-39 and calculated using 2-?Ct method.Samples derived from blood and saliva were collected from 6 patients with OSCC before surgical operation.q PCR assay was used to determine the expression of miR-21-5p in serum and saliva-derived EVs from OSCC patients.Differential relative expression of miRNA compared between 11 healthy controls and 17 OSCC patients.Receiver-operating characteristics(ROC)curves were constructed to evaluate the power of two up-regulated miRNAs as non-invasive biomarkers for diagnostic accuracy.The analysis of miRNA relative expression between pre-operation and post-operation were detected in 7 patients in the same way.Results: TEM analysis demonstrated the characteristic shape(round shaped cystic vesicle)and diameter(~100nm)of EVs,and western blot analyses were verified the expression of typical marker of EVs.The protein concentrations of OSCC patient-derived EVs were significantly increased compared to those from healthy controls.Total RNA in salivary EVs had no significant difference between healthy controls and OSCC patients.It was reported that miR-21-5p,miR-10b-5p,miR-486-5p,miR-412-3p and miR-512-3p in EVs have potential to be non-invasive OSCC biomarkers.The relative expression of miR-21-5p in EVs from blood was higher than that in saliva samples,and the trend was basically the same.The value of relative expression levels of miR-21-5p in EVs from blood and saliva were plotted as scatter plots,and R2 of trend line was calculated.R2=0.6951.The Pearson correlation coefficient was calculated by SPSS 20.0 software,and the correlation coefficient was 0.834,p<0.05.The q PCR results showed a significant upregulated relative expression of miR-21-5p and miR-10b-5p in OSCC patients with respect to healthy controls(p<0.05).MiR-486-5p,miR-412-3p,miR-512-3p in EVs showed no significant difference between OSCC patients and healthy controls.The area under ROC curve(95%confidence interval,p)of EVs miR-21-5p and miR-10b-5p for distinguishing the OSCC patients with health control were 0.936(0.849~1.023,p=0.0001)and 0.807(0.646~0.967,p=0.0069).The analysis of EV miRNA relative expression between pre-and post-operation were measured.MiR-21-5p of EVs in 6 OSCC patients were down-regulated one month significantly after operation.However,miR-21-5p in EV showed upexpression after one month and downexpression after three months in one patient.There no consistent tendency for the changes of miR-512-3p,miR-486-5p,miR-412-3p,and miR-10b-5p in pre-and post-operation samples.Conclusions: 1.MiR-21-5p and miR-10b-5p in salivary EVs might be potential biomarkers in OSCC diagnosis.2.MiR-21-5p in salivary EVs might be a postoperative monitoring biomarker in OSCC patients. |
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