Screening Of MiRNAs Differentially Expressed In Serum Exosomes And The Effect Of MiR-941 On Cell Function In Laryngeal Squamous Cell Carcinoma

Background:Laryngeal squamous cell carcinoma(LSCC)is one of the most commonly seen malignant head and neck tumors,with a high incidence in northern China,including Shanxi Province.After decades of efforts,the treatment of LSCC has made great progress,but the 5-year survival rate has not increased.In LSCC,early detection is very difficult given the anatomical location of the larynx and the tendency of a tumor to be more hidden.LSCC patients usually have no overt clinical symptoms at the early stages and cannot be diagnosed in time,which greatly affects the treatment effect and prognosis of patients.At present,LSCC is predominantly diagnosed via endoscopy and pathological examination,and the invasive detection procedure affects the popularity and dynamic monitoring of this examination.Therefore,it is urgent to develop a rapid,minimally invasive and highly sensitive diagnostic method and reliable biomarkers for clinically diagnosing LSCC.Exosomes are membranous vesicles with a diameter of 30–150 nm that contain proteins,lipids,and nucleic acids derived from their parental cells.They are released into circulating blood,which is of clinical interest due to being minimally invasive and easily repeatable.MicroRNAs(miRNAs)are ubiquitous non-coding RNA molecules in eukaryotes,which are usually abnormally expressed in tumors.As tumor suppressors or oncogenes,miRNAs play an important regulatory role in the occurrence and development of tumors.Circulating exosomal miRNAs have been reported in most tumors as potential biomarkers for tumor screening,disease course monitoring,therapeutic efficacy observation and prognosis evaluation.In the case of circulating exosomal miRNAs,they are protected against nuclease digestion and are less susceptible to the influence of the extracellular environment;thus,they are able to provide more reliable insights.Moreover,since exosomal miRNA composition and abundance is determined by the state of the parental cell,these miRNAs can better reflect the functional status of the organ of origin.Additionally,some miRNAs are enriched in exosomes,which improves the detection of low abundance miRNAs.In laryngeal cancer,there are few related studies,and we have not retrieved reports of miRNA expression profiles in peripheral blood exosomes.The present study was designed to screen serum exosomal miRNA profiles based on the high-throughput sequencing and evaluate meaningful miRNA.Then,the value of miRNA for LSCC diagnosis was further assessed,and the biological function of miRNA in the malignant process of LSCC was explored.Based on these analyses,this study would provide references for the discovery of diagnostic markers and new therapeutic targets for LSCC.Objective:The aim of this study was to establish the miRNA profiles of the serum exosome in LSCC,screen out differentially expressed miRNAs,evaluate the monitoring value of them in LSCC diagnosis,and finally explore the biological function of the key miRNA in the malignant progress of LSCC.Methods:1.Evaluation of isolation methods of serum exosomesSerum samples from 3 LSCC patients were utilized and four isolation methods,including Ultracentrifugation(UC),ExoQuick Solution(EQ),8% Polyethylene Glycol(PEG1)and 8% PEG + 5% PEG(PEG2),were used to isolate serum exosomes in these samples.The advantages and disadvantages of the four methods were evaluated from the morphology,particle size,concentration,protein markers and miRNA profiles of the extracted exosomes.(1)Transmission electron microscopy(TEM)was used to observe the shape and particle size of exosomes.Nanoparticle Tracking Analysis(NTA)was used to detect the particle size and concentration.Western Blot(WB)was used to detect the expression of exosomal protein markers.(2)Serum exosomal RNA was extracted and RNA-seq was performed to examine miRNA profiles of the serum exosomes extracted by these four methods.Then,similarities between the four miRNA profiles were examined.2.Screening of miRNAs differentially expressed in serum exosomes in LSCC patientsSerum exosomes were extracted from 6 LSCC patients and 6 healthy controls(HCs)using the EQ method.(1)Exosomes were extracted from the serum of the same volume,then the expression of exosome-related proteins was analyzed by Exo-check antibody array,and the secretion levels of serum exosomes between the two groups were compared by TEM and NTA measurements.(2)Serum exosomal RNA of these 12 samples was extracted and RNA sequencing(RNA-seq)was performed.The differentially expressed miRNAs between the two groups were identified.Target genes of these differentially expressed miRNAs were predicted using software miRanda,PITA and RNAhybrid,and their statistical enrichment was examined within GO and KEGG pathways.3.Identification of endogenous references in serum exosomes of LSCC patients and HCs(1)Candidate reference genes were selected based on our RNA-seq data and the literature.MiRNAs with stable expression based on RNA-seq data,having a small coefficient of variation(CV)value and having a moderate expression level were evaluated as candidate reference miRNAs.First,miRNAs with significantly different expression levels between the two groups(P < 0.05),or if a mean TPM < 1 was obtained,were excluded.Second,mean TPMs were compared between the two groups and miRNAs with a ratio < 0.75 or > 1.3 were excluded.Finally,CVs were calculated and ranked from smallest to largest,and the top eight miRNAs with a TPM > 50 were selected as candidate internal reference genes.In addition,two previously published endogenous controls,U6 and miR-16-5p,were selected as candidate internal reference genes.(2)These candidate references were then further evaluated using qRT-PCR in another independent samples.Candidate reference expression stability was then statistically analyzed using several statistical algorithms,including BestKeeper,NormFinder,geNorm,delt Ct method,and RefFinder.4.Validation of deferentially expressed miRNAs and ROC curve analysis(1)9 miRNAs that were upregulated and highly expressed(TPM ?50)in serum exosomes of LSCC patients were selected for qRT-PCR verification in another independent set that included 50 LSCC and 25 HC individuals.(2)ROC curve was used to analyze the diagnostic efficacy of miR-941 and miR-27a-5p.(3)The expression of miR-941 in the RNA-seq data of LSCC tissues was analyzed.(4)The correlation between serum exosomal miR-941 and pathological parameters of LSCC patients was analyzed.(5)The expression of miR-941 in head and neck squamous cell carcinoma(HNSCC)and various other tumors in the YM500v3 database was analyzed.5.Effect of miR-941 on the function of LSCC cell lines(1)The intracellular and extracellular miR-941 levels were detected in two LSCC cell lines,FD-LSC-1 and Tu 686,and a normal oral keratinocyte line,HOK,by qRT-PCR.(2)FD-LSC-1 and Tu 686 cells were transfected by Lipofectamine 3000 with miR-941 mimics or inhibitor,with the transfection efficiency verified using qRT-PCR.(3)CCK8 assay and Transwell invasion assay were used to analyze the changes of proliferation and invasion ability of FD-LSC-1 and Tu 686 cells transfected with miR-941 mimics or inhibitor.Results:1.Evaluation of isolation methods of serum exosomesTEM measurements showed that all the methods enriched exosomes with a typical cup-shaped or Concave disc-shaped morphology.NTA measurements showed that for the EQ method,the vesicles harvested at the peak level generally had a modal size of less than 150 nm,while the others were above 150 nm.Furthermore,the UC and PEG2 methods displayed multiple particle size peaks,with some larger particles > 300 nm also noted.Additionally,the EQ method was shown to provide the highest exosome concentration of the methods,followed by PEG1,PEG2 and UC.Exosomal markers,CD63,CD81 and TSG101,were examined via Western blot and were found to be enriched for all 4 methods.However,while the same amount of total protein was loaded into the gel,expression levels for these markers were higher in samples extracted with the EQ and PEG1 methods relative to the UC and PEG2 methods.(2)Similarities between the four miRNA profiles identified by RNA-seq were examined and a Pearson correlation coefficient above 0.90 was obtained,with a total of 571(UC),1094(EQ),1079(PEG1),and 896(PEG2)miRNAs detected.2.Screening of miRNAs differentially expressed in serum exosomes in LSCC patients(1)Exo-check antibody array showed that for both groups CD81,CD63,ALIX,FLOT1,ICAM1,EpCam,ANXA5 and TSG101 were expressed,but GM130,a cellular contamination marker,was not expressed.Next,serum exosome levels were examined using TEM and NTA.The TEM analysis showed that LSCC samples have higher exosome levels than HC samples when comparing equal serum volumes.NTA showed that the relative exosome concentration in LSCC serum was significantly higher than the levels in HCs(P < 0.05).(2)Across samples,Agilent2100 and microelectrophoresis showed that the RNAs were predominantly small RNA species,with no detectable 18 S or 28 S ribosomal RNA.(3)RNA-seq identified a total of 1608 mature miRNAs from LSCC patients and HCs,including 1496 known miRNAs and 112 new miRNAs predicted by softwares.With the differentially expressed cut-off threshold set to P < 0.05 and a | log2 fold change | of ? 0.5,the expression levels of 75 miRNAs were significantly different between the two groups.The results identified 34 upregulated and 41 downregulated miRNAs in LSCC.Target genes for the differentially expressed miRNAs were predicted.GO analysis revealed that the predicted target genes were mainly involved in biological pathways or functions,such as single organism process,localization,cellular component organization or biogenesis,molecular function and binding.Pathway analysis revealed that they were mainly involved in tumor related pathways,including MAPK,Ras,and FAK signaling pathways.3.Identification of endogenous references in serum exosomes of LSCC patients and HCsA total of 8 miRNAs from the RNA-seq data were selected as candidate internal reference miRNAs,including miR-30a-5p,miR-532-5p,miR-181a-5p,miR-425-5p,miR-363-3p,miR-424-3p,miR-181b-5p and miR-181a-2-3p,respectively.These 10 candidate internal reference genes,including U6 and miR-16-5p,were detected by qRT-PCR,and their expression stability was evaluated by five algorithms.Finally,miR-30a-5p,miR-532-5p and U6 were selected as the best combination of internal reference genes.4.Validation of deferentially expressed miRNAs and ROC curve analysis(1)miR-941(P < 0.001)and miR-27a-5p(P < 0.01)were the serum exosomal miRNAs with statistically significant differences in expression levels between the LSCC patients and the HCs,and their expression trends were consistent with the RNA-seq data.There was no statistical difference in the expression levels of the remaining 7 miRNAs between the two groups.(2)The area under the ROC curve(AUC)value of miR-941 for LSCC diagnosis was 0.797(P < 0.001),with a 95% confidence interval = 0.676–0.918.miR-27a-5p had an AUC = 0.672,which had low diagnostic efficiency and had not been further studied.Taking the ?Ct value of serum exosomal miR-941 as 5.139 as the diagnosis point,the sensitivity and specificity for the prediction of LSCC were 82% and 72%,respectively,and the Yoden index was the highest,which was 0.54;when the cut-off value of ?Ct was 5.381,the diagnostic sensitivity and specificity were 92% and 60%,respectively,and the Yoden index was 0.52.(3)To further examine the expression level of miR-941 in LSCC tissues,miRNAs profiles for 57 pairs of LSCC tissues and corresponding normal tissues that we previously sequenced were examined and miR-941 was found to be 2.1-fold upregulated in LSCC tissues(P < 0.001).(4)when examining miR-941 expression within the Ym500v3 database,in 456 HNSCC tumor tissues and 43 cases of normal tissues,miR-941 was found to be 2.38-fold upregulated in HNSCC tumor tissues(P = 0.0014).Similarly,miR-941 was shown to be significantly highly expressed in most tumor tissues relative to normal tissues,including lung squamous cell carcinoma,esophageal cancer,cervical squamous cell carcinoma,skin cutaneous melanoma,liver hepatic carcinoma,and breast carcinoma.(5)The expression levels of serum exosomal miR-941 in LSCC patients with supraglottic location,stage T3+T4,cervical lymph node metastasis,stage III+?,and poor differentiation were higher than that in patients with glottis location,stage T1+T2,no cervical lymph node metastasis,and medium/high differentiation,respectively(P > 0.05).Although there was no significant difference,it was found that the miR-941 expression level was related to the disease progression and malignant degree of LSCC.Furthermore,data from public database Kaplan-Meier plotter showed that upregulated miR-941 was significantly correlated a poor outcome for HNSCC patients(P = 0.0016).5.Effect of miR-941 on the function of LSCC cell lines(1)The expression levels of miR-941 in FD-LSC-1 and Tu 686 cells were 5.51(P < 0.01)and 2.11(P < 0.01)times that in HOK cells,respectively.The expression levels of miR-941 in exosomes of FD-LSC-1 and Tu 686 cells were 2.20(P < 0.05)and 5.46(P < 0.01)times that in HOK cell exosomes,respectively.(2)In CCK8 proliferation assay,the cell growth rate of miR-941 mimics group was significantly faster than that of mimics NC group after overexpression of miR-941(P < 0.01),indicating that the proliferative ability of FD-LSC-1 and Tu 686 cells was enhanced.On the contrary,the proliferative ability of cells transfected with miR-941 inhibitor decreased.(3)In Transwell invasion assay,the number of transferred cells in miR-941 mimics group was significantly more than that in mimics NC group after overexpression of miR-941(P < 0.01),indicating that the invasive ability of FD-LSC-1 and Tu 686 cells was enhanced.On the contrary,the invasive ability of cells transfected with miR-941 inhibitor decreased.Conclusion:1.The ExoQuick method is a high-yield,high-efficiency and time-saving method for extracting serum exosomes.2.LSCC patients had higher serum exosome levels than HCs.The expression levels of serum exosomal miRNAs were significantly different between the two groups.Target genes for the differentially expressed miRNAs were mainly involved in tumor related pathways.3.miR-30a-5p,miR-532-5p and U6 were the best combination of internal reference genes for serum exosomes in LSCC patients and HCs.4.Serum exosomal miR-941 may serve as a promising biomarker for diagnosing LSCC,and also has the potential as a therapeutic target for LSCC.5.miR-941 plays a role as an oncogene in the malignant progression of LSCC.