| Background:Oral squamous cell carcinoma（OSCC）is the most common tumor in oral and maxillofacial region.Although the diagnosis and treatment methods have been greatly improved at this stage,the 5-year survival rate of patients is still about 50%,and the recurrence rate is 20%.Therefore,early detection,early diagnosis and early treatment are of great significance for OSCC patients.At present,the clinical diagnosis of OSCC is mainly routine examination and some auxiliary examinations.With the development of molecular biology technology,the discovery of new tumor markers has high clinical value for the diagnosis and treatment of OSCC.Transcriptome sequencing has the characteristics of fast,comprehensive and high sensitivity.It can obtain the sequence information and expression information of almost all transcripts of specific cells or tissues in a certain state.At present,transcriptome sequencing technology has been widely used in the screening and analysis of a variety of human cancer markers.However,there is no specific tumor biomarker for early de-tection of OSCC.Objective:1.Using transcriptome sequencing technology to screen the differentially ex-pressed genes of OSCC to find new early diagnostic markers for OSCC screening.2.To study the function and potential mechanism of MCM5 in OSCC cell line（SCC-15）.Methods:1.The cancer tissues and paracancerous tissues of OSCC patients were sequenced by transcriptome sequencing.Differential expression genes（DEGs）were screened with P<0.05 and|fold change（FC）|>2.Using gene ontology（GO）enrichment analysis and Kyoto Encyclopedia of genes and genomes（KEGG）approach to analyze the bio-logical information of DEGs,identify the way of significant enrichment of DEGs,search for key genes and pathways,and carry out RT-qPCR experiments on seven of the DEGs（AQP5,LYZ,STATH,Cdc7,MCM5,E2F1,CCND2）to further verify the transcriptome sequencing results.2.On the basis of transcriptome sequencing,in order to further explore the cell function of MCM5,the MCM5 was knocked down by transient transfection in OSCC cell line SCC-15,and the effects of knockdown on SCC-15 cell proliferation,clonal formation,cell migration and cell cycle were observed by CCK-8,clonal formation,cell scratch test,flow cytometry,RT-qPCR and Western blot.Furthermore,the role and potential mechanism of MCM5 in SCC-15 cells were clarified.Results:1.Compared with paracancerous tissue,there are 832 DEGs in OSCC,416 of which are up-regulated and 416 are down regulated.DAVID comprehensive analysis tool was used to carry out GO annotation on the differentially expressed target genes（P<0.05）.The most commonly predicted GO terms of DEGs are“single organization process”、“binding”、“extracellular part”、“extracellular”and“organelle”,while the significant pathways in KEGG pathway include“pathways in cancer”、“cytokine-cytokine receptor interaction”、“cell cycle”、“chemokine signaling pathway”and“microRNAs in cancer”.RT-qPCR showed that AQP5,LYZ and STATH were signifi-cantly down-regulated at the transcription level and correlated with"salivary secretion",while Cdc7 and MCM5 were significantly up-regulated and correlated with"cell cycle",while the expression of E2F1 and CCND2 was not statistically significant.2.Knockdown of MCM5 could significantly inhibit the proliferation and clonal formation of SCC-15 cells,but had no significant effect on cell migration.In addition,silencing MCM5 could induce G2/M phase arrest in SCC-15 cells.CyclinE was sig-nificantly down regulated in transcription and protein levels（P<0.05）.P21 was signif-icantly up regulated in transcription and protein levels（P<0.05）.Conclusion:1.There are 832 significantly different genes in OSCC,416 are up-regulated,416 are down regulated.These genes are mainly enriched in“cancer pathway”、“cytokine-cytokine receptor interaction”、“cell cycle”、“chemokine signaling pathway”and“mi-croRNAs in cancer”.The expression of Cdc7 and MCM5 in OSCC was significantly up-regulated,and the expression of AQP5,STATH and LYZ in OSCC was significantly down regulated,which is expected to be a potential diagnostic marker or drug treatment target of OSCC.2.Knockdown of MCM5 in SCC-15 can inhibit cell proliferation by inducing G2/M phase arrest,in which cyclin E and p21 may play an important role. |
