| ObjectiveOur study aims to screen the serum tumor marker of oral squamous cell carcinoma(OSCC)and to explore the effects of AC007271.3 on its roles and mechanisms in OSCC in vitro.Material and Methods1.LncRNAs expression was measured by microarray analysis in three sets of OSCC and paired normal mucosal tissues.The potential IncRNAs involved in OSCC development were investigated by bioinformatics and verification experiments.2.We analyzed the expression of AC007271.3 in OSCC tissues and paired serum samples,and in OSCC cells,thus found the specific expression of AC007271.3 in OSCC.3.We determined the expression of AC007271.3,SCCA,TSGF,CEA and PD-L1 in a case-control study of 80 OSCC cases and 70 controls by qPCR and ELISA assay.Receiver operating characteristics(ROC),decision curve analysis(DCA)and combined detection of these potential biomarkers were performed to screen for OSCC biomarkers.Kaplan-Meier survival curve analysis was performed to assess overall survival(OS)time in patients.4.The localization of AC007271.3 in OSCC cells was confirmed by fluorescence in situ hybridization(FISH)assay.Subsequently,we explored the function and molecular mechanism of AC007271.3 in OSCC cells by CCK8 assay,colony formation assay,wound healing assay,matrigel invasion assay and flow cytometry analysis,respectively.5.Immunofluorescence(IF)assay and western blotting(WB)analysis were conducted to investigate the activity of the Wnt/?-catenin signaling pathway and the proteins involved in the pathway.Results1.A total of 691 1ncRNAs(433 upregulated and 258 downregulated)and 816 mRNAs(342 upregulated and 474 downregulated)were detected to be differentially expressed.Based on Gene Ontology and KGEE pathway analysis and qPCR verification,we selected four differentially expressed lncRNAs(AC007271.3,AC007182.6,LOC283481 and RP11-893F2.9)in OSCC tissues compared with normal controls(n=30,P<0.05).2.We demonstrated that the expression of AC007271.3 was significantly up-regulated in OSCC tissues and cell lines.Serum AC007271.3 expression significantly decreased after surgery(P=0.002),and a significant correlation was observed between serum and tissue AC007271.3(r=0.639,P<0.001).3.Expanded case-control study showed that the aberrant AC007271.3 levels in OSCC patients were associated with clinical stage,differentiation and lymph node metastasis.The combination of AC007271.3 and SCCA(AUC=0.902,P<0.001)showed a significant improvement compared-with SCCA or AC007271.3 alone,especially at an early stage.High expression of AC007271.3 and SCCA indicate a poor prognosis.Additionally,serum AC007271.3,SCCA and TSGF levels could discriminate OSCC from normal controls with sensitivity(77.6%,55.0%,63.3%,respectively)and specificity(84.5%,93.3%,66.7%,respectively).4.AC007271.3 was expressed in both cytoplasm and nucleus,mainly in the latter.In vitro,we showed that silencing of AC007271.3 suppressed cell growth,proliferation,migration and invasion,and induced apoptosis in OSCC cell lines,while overexpression of AC007271.3 has the opposite effects.5.Increasing expression of AC007271.3 in OSCC cells activated the Wnt/?-catenin signaling pathway activity by upregulating the expression of ?-catenin,CyclinD1,c-myc and Bcl-2,while silencing of AC007271.3 in OSCC cells has the opposite effects.ConclusionOur study have identified AC007271.3 as a tumor marker for early diagnosis and prognosis of OSCC,and AC007271.3,SCCA and TSGF could be circulating biomarkers for OSCC.AC007271.3 can promote cell growth,proliferation,migration and invasion,and affects cell apoptosis in OSCC via activation of Wnt/p-catenin signaling pathway.These findings provide evidence that AC007271.3 could be a novel biomarker and therapeutic target in OSCC. |
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