| Background and objectivesOral cancer is one of the most common malignant tumors of the head and neck,and squamous cell carcinoma(SCC)is the most common histological type.About 90%of cases in oral cancer are classified as oral squamous cell carcinoma(OSCC).Smoking and alcohol consumption are its main causative factors,and the incidence is increasing overall in the world.Although early diagnosis is relatively easy,most patients are already in middle or late stage when diagnosis.Surgery is the treatment of choice,although it is able to control tumor development,the rich vasculature and complex anatomy of the oral and maxillofacial region make about 1/3 of patients still experience recurrence and distant metastasis after conventional surgery or radiotherapy and require retreatment.Once OSCC spread and metastasized,or invaded important anatomical sites,patients in middle and late stages would lose the opportunity of surgical treatment.Thus,palliative treatment becomes a common treatment option,but radiotherapy has a large impact,chemotherapy is easily resistant,its prognosis is poor,and the mortality rate is up to 50%.However,the mechanism of OSCC is not fully understood,therefore,further research of its pathogenesis is urgently needed for the treatment of OSCC.The tumor microenvironment(TME)plays an important role in cancer cell initiation,progression,and metastasis,therefore modulating the biochemical components of TME is known as a novel therapeutic strategy.Recent studies have shown that adenosine triphosphate(ATP)in TME is significant during tumor progression.ATP is one of the major biochemical components of TME and has a clear role in promoting tumor proliferation,invasion,and migration.In recent years,ATP has gradually become a research hotspot as an important tumor extracellular signaling molecule.Current studies on ATP in cancer are mainly focused on a few tumors,such as prostate,breast,rectal,bladder,and so on.Whether ATP is universal as a signaling molecule needs further investigation,and its molecular regulatory role in OSCC remains to be studied,therefore,this study aims to explore the regulatory effect of ATP on the biological behavior of OSCC cells,and to elucidate the possible mechanism,hoping to provide new ideas for OSCC tumor therapy.Materials and MethodsPart 1 Evaluation of the effects of ATP on the biological behavior of OSCC cells CAL27 and SCC15 cell lines were resuspended.0,50,100,200 μM concentrations of ATP were used to treat CAL27 and SCC15 cells.CCK8 assay,scratch assay,Transwell assay,and clonogenic assay were used to determine the effects of extracellular ATP on the biological behaviors of proliferation,invasion,and migration of OSCC cells.The expression of the related invasion and migration transcription factors Snail,MMP2,MMP9,E-cadherin and Vimentin in the cells was measured by quantitative real time polymerase chain reaction(qRT-PCR).Part 2 Elucidating the relationship between ATP promoted OSCC cell invasion and migration and the P2Y2-Src-EGFR axis2.1 P2Y2,SRC and EGFR expression in OSCC cancer tissuesThe TCGA-HNSC database was downloaded,and 520 OSCC samples and 44 control samples were screened for bioinformatics analysis to elucidate the expression differences of P2Y2,Src and EGFR between OSCC tissues and control samples.The correlation of P2Y2 with Src and EGFR was analyzed from the TIMER database.Protein interaction analysis of P2Y2 was performed using STRING database.Western blot and qRT-PCR experiments were performed to examine the expression differences of P2Y2,Src and EGFR in OSCC tissues.2.2 P2Y2,SRC and EGFR expression in OSCC cellsWestern blot and qRT-PCR experiments were performed to detect the differences of P2Y2,Src and EGFR expression in ATP treated OSCC cells.Part 3 Probing the molecules by which ATP regulates OSCC cell invasion andmigration through the P2Y2-Src-EGFR axis 3.1 P2Y2 is involved in the effects of ATP on OSCC cell invasion and migration P2Y2 expression in OSCC cells was knocked down by siRNA,Western blot and qRTPCR were employed to detect the transfection efficiency,and to screen the optimal siRNA sequence.CCK8 assay,scratch assay,Transwell assay and clonogenic assay were used to examine the effects of P2Y2 knockdown on OSCC cell proliferation,migration and invasion.In vivo nude mice tumorigenesis assay was performed to examine the effect of P2Y2 knockdown on OSCC transplanted tumor growth.The expression changes of P2Y2 knockdown on cell migration and migration related factors gene Snail,MMP2,MMP9,E-cadherin and Vimentin in OSCC were determined by qRT-PCR.3.2 ATP promotes OSCC cell invasion and migration through the P2Y2-Src-EGFR axas.The effects of P2Y2 knockdown on the expression of Src and EGFR induced by ATP in OSCC cells were determined by Western blot.The effect of Src inhibitor(Dasatinib)on EGFR expression in OSCC cells induced by ATP was detected by Western blot.3.3 PI3K/AKT signaling is involved in ATP regulated OSCC cell invasion and migrationBased on transcriptome data of oral squamous cell carcinoma in TCGA database,GSEA enrichment analysis was performed to explore the correlation between P2Y2 and PI3K/AKT signaling pathway.Western blot was used to detect the effects of PI3K and AKT expression after ATP stimulation in OSCC cells.The effects of P2Y2 knockdown,PI3K and AKT expression in cells were determined by Western blot.Western blot assay was used to analyze the expression of PI3K and AKT in OSCC cells induced by ATP after inhibition of Src and EGFR expression.Clonogenic assay,Transwell assay and scratch assay were performed to elucidate the regulatory effects of ATP on OSCC cell invasion and migration after PI3K and AKT inhibition.ResultsPart 1 ATP promotes OSCC cell proliferation,invasion and migrationThe CCK8 assay results indicated that OSCC cell viability was the strongest when the ATP concentration was 100 μM,and the ATP concentration of 100 μM was chosen as the concentration for subsequent experiments.Cell scratch assay,Transwell assay,and clonogenic assay showed that ATP promoted OSCC cell invasion and migration.qRTPCR assays identified increased expression of invasion and migration related factors Snail,MMP2,Vimentin and MMP9 and decreased expression of E-cadherin in OSCC cells.Part 2 ATP promotes OSCC cell invasion and migration in close association with the P2Y2-Src-EGFR axis2.1 P2Y2,Src and EGFR expression in OSCC cancer tissuesBioinformatics results showed that Src and EGFR were highly expressed in OSCC tissues compared with paracancerous tissues,and the expression of P2Y2 was highly correlated with that of Src and EGFR.Protein interaction analysis indicated that P2Y2,Src and EGFR proteins also interacted with each other.The results of Western blot and qRT-PCR experiments demonstrated that P2Y2,Src and EGFR were highly expressed in OSCC tissues and cells compared with oral healthy tissues and cells.2.2 P2Y2,Src and EGFR expression in OSCC cellsWestern blot and qRT-PCR experiments showed that P2 Y2,Src and EGFR were highly expressed in ATP treated OSCC cells compared with normal oral mucosa cells.The elevation of P2Y2,Src and EGFR was dose-and time-dependent.Part 3 ATP promotes OSCC cell invasion and migration by activating the PI3K/AKT pathway through the P2Y2-Src-EGFR axis3.1 P2Y2 is involved in the effects of ATP on OSCC cell invasion and migration In vitro scratch assay,Trans well assay and clonogenic assay showed that P2Y2 knockdown significantly inhibited OSCC cell proliferation,invasion and migration.The results of in vivo tumorigenesis experiments in nude mice indicated that P2Y2 knockdown could significantly inhibit OSCC transplanted tumor growth.qRT-PCR experiments revealed that P2Y2 knockdown resulted in the suppression of invasion and migration related factors Snail,MMP2,MMP9,and E-cadherin expression in OSCC cells.3.2 ATP promotes OSCC cell invasion and migration through the P2Y2-Src-EGFR axisWestern blot showed that knockdown of P2Y2 attenuated ATP induced Src and EGFR expression in OSCC cells compared with the control group.Western blot demonstrated that the Src inhibitor(Dasatinib)decreased the expression of EGFR compared with the control group.3.3 PI3K/AKT signaling is involved in ATP regulated OSCC cell invasion and migrationBioinformatics results showed that the PI3K/AKT pathway was significantly activated in OSCC with high P2Y2 expression.Western blot demonstrated that ATP stimulated elevated PI3K and AKT expression in OSCC cells.P2Y2 knockdown led to a marked reduction in the expression of p-PI3K and p-AKT.Inhibition of Src and EGFR expression resulted in significantly reduced expression of p-PI3K and p-AKT.Clonogenic assays,Trans well assays,and scratch assays revealed that the use of PI3K and AKT inhibitors significantly inhibited ATP induced proliferation,invasion,and migration of OSCC cells.Conclusion1.P2Y2 activation by ATP promotes OSCC cell proliferation,invasion and migration.2.The mRNA and protein of Src and EGFR are highly expressed in OSCC tissues and cells,and activation of the PI3K/AKT pathway by the P2Y2-Src-EGFR axis promotes OSCC cell invasion and migration.3.As an important signaling molecule in TME,ATP plays a significant role in the proliferation,invasion,and migration of tumors.This study shows that ATP promotes OSCC cell invasion and migration by activating the PI3K/AKT signaling pathway through the P2Y2-Src-EGFR axis. |
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