The Role Of TROP2 In Nead And Neck Tumors And Its Regulatory Mechanism

Objective:Head and Neck Squamous Cell Carcinoma(HNSCC)is one of the most common malignant tumors in the world.Although the treatment for head and neck squamous cell carcinoma has improved significantly in recent years,the recurrence and mortality rate remain high,underlining the importance to better understand the molecular basis of tumor characteristics.Therefore,investigating the pathogenesis of HNSCC and trying to find molecular targets that can accurately predict its prognosis are of great intersting.TROP2(trophoblast cell surface antigen 2),has been clearly demonstrated to be overexpressed in a variety of epithelial-derived human malignancies,including laryngeal and nasopharyngeal carcinomas.Interestingly,the high expression of TROP2 has been noted to be associated with cancer progression and poor prognosis.However,the underlying mechanisms of TROP2 in carcinogenesis,particularly its upstream regulatory factors,and the downstream signal transduction pathways remain largely unknown.Circular RNAs(circRNAs)and microRNAs(miRNAs)have attracted much attention in recent years.As key regulators of various diseases,they are also involved in tumorigenesis and invasion.However,the function of most circRNAs and miRNAs are still need to be elucidated.In the current study,we will explore the expression level of TROP2 in HNSCC and correlate their expression with patient clinical outcome.The correlations of dysregulated TROP2 with tumor cell proliferation,invasion and metastasis will be investigated by siRNA silencing TROP2 in vitro and in vivo.Finally,the regulatory effects of circRNA,miRNA on TROP2 targeted downstream signal transduction pathways will be evaluated.Method:The expression levels of TROP2 in 42 laryngeal and hypopharyngeal carcinoma patient samples were detected by immunohistochemical methods.Kaplan-Meier survival curve,Log-rank test,univariate and multivariate Cox regression analysis models were used to analyze TROP2 expression with patient clinical outcome.The correlations of TROP2 expression with patient overall survival and disease-free survival were evaluated,and adjusted with clinical pathological data.By qRT-PCR,two HNSCC cell lines: FaDu(human hypopharyngeal squamous cancer)and SCC-9(human tongue squamous cell carcinoma)were selected for silencing TROP2 studies.Silencing TROP2 cell lines(Fadu and SCC-9)were constructed by transfecting siRNA with LipofectAMINE;Effects of TROP2 on cell proliferation,migration and invasion were evaluated by qRT-PCR,western blot,MTS assay,Transwell chamber and Matrigel invasion chamber assays in vitro.Tumor formation experiments in SCID mice were performed after Fadu cells transfected with siRNA to TROP2.Tumor growth were monitored and Ki-67 expression levels in the tumor-bearing mouse was detected by immunohistochemistry.By utilized five gene target-prediction softwares,the potential miRNAs that target TROP2 were selected and their biological effects were analyzed by KEGG and GO pathways.By qRT-PCR,Western blot and miRNA mimics transfection methods,a potential miRNA targeted to TROP2 was selected(miR-488-3p).A TROP2 3’UTR binding site and mutation binding site recombinant plasmids were constructed by vector cloning technology.A double luciferase gene report assay was used to verify the direct targeting of miRNA(miR-488-3p)to TROP2.The PI3K/AKT and MAPK pathway related proteins e.g.AKT,ERK1/2,AP-1 were detected by Western blot after silencing of TROP2.Finally,CircRNA database and miRNA target gene database were used to select circRNA(circ-0000495)that can specifically bind to miR-488-3p.The stability of circRNA was verified by qRT-PCR after cells were treated by RNase;the direct targeting effect of circRNA on miR-488-3p was verified by a double luciferase gene report experiment.Result:TROP2 overexpresses in HNSCC and associates with tumor recurrence.TROP2 was highly expressed in HNSCC tissues,and the expression levels in therelapse group were significantly higher than that in the non-relapse group(p =0.05);Kaplan-Meier survival curve showed that patients with higher TROP2 expression level associated with worse overall survival(OS)(p= 0.027)as well as with worse disease-free survival(DFS)(p = 0.015),indicating that high expression of TROP2 is closely related to recurrence,worse overall survival and worse disease-free survival.The univariate and multivariate Cox regression analysis suggested that TROP2 overexpression was an independent prognostic factor for both OS and DFS(p=0.031 and p=0.018,respectively),suggesting predictive and prognostic roles of TROP2 in the development of HNSCC.Silencing TROP2 inhibits cell proliferation,migration,and invasion in vitro and suppresses tumor growth in vivo.TROP2 was significantly upregulated in three HNSCC cell lines.Fadu and SCC-9 were selected for the in vitro silencing of TROP2 studies.Through the siRNA interference technology,specific anti-TROP2 siRNA cells were successfully constructed by a lipofectamine transfection method.siRNA specifically against TROP2 was then transfected into Fadu and SCC-9 cells.The results showed that at 48 hours after transfection,the expression of TROP2 mRNA in both cell lines was significantly reduced,by 38% and 24%,respectively(p<0.05),Those reductions were further confirmed by western blot analysis.Depleted TROP2 led to significant decreases in cell viability for both cell lines,particularly at 48 hours post-transfection(p<0.05);knockdown of TROP2 resulted in a significant reduction in cell migration(48%)and invasion(55%)of Fadu cells compared to that of the scramble control.A similar reduction in cell migration and invasion was also observed in SCC-9 cells.Furthermore,TROP2 knockdown cells showed significantly delayed tumor growth compared with mice injected with control(p=0.05),and the difference is significant over time(p<0.05).In addition,the expression of tumor cell proliferation marker Ki-67 in mouse transplanted tumors was significantly lower than that in the control group after silencing TROP2(p<0.01),indicating that silencing TROP2 inhibits tumor growth.miR-488-3p directly target TROP2 and TROP2 induces tumor growth by regulating PI3 K / AKT and MAPK signaling pathways.Three tumor suppressor miRNAs were selected as potentially targets to TROP2.The baseline expression of each miRNAs assays demonstrated that miR-488-3p and miR-15a-3p were indeedunder-expressed in both Fadu and SCC-9 cells,Mimics miR-488-3p resulted in down-regulation of TROP2 in both Fadu and SCC-9 cells,indicating that miR-488-3p is highly probable to target TROP2 in HNSCC cells.Several reporter vectors carrying the predicted miR-488-3p and the 3’-UTR of TROP2 binding site(s)or 3’-UTR of TROP2 mutation binding sites,which downstream of a firefly luciferase gene in the pMIR-Report vector were successfully constructed.Either Fadu or SCC-9 cells were co-transfected with mimic miR-488-3p(or a scramble control)and pmiR-TROP2-3’UTR(or a mutated pmiR-TROP2-3’ UTR).The luciferase reporter that contained the TROP2 3’ UTR was significantly decreased by mimic miR-488-3p,whereas the mutated reporter was not affected,demonstrating that miR-488-3p are direct target TROP2.In addition,the levels of AKT,p44 / 42(ERK1 / 2),and AP-1 were reduced after silencing TROP2,while the levels of AKT and ERK1 / 2 remained unchanged,indicating that TROP2 regulates HNSCC development through PI3K/AKT and MAPK signal transduction pathways.Circ-0000495 interactes TROP2 by sponging miR-488-3p and upregulates TROP2 expression.CircRNA database and miRNA target gene database predicted that circ-0000495 has potential targeting effect on miR-488-3p;circ-0000495 is significantly overexpressed in three head and neck squamous carcinoma cell lines(Fadu,SCC22,and SCC-9);After RNase R treatment,the linear RNAs were significantly reduced,but the circular RNAs were more stable and resisted to RNase R;When miR-488-3p mimics was transfected into Fadu cells,it was found that miR-488-3p increased significantly and TROP2 expression decreased significantly(p<0.05),while the expression of circ-0000495 did not change.A dual‐luciferase reporter assay demonstrated decresesing luciferase activity in circ-0000495 after transfected with mimics of miR-488-3p(wild type),indicating that circ-0000495 could directly bind to miR-488-3p.Conclusion:This study revealed that the high expression of TROP2 is closely related to the recurrence,worse overall survival and disease-free survival and is an independentprognostic factor for HNSCC patients.In vitro and in vivo experiments have shown that TROP2 can significantly regulate the tumor cell proliferation,invasion,metastasis and affect tumor formation in vivo.Based on our best knowledge,this is the first study to explore a novel underlying machenism,by which up-regulated circ-0000495 in cancer cells reduces miR-488-3p expression by sponge-like adsorption of specific targets.The down-regulated miR-488-3p then releases the inhibitory effect on TROP2,which causes the up-regulated expression of TROP2 in the tumor cells.Furthermore,the up-regulated TROP2 regulates the occurrence,development and metastasis of HNSCC through PI3 K / AKT and MAPK signaling pathways.Thus,a comprehensive understanding of the circ-0000495-miR-488-3pTROP2 regulatory pathways will open up new strategy for the treatment of head and neck squamous cell carcinoma.