| Background and PurposeNPC is sensitive to radiotherapy;thus,this treatment strategy has greatly improved local control of lesions and the 5-year survival rate in patients with NPC.However,frequent local recurrence,metastasis,and poor prognosis remain major challenges in the clinical treatment of NPC because the mechanisms related to NPC progression are not completely understood.Recent studies have reported that multiple miRNAs are abnormally expressed in patients with NPC and contribute to the development and progression of NPC.MiR-124-3p acts as a tumor suppressor in some cancer types,Nevertheless,the potential function and mechanism of tumor suppression remain unclear.In this study,we will explore the biological function and mechanism of miR-124-3p in NPC,and explore the target genes regulated by miR-124-3p and the mechanism through which it exerts its tumor suppression effect.The elucidation of this issue will further deepen the understanding of the pathogenesis of NPC.Methods and Results1.The expression level of miR-124-3p in nasopharyngeal carcinoma cell lines was detected by qPCR.Compared with normal nasopharyngeal epithelial cell line NP69,miR-124-3p was significantly down-regulated in NPC cell lines HK1,S26,S18,6-10b,and CNE2(p<0.05),especially 6-1 OB and CNE2 cell lines.2.The expression of miR-124-3p in patients with NPC was detected by in situ hybridization.Correlation analysis of miR-124-3p expression with clinical pathological features of NPC。There was no significant difference between the expression of miR-124-3p and age,gender,T stage,N stage and clinical stage(all p>0.05).The Kaplan-Meier method was used for prognostic analysis to explore the relationship between miR-124-3p expression and prognosis.Compared with patients with lower expression of miR-124-3p,NPC patients with higher expression of miR-124-3p had significantly longer 5-year OS and 5-year RFS.Cox univariate and multivariate regression analysis showed that miR-124-3p expression,clinical stage was correlated with overall survival(p<0.05),while age and gender had no correlation with overall survival(p>0.05).Univariate analysis of recurrence-free survival showed that miR-124-3p expression,clinical stage was correlated with recurrence-free survival(p<0.05),while age,gender had no correlation with recurrence-free survival(p>0.05).However,multivariate analysis showed that clinical stage,age,and gender were associated with recurrence-free survival(p<0.05),while miR-124-3p expression was not associated with recurrence-free survival(p>0.05).3.To study the effect of miR-124-3p over expression or down expression on the expression of target genes,the expression of target gene proteins was analyzed by qPCR and Western blotting.Exploration of the effect of overexpression of target genes on the proliferation of NPC cells by Cell Counting Kit-8(CCK-8)assay and colony formation.After overexpression of miR-124-3p,cell proliferation were significantly decreased in the miR-124-3p mimic group compared with those in the mimic negative control(NC)group(p<0.05).Similarly,after overexpression of miR-124-3p,colony formation were significantly decreased in the miR-124-3p mimic group compared with those in the mimic negative control(NC)group(p<0.05).Overexpression of miR-124-3p in CNE2 and 6-1 OB cells caused a decrease in cyclin D3 and cyclin-dependent kinase 4 protein levels(CDK4).After down expression of miR-124-3p,cell proliferation were significantly increased in the miR-124-3p mimic group compared with those in the mimic negative control(NC)group(p<0.05).Similarly,after down expression of miR-124-3p,colony formation were significantly increased in the miR-124-3p inhibitor group compared with those in the mimic negative control(NC)group(p<0.05).Down expression of miR-124-3p in S18 and HK1 cells caused an increase in cyclin D3 and cyclin-dependent kinase 4 protein levels(CDK4).4.miR-124-3p inhibits xenograft tumor growth in a mouse xenograft model.Compared with the control group,miR-124-3p overexpression resulted in a smaller size of the subcutaneous tumor in mice.And the mean tumor volume for miR-124-3p overexpression group was significantly smaller than NC group.Additionally,the weight of tumor tissue was lower in the miR-124-3p overexpression group than that of the NC group.Moreover,qRT-PCR analysis suggested that miR-124-3p expression was obviously increased in tumor tissue of miR-124-3p overexpressed nude mice.5.Prediction and validation of target genes of miR-124-3p.To explore the potential target of miR-124-3p,the target genes of miR-124-3p were investigated using TargetScan(http://www.targetscan.org/).The results showed that the 3’-UTR sequence in PCDH8 may be a binding target of miR-124-3p.Dual-luciferase assay showed that miR-124-3p targets the 3’-UTR of PCDH8,thereby regulating the transcriptional activity of PCDH8.6.miR-124-3p positively regulates the expression of PCDH8.The expression of PCDH8 in CNE2 and 6-10B cells were significantly upregulated in cells transfected with miR-124-3p mimic compared with those in the mimic NC group.The expression of PCDH8 in CNE2 and 6-10B cells were significantly downregulated in cells transfected with miR-124-3p inhibition compared with those in the mimic NC group.In mouse xenograft models,western blotting assay showed that the protein of PCDH8 was increased when miR-124-3p overexpressed.7.Overexpression of PCDH8 inhibits the proliferation of NPC cells.CCK8 assays showed that treatment with miR-124-3p inhibitor markedly promoted cell proliferation compared with those in the miR-124-3p inhibitor NC group.The cell proliferation was inhibited in the infected miR-124-3p inhibitor and PCDH8 plasmid group compared with those in the inhibitor vector.Colony-forming assays showed that the number of cell clones was significantly inhibited in the infected miR-124-3p inhibitor and PCDH8 plasmid group compared with those in the inhibitor vector(P<0.05).8.The expression of miR-124-3p in patients with NPC was detected by in situ hybridization。Correlation analysis of PCDH8 expression with clinical pathological features of NPC.There was no significant difference between PCDH8 expression and age,gender,T stage,N stage and clinical stage of NPC patients(p>0.05).The Kaplan-Meier method was used for prognostic analysis to explore the relationship between miR-124-3p expression and prognosis.Compared with patients with lower expression of PCDH8,NPC patients with higher expression of PCDH8 had significantly longer 5-year OS and 5-year RFS.Cox univariate and multivariate regression analysis showed that PCDH8 expression,gender,age were not correlated with overall survival and recurrence-free survival(p>0.05);while clinical stage was significantly associated with overall survival and recurrence-free survival(p<0.05).9.miR-124-3p inhibited NPC cell proliferation and growth by inactivating the PI3K/AKT/mTOR signaling pathway.Notably,PI3K,phospho-AKT(Thr308),phospho-mTOR(Ser2448),p70 S6 kinase,S6,4E-BP1,and phospho-4E-BP1(Thr37/46)levels decreased after miR-124-3p overexpression in CNE2 and 6-1 OB cells.In contrast,after inhibition of miR-124-3p expression in S18 and HK1 cells,PI3K,phospho-AKT(Thr308),phospho-mTOR(Ser2448),p70 S6 kinase,S6,4E-BP1,and phospho-4E-BP1(Thr37/46)levels were increased compared with those in the NC group.Conclusions1.miR-124-3p was significantly downregulated in NPC cell lines,miR-124-3p were positively correlated with the prognosis.2.Overexpression of miR-124-3p decreased NPC cell proliferation and colony formation abilities in vitro and in vivo.3.Overexpression of PCDH8 can reverse the effect of mir-124-3p inhibitor on the proliferation of NPC cell.4.miR-124-3p played a tumor suppressor by directly interacting with PCDH8 and inhibiting the activation of the phosphatidylinositol 3-kinase(PI3K)/AKT/mammalian target of rapamycin(mTOR)signaling pathway. |
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